Combination therapy for treatment of cancer

Abstract

To provide a novel improved therapeutic approach for the treatment of cancer.SOLUTION: The invention relates to a combination of an immuno-oncology agent with Omomyc, a functionally equivalent variant thereof, a conjugate comprising Omomyc or the functionally equivalent variant, a polynucleotide encoding the polypeptides, a vector comprising the polynucleotide and a cell capable of secreting the polypeptide or the conjugate. The invention also relates to pharmaceutical compositions containing the combination of the invention and to their medical uses, particularly their uses in the treatment of cancer.SELECTED DRAWING: None

Description

[Technical field]

[0001] The present invention relates to the field of cancer, more particularly to a combination comprising a polypeptide and a cancer immunotherapy agent, and to its use in the field of medicine, more particularly in the prevention and / or treatment of cancer. [Background technology]

[0002] An ideal anticancer drug should target non-redundant functions that are persistently necessary for tumor maintenance but dispensable for the maintenance and function of any normal tissue. Thus, the most common logic is to target gene products that are specifically mutated in cancer, based on the idea that these mutant molecules are likely the "drivers" of cancer and are probably not of great importance to normal tissues. For these reasons, much attention has been directed to classifying recurrent lesions in specific cancer types. Unfortunately, this approach has several problems. First, most human solid tumors undergo episodes of genomic instability and exhibit mutational noise that can obscure "driver" mutations and their associated effector pathways. Second, cancer is the end result of a process that involves transitions through multiple evolutionary bottlenecks. Each bottleneck may require a specific type of mutation, but those functions are then dispensable for tumor maintenance and, as a result, are not good therapeutic targets after that point in the tumor's development.

[0003] Myc is a basic helix-loop-helix leucine zipper (b-HLH-LZ) protein involved in growth control and cancer that operates in a network with the structurally related proteins Max, Mad, and Mnt. Myc / Max dimers activate gene transcription and induce cell proliferation or apoptosis. Mad / Max and Mnt / Max complexes function as repressors, causing cell growth arrest and differentiation. All dimers recognize the same DNA consensus site, the CACGTG E-box.

[0004] Myc is tightly regulated in normal cells, with levels higher in proliferating cells and lower in non-proliferating cells. Abnormally high and / or deregulated Myc activity is causally linked to most cancers and is often associated with aggressive, poorly differentiated, angiogenic tumors. Deregulation of Myc expression results from overexpression due to gene amplification, loss of transcriptional control, impaired degradation, or increased stabilization. This results in abnormal proliferation, increased survival, altered metabolism, angiogenesis, and inflammation, all of which represent key features of cancer. Multiple studies have demonstrated a critical role for Myc in controlling intracellular and extracellular aspects of tumorigenesis, suggesting that targeting Myc function may be therapeutically valuable.

[0005] It is known that downregulation of Myc by BET bromodomain inhibitors results in the remission of multiple tumor types. While this approach shows good potential, it also presents some limitations, such as toxicity and numerous off-target effects. Many small molecules that disrupt Myc / Max interactions have shown low specificity in cells.

[0006] However, Myc inhibitors are not yet clinically available, and their design presents various caveats: first, Myc is a nuclear transcription factor and, as a result, is more difficult to reach than membrane or cytoplasmic molecules; second, Myc does not have an enzymatic "active site" that can be targeted; and third, the Myc family includes three distinct proteins, c-Myc, N-Myc, and L-Myc, which are functionally redundant under certain conditions, and therefore all of them require simultaneous inhibition. Moreover, there have been concerns that Myc inhibition may induce severe side effects by inhibiting the proliferation of normal cells. For all these reasons, the creation of Myc inhibitors is a challenging task.

[0007] Omomyc is a dominant negative MYC mutant that contains the b-HLH-LZ domain of Myc and has four amino acid substitutions in the leucine zipper of Myc (Soucek, L. et al., 1998, Oncogene 17, 2463-2472; Soucek, L. et al. (2002), Cancer Res 62: 3507-3510). The amino acid substitutions E61T, E68I, R74Q, and R75N ​​confer altered dimerization specificity to the protein, which retains the ability to bind its natural partner Max and to form homodimers with itself, as well as to form heterodimers with wild-type c-Myc, N-Myc, and L-Myc.

[0008] These properties allow Omomyc to prevent Myc-dependent gene transactivation function both in vitro and in vivo by abolishing the ability of Myc to bind to its DNA recognition binding site E box. At the same time, Omomyc strongly enhances Myc-induced apoptosis in a manner that depends on the Myc expression level, thereby enhancing Myc transrepression activity. Thus, Omomyc prevents Myc from binding to promoter E-boxes and transactivating target genes while retaining Miz-1-dependent binding to promoters and transrepression. In the presence of Omomyc, the Myc interactome opens a channel to repression and its activity switches from pro-oncogenic to tumor suppressive.

[0009] In EP2801370A1 it was demonstrated that the Omomyc peptide itself can be efficiently transduced across the cell membrane as well as translocate to the nucleus where it exerts its tumor suppressor effects. Summary of the Invention

[0010] However, given the current state of the art, there remains a need to develop new and improved therapeutic approaches for the treatment of cancer.

[0011] In a first aspect, the present invention provides a method for producing ... composition comprising the steps of: i) the group consisting of: a) a polypeptide comprising the sequence of SEQ ID NO:1 or a functionally equivalent variant thereof; b) a conjugate comprising a polypeptide comprising the sequence of SEQ ID NO:1 or a functionally equivalent variant thereof and a chemical moiety that promotes the cellular uptake of said polypeptide or a functionally equivalent variant thereof; c) a polynucleotide encoding the polypeptide of a) or the conjugate of b); d) a vector comprising the polynucleotide of c), and e) a cell capable of secreting the polypeptide of a) or the conjugate of b) into the culture medium; and a first component selected from ii) a second component that is an immuno-oncology agent; The present invention relates to a combination comprising:

[0012] In a second aspect, the present invention relates to a pharmaceutical composition comprising a pharma- ceutically effective amount of the combination according to the invention and a pharma- ceutically acceptable excipient.

[0013] In a third aspect, the present invention relates to a combination according to the invention or a pharmaceutical composition according to the invention for use in medicine.

[0014] In a fourth aspect, the present invention relates to a combination according to the invention or a pharmaceutical composition according to the invention for use in the prevention and / or treatment of cancer. [Brief description of the drawings]

[0015] [Figure 1]Intranasal administration of Omomyc recruits T cells to tumor sites. Mice bearing KRasG12D-driven NSCLC were treated with Omomyc intranasally 4 times per week for 4 weeks. (A) Omomyc administration induced T cell recruitment to the tumor site as early as 1 week after treatment initiation, where T cells remained throughout treatment. * represents p<0.05; ** represents p<0.01. (B) FACS analysis showing that Omomyc induced recruitment of CD4 T cells to tumors, and in particular activated CD4 T cells that displayed higher levels of PD-1 and both PD-1 and Tim-3 molecules. Omomyc also induces the expression of T regulatory cells (Tregs). [Diagram 2] Systemic administration of Omomyc recruits T cells to the tumor site. Syngeneic mice were inoculated subcutaneously with Kras / p53 mutant NSCLC MuH-163 cell lines. Mice were treated systemically with Omomyc for 3 weeks. Omomyc recruited more CD4+ T cells to the tumor site (A) compared to their vehicle counterparts (B), and significantly more CD4 and CD8 T cells expressed both PD-1 and Tim-3 molecules. ** indicates p<0.01. [Diagram 3] Omomyc in combination with anti-PD-1 recruits CD4+PD-1+Tim-3- T cells to tumors. Mice bearing KRasG12D-driven NSCLC were treated intranasally with Omomyc four times a week and intraperitoneally with anti-PD-1 (250 μg) once a week for four weeks. Omomyc in combination with anti-PD-1 induced recruitment of CD4+PD-1+Tim-3- T cells to tumor sites. [Figure 4] Omomyc in combination with anti-PD-1 induces IFN-γ production. Combination therapy with Omomyc and anti-PD-1 induced IFN-γ production by intratumoral CD4 (A) and CD8 (B) T cells. [Diagram 5]Combination of Omomyc with anti-PD-1 antibody synergistically increases the percentage of healthy lungs and recruits T cells to tumor sites. Mice with KRasG12D-driven NSCLC were treated with Omomyc intranasally four times a week and anti-PD-1 antibody intraperitoneally once a week. (A) Animals treated with Omomyc in combination with anti-PD-1 showed an increased percentage of healthy lungs compared to vehicle and treatment alone. (B) Representative transverse plane CT images from each experimental group taken at the start of treatment and at the endpoint. Dark areas correspond to healthy lungs and grey areas correspond to affected lungs. (C) FACS analysis showed that Omomyc and anti-PD-1 administered in combination induced T cell recruitment to tumor sites, especially CD4 T cells and Th1 / Th17c cells. * represents p<0.05; ** represents p<0.01; *** represents p<0.0001. [Figure 6] Combination of Omomyc with anti-CTLA-4 antibody synergistically reduces tumor growth and recruits anti-tumor T cells to the tumor site. Mice with KRasG12D-driven NSCLC were treated with Omomyc intranasally four times a week and anti-CTLA-4 antibody intraperitoneally once a week. (A) Animals treated with Omomyc in combination with anti-CTLA-4 showed reduced tumor growth compared to vehicle and treatment alone. Table shows the mean tumor growth for all treatment groups. (B) FACS analysis showed that Omomyc and anti-CTLA-4 administered in combination induced T cell recruitment to the tumor site, specifically both CD4+ and CD8PD-1+ T cells. * represents p<0.05; ** represents p<0.01; *** represents p<0.0001. [Figure 7]Sequential combination of Omomyc with anti-PD-1 antibody synergistically recruits anti-tumor T cells to tumor sites. Mice bearing KRasG12D-driven NSCLC were treated with Omomyc intravenously once every 4 days for 10 days, and then with anti-PD-1 antibody intraperitoneally once a week. FACS analysis showed that sequential treatment with Omomyc and then anti-PD-1 induced T cell recruitment to tumor sites, particularly CD4 T cells expressing both PD-1 and Tim-3 molecules, and Th1 / Th17 T cells expressing PD-1. * represents p<0.05; ** represents p<0.01. [Figure 8] Combination of Omomyc with anti-PD-1 antibody synergistically recruits T cells to tumor sites. Mice with KRasG12D / p53-driven NSCLC were simultaneously treated with Omomyc (intravenously) and anti-PD-1 (intraperitoneally) once a week. (A) IHC staining showed that co-treatment of Omomyc and anti-PD-1 significantly recruited T cells to tumor sites. (B) FACS analysis showed that treatment with Omomyc with anti-PD-1 induced total immune cell recruitment to tumor sites. * represented p<0.05; ** represented p<0.01. [Figure 9] High expression of CD3, CD4, IL-17, and IFN-γ correlates with higher survival. Representative Kaplan-Meier curves for NSCLC patients considering expression of CD3, CD4, IL-17, and IFN-γ. The table below the graph shows the survival in the upper quartile. Graphs were generated using the Kaplan-Meier plotter http: / / kmplot.com / analysis / index.php?p=background. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0016] The present invention relates to the provision of novel therapeutic combinations for the prevention and treatment of cancer.

[0017] Unless otherwise defined, all technical terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.

[0018] All embodiments disclosed in relation to one aspect of the invention are also applicable to the other aspects.

[0019] Combinations and pharmaceutical compositions of the present invention The definitions provided in this aspect, together with all other aspects of the invention, are equally applicable to the invention as a whole.

[0020] We demonstrated that intranasal and systemic administration of Omomyc recruited T cells to tumor sites (Figures 1 and 2). Thus, Omomyc may be useful in the treatment of cancer in combination with cancer immunotherapy. Moreover, the combination of Omomyc and cancer immunotherapy was found to have a synergistic effect in the treatment of cancer. For example, the combination of Omomyc and anti-PD-1 therapy significantly increased the recruitment of PD-1-expressing CD4+ T cells, but not Tim-3, to tumor sites compared with both vehicle and anti-PD-1 only treated groups (Figure 3). Furthermore, the combination of Omomyc and anti-PD-1 therapy significantly induced the production of interferon-γ (IFN-γ) by both CD4+ helper and CD8+ cytotoxic intratumoral T cells compared with their vehicle counterparts (Figure 4), which was not observed in either the Omomyc or anti-PD-1 treated groups. Recruitment of T cells to tumor sites results in a synergistic increase in the proportion of healthy lungs when subjects with lung cancer are treated (Figure 5). This synergistic effect is maintained regardless of route of administration, dose, and dosing regimen (Figures 7 and 8). It was also found that the combination of Omomyc with anti-CTLA-4 therapy synergistically reduced tumor growth and recruited anti-tumor T cells to tumor sites (Figure 6).

[0021] Thus, in a first aspect, the present invention comprises: i) the group consisting of: a) a polypeptide comprising the sequence of SEQ ID NO:1 or a functionally equivalent variant thereof; b) a conjugate comprising a polypeptide comprising the sequence of SEQ ID NO:1 or a functionally equivalent variant thereof and a chemical moiety that promotes the cellular uptake of said polypeptide or a functionally equivalent variant thereof; c) a polynucleotide encoding the polypeptide of a) or the conjugate of b); d) a vector comprising the polynucleotide of c), and e) a cell capable of secreting the polypeptide of a) or the conjugate of b) into the culture medium. and a first component selected from ii) a second component that is an immuno-oncology agent; The present invention relates to a combination comprising:

[0022] According to the present invention, the expression "combination" refers to various combinations of compounds (i) and (ii), for example in compositions formulated as a single preparation, in combined mixtures composed of separate preparations of each component, such as "tank mixes" that can be combined for combined use as a combined preparation, as well as in combined use of single active ingredients in a continuous manner, i.e., when applied alternately in a reasonably short period of time, such as a few hours or days, or in simultaneous administration. In the present invention, compound (i) means a therapeutically effective amount of a polypeptide comprising the sequence of SEQ ID NO: 1 or a functionally equivalent variant thereof, or a conjugate comprising a polypeptide comprising the sequence of SEQ ID NO: 1 or a functionally equivalent variant thereof and a chemical moiety that promotes the intracellular uptake of said polypeptide or said functionally equivalent variant thereof, or a polynucleotide that codes for said polypeptide or said conjugate, or a vector that comprises said polynucleotide, or a cell that can secrete said polypeptide or said conjugate into the medium. In the present invention, compound (ii) means a therapeutically effective amount of a cancer immunotherapy agent. Preferably, the order of application of compounds (i) and (ii) is not essential in working the present invention.

[0023] The combination may be a kit-of-part, where each component is formulated or packaged separately.

[0024] The combination of compounds (i) and (ii) can be formulated for simultaneous, separate, or sequential administration. In particular, when administration is not simultaneous, the compounds are administered in close time proximity to each other. Moreover, the compounds can be administered in the same or different dosage forms or by the same or different administration routes, for example, one compound can be administered orally and the other compound can be administered intravenously. Preferably, compound (i) is administered intranasally and compound (ii) is administered systemically, more preferably parenterally, even more preferably intraperitoneally. In another embodiment, compound (i) is administered intravenously and compound (ii) is administered parenterally, even more preferably intraperitoneally.

[0025] The combination of the two compounds (i) and (ii) is - as a combination, which is part of the same pharmaceutical formulation and in which the two compounds are usually administered simultaneously; - each substance may be administered together with the other as a combination of two units allowing for simultaneous, sequential or separate administration.

[0026] In a particular embodiment, compound (i) of the combination of the invention is administered independently from compound (ii), ie in two units, but simultaneously.

[0027] In another particular embodiment, compound (i) of the combination of the invention is administered first, followed by compound (ii), i.e. compound (ii) is administered separately or sequentially.

[0028] In yet another specific embodiment, compound (ii) of the combination of the present invention is administered first, and then compound (i), i.e., compound (i) is administered separately or sequentially, as defined.When administered separately, compounds (i) and (ii) of the combination of the present invention can be administered within a period of time from each other, for example, within 1 hour, within 2 hours, within 3 hours, within 4 hours, within 5 hours, within 6 hours, within 7 hours, within 8 hours, within 9 hours, within 10 hours, within 11 hours, within 12 hours, within 13 hours, within 14 hours, within 15 hours, within 16 hours, within 17 hours, within 18 hours, within 19 hours, within 20 hours, within 21 hours, within 22 hours, within 23 hours, or within 24 hours from each other. In another embodiment, compound (i) and (ii) of the combination of the present invention can be administered within 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, 14 days, 15 days, 16 days, 17 days, 18 days, 19 days, 20 days, 21 days, 22 days, 23 days, or 24 days from each other, preferably within 1 day from each other, more preferably within 10 days from each other.In a preferred embodiment, compound (ii) is administered 10 days after the first administration of compound (i).In some embodiments, the administration of the first compound is stopped before starting the administration of the second compound.

[0029] In another aspect, the present invention relates to a combination or pharmaceutical composition comprising a synergistically effective amount of a first compound according to the first aspect of the invention and a cancer immunotherapy agent.

[0030] In a preferred embodiment, the compound (i) of the invention is a polypeptide comprising the sequence of SEQ ID NO: 1 or a functionally equivalent variant thereof.

[0031] The terms "polypeptide" and "peptide" are used interchangeably herein to mean a polymer of amino acids of any length. The polypeptide of the invention may contain modified amino acids and it may be interrupted by non-amino acids. In a preferred embodiment, the polypeptide is exclusively formed by amino acids. Preferably, the polypeptide forming item (i) of the combination has a length of between 80 and 500 amino acids, more preferably between 80 and 300 amino acids, more preferably between 80 and 250 amino acids, more preferably between 80 and 150 amino acids, even more preferably between 80 and 130 amino acids, preferably between 90 and 130 amino acids, preferably not more than 125 amino acids, more preferably not more than 100 amino acids. In a preferred embodiment, the polypeptide has a length of between 90 and 98 amino acids, preferably between 90 and 95 amino acids, more preferably 91 amino acids.

[0032] The term "amino acid" refers to naturally occurring and synthetic amino acids, as well as amino acid analogs and amino acid mimetics that function in a manner similar to the naturally occurring amino acids. Moreover, the term "amino acid" encompasses both D- and L-amino acids (stereoisomers). Preferably, the amino acids are L-amino acids.

[0033] The term "natural amino acids" or "naturally occurring amino acids" includes the 20 naturally occurring amino acids; amino acids that are often post-translationally modified in vivo, such as, for example, hydroxyproline, phosphoserine, and phosphothreonine; and other unusual amino acids, such as, but not limited to, 2-aminoadipic acid, hydroxylysine, isodesmosine, norvaline, norleucine, and ornithine.

[0034] As used herein, the term "unnatural amino acid" or "synthetic amino acid" refers to a carboxylic acid or a derivative thereof that is structurally related to the natural amino acids, but is substituted with an amino group at position "a". Illustrative non-limiting examples of modified or uncommon amino acids include 2-aminoadipic acid, 3-aminoadipic acid, β-alanine, 2-aminobutyric acid, 4-aminobutyric acid, 6-aminocaproic acid, 2-aminoheptanoic acid, 2-aminoisobutyric acid, 3-aminoisobutyric acid, 2-aminopimelic acid, 2,4-diaminobutyric acid, desmosine, 2,2′-diaminopimelic acid, 2,3-diaminopropionic acid, N-ethylglycine, N-ethylasparagine, hydroxylysine, allohydroxylysine, 3-hydroxyproline, 4-hydroxyproline, isodesmosine, alloisoleucine, N-methylglycine, N-methylisoleucine, 6-N-methyl-lysine, N-methylvaline, norvaline, norleucine, ornithine, and the like.

[0035] The polypeptides of the present invention may also include non-amino acid moieties attached to the peptide, such as hydrophobic moieties (various linear, branched, cyclic, polycyclic, or heterocyclic carbohydrates and carbohydrate derivatives); various protecting groups attached to the termini of the compounds to reduce degradation. Suitable protecting functional groups are described in Green and Wuts, "Protecting Groups in Organic Synthesis", John Wiley and Sons, Chapters 5 and 7, 1991.

[0036] Chemical (non-amino acid) groups present in the polypeptide may be included to improve various physiological properties, such as reduced degradation or clearance; reduced repulsion by various cellular pumps, improved modes of administration, increased specificity, increased affinity, increased stability, bioavailability, solubility, reduced toxicity, etc.

[0037] "Mimetic" includes molecules that mimic the chemical structure of a peptide structure and retain the functional properties of that peptide structure. Approaches to designing peptide analogs, derivatives, and mimetics are known in the art.

[0038] In one embodiment, the polypeptide of the present invention is a polypeptide consisting of SEQ ID NO: 1 or a functionally equivalent variant of SEQ ID NO: 1, preferably a polypeptide consisting of the sequence of SEQ ID NO: 1. SEQ ID NO: 1 is TEENVKRRTHNVLERQRRNELKRSFFALRDQIPELENNEKAPKVVILKKATAYILSVQAETQKLISEIDLLRKQNEQLKHKLEQLRNSCA (SEQ ID NO: 1) Corresponds to.

[0039] The polypeptide of sequence SEQ ID NO: 1 corresponds to the Omomyc protein sequence. The term "Omomyc" as used herein is a polypeptide consisting of a mutated version of the bHLHZip domain of Myc with the mutations E61T, E68I, R74Q, and R75N ​​(wherein the numbering of the mutation positions is given with respect to the sequence of the Myc region corresponding to amino acids 365 to 454 of the polypeptide defined in the NCBI database under accession number NP_002458, released on March 15, 2015). The sequence of c-Myc provided in the NCBI database under accession number NP_002458 is shown below (SEQ ID NO: 2), where the region from which Omomyc is derived is underlined: TIFF2025063152000001.tif62161

[0040] Omomyc also contains the M2 domain of c-Myc (see Dang and Lee, Mol. Cell. Biol., 1988, 8:4048-4054), which has the sequence RQRRNELKRSF (sequence number 3) (double underlined above), which corresponds to a nuclear localization signal.

[0041] Omomyc is characterized in that it exhibits increased dimerization ability with all three oncogenic Myc proteins (c-Myc, N-Myc, and L-Myc). Omomyc can be derived from the bHLHZip domain of any Myc protein known in the art, as long as the mutation resulting in tumor suppression effect is preserved. Thus, the Omomyc that can be used in the present invention can be derived from any mammal, such as, but not limited to, livestock and farm animals (cow, horse, pig, goat, sheep, dog, cat, or rodent), primate, and human. Preferably, the Omomyc protein can be derived from human Myc protein (accession number NP_002458, released March 12, 2019).

[0042] The term "Myc" as used herein refers to a family of transcription factors including c-Myc, N-Myc, and L-Myc. Myc protein activates the expression of many genes by binding at the consensus sequence CACGTG (which recruits enhancer box sequence or E-box and histone acetyl-transferase or HAT). However, Myc can also function as a transcription repressor. It inhibits the expression of Miz-1 target genes by binding Miz-1 transcription factor and displacing p300 coactivator. Myc also has a direct role in controlling DNA replication.

[0043] Myc b-HLH-LZ or Myc basic region helix-loop-helix leucine zipper domain refers to the region that determines Myc dimerization with Max protein and binding to Myc-target genes. This region corresponds to amino acids 365-454 of human Myc and is characterized by two alpha helices connected by a loop (Nair, SK, & Burley, SK, 2003, Cell, 112: 193-205).

[0044] In a preferred embodiment, the polypeptide of the present invention is a polypeptide comprising, consisting of, or consisting essentially of SEQ ID NO:4 as shown below. MTEENVKRRTHNVLERQRRNELKRSFFALRDQIPELENNEKAPKVVILKKATAYILSVQAETQKLISEIDLLRKQNEQLKHKLEQLRNSCA (SEQ ID NO: 4)

[0045] In this context, "consisting essentially of" means that the specified molecule does not contain any additional sequences that alter the activity of SEQ ID NO:4.

[0046] Preferably, the polypeptide consists of SEQ ID NO:4.

[0047] The term "functionally equivalent variant" refers to any polypeptide resulting from the insertion or addition of one or more amino acids and / or the deletion of one or more amino acids and / or the conservative substitution of one or more amino acids with respect to the polypeptide of SEQ ID NO: 1, and / or any polypeptide resulting from chemical modification of the polypeptide of SEQ ID NO: 1 and which substantially preserves the tumor suppressor activity of SEQ ID NO: 1. Preferably, functionally equivalent variant refers to any polypeptide resulting from the insertion or addition of one or more amino acids and / or the deletion of one or more amino acids and / or any polypeptide resulting from the conservative substitution of one or more amino acids with respect to the polypeptide of SEQ ID NO: 1 and which substantially preserves the tumor suppressor activity of SEQ ID NO: 1, more preferably any polypeptide resulting from the insertion or addition of one or more amino acids with respect to the polypeptide of SEQ ID NO: 1.

[0048] Those skilled in the art will understand that preservation of tumor suppressor activity requires that the variant can dimerize with Myc and / or its essential partner p21 / p22Max and inhibit Myc activity, that it can translocate across the cell membrane, and that it can translocate across the nuclear envelope. In some embodiments, functionally equivalent variants of the polypeptides of the present invention do not homodimerize as much as Omomyc, or are not forced into homodimers by disulfide bond formation. In particular, disulfide bridge formation in the homodimeric form of certain embodiments of the polypeptides of the present invention is less than in the polypeptide Omomyc.

[0049] "Less homodimerization" as used herein relates to a lower ability of the polypeptide of the invention to form homodimers even under reducing conditions. In a preferred embodiment, the ability is at least 5% less, at least 10% less, at least 15% less, at least 20% less, at least 25% less, at least 30% less, at least 35% less, at least 40% less, at least 45% less, at least 50% less, at least 55% less, at least 60% less, at least 65% less, at least 70% less, at least 75% less, at least 80% less, at least 85% less, at least 90% less, at least 95% less than the ability of Omomyc to form homodimers.

[0050] As used herein, reducing conditions refer to the presence of a reducing agent, i.e., a compound that donates electrons to another chemical species in a redox chemical reaction. Illustrative non-limiting examples of reducing agents are DTT (dithiothreitol), b-mercaptoethanol, or TCEP (tris(2-carboxyethyl)phosphine). It is possible that the amount of homodimers is the same in vitro, and the difference between functionally equivalent variants and Omomyc is only present in cells in the presence of heterodimerization partners, where the absence of disulfides allows for a higher potential formation of heterodimers.

[0051] As an illustrative, non-limiting example, several assays can be used to identify peptide homodimerization by thermal denaturation monitored by circular dichroism, so that dimerization can be detected by quantification of folding and thermal stability.

[0052] Suitable functionally equivalent variants include polypeptides that consist essentially of the polypeptide of SEQ ID NO: 1. In this context, "consisting essentially of" means that the specified molecule does not contain any additional sequences that would alter the activity of SEQ ID NO: 1.

[0053] In a preferred embodiment, a functionally equivalent variant of SEQ ID NO: 1 is a polypeptide resulting from the insertion or addition of one or more amino acids with respect to the polypeptide of SEQ ID NO: 1. In one embodiment, the functionally equivalent variant results from the insertion of less than 10 amino acids, more preferably less than 5 amino acids, more preferably from the insertion of one amino acid. In a preferred embodiment, the functionally equivalent variant results from the insertion of one amino acid, which is methionine.

[0054] In another embodiment, a functionally equivalent variant of SEQ ID NO: 1 is a polypeptide resulting from the deletion of one or more amino acids with respect to the polypeptide of SEQ ID NO: 1. In one embodiment, the functionally equivalent variant results from the deletion of fewer than 10 amino acids, more preferably fewer than 5 amino acids, and more preferably from the deletion of one amino acid.

[0055] Suitable functional variants of targeting peptides are those that exhibit a degree of identity with respect to the peptide of SEQ ID NO: 1, such as greater than about 25% amino acid sequence identity, for example, 25%, 30%, 40%, 50%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% amino acid sequence identity. The degree of identity between two polypeptides is determined using computer algorithms and methods well known to those of skill in the art. Preferably, the identity between two amino acid sequences is determined by using the BLASTP algorithm described above (BLAST Manual, Altschul, S., et al., NCBI NLM NIH Bethesda, Md. 20894, Altschul, S., et al., J. Mol. Biol. 1990;215: 403-410). In a preferred embodiment, the sequence identity is determined over the entire length of the polypeptide of SEQ ID NO: 1, or over the entire length of a variant, or both.

[0056] Functionally equivalent variants of the polypeptides of the invention may also contain post-translational modifications, such as glycosylation, acetylation, isoprenylation, myristoylation, proteolytic processing, and the like.

[0057] In another embodiment, suitable functional variants of targeting peptides are those in which one or more positions in the polypeptide of the present invention contain amino acids that are conservative substitutions of the amino acids present in the proteins mentioned above. "Conservative amino acid substitutions" are made by replacing one amino acid with another amino acid that has similar structure and / or chemical properties. For example, each of the following six groups contains amino acids that are conservative substitutions for each other: 1) alanine (A), serine (S), threonine (T); 2) aspartic acid (D), glutamic acid (E); 3) asparagine (N), glutamine (Q); 4) arginine (R), lysine (K); 5) isoleucine (I), leucine (L), methionine (M), valine (V); and 6) phenylalanine (F), tyrosine (Y), tryptophan (W). The selection of such conservative amino acid substitutions is within the skill of the art and is described, for example, by Dordo et al., (J. Mol. Biol, 1999, 217;721-739) and Taylor et al., (J. Theor. Biol., 1986, 119:205-218).

[0058] It will be understood that functionally equivalent variants of Omomyc contain mutations at positions corresponding to the mutations E61T, E68I, R74Q, and R75N ​​found in Omomyc derived from human c-Myc. The positions at which mutations must occur in the functionally equivalent variants can be identified by multiple sequence alignment of the various Myc sequences, and by alignment of positions corresponding to positions 61, 68, 74, and 75 in the sequence of Omomyc derived from human c-Myc. In one embodiment, a functionally equivalent variant of Omomyc contains mutations at positions corresponding to the mutations E61T, E68I, R74Q, and R75N ​​found in Omomyc derived from human c-Myc.

[0059] In another embodiment, a functionally equivalent variant of Omomyc contains mutations at positions corresponding to E61, E68, R74, and R75 within the Omomyc sequence, where E61 is mutated to E61A or E61S; E68 is mutated to E68L, E68M, or E68V; R74 is mutated to R74N; and R75 is mutated to R75Q.

[0060] Multiple sequence alignment is an extension of pairwise alignment to incorporate more than two sequences at a time. Multiple alignment methods align all of the sequences in a given query set. Preferred multiple sequence alignment programs (and their algorithms) are ClustalW, Clustal2W, or ClustalW XXL (see Thompson et al. (1994) Nucleic Acids Res 22:4673-4680). Once the sequences of c-Myc from different organisms or variants are compared (aligned) as described herein, a skilled technician can easily identify the positions in each sequence that correspond to the positions E61T, E68I, R74Q, and R75N ​​found in Omomyc, and can introduce mutations into Omomyc variants that correspond to the mutations E61T, E68I, R74Q, and R75N ​​found in Omomyc from human c-Myc.

[0061] Suitable assays for whether a polypeptide can be considered a functionally equivalent variant of Omomyc include, but are not limited to, the following. - assays measuring the ability of the polypeptide to form dimeric complexes with Max and Myc, such as, for example, reporter gene expression-based assays as described by Soucek et al. (Oncogene, 1998, 17: 2463-2472), as well as PLA (protein ligation assay) or co-immunoprecipitation methods, - an assay that measures the ability of a polypeptide to bind to Myc / Max recognition sites (CACGTG sites) in DNA, such as the electrophoretic mobility shift assay (EMSA) described by Soucek et al. (supra); - an assay measuring the ability to inhibit Myc-induced transactivation, such as, for example, an assay based on the expression of a reporter gene under the control of a Myc / Max specific DNA binding site as described by Soucek et al. (supra), - an assay based on the ability of the polypeptide to inhibit the proliferation of cells expressing the Myc oncogene, as described by Soucek et al. (supra); - Assays that measure the ability of a polypeptide to enhance Myc-induced apoptosis, such as the assay described by Soucek et al. (Oncogene, 1998: 17, 2463-2472). In addition, any assay commonly known in the art for assessing apoptosis in cells can be used, such as Hoechst staining, propidium iodide (PI) or Annexin V staining, trypan blue, DNA laddering / fragmentation, and TUNEL.

[0062] In preferred embodiments, a polypeptide is considered to be a functionally equivalent variant of Omomyc if it exhibits at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or 100% of the activity of the original Omomyc in one or more of the above assays.

[0063] In certain embodiments, functionally equivalent variants of the polypeptide of SEQ ID NO:1 include the polypeptide of SEQ ID NO:1 in which residue X at position 89 of SEQ ID NO:1 is not a cysteine. Preferably, residue X at position 89 of SEQ ID NO:1 is an aliphatic amino acid, or a sulfurized amino acid, or a dicarboxyl amino acid or its amide, or an amino acid having two basic groups, or an aromatic amino acid, or a cyclic amino acid, or a hydroxylated amino acid. More preferred is an amino acid selected from serine, threonine, and alanine, preferably serine and alanine.

[0064] Preferred functionally equivalent variants of SEQ ID NO:1 having a residue X at position 89 of SEQ ID NO: which is not a cysteine ​​are disclosed in the table below.

[0065] [Table 1]

[0066] Thus, in a preferred embodiment, a functionally equivalent variant of the polypeptide of SEQ ID NO:1 is selected from the group consisting of SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, and SEQ ID NO:10.

[0067] Furthermore, functionally equivalent variants of Omomyc are also capable of transducing cells after the variant contacts the cells, It will be understood that functionally equivalent variants of Omomyc include the protein transduction domain found in native Omomyc or another functional protein transduction domain.

[0068] In preferred embodiments, a polypeptide is considered to be a functionally equivalent variant of SEQ ID NO:1 if it is capable of transducing target cells at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or 100% as efficiently as SEQ ID NO:1.

[0069] Furthermore, functionally equivalent variants of SEQ ID NO:1 are also capable of translocating to the nucleus of targeted tumor cells.

[0070] In preferred embodiments, a polypeptide is considered to be a functionally equivalent variant of SEQ ID NO:1 if it is capable of translocating into the nucleus of a target tumor cell at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or 100% as efficiently as SEQ ID NO:1.

[0071] A suitable assay for identifying whether a polypeptide is a functionally equivalent variant of SEQ ID NO: 1 with respect to its ability to translocate across the cell membrane to the nucleus includes double labeling of cells with a reagent specific for the polypeptide and a dye that specifically labels the nucleus of the cell, such as DAPI or Hoechst dye. Detection of the polypeptide of the invention can be performed by confocal or fluorescence microscopy.

[0072] In another preferred embodiment, compound (i) of the present invention is a conjugate comprising a polypeptide comprising the sequence of SEQ ID NO:1 or a functionally equivalent variant thereof and a chemical moiety that promotes cellular uptake of the polypeptide or a functionally equivalent variant thereof.

[0073] The term "conjugate," as used herein, means two or more compounds covalently linked together in such a way that the function of each compound in the conjugate is maintained.

[0074] The term "chemical moiety" refers to any chemical compound that contains at least one carbon atom. Examples of chemical moieties include, but are not limited to, any peptide chain enriched with hydrophobic amino acids and hydrophobic chemical moieties.

[0075] In a preferred embodiment, a conjugate according to the invention comprises at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, or more chemical moieties that facilitate cellular uptake of the polypeptide or a functionally equivalent variant of the polypeptide.

[0076] In one embodiment, the chemical moiety that enhances cellular uptake of the polypeptide is a lipid or fatty acid.

[0077] Fatty acids are generally molecules that contain a carbon chain with an acidic moiety (e.g., carboxylic acid) at the end of the chain. The carbon chain of a fatty acid may be of any length, but it is preferred that the length of the carbon chain is at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or more carbon atoms, as well as any range derivable therein. In certain embodiments, the length of the carbon chain is 4 to 18 carbon atoms in the chain portion of the fatty acid. In certain embodiments, the carbon chain of the fatty acid may contain an odd number of carbon atoms, although in certain embodiments, an even number of carbon atoms in the chain may be preferred. Fatty acids that contain only single bonds in the carbon chain are said to be saturated, while fatty acids that contain at least one double bond in the carbon chain are said to be unsaturated. The fatty acid may be branched, but in a preferred embodiment of the invention, it is unsaturated. Specific fatty acids include, but are not limited to, linoleic acid, oleic acid, palmitic acid, linolenic acid, stearic acid, lauric acid, myristic acid, arachidic acid, palmitoleic acid, and arachidonic acid.

[0078] In a preferred embodiment, the chemical moiety that promotes cellular uptake of a polypeptide comprising the sequence of SEQ ID NO:1 or a functionally equivalent variant thereof is a cell membrane-permeable peptide sequence, in which case the conjugate will comprise a fusion protein comprising a polypeptide comprising SEQ ID NO:1 or a functionally equivalent variant thereof and a cell membrane-permeable peptide sequence.

[0079] The term "fusion protein" refers to a protein produced by genetic techniques, consisting of two or more functional domains from different proteins. Fusion proteins can be obtained in conventional ways, for example by gene expression of a nucleotide sequence encoding said fusion protein in a suitable cell. It will be understood that said cell membrane permeable peptide refers to a cell membrane permeable peptide that is different from the cell membrane permeable peptide that forms part of a polypeptide comprising SEQ ID NO:1 or a functionally equivalent variant of SEQ ID NO:1.

[0080] The term "cell membrane penetrating peptide sequence" is used herein interchangeably with "CPP", "protein transduction domain", or "PTD". It refers to a peptide chain of various lengths that directs the trafficking of a protein within a cell. The delivery process into the cell is generally triggered by endocytosis, but the peptide can also be internalized into the cell by direct membrane translocation. CPPs typically have an amino acid composition that contains a high relative abundance of positively charged amino acids, such as lysine or arginine, or have sequences that contain an alternating pattern of polar / charged and non-polar hydrophobic amino acids.

[0081] Examples of CPPs that can be used in the present invention include, but are not limited to, a CCP found in Drosophila antennapedia protein (RQIKIWFQNRRMKWKK, SEQ ID NO: 13), a CCP found in herpes simplex virus 1 (HSV-1) VP22 DNA binding protein (DAATATRGRSAASRPTERPRAPARSASRPRRPVE, SEQ ID NO: 14), a CPP of Bac-7 (RRIRRPRPPRLPRPRPRPLPFPRPG; SEQ ID NO: 15), and a CPP of HIV-1 consisting of amino acids 49-57 (RKKRRQRRR, SEQ ID NO: 16), amino acids 48-60 (GRKKRRQRRRTPQ, SEQ ID NO: 17), and amino acids 47-57 (YGRKKRRQRRR; SEQ ID NO: 18). CPP of TAT protein; CPP of S413-PV peptide (ALWKTLLKKVLKAPKKKRKV; SEQ ID NO: 19), CPP of penetratin (RQIKWFQNRRMKWKK; SEQ ID NO: 20), CPP of SynB1 (RGGRLSYSRRRFSTSTGR; SEQ ID NO: 21), CPP of SynB3 (RRLSYSRRRF; SEQ ID NO: 22), CPP of PTD-4 (PIRRRKKLRRLK; SEQ ID NO: 23), CPP of PTD-5 (RRQRRTSKLMKR; SEQ ID NO: 24), CPP of FHV Coat-(35-49) (RRRRNRTRRNRRRVR; SEQ ID NO: 25), CPP of BMV Gag-(7-25) (KMTRAQRRAAARRNRWTAR; SEQ ID NO: 26), CPP of HTLV-II A CPP of Rex-(4-16) (TRRQRTRRARRNR; SEQ ID NO: 27), a CPP of D-Tat (GRKKRRQRRRPPQ; SEQ ID NO: 28), a CPP of R9-Tat (GRRRRRRRRRPPQ; SEQ ID NO: 29), a CPP of MAP (KLALKLALKLALALKLA; SEQ ID NO: 30), a CPP of SBP (MGLGLHLLVLAAALQGAWSQPKKKRKV; SEQ ID NO: 31), a CPP of FBP (GALFLGWLGAAGSTMGAWSQPKKKRKV; SEQ ID NO: 32), a CPP of MPG (ac-GALFLGFLGAAGSTMGAWSQPKKKRKV-cya;CPP of MPG(ENLS) (ac-GALFLGFLGAAGSTMGAWSQPKSKRKV-cya; SEQ ID NO: 34), CPP of Pep-1 (ac-KETWWETWWTEWSQPKKKRKV-cya; SEQ ID NO: 35), CPP of Pep-2 (ac-KETWFETWFTEWSQPKKKRKV-cya; SEQ ID NO: 36), a polyarginine sequence having the structure RN (wherein N is between 4 and 17), the sequence GRKKRRQRRR (SEQ ID NO: 37), the sequence RRRRRRLR (SEQ ID NO: 38), RRQR RTSKLMKR sequence (SEQ ID NO: 39); Transportan GWTLNSAGYLLGKINLKALAALAKKIL (SEQ ID NO: 40); KALAWEAKLAKALAKALAKHLAKALAKALKCEA (SEQ ID NO: 41); RQIKIWFQNRRMKWKK (SEQ ID NO: 42), YGRKKRRQRRR sequence (SEQ ID NO: 43); RKKRRQRR sequence (SEQ ID NO: 44); YARAAARQARA sequence (SEQ ID NO: 45); THRLPRRRRRR sequence (SEQ ID NO: 46); GGRRARRRRRR sequence (SEQ ID NO: 47).

[0082] In a preferred embodiment, the cell membrane-permeable peptide is not an endogenous peptide contained in SEQ ID NO:1.

[0083] In a preferred embodiment, the CPP is a CPP of the HIV-1 TAT protein consisting of amino acids 49 to 57 (RKKRRQRRR, SEQ ID NO: 16). In another preferred embodiment, the CPP is the GRKKRRQRRR sequence (SEQ ID NO: 37) or RRRRRRLR (SEQ ID NO: 38). In another embodiment, the CPP is the GRKKRRQRRR sequence (SEQ ID NO: 37) or RRRRRRRR (SEQ ID NO: 65).

[0084] In some embodiments, the CPP is a CPP described in WO2019 / 018898, the contents of which are incorporated by reference in their entirety.

[0085] In one embodiment, the cell membrane permeable peptide sequence is fused to the N-terminus of the polypeptide of the present invention or a functionally equivalent variant of the polypeptide. In another embodiment, the cell membrane permeable peptide is fused to the C-terminus of the polypeptide of the present invention or a functionally equivalent variant of the polypeptide.

[0086] In a preferred embodiment, the conjugate or fusion protein of the combination according to the present invention comprises, in addition to the own cell membrane permeable peptide found in the polypeptide of SEQ ID NO:1 or a functionally equivalent variant of said polypeptide, at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, or more additional cell membrane permeable peptides.

[0087] A preferred fusion protein of the present invention is the polypeptide Omomycin, defined below. * TAT and Omomyc * Contains LZArg.

[0088] [Table 2]

[0089] Thus, in a preferred embodiment, the fusion protein is a polypeptide selected from SEQ ID NOs:11 and 12.

[0090] Suitable assays for determining whether a conjugate preserves the cell membrane translocation ability of Omomyc include, but are not limited to, assays that measure the ability of the conjugate to transduce cells in culture, which are based on contacting the conjugate with cultured cells and detecting the presence of the conjugate at an intracellular location.

[0091] In another preferred embodiment, the conjugate of the combination of the present invention further comprises an additional nuclear localization signal.

[0092] The term "nuclear localization signal" (NLS), as used herein, refers to an amino acid sequence of about 4 to 20 amino acid residues in length that serves to direct a protein to the nucleus. Typically, nuclear localization sequences are rich in basic amino acids, and exemplary sequences are well known in the art (Gorlich D. (1998) EMBO 5.17:2721-7). In some embodiments, the NLS is selected from the group consisting of SV40 large T antigen NLS (PKKKRKV, SEQ ID NO: 48); nucleoplasmin NLS (KRPAATKKAGQAKKKK, SEQ ID NO: 49); CBP80 NLS (RRRHSDENDGGQPHKRRK, SEQ ID NO: 50); HIV-I Rev protein NLS (RQARRNRRRWE, SEQ ID NO: 51); HTLV-I Rex (MPKTRRRPRRSQRKRPPT, SEQ ID NO: 52); hnRNP A NLS (NQSSNFGPMKGGNFGGRSSGPYGGGGQYFKPRNQGGY, SEQ ID NO: 53); rpL23a NLS (VHSHKKKKIRTSPTFTTPKTLRLRRQPKYPRKSAPRRNKLDHY, SEQ ID NO: 54). In one embodiment of the invention, the nuclear localization signal comprises the motif K(K / R)X(K / R) (SEQ ID NO: 55).

[0093] In an even more preferred embodiment, the nuclear localization signal is selected from the group consisting of PKKKRKV (SEQ ID NO: 48), PAAKRVKLD (SEQ ID NO: 56), and KRPAATKKAGQAKKKK (SEQ ID NO: 49).

[0094] In another preferred embodiment, the NLS may be N- or C-terminal to a conjugate or fusion protein comprising the polypeptide of SEQ ID NO:1 or a functionally equivalent variant thereof.

[0095] Those skilled in the art will understand that it may be desirable for the conjugate of the present invention to further comprise one or more flexible peptides connecting the polypeptide comprising SEQ ID NO:1 or its functionally equivalent variant to the cell membrane penetrating peptide sequence and / or NLS. Thus, in a particular embodiment, the polypeptide comprising the sequence of SEQ ID NO:1 or its functionally equivalent variant is directly connected to the cell membrane penetrating peptide sequence. In another particular embodiment, the polypeptide comprising the sequence of SEQ ID NO:1 or its functionally equivalent variant is connected to the cell membrane penetrating peptide sequence by a flexible peptide. In an embodiment, the polypeptide comprising SEQ ID NO:1 or its functional variant is directly connected to the NLS. In another embodiment, the polypeptide comprising the sequence of SEQ ID NO:1 or its functionally equivalent variant is connected to the NLS by a flexible peptide.

[0096] In a particular embodiment, the polypeptide of the conjugate according to the invention is directly connected to the cell membrane penetrating peptide sequence and the NLS.

[0097] In one embodiment, the NLS is one of the NLSs that appear endogenously in the Myc sequence, such as the M1 peptide (PAAKRVKLD, SEQ ID NO:56) or the M2 peptide (RQRRNELKRSF, SEQ ID NO:57).

[0098] In another embodiment, an additional NLS refers to an NLS that is different from the endogenous NLS found in a polypeptide comprising SEQ ID NO:1 or a functionally equivalent variant of SEQ ID NO:1.

[0099] In a preferred embodiment, a conjugate or fusion protein according to the invention comprises at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10 NLSs in addition to the endogenous NLSs found in the polypeptide of the invention or a functionally equivalent variant thereof.

[0100] In another particular embodiment, the polypeptide of the conjugate for use according to the invention is connected to the cell membrane penetrating peptide sequence by a first flexible peptide linker and to the NLS by a second flexible peptide linker.

[0101] As used herein, the term "flexible peptide", "spacer peptide" or "linker peptide" refers to a peptide that is covalently linked to two proteins or moieties, but is not part of either polypeptide, and allows the movement of one relative to the other without causing substantial adverse effects on the function of either of the proteins or moieties. Thus, the flexible linker does not affect the tumor tracking activity of the polypeptide sequence, the cell membrane penetrating activity of the cell membrane penetrating peptide, or the nuclear localization ability of the NLS.

[0102] The flexible peptide comprises at least 1 amino acid, at least 2 amino acids, at least 3 amino acids, at least 4 amino acids, at least 5 amino acids, at least 6 amino acids, at least 7 amino acids, at least 8 amino acids, at least 9 amino acids, at least 10 amino acids, at least 12 amino acids, at least 14 amino acids, at least 16 amino acids, at least 18 amino acids, at least 20 amino acids, at least 25 amino acids, at least 30 amino acids, at least 35 amino acids, at least 40 amino acids, at least 45 amino acids, at least 50 amino acids, at least 60 amino acids, at least 70 amino acids, at least 80 amino acids, at least 90 amino acids, or about 100 amino acids. In some embodiments, the flexible peptide will allow movement of one protein relative to the other protein to increase the solubility of the protein and / or improve its activity. Suitable linker regions include polyglycine regions, the GPRRRR sequence (SEQ ID NO:58) of a combination of glycine, proline, and alanine residues.

[0103] In a particular embodiment, the conjugate according to the invention comprises a tag attached to the conjugate or to the C-terminal or N-terminal domain of the polypeptide or fusion protein or variants thereof. Typically, the tag is a peptide or amino acid sequence that can be used in the isolation or purification of the fusion protein. Thus, the tag can bind to one or more ligands, for example, one or more ligands of an affinity matrix, such as a high affinity chromatographic support or beads. An example of such a tag is nickel (Ni 2+ ) or cobalt (Co 2+ The His tag has the desirable property of being denatured to most proteins and capable of binding to its ligand under conditions that disrupt most protein-protein interactions. It can therefore be used to remove a bait protein tagged with H6 after disruption of a protein-protein interaction in which the bait protein participates.

[0104] Additional illustrative, non-limiting examples of tags useful for isolating or purifying a conjugate or a polypeptide comprising SEQ ID NO:1 or a variant or fusion protein thereof include Arg tags, FLAG tags (DYKDDDDK; SEQ ID NO:59), Strep tags (WSHPQFEK, SEQ ID NO:60), epitopes that can be recognized by an antibody, such as a c-myc tag (recognized by an anti-c-myc antibody), HA tags (YPYDVPDYA, SEQ ID NO:61), V5 tags (GKPIPNPLLGLDST, SEQ ID NO:62), SBP tags, S tags, calmodulin-binding peptides, cellulose-binding domains, chitin-binding domains, glutathione S-transferase tags, maltose-binding proteins, NusA, TrxA, DsbA, Avi-tags, and the like (Terpe K., Appl. Microbiol. Biotechnol. 2003, 60:523-525), amino acid sequences such as AHGHRP (SEQ ID NO:63) or PIHDHDHPHLVIHSGMTCXXC (SEQ ID NO:64), and β-galactosidase.

[0105] The tag can be used to isolate or purify the fusion protein, if desired.

[0106] In another preferred embodiment, compound (i) of the present invention is a polynucleotide encoding the polypeptide or fusion protein disclosed above. In a preferred embodiment, compound (i) of the present invention is a polynucleotide encoding a polypeptide comprising the sequence of SEQ ID NO: 1 or a functionally equivalent variant thereof. In another embodiment, compound (i) of the present invention is a polynucleotide encoding a conjugate comprising a polypeptide comprising the sequence of SEQ ID NO: 1 or a functionally equivalent variant thereof and a chemical moiety that promotes the intracellular uptake of the polypeptide or its functionally equivalent variant thereof, more preferably a polynucleotide encoding a fusion protein between a polypeptide comprising the sequence of SEQ ID NO: 1 or a functionally equivalent variant thereof and a cell membrane permeable peptide sequence.

[0107] The terms "polynucleotide", "nucleic acid" and "nucleic acid molecule" are used interchangeably to mean a polymeric form of nucleotides of any length. Polynucleotides can contain deoxyribonucleotides, ribonucleotides, and / or their analogs. Nucleotides can have any three-dimensional structure and perform any function, known or unknown. The term "polynucleotide" encompasses, for example, single-stranded, double-stranded, and triple-helical molecules, genes or gene fragments, exons, introns, mRNA, tRNA, rRNA, ribozymes, cDNA, recombinant polynucleotides, branched polynucleotides, plasmids, vectors, isolated DNA of any sequence, isolated RNA of any sequence, nucleic acid probes, and primers. The nucleic acid molecules of the present invention can include modified nucleic acid molecules in addition to naturally occurring nucleic acid molecules. As used herein, mRNA means RNA that can be translated in a cell.

[0108] In a preferred embodiment, the polynucleotide of the invention is mRNA.

[0109] The mRNA can be chemically synthesized, obtained by in vitro transcription, or synthesized in vivo in the target cell. The nucleotide sequences forming the polynucleotides encoding the conjugates or fusion proteins of the invention are in the same reading frame correct for their expression.

[0110] In a preferred embodiment, component (i) of the combination of the invention is an mRNA encoding a polypeptide consisting of the sequence of SEQ ID NO: 1 or a functionally equivalent variant of SEQ ID NO: 1 or a polypeptide consisting of SEQ ID NO: 4.

[0111] In another embodiment, component (i) of the combination of the invention is a vector comprising a polynucleotide of the invention.

[0112] The term "vector" as used herein means a nucleic acid sequence comprising the necessary sequences for the production of a polypeptide encoded by a polynucleotide of the present invention after transcription and translation of the sequence in a cell. The sequence is operably linked to additional segments that provide for autonomous replication in a host cell of interest. Preferably, the vector is an expression vector, which is defined as a vector that comprises a region operably linked to the nucleic acid of the present invention in addition to a region for autonomous replication in a host cell, and is capable of enhancing the expression of the product of the nucleic acid according to the present invention. The vector of the present invention can be obtained by techniques well known in the art.

[0113] Examples of vectors include, but are not limited to, viral vectors, naked DNA or RNA expression vectors, plasmids, cosmids or phage vectors, DNA or RNA expression vectors associated with cationic condensing agents, DNA or RNA expression vectors encapsulated in liposomes, and certain eukaryotic cells, e.g. Suitable vectors comprising the polynucleotide of the invention include expression vectors in prokaryotes, such as pUC18, pUC19, pBluescript and its derivatives, mp18, mp19, pBR322, pMB9, ColE1, pCR1, RP4, phage and "shuttle" vectors, such as pSA3 and pAT28, expression vectors in yeast, such as 2 micron plasmid type vectors, integration plasmids, YEP vectors, centromeric plasmids and the like, expression vectors in insect cells, such as vectors of the pAC series and pVL series, expression vectors in plants, such as vectors of the series pIBI, pEarleyGate, pAVA, pCAMBIA, pGSA, pGWB, pMDC, pMY, pORE and the like, as well as superior eukaryotic expression vectors based on viral vectors (adenoviruses, viruses associated with adenoviruses, etc. and retroviruses, particularly lentiviruses), as well as non-viral vectors, such as vectors derived from pSilencer4.1-CMV (Ambion), pcDNA3, pcDNA3.1 / hyg, pHCMV / Zeo, pCR3.1, pEFl / His, pIND / GS, pRc / HCMV2, pSV40 / Zeo2, pTRACER-HCMV, pUB6 / V5-His, pVAXl, pZeoSV2, pCI, pSVL, pKSV-10, pBPV-1, pML2d, and pTDT1. In a preferred embodiment, the polynucleotide of the invention is comprised in a vector selected from the group consisting of pEGFP or pBabe retroviral vectors and pTRIPZ or pSLIK lentiviral vectors.

[0114] The vectors of the invention may be used to transform, transfect or infect a cell which can be transformed, transfected or infected by the vector. The cell may be a prokaryotic or eukaryotic cell.

[0115] Preferably, the vector comprises a polynucleotide of the present invention operatively linked to a sequence that regulates the expression of the polynucleotide of the present invention. The regulatory sequence for use in the present invention may be a nuclear promoter or an enhancer sequence and / or other control sequence that increases the expression of the heterologous nucleic acid sequence. In principle, any promoter can be used in the present invention, provided that it is compatible with the cell in which the polynucleotide is to be expressed. Thus, suitable promoters for implementing the present invention include, but are not necessarily limited to, constitutive promoters such as derivatives of eukaryotic viral genomes, such as polyoma virus, adenovirus, SV40, CMV, avian sarcoma virus, hepatitis B virus, metallothionein gene promoter, herpes virus thymidine kinase gene promoter, retroviral LTR regions, immunoglobulin gene promoters, actin gene promoters, EF-1α gene promoters, and inducible promoters in which protein expression is dependent on the addition of a molecule or exogenous signal, such as the tetracycline system, the NFκB / UV light system, the Cre / Lox system, and heat shock gene promoters, the regulatable RNA polymerase II promoters described in WO2006 / 135436, and tissue-specific promoters.

[0116] In another embodiment, component (i) of the combination of the invention is a cell capable of secreting a polypeptide of the invention or a conjugate of the invention, preferably a polypeptide of the invention, or a fusion protein of the invention, into the culture medium.

[0117] Suitable cells capable of secreting the polypeptide of the present invention include, but are not limited to, cardiomyocytes, adipocytes, endothelial cells, epithelial cells, lymphocytes (B cells and T cells), mast cells, eosinophils, endothelial cells, primary cultures of isolated cells from different organs, preferably cells isolated from the islets of Langerhans, hepatocytes, leukocytes, such as mononuclear leukocytes, mesenchymal, umbilical cord, or adult (skin, lung, kidney, and liver), osteoclasts, chondrocytes, and other connective tissue cells. Also suitable are cells of established cell lines, such as JurkatT cells, NIH-3T3, CHO, Cos, VERO, BHK, HeLa, COS, MDCK, 293, 3T3 cells, C2C12 myoblasts, and W138 cells. Those skilled in the art will understand that cells capable of secreting the polypeptide of the present invention into the culture medium may be found to form microparticles or microcapsules, so that the cells spend a more useful life in the patient. Suitable materials for the formation of the microparticulate objects of the present invention include any biocompatible synthetic polymeric material that allows for the continuous secretion of a therapeutic agent and serves as a support for the cells. Thus, the biocompatible polymeric material can be, for example, a thermoplastic polymer or a hydrogel polymer.Among the thermoplastic polymers, the inventors have selected acrylic acid, acrylamide, 2-aminoethyl methacrylate, poly(tetrafluoroethylene-cohexafluoropropylene), methacrylic acid-(7-coumaroxy)ethyl ester acid, N-isopropylacrylamide, polyacrylic acid, polyacrylamide, polyamidoamine, poly(amino)-p-xylylene, poly(chloroethyl vinyl ether), polycaprolactone, poly(caprolactone-co-trimethylene carbonate), poly(carbonate urea)urethane, poly(carbonate)urethane, polyethylene, polyethylene and acrylamide copolymers, polyethylene glycol, polyethylene glycol methacrylate, poly(vinyl alcohol), ... Poly(ethylene terephthalate), poly(4-hydroxybutyl acrylate), poly(hydroxyethyl methacrylate), poly(N-2-hydroxypropyl methacrylate), poly(lactic acid-glycolic acid), poly(L-lactic acid), poly(gamma-methyl, L-glutamic acid), poly(methyl methacrylate), poly(propylene fumarate), poly(propylene oxide), polypyrrole, polystyrene, poly(tetrafluoroethylene), polyurethane, polyvinyl alcohol, ultra high molecular weight polyethylene, 6-(p-vinylbenzamido)-hexanoic acid Np-vinylbenzyl-D-malonamide, and copolymers comprising two or more of such polymers. Among the hydrogel type polymers we have the natural materials alginate, agarose, collagen, starch, hyaluronic acid, bovine serum albumin, cellulose and its derivatives, pectin, chondroitin sulfate, fibrin and fibroin, as well as synthetic hydrogels such as Sepharose® and Sephadex®.

[0118] Compound (ii) of the combination of the present invention is an immuno-cancer agent.

[0119] As used herein, the term "immuno-cancer agent" refers to an agent that is effective in enhancing, stimulating and / or upregulating immune response in a subject. In some embodiments, the administration of an immuno-cancer agent with compound (i) of the combination of the present invention has a synergistic effect in the treatment of cancer.

[0120] The cancer immunotherapy agent may be, for example, a small molecule drug, an antibody, or a biomolecule or small molecule. Examples of biological cancer immunotherapy agents include, but are not limited to, cancer vaccines, antibodies, and cytokines. In some embodiments, the antibody is a monoclonal antibody. In some embodiments, the monoclonal antibody is a humanized or human antibody.

[0121] In some embodiments, the cancer immunotherapy agent is a cytokine.

[0122] "Cytokines" are understood as peptides of different sizes and molecular weights that synthesize cells of the immune system for the purpose of regulating immune responses, and they can be hormones, growth factors, necrosis factors, chemokines, etc. They can be of natural origin or derived from recombinant cell cultures and biologically active equivalents of native sequence cytokines. Exemplary cytokines can be cytokines that inhibit T cell activation, such as IL-6, IL-10, TGF-β, VEGF, and other immunosuppressive cytokines; or cytokines that stimulate T cell activation to stimulate immune responses. Their conjugation with antibodies results in immunocytokines. In some embodiments, the cytokine is recombinant human interleukin-15 (rhIL-15), recombinant human interleukin-12 (rhIL-12), a fusion complex composed of a synthetic form of endogenous IL-15 complexed to a soluble IL-15 binding protein, the IL-15 receptor alpha chain (IL15:sIL-15RA), such as IL-NM12 (Neumedicines Inc.) or heterodimeric IL-15 (hetIL-15, Novartis / Admune).

[0123] In another embodiment, the cytokine is IL2, IL7, IL12, IL15, IL21, IL1, IL3, IL4, IL5, IL6, IL8, CXCL8, IL9, II, 10, ILII, IL13, IL14, IL16, IL17, IL18, IL19, IL20, IL22, IL23, IL25, IL26, IL27, IL28, IL29, IL30, 11,31, 1L32, IL33, 11,35, IL36, GM-CSF, I FN-gamma, IL-1α / IL-lFl, IL-1β / IL-lF2, IL-12p70, IL-12 / IL-35p35, IL-13, IL-17 / 1L-17A, IL-17A / F heterodimer, IL-17F, IL-18 / IL-1F4, 1L-23, IL-24, IL-32, TL-32β, IL-32γ, iL-33, LAP (TGF-β1), lymphotoxin-α / TNF-β, TGF-β, TNF-α, TRANCE / TNFSFl l / RANK L, and any combination thereof.

[0124] In a preferred embodiment, the cancer immunotherapy agent is not a cytokine. Thus, in a preferred embodiment, cytokines are excluded from the scope of the present invention. Preferably, cytokines excluded from the scope of the present invention are TNF factor alpha, INF-gamma, GM-GSF factor, and IL-2.

[0125] In another preferred embodiment, cytokines are excluded from the scope of the present invention only if component (i) of the combination is (i)(a) or (i)(b). Thus, in one embodiment, when component (i) of the combination is a polypeptide comprising the sequence of SEQ ID NO: 1 or a functionally equivalent variant thereof or a conjugate comprising a polypeptide comprising the sequence of SEQ ID NO: 1 or a functionally equivalent variant thereof and a chemical moiety that promotes the cellular uptake of the polypeptide or a functionally equivalent variant thereof, the cancer immunotherapy is not a cytokine, preferably not a cytokine selected from the group consisting of TNF-α, INF-γ, GM-GSF, and IL-2.

[0126] In some embodiments, the cancer immunotherapy agent is (i) an agonist of a stimulatory (including costimulatory) receptor, or (ii) an antagonist of an inhibitory (including costimulatory) signal on T cells, both of which result in amplification of antigen-specific T cell responses.

[0127] Certain stimulatory and inhibitory molecules are members of the immunoglobulin superfamily (IgSF). One important family of membrane-bound ligands that bind to costimulatory or co-inhibitory receptors is the B7 family, which includes B7-1, B7-2, B7-H1 (PD-L1), B7-DC (PD-L2), B7-H2 (ICOS-L), B7-H3, B7-H4, B7-H5 (VISTA), and B7-H6.

[0128] Another family of membrane-bound ligands that bind to costimulatory or coinhibitory receptors is the TNF family of molecules that bind to cognate TNF receptor family members, including CD40 and CD40L, OX-40, OX-40L, CD70, CD27L, CD30, CD30L, 4-1BBL, CD137 (4-1BB), TRAIL / Apo2-L, TRAILR1 / DR4, TRAILR2 / DR5, TRAILR3, TRAILR4, and OPG. , RANK, RANKL, TWEAKR / Fn14, TWEAK, BAFFR, EDAR, XEDAR, TACI, APRIL, BCMA, LTR, LIGHT, DcR3, HVEM, VEGI / TL1A, TRAMP / DR 3, including EDAR, EDA1, XEDAR, EDA2, TNFR1, lymphotoxin α / TNFβ, TNFR2, TNFα, LTβR, lymphotoxin α1β2, FAS, FASL, RELT, DR6, TROY, NGFR.

[0129] In some embodiments, the combination of compound (i) of the present invention and an immuno-oncology agent is capable of stimulating a T cell response. In some embodiments, the cancer immunotherapy agent is (i) an antagonist of a protein that inhibits T cell activation (e.g., an immune checkpoint inhibitor), such as CTLA-4, PD-1, PD-L1, PD-L2, LAG-3, TIM-3, Galectin 9, CEACAM-1, BTLA, CD69, Galectin-1, TIGIT, CD113, GPR56, VISTA, 2B4, CD48, GARP, PD1H, LAIR1, TIM-1, and TIM-4; or (ii) an agonist of a protein that stimulates T cell activation, such as B7-1, B7-2, CD28, 4-1BB (CD137), 4-1BBL, ICOS, ICOS-L, OX40, OX40L, GITR, GITRL, CD70, CD27, CD40, DR3, and CD28H.

[0130] In some embodiments, the cancer immunotherapy agent is an antagonist of an inhibitory receptor on NK cells or an agonist of an activating receptor on NK cells. In some embodiments, the cancer immunotherapy agent is an antagonist of KIR, such as lirilumab.

[0131] In some embodiments, the cancer immunotherapy agent is an agent that inhibits or depletes macrophages or monocytes, including, but not limited to, CSF-1R antagonists, such as CSF-1R antagonist antibodies, including RG7155 (WO11 / 70024, WO11 / 107553, WO11 / 131407, WO13 / 87699, WO13 / 119716, WO13 / 132044) or FPA-008 (WO11 / 140249; WO13169264; WO14 / 036357).

[0132] In some embodiments, the immuno-oncology agent is selected from agonistic agents that ligate positive co-stimulatory receptors, blocking agents that attenuate signaling by inhibitory receptors, antagonists, and one or more agents that systemically increase the frequency of anti-tumor T cells, agents that overcome different immune suppressive pathways within the tumor microenvironment (e.g., blocking inhibitory receptor ligation (e.g., PD-L1 / PD-1 interactions), depleting or inhibiting Tregs (e.g., using anti-CD25 monoclonal antibodies (e.g., daclizumab) or by ex vivo anti-CD25 bead depletion), inhibiting metabolic enzymes such as IDO, or reversing / preventing T cell energetics or exhaustion), and agents that cause innate immune activation and / or inflammation at the tumor site.

[0133] The term "cytotoxic T-lymphoid associated protein 4" (abbreviated as "CTLA-4" and also known as cluster of differentiation 152 (CD152)) as used herein refers to a protein receptor that functions as an immune checkpoint. CTLA-4 is a member of the immunoglobulin superfamily that is expressed by activated T cells and transmits inhibitory signals to T cells. CTLA-4 is homologous to the T cell costimulatory protein CD28, and both molecules bind to CD80 and CD86, called B7-1 and B7-2, respectively, on antigen-presenting cells. CTLA-4 binds CD80 and CD86 with stronger affinity and avidity than CD28, thereby allowing them to outcompete CD28 for its ligand. CTLA-4 transmits inhibitory signals to T cells, while CD28 transmits stimulatory signals. CTLA-4 is also found in regulatory T cells (Tregs) and contributes to their inhibitory function. T cell activation by the T cell receptor and CD28 leads to increased expression of CTLA-4. The CTLA-4 protein is encoded by the CTLA-4 gene in humans (Ensembl ref:ENSG00000163599). Normally, after T cell activation, CTLA-4 is upregulated on the cell membrane where it functions to downregulate T cell function by various mechanisms, such as by outcompeting CD28 for its ligand B7, and further preventing costimulation by inducing T cell cycle arrest (Postow et al (2015) J. Clinical oncology, Vol. 33, pages 1974-1983; Pardoll, D. et al (2012), Nature Reviews Cancer 12, 252-264).

[0134] In some embodiments, the cancer immunotherapy agent is a CTLA-4 antagonist. The term "CTLA-4 antagonist" as used herein means, without limitation, any chemical compound or drug or biological molecule that blocks the binding of CTLA-4 to its ligands B7-1 and / or B7-2. In the context of the present invention, it is understood that when a subject (e.g., a human individual) is treated with a CTLA-4 antagonist (e.g., a CTLA-4 antibody), the CTLA-4 antagonist blocks the binding of (human) CTLA-4 to (human) B7-1 and / or B7-2.

[0135] Non-limiting examples of CTLA-4 antagonist compounds currently contemplated for clinical use in the treatment of cancer include antagonist antibodies against CTLA-4.

[0136] In some embodiments, the CTLA-4 antagonist is a CTLA-4 antagonist antibody. In some embodiments, the CTLA-4 antagonist antibody is YERVOY (ipilimumab) or tremelimumab.

[0137] Other non-limiting examples of CTLA-4 antagonists include immunoadhesins (also known as fusion proteins), which are compounds that can specifically bind to CTLA-4 and block its binding to B7-1 and / or B7-2.

[0138] The term "programmed death-1 (PD-1)" receptor, as used herein, refers to an immunoinhibitory receptor belonging to the CD28 family. In humans, PD-1 is encoded by the PDCD1 gene. PD-1 is predominantly expressed on previously activated T cells in vivo and binds to two ligands, PD-L1 and PD-L2. The term "PD-1", as used herein, encompasses human PD-1 (hPD-1), variants, isoforms, and cognate species of hPD-1, as well as analogs that share at least one common epitope with hPD-1. The complete hPD-1 sequence can be found in GENBANK Accession No. U64863. PD-1 is expressed on immune cells, such as activated T cells (including effector T cells), B cells, myeloid cells, thymocytes, and natural killer (NK) cells (Suya Dai et al., (2014) Cellular Immunology, Vol:290, pages 72-79; Gianchecchi et al., (2013), Autoimmun. Rev. 12 1091-1 100).

[0139] In some embodiments, the cancer immunotherapy agent is a PD-1 antagonist. The term "PD-1 antagonist" as used herein means, without limitation, any chemical compound or drug or biological molecule (e.g., an antibody) that blocks the binding of PD-L1 expressed on a cancer cell to PD-1 expressed on an immune cell (T cell, B cell, or NKT cell) and / or blocks the binding of PD-L2 expressed on a cancer cell to an immune cell expressing PD-1. In the context of the present invention, when a subject (e.g., a human individual) is treated with a PD-1 antagonist (e.g., a PD-1 antibody), the PD-1 antagonist blocks the binding of (human) PD-L1 to (human) PD-1 or blocks the binding of (human) PD-L2 to (human) PD-1, preferably blocks the binding of both (human) PD-L1 and PD-L2 to (human) PD-1. The human PD-1 amino acid sequence can be found at NCBI Locus No.: NP_005009. The human PD-L1 and PD-L2 amino acid sequences can be found at NCBI Locus No.: NP_054862 and NP_079515, respectively.

[0140] Non-limiting examples of PD-1 antagonists are antibodies against PD-1 (called PD-1 antibodies or anti-PD-1 antibodies), such as PD-1 monoclonal antibodies (mAbs), or antigen-binding fragments thereof, that specifically bind to PD-1, preferably human PD-1. The mAbs may be human, humanized, or chimeric antibodies, and may contain human constant regions. Non-limiting examples of PD-1 antagonist compounds include PD1 antibodies, such as nivolumab (Opdivo®, Bristol-Myers Squibb), pembrolizumab (Keytruda®, Merck), BGB-A317, and others, such as PDR001 (Novartis). Other non-limiting examples of PD-1 antagonists include pidilizumab (Cure Tech), AMP-224 (GlaxoSmithKline), AMP-514 (GlaxoSmithKline), PDR001 (Novartis), and cemiplimab (Regeneron and Sanofi). Additional PD-1 antagonists also include any of the anti-PD-1 antibodies described in US8008449, US7521051, and US8354509.

[0141] Other non-limiting examples of PD-1 antagonists include immunoadhesins (also known as fusion proteins), which are compounds that can specifically bind to PD-1 and block its binding to PD-L1. Examples of immunoadhesin molecules that specifically bind to PD-1 are described in WO2010 / 027827, US2016 / 0304969, and WO2011 / 066342. For example, a non-limiting example of a fusion protein that can be used as a PD-1 antagonist in the present invention is AMP-224 (a recombinant B7-DC Fc fusion protein that is composed of the extracellular domain of PD-1 ligand programmed cell death ligand 2 (PD-L2, B7-DC) and the Fc region of human immunoglobulin (Ig) G1).

[0142] The term "antibody" (e.g., PD-1 and CTLA-4 antibodies), as used herein, refers to any form of antibody and fragments thereof that exhibits a desired biological or binding activity (e.g., blocks the binding of PD-1 to its ligand or blocks the binding of CTLA-4 to its ligand, as described above). It is therefore used in the broadest sense and specifically encompasses, but is not limited to, monoclonal antibodies (including full-length monoclonal antibodies) and fragments thereof, polyclonal antibodies and fragments thereof, multispecific antibodies (e.g., bispecific antibodies) and fragments thereof, humanized and fully human antibodies and fragments thereof, chimeric antibodies and fragments thereof, and camelized single domain antibodies and fragments thereof.

[0143] In some embodiments, the cancer immunological agent is an antibody or antigen-binding portion thereof that specifically binds to the programmed death-1 (PD-1) receptor and inhibits PD-1 activity. In some embodiments, the PD-1 antagonist is a PD1 antagonist antibody. In some embodiments, the PD1 antagonist antibody is OPDIVO (nivolumab), KEYTRUDA (pembrolizumab), or MEDI-0680 (AMP-514; WO2012 / 145493). In some embodiments, the cancer immunological agent can be pidilizumab (CT-011). In some embodiments, the cancer immunological agent is a recombinant protein composed of the extracellular domain of PD-L2 (B7-DC) fused to the Fc portion of IgG1, called AMP-224.

[0144] In some embodiments, the cancer immunotherapy agent is a PD-L1 antagonist. In some embodiments, the PD-L1 antagonist is a PD-L1 antagonist antibody. In some embodiments, the PD-L1 antibody is MPDL3280A (RG7446; WO2010 / 077634), durvalumab (MEDI4736), BMS-936559 (WO2007 / 005874), and MSB0010718C (WO2013 / 79174).

[0145] In some embodiments, the cancer immunotherapy agent is a LAG-3 antagonist. In some embodiments, the LAG-3 antagonist is a LAG-3 antagonist antibody. In some embodiments, the LAG-3 antagonist antibody is BMS-986016 (WO10 / 19570, WO14 / 08218), or IMP-731 or IMP-321 (WO08 / 132601, WO009 / 44273).

[0146] In some embodiments, the cancer immunotherapy agent is a CD137 (4-1BB) agonist. In some embodiments, the CD137 (4-1BB) agonist is a CD137 agonist antibody. In some embodiments, the CD137 antibody is urelumab or PF-05082566 (WO12 / 32433).

[0147] In some embodiments, the cancer immunotherapy agent is a GITR agonist. In some embodiments, the GITR agonist is a GITR agonist antibody. In some embodiments, the GITR antibody is BMS-986153, BMS-986156, TRX-518 (WO006 / 105021, WO009 / 009116) or MK-4166 (WO11 / 028683).

[0148] In some embodiments, the cancer immunotherapy agent is an indoleamine (2,3)-dioxygenase (IDO) antagonist. In some embodiments, the (IDO) antagonist is selected from epacadostat (INCB024360, Incyte); indoximod (NLG-8189, NewLink Genetics Corporation); capmanitib (INC280, Novartis); GDC-0919 (Genentech / Roche); PF-06840003 (Pfizer); BMS:F001287 (Bristol-Myers Squibb); Phy906 / KD108 (Phytoceutica); enzymes that break down kynurenine (kynureninase (KYNase), Kyn Therapeutics); and NLG-919 (WO09 / 73620, WO009 / 1156652, WO11 / 56652, WO12 / 142237).

[0149] In some embodiments, the cancer immunotherapy agent is an OX40 agonist. In some embodiments, the OX40 agonist is an OX40 agonist antibody. In some embodiments, the OX40 antibody is MEDI-6383 or MEDI-6469.

[0150] In some embodiments, the cancer immunotherapy agent is an OX40L antagonist. In some embodiments, the OX40L antagonist is an OX40L antagonist antibody. In some embodiments, the OX40L antagonist is RG-7888 (WO06 / 029879).

[0151] In some embodiments, the cancer immunotherapy agent is a CD40 agonist. In some embodiments, the CD40 agonist is a CD40 agonist antibody. In some embodiments, the cancer immunotherapy agent is a CD40 antagonist. In some embodiments, the CD40 antagonist is a CD40 antagonist antibody. In some embodiments, the CD40 antibody is lucatumumab or dacetuzumab.

[0152] In some embodiments, the cancer immunotherapy agent is a CD27 agonist. In some embodiments, the CD27 agonist is a CD27 agonist antibody. In some embodiments, the CD27 antibody is varlilumab.

[0153] In some embodiments, the cancer immunotherapy agent is MGA271 (directed against B7H3) (WO11 / 109400).

[0154] In some embodiments, the cancer immunoagent is abagovomab, adecatumumab, afutuzumab, alemtuzumab, anatumomab mafenatox, apolizumab, atezolizumab, avelumab, blinatumomab, BMS-936559, catumaxomab, durvalumab, epacadostat, epratuzumab, indoximod, inotuzumab ozogamicin, intetumumab, ipilimumab, isatuximab, lambrolizumab, MED14736, MPDL3280A, nivolumab, obinutuzumab, ocaratuzumab, ofatumumab, olaratumab, pembrolizumab, pidilizumab, rituximab, ticilimumab, samalizumab, or tremelimumab.

[0155] In some embodiments, the cancer immunotherapy is an immunostimulant.For example, antibodies that block the PD-1 and PD-L1 inhibitory axis can unleash activated tumor-reactive T cells and have been shown in clinical trials to induce sustained antitumor responses in an increasing number of tumor tissue structures, including some tumor types that have traditionally been considered immunotherapy-sensitive.The anti-PD-1 antibody nivolumab (also known as Opdivo®, Bristol-Myers Squibb, ONO-4538, MDX1106, and BMS-936558) has shown the potential to improve the overall survival of patients with RCC who have experienced disease progression during or after previous antiangiogenic therapy.

[0156] In some embodiments, the immunomodulatory therapeutic specifically induces apoptosis of tumor cells. Approved immunomodulatory therapeutics that may be used in the present invention include pomalidomide (Pomalyst®, Celgene); lenalidomide (Revlimid®, Celgene); ingenol mebutate (Picato®, LEOPharma).

[0157] In some embodiments, the cancer immunological agent is a cancer vaccine, hi some embodiments, the cancer vaccine is selected from sipuleucel-T (Provenge®, Dendreon / Valeant Pharmaceuticals) and talimogene laherparepvec (Imlygic®, BioVex / Amgen, formerly known as T-VEC). In some embodiments, the cancer immunotherapy is an oncolytic virotherapy, such as pexaVec (JX-594, SillaJen / formerly Jennerex Biotherapeutics), pelareorep (Reolysin®, Oncolytics Biotech), enadenotsilieff (NG-348, PsiOxus, formerly known as ColoAd1), ONCOS-102 (Targovax / formerly Oncos), vaccinia viruses engineered to express β-galactosidase (β-gal) / β-glucoronidase and / or β-gal / human sodium iodide symporter (hNIS), such as GL-ONC1 (GLV-1h68 / GLV-1h153, Genelux GmbH) and CG0070 (Cold The adenovirus may be selected from among those engineered to express GM-CSF, such as adenoviruses engineered to express GM-CSF, such as those from Genesys.

[0158] In some embodiments, the cancer immunoagent is selected from JX-929 (SillaJen / formerly Jennerex Biotherapeutics), TG01 and TG02 (Targovax / formerly Oncos), TILT-123 (TILT Biotherapeutics), and VSV-GP (ViraTherapeutics).

[0159] In some embodiments, the cancer immunotherapy agent is a T cell engineered to express a chimeric antigen receptor (CAR). Such a T cell engineered to express a chimeric antigen receptor is called a CAR-T cell. CAR is composed of a single chain variable fragment (scFv) derived from a monoclonal antibody specific for a cell surface antigen and fused to an endodomain that is the functional end of a T cell receptor (TCR), such as the CD3-ζ signaling domain from TCR, which can generate an activation signal in T lymphocytes. Upon antigen binding, such a CAR binds to an endogenous signaling pathway in effector cells and generates an activation signal similar to that initiated by the TCR complex.

[0160] For example, in some embodiments, the CAR-T cell is one of those described in US8,906,682 (June; incorporated herein by reference in its entirety), which discloses CAR-T cells engineered to include an extracellular domain with an antigen-binding domain (e.g., a domain that binds to CD19) fused to the intracellular signaling domain of the T cell antigen receptor complex zeta chain (e.g., CD3zeta). When expressed in T cells, the CAR can redirect antigen recognition based on antigen-binding specificity. In the case of CD19, the antigen is expressed on malignant B cells. More than 200 clinical trials using CAR-T in various indications are currently underway [https: / / clinicaltrials.gov / ct2 / results?term=chimeric+antigen+receptors&pg=1].

[0161] In some embodiments, the immunostimulant is an activator of retinoic acid receptor-related orphan receptor gamma (RORγt). RORγt is a transcription factor that plays an important role in the differentiation and maintenance of type 17 effector subsets of CD4+ (Th17) and CD8+ (TC17) T cells, as well as in the differentiation of IL-17 expressing innate immune cell subpopulations such as NK cells. In some embodiments, the activator of RORγt is LYC-55716 (Lycera), which is currently being evaluated in clinical trials (NCT02929862) for the treatment of solid tumors.

[0162] In some embodiments, the immunostimulant is a toll-like receptor (TLR) agonist or activator.Suitable TLR activators include TLR9 agonists or activators, such as SD-101 (Dynavax).The TLR9 agonists or activators that can be used in the present invention include motolimod (VTX-2337, VentiRx Pharmaceuticals).

[0163] Other cancer immunotherapy agents that may be used in the present invention include urelumab (BMS-663513, Bristol-Myers Squibb), an anti-CD137 monoclonal antibody; varlilumab (CDX-1127, Celldex Therapeutics), an anti-CD27 monoclonal antibody; BMS-986178 (Bristol-Myers Squibb), an anti-OX40 monoclonal antibody; lirilumab (IPH2102 / BMS-986015, Innate Pharma, Bristol-Myers Squibb), an anti-KIR monoclonal antibody; monalizumab (IPH2201, Innate Pharma, AstraZeneca), an anti-NKG2A monoclonal antibody; andecaliximab (GS-5745, Gilead Sciences), an anti-MMP9 antibody; MK-4166 (Merck & Co., Inc., 2011). Co.), and anti-GITR monoclonal antibody.

[0164] In some embodiments, the immunostimulant is selected from elotuzumab, mifamurtide, agonists or activators of Toll-like receptors, and activators of RORγt.

[0165] In some embodiments, the cancer immunotherapy is selected from those described in Jerry L. Adams et al., "Big opportunities for small molecules in immuno-oncology," Cancer Therapy 2015, Vol. 14, pages 603-622, the contents of which are incorporated herein by reference in their entirety. In some embodiments, the cancer immunotherapy is selected from the examples described in Table 1 of Jerry L. Adams et al. In some embodiments, the cancer immunotherapy is a small molecule targeting cancer immunotherapy selected from those listed in Table 2 of Jerry L. Adams et al. In some embodiments, the cancer immunotherapy is a small molecule drug selected from those listed in Table 2 of Jerry L. Adams et al.

[0166] In some embodiments, the cancer immunotherapy agent is selected from the small molecule cancer immunotherapy agents described in Peter L. Toogood, "Small molecule immuno-oncology therapeutic agents," Bioorganic & Medicinal Chemistry Letters 2018, Vol. 28, pages 319-329, the contents of which are incorporated herein by reference in their entirety. In some embodiments, the cancer immunotherapy agent is an agent that targets a pathway as described in Peter L. Toogood.

[0167] In some embodiments, the cancer immunological agent is selected from those described in Sandra L. Ross et al., "Bispecific T cell engager (BiTE®) antibody constructs can mediate bystander tumor cell killing", PLoS ONE 12(8):e0183390, the contents of which are incorporated herein by reference in their entirety. In some embodiments, the cancer immunological agent is a bispecific T cell engaging (BiTE®) antibody construct. In some embodiments, the bispecific T cell engaging (BiTE®) antibody construct is a CD19 / CD3 bispecific antibody construct. In some embodiments, the bispecific T cell engaging (BiTE®) antibody construct is an EGFR / CD3 bispecific antibody construct. In some embodiments, the bispecific T cell engaging (BiTE®) antibody construct activates T cells. In some embodiments, the bispecific T cell engaging (BiTE®) antibody construct activates T cells, which release cytokines that induce upregulation of intercellular adhesion molecule 1 (ICAM-1) and FAS on bystander cells. In some embodiments, the bispecific T cell engaging (BiTE®) antibody construct activates T cells, which results in induction of lysis of bystander cells. In some embodiments, the bystander cells are in a solid tumor. In some embodiments, the bystander cells that are lysed are in the vicinity of the BiTE®-activated T cells. In some embodiments, the bystander cells comprise tumor-associated antigen (TAA) negative cancer cells. In some embodiments, the bystander cells comprise EGFR negative cancer cells. In some embodiments, the cancer immunological agent is an antibody that blocks the PD-L1 / PD1 axis and / or CTLA4. In some embodiments, the cancer immunological agent is ex vivo expanded tumor infiltrating T cells.In some embodiments, the cancer immunotherapy agent is a bispecific antibody construct or a chimeric antigen receptor (CAR) that directly links T cells to tumor-associated surface antigens (TAA).

[0168] In another embodiment, the cancer immunotherapy agent of the combination of the invention is an antagonist of a protein that inhibits T cell activation or an immune checkpoint inhibitor.

[0169] The term "checkpoint inhibitor" as used herein refers to a drug that is useful in preventing cancer cells from evading the immune system of patients.One of the main mechanisms of antitumor immune destruction is known as "T cell exhaustion", which occurs as a result of chronic exposure to antigens, leading to the upregulation of inhibitory receptors.These inhibitory receptors act as immune checkpoints to prevent uncontrolled immune responses.

[0170] PD-1 and co-inhibitory receptors, such as cytotoxic T lymphocyte antigen 4 (CTLA-4), B and T lymphocyte attenuator (BTLA; CD272), T cell immunoglobulin and mucin domain-33 (Tim-3), lymphocyte activation gene-3 (Lag-3; CD223), and others, are often referred to as checkpoint regulators. They function as molecular "gatekeepers" that allow extracellular signals to determine whether cell cycle progression and other intracellular signaling processes should continue.

[0171] In some embodiments, the immune checkpoint inhibitor is an antibody that binds to PD-1, which binds to the programmed cell death 1 receptor (PD-1) and thereby prevents the receptor from binding to the inhibitory ligand PDL-1, thereby abolishing the ability of the tumor to suppress the host anti-tumor immune response.

[0172] In one embodiment, the checkpoint inhibitor is a biological therapeutic agent or a small molecule.In another embodiment, the checkpoint inhibitor is a monoclonal antibody, a humanized antibody, a fully human antibody, a fusion protein, or a combination thereof.In a further embodiment, the checkpoint inhibitor inhibits checkpoint proteins selected from CTLA-4, PDL1, PDL2, PD1, B7-H3, B7-H4, BTLA, HVEM, TIM3, GAL9, LAG3, VISTA, KIR, 2B4, CD160, CGEN-15049, CHK1, CHK2, A2aR, B-7 family ligand, or a combination thereof. In additional embodiments, the checkpoint inhibitor interacts with a ligand of a checkpoint protein selected from CTLA-4, PDL1, PDL2, PD1, B7-H3, B7-H4, BTLA, HVEM, TIM3, GAL9, LAG3, VISTA, KIR, 2B4, CD160, CGEN-15049, CHK1, CHK2, A2aR, B-7 family ligand, or a combination thereof. In certain embodiments, the checkpoint inhibitor is an immunostimulant, a T cell growth factor, an interleukin, an antibody, a vaccine, or a combination thereof. In further embodiments, the interleukin is IL-7 or IL-15. In certain embodiments, the interleukin is glycosylated IL-7. In additional embodiments, the vaccine is a dendritic cell (DC) vaccine.

[0173] Checkpoint inhibitors include any agent that blocks or inhibits the inhibitory pathway of the immune system in a statistically significant manner. Such inhibitors may include small molecule inhibitors, or may include antibodies or antigen-binding fragments thereof that bind to and block or inhibit immune checkpoint receptors, or antibodies that bind to and block or inhibit immune checkpoint ligands. Exemplary checkpoint molecules that can be targeted for blocking or inhibition include, but are not limited to, CTLA-4, PDL1, PDL2, PD1, B7-H3, B7-H4, BTLA, HVEM, GAL9, LAG3, TIM3, VISTA, KIR, 2B4 (belonging to the CD2 family of molecules and expressed in all NK cells, gamma delta cells, and memory CD8+ (alpha beta) cells), CD160 (also called BY55), CGEN-15049, CHK1 and CHK2 kinases, A2aR, and various B7 family ligands. B7 family ligands include, but are not limited to, B7-1, B7-2, B7-DC, B7-H1, B7-H2, B7-H3, B7-H4, B7-H5, B7-H6 and B7-H7. Checkpoint inhibitors include antibodies or antigen-binding fragments thereof, other binding proteins, biotherapeutics, or small molecules that bind to and block or inhibit the activity of one or more of CTLA-4, PDL1, PDL2, PD1, BTLA, HVEM, TIM3, GAL9, LAG3, VISTA, KIR, 2B4, CD160, and CGEN-15049. Exemplary immune checkpoint inhibitors include tremelimumab (a CTLA-4 blocking antibody), anti-OX40, PD-Ll monoclonal antibody (anti-B7-Hl; MEDI4736), MK-3475 (a PD-1 blocker), nivolumab (an anti-PDl antibody), CT-011 (an anti-PDl antibody), BY55 monoclonal antibody, AMP224 (an anti-PDLl antibody), BMS-936559 (an anti-PDLl antibody), MPLDL3280A (an anti-PDLl antibody), MSB0010718C (an anti-PDLl antibody), and ipilimumab (an anti-CTLA-4 checkpoint inhibitor).Checkpoint protein ligands include, but are not limited to, PD-L1, PD-L2, B7-H3, B7-H4, CD28, CD86, and TIM-3.

[0174] In certain embodiments, the immune checkpoint inhibitor is selected from a PD-1 antagonist, a PD-L1 antagonist, and a CTLA-4 antagonist. In some embodiments, the checkpoint inhibitor is selected from the group consisting of nivolumab (Opdivo®), ipilimumab (Yervoy®), and pembrolizumab (Keytruda®). In some embodiments, the checkpoint inhibitor is selected from nivolumab (anti-PD-1 antibody, Opdivo®, Bristol-Myers Squibb); pembrolizumab (anti-PD-1 antibody, Keytruda®, Merck); ipilimumab (anti-CTLA-4 antibody, Yervoy®, Bristol-Myers Squibb); durvalumab (anti-PD-L1 antibody, Imfinzi®, AstraZeneca); and atezolizumab (anti-PD-L1 antibody, Tecentriq®, Genentech).

[0175] In some embodiments, the checkpoint inhibitor is selected from the group consisting of lambrolizumab (MK-3475), nivolumab (BMS-936558), pidilizumab (CT-011), AMP-224, MDX-1105, MEDI4736, MPDL3280A, BMS-936559, ipilimumab, ipilimumab, IPH2101, pembrolizumab (Keytruda®), and tremelimumab.

[0176] In some embodiments, the immune checkpoint inhibitor is REGN2810 (Regeneron), an anti-PD-1 antibody that has been tested in patients with basal cell adenocarcinoma (NCT03132636); NSCLC (NCT03088540); cutaneous squamous cell carcinoma (NCT02760498); lymphoma (NCT02651662); and melanoma (NCT03002376); pidilizumab (CureTech), also known as CT-011, an antibody that binds to PD-1, in clinical trials for diffuse large B-cell lymphoma and multiple myeloma; avelumab (Bavencio®, Pfizer / Merck), also known as MSB0010718C, a fully human IgG1 anti-PD-L1 antibody in clinical trials for non-small cell lung cancer, Merkel cell carcinoma, mesothelioma, solid tumors, renal cancer, ovarian cancer, bladder cancer, head and neck cancer, and gastric cancer. or PDR001 (Novartis), an inhibitory antibody that binds to PD-1, in clinical trials for non-small cell lung cancer, melanoma, triple-negative breast cancer, and advanced or metastatic solid tumors. Tremelimumab (CP-675,206; Astrazeneca) is a fully human monoclonal antibody against CTLA-4 that is being studied in clinical trials for several indications, including mesothelioma, colorectal cancer, renal cancer, breast cancer, lung and non-small cell lung cancer, pancreatic ductal adenocarcinoma, pancreatic cancer, germ cell carcinoma, squamous cell carcinoma of the head and neck, hepatocellular carcinoma, prostate cancer, endometrial cancer, metastatic cancer of the liver, liver cancer, large B-cell lymphoma, ovarian cancer, cervical cancer, metastatic thyroid anaplastic carcinoma, urothelial carcinoma, fallopian tube cancer, multiple myeloma, bladder cancer, soft tissue sarcoma, and melanoma. AGEN-1884 (Agenus) is an anti-CTLA4 antibody being studied in Phase 1 clinical trials for advanced solid tumors (NCT02694822).

[0177] In some embodiments, the checkpoint inhibitor is an inhibitor of T cell immunoglobulin mucin containing protein 3 (TIM-3). TIM-3 inhibitors that can be used in the present invention include TSR-022, LY3321367, and MBG453. TSR-022 (Tesaro) is an anti-TIM-3 antibody that is being studied in solid tumors (NCT02817633). LY3321367 (Eli Lilly) is an anti-TIM-3 antibody that is being studied in solid tumors (NCT03099109). MBG453 (Novartis) is an anti-TIM-3 antibody that is being studied in advanced malignancies (NCT02608268).

[0178] In some embodiments, the checkpoint inhibitor is an inhibitor of T cell immunoreceptor TIGIT, which is an immunoreceptor on certain T cells and NK cells and has Ig and ITIM domains.The TIGIT inhibitor that can be used in the present invention includes BMS-986207 (Bristol-Myers Squibb), anti-TIGIT monoclonal antibody (NCT02913313); OMP-313M32 (Oncomed); and anti-TIGIT monoclonal antibody (NCT03119428).

[0179] In some embodiments, the checkpoint inhibitor is an inhibitor of lymphocyte activation gene-3 (Lag-3). LAG-3 inhibitors that can be used in the present invention include BMS-986016 and REGN3767 and IMP321. BMS-986016 (Bristol-Myers Squibb), an anti-LAG-3 antibody, is being studied in glioblastoma and gliosarcoma (NCT02658981). REGN3767 (Regeneron), also an anti-LAG-3 antibody, is being studied in malignant tumors (NCT03005782). IMP321 (Immutep SA) is a LAG-3-Ig fusion protein, which is being studied in melanoma (NCT02676869); adenocarcinoma (NCT02614833); and metastatic breast cancer (NCT00349934).

[0180] Checkpoint inhibitors that can be used in the present invention include OX40 agonists. OX40 agonists that have been studied in clinical trials include PF-04518600 / PF-8600 (Pfizer), an anti-OX40 agonist antibody in metastatic renal cancer (NCT03092856) and advanced carcinomas and neoplasms (NCT02554812; NCT05082566); GSK3174998 (Merck), an anti-OX40 agonist antibody in phase 1 cancer trials (NCT02528357); and anti-OX40 agonist antibody in advanced solid tumors (NCT02318394 and NCT02705482). These include MEDI0562 (Medimmune / AstraZeneca), an anti-OX40 agonist antibody in patients with colorectal cancer (NCT02559024), breast cancer (NCT01862900), head and neck cancer (NCT02274155), and metastatic prostate cancer (NCT01303705); and BMS-986178 (Bristol-Myers Squibb), an anti-OX40 agonist antibody in advanced cancers (NCT02737475).

[0181] The checkpoint inhibitor that can be used in the present invention includes CD137 (also called 4-1BB) agonist.The CD137 agonist that is being studied in clinical trials includes utomirumab (PF-05082566, Pfizer), an anti-CD137 agonist antibody in diffuse large B-cell lymphoma (NCT02951156) and advanced cancer and neoplasm (NCT02554812 and NCT05082566);Urelumab (BMS-663513, Bristol-Myers Squibb), an anti-CD137 agonist antibody in melanoma and skin cancer (NCT02652455) and glioblastoma and gliosarcoma (NCT02658981).

[0182] The checkpoint inhibitor that can be used in the present invention includes CD27 agonist.The CD27 agonist that is being studied in clinical trials includes vallilumab (CDX-1127, Celldex Therapeutics), an anti-CD27 agonist antibody, in squamous cell head and neck cancer, ovarian carcinoma, colorectal cancer, renal cell carcinoma and glioblastoma (NCT02335918); lymphoma (NCT01460134); and glioma and astrocytoma (NCT02924038).

[0183] Checkpoint inhibitors that can be used in the present invention include glucocorticoid-induced tumor necrosis factor receptor (GITR) agonists. GITR agonists that have been studied in clinical trials include TRX518 (Leap Therapeutics), an anti-GITR agonist antibody, in melanoma and other malignant solid tumors (NCT01239134 and NCT02628574); These include GWN323 (Novartis), an anti-GITR agonist antibody in advanced cancers (NCT02740270); INCAGN01876 (Incyte / Agenus), an anti-GITR agonist antibody in advanced cancers (NCT02697591 and NCT03126110); MK-4166 (Merck), an anti-GITR agonist antibody in solid tumors (NCT02132754), and MEDI1873 (Medimmune / AstraZeneca), an agonistic hexameric GITR-ligand molecule with a human IgG1 Fc domain, in advanced solid tumors (NCT02583165).

[0184] The checkpoint inhibitor that can be used in the present invention includes inducible T cell costimulator (ICOS, also known as CD278) agonist.The ICOS agonist that is being studied in clinical trials includes MEDI-570 (Medimmune), an anti-ICOS agonist antibody in lymphoma (NCT02520791);GSK3359609 (Merck), an anti-ICOS agonist antibody in phase 1 (NCT02723955);JTX-2011 (Jounce Therapeutics), an anti-ICOS agonist antibody in phase 1 (NCT02904226).

[0185] Checkpoint inhibitors that may be used in the present invention include killer IgG-like receptor (KIR) inhibitors. KIR inhibitors being studied in clinical trials include lirilumab (IPH2102 / BMS-986015, Innate Pharma / Bristol-Myers Squibb), an anti-KIR antibody, in leukemia (NCT01687387, NCT02399917, NCT02481297, NCT02599649), multiple myeloma (NCT02252263), and lymphoma (NCT01592370); IPH2101 (1-7F9, Innate Pharma) in myeloma (NCT01222286 and NCT01217203); and IPH4102 (Innate Pharma), an anti-KIR antibody that binds to the three domains of the long cytoplasmic tail (KIR3DL2) in lymphoma (NCT02593045).

[0186] Checkpoint inhibitors that may be used in the present invention include CD47 inhibitors of the interaction between CD47 and signal regulatory protein alpha (SIRPa). CD47 / SIRPa inhibitors that have been investigated in clinical trials include ALX-148 (Alexo Therapeutics), an antagonist variant of (SIRPa) that binds to CD47 and prevents CD47 / SIRPa-mediated signaling, in Phase 1 (NCT03013218); TTI-621 (SIRPa-Fc, Trillium Therapeutics), a soluble recombinant fusion protein made by linking the N-terminal CD47 binding domain of SIRPa to the Fc domain of human IgG1, which acts by binding human CD47 and preventing it from delivering its "don't eat me" signal to macrophages, in Phase 1 clinical trials (NCT02890368 and NCT02663518). Therapeutics; CC-90002 (Celgene), an anti-CD47 antibody in leukemia (NCT02641002); and Hu5F9-G4 (Forty Seven, Inc.) in colorectal neoplasia and solid tumors (NCT02953782), acute myeloid leukemia (NCT02678338), and lymphoma (NCT02953509). In a preferred embodiment, the checkpoint inhibitor is a CD47 inhibitor.

[0187] The checkpoint inhibitor that can be used in the present invention includes CD73 inhibitor.The CD73 inhibitor that is being studied in clinical trials includes MEDI9447 (Medimmune), which is an anti-CD73 antibody in solid tumors (NCT02503774); and BMS-986179 (Bristol-Myers Squibb), which is an anti-CD73 antibody in solid tumors (NCT02754141).

[0188] Checkpoint inhibitors that can be used in the present invention include agonists of stimulator of interferon genes protein (STING, also known as transmembrane protein 173, or TMEM173). Agonists of STING that have been studied in clinical trials include MK-1454 (Merck), an agonist synthetic cyclic dinucleotide in lymphoma (NCT03010176); and ADU-S100 (MIW815, Aduro Biotech / Novartis), an agonist synthetic cyclic dinucleotide in phase 1 (NCT02675439 and NCT03172936).

[0189] The checkpoint inhibitor that can be used in the present invention includes CSF1R inhibitor.The CSF1R inhibitor that has been studied in clinical trials includes pexidartinib (PLX3397, Plexxikon), a CSF1R small molecule inhibitor in colorectal cancer, pancreatic cancer, metastatic cancer and advanced cancer (NCT02777710) and melanoma, non-small cell lung cancer, squamous cell head and neck cancer, gastrointestinal mesenchymal tumor (GIST) and ovarian cancer (NCT02452424); and pancreatic cancer (NCT03153410), melanoma (NCT03101254), and IMC-CS4 (LY3022855, Lilly), an anti-CSF-1R antibody in solid tumors (NCT02718911); and BLZ945 (4-[2((1R,2R)-2-hydroxycyclohexylamino)-benzothiazol-6-yloxyl]-pyridine-2-carboxylic acid methylamide, Novartis), an orally available inhibitor of CSF1R in advanced solid tumors (NCT02829723).

[0190] The checkpoint inhibitor that can be used in the present invention includes NKG2A receptor inhibitor.The NKG2A receptor inhibitor that is being studied in clinical trials includes monalizumab (IPH2201, InnatePharma), an anti-NKG2A antibody in head and neck neoplasms (NCT02643550) and chronic lymphocytic leukemia (NCT02557516).

[0191] In some embodiments, the immune checkpoint inhibitor is selected from nivolumab, pembrolizumab, ipilimumab, avelumab, durvalumab, atezolizumab, or pidilizumab.

[0192] In a preferred embodiment, the antagonist of a protein that inhibits T cell activation is selected from anti-PD-1 and anti-CTLA-4.

[0193] In a preferred embodiment, the cancer immunotherapy agent is a CTLA-4 antagonist, preferably a CTLA-4 antibody, more preferably ipilimumab or tremelimumab.

[0194] In a more preferred embodiment, the protein antagonist that inhibits T cell activation is anti-PD-1. In a preferred embodiment, the anti-PD-1 is an antibody or antigen-binding portion thereof, preferably an antibody selected from the group consisting of OPDIVO (nivolumab), KEYTRUDA (pembrolizumab), MEDI-0680 (AMP-514; WO2012 / 145493), and pidilizumab (CT-011). In another preferred embodiment, the anti-PD-1 is a recombinant protein called AMP-224, which is composed of the extracellular domain of PD-L2 (B7-DC) fused to the Fc portion of IgG1.

[0195] In one embodiment, the combination of the present invention is a conjugate between components (i) and (ii) of the combination of the present invention, in particular a conjugate between a polypeptide comprising the sequence of SEQ ID NO: 1 or a functionally equivalent variant thereof and an immuno-cancer agent.

[0196] In some embodiments, the conjugate between components (i) and (ii) is mediated by a non-cleavable linker. In some embodiments, the conjugate between components (i) and (ii) is mediated by a cleavable linker. Exemplary non-cleavable and cleavable linkers are described in US8088387, US8142784, WO2013075048, US6630579, US8512707, US9120854, US9023351, US20160095938, US9446146, WO2005009369, US5773001, US6214 345, US10111954, US8153768, US7829531, US20160082119, WO2018218004, US8568728, WO2015057699, US20170182181, and US9198979, the contents of each of which are incorporated herein by reference in their entirety.

[0197] In another aspect, the present invention relates to a pharmaceutical composition comprising a pharma- ceutically effective amount of a combination of the present invention together with a pharma- ceutically acceptable excipient.

[0198] As used herein, the expression "pharmaceutical composition" relates to a formulation adapted for administering a predetermined dose of one or more therapeutically useful agents to a cell, a group of cells, an organ, a tissue, or an animal in which cell division is unregulated, such as, for example, cancer.

[0199] The pharmaceutical composition of the present invention comprises a pharmacologic effective amount of the combination according to the present invention and a pharmacologic active carrier.The pharmaceutical composition of the present invention comprises a polypeptide comprising the sequence of SEQ ID NO:1, its functionally equivalent variant, a conjugate according to the present invention, a polynucleotide encoding said polypeptide or said conjugate, a vector comprising said polynucleotide, or a cell capable of secreting said polypeptide or said conjugate into culture medium, and a cancer immunotherapy agent.The suitable functionally equivalent variant of the polypeptide of SEQ ID NO:1, suitable conjugate, fusion protein, polynucleotide, vector, or cell for use in the pharmaceutical composition according to the present invention are as defined above.

[0200] The expression "pharmacologically effective amount" as used herein is understood as an amount capable of providing a therapeutic effect and can be determined by those skilled in the art by commonly used means. The amount of Omomyc polypeptide, its functionally equivalent variant, the conjugate, fusion protein, polynucleotide, vector, cell, or cancer immunological agent that may be combined in the pharmaceutical composition according to the present invention will vary depending on the subject and the specific mode of administration. Those skilled in the art will understand that the dosage can also be determined by the guidance of Goodman and Goldman's The Pharmacological Basis of Therapeutics, Ninth Edition (1996), Appendix II, pp. 1707-1711 and Goodman and Goldman's The Pharmacological Basis of Therapeutics, Tenth Edition (2001), Appendix II, pp. 475-493.

[0201] Appropriate dosages of the active ingredients in the pharmaceutical composition will vary depending on the type of cancer being treated, the severity and course of the disease, whether the composition is being administered for prophylactic or therapeutic purposes, medical history, the patient's medical history and response to the peptide or polypeptide, and the discretion of the attending physician.

[0202] An amount of a polypeptide comprising the sequence of SEQ ID NO:1, a functionally equivalent variant thereof, a fusion protein, a conjugate, a polynucleotide, a vector, or a cell is administered to a patient at one time or over a series of treatments, as appropriate. Depending on the type and severity of the disease, an appropriate dosage level will generally be about 0.01 to 500 mg / kg (of patient's body weight) / day, which can be administered in a single dose or multiple doses. Preferably, the dosage level will be about 0.1 to about 250 mg / kg / day, more preferably about 0.5 to about 100 mg / kg / day.

[0203] In a preferred embodiment, the amount of the first component is about 3.75 mg / kg (of subject's body weight) / day, preferably administered intranasally, preferably four times per week. In a preferred embodiment, the amount of the first component is about 8 mg / m, preferably administered intranasally, preferably four times per week. 2 / day to 15 mg / m 2 / day, preferably 10 mg / m 2 / day to 12 mg / m 2 / day, more preferably 11.25 mg / m 2 / day amount.

[0204] In a preferred embodiment, the amount of the first component is about 50 mg / kg (of the subject's body weight) / day, preferably administered intravenously, preferably twice weekly. In a preferred embodiment, the amount of the first component is about 100 mg / m, preferably administered intravenously, preferably twice weekly. 2 / day to 200 mg / m 2 / day, preferably 125 mg / m 2 / day to 175 mg / m 2 / day, preferably 140 mg / m 2 / day to 160 mg / m 2 / day, more preferably 150 mg / m 2 / day amount.

[0205] Suitable dosage levels may be from about 0.01 mg / kg / day to 250 mg / kg / day, from about 0.05 mg / kg / day to 100 mg / kg / day, or from about 0.1 mg / kg / day to 50 mg / kg / day. Within this range, dosages may be from 0.05 mg / kg / day to 0.5 mg / kg / day, 0.5 mg / kg / day to 5 mg / kg / day, or 5 mg / kg / day to 50 mg / kg / day. For oral administration, the composition is preferably provided in the form of a tablet containing 1.0 milligram to 1000 milligrams of active ingredient, in particular 1.0 milligram, 5.0 milligram, 10.0 milligram, 15.0 milligram, 20.0 milligram, 25.0 milligram, 50.0 milligram, 75.0 milligram, 100.0 milligram, 150.0 milligram, 200.0 milligram, 250.0 milligram, 300.0 milligram, 400.0 milligram, 500.0 milligram, 600.0 milligram, 750.0 milligram, 800.0 milligram, 900.0 milligram, and 1000.0 milligrams of active ingredient, for symptomatic adjustment of the dosage to the patient being treated. The compound may be administered in a dosage regimen of 1 to 4 times per day, preferably 1 or 2 times per day.

[0206] In some embodiments, the combination or composition can be administered once a week, twice a week, three times a week, four times a week, five times a week, six times a week, seven times a week. In some embodiments, the combination or composition can be administered once a week. In another embodiment, the combination or composition can be administered twice a week. In another embodiment, the combination or composition can be administered four times a week. In another preferred embodiment, the first component of the combination or composition is administered four times a week, and the second component of the combination or composition is administered once a week. In another embodiment, the first component of the combination or composition is administered twice a week, and the second component of the combination or composition is administered once a week. Both compounds can be administered simultaneously or sequentially. If the compounds are administered sequentially, administration of the first compound is stopped before starting administration of the second compound.

[0207] The duration of treatment can be at least 1 week, at least 2 weeks, at least 3 weeks, at least 4 weeks, at least 5 weeks, at least 6 weeks, at least 7 weeks, at least 8 weeks, at least 9 weeks, at least 10 weeks or more.Preferably, the duration of treatment is at least 4 weeks.In another embodiment, the duration of treatment is at least 3 weeks.

[0208] The amount of the cancer immunotherapy will vary depending on the particular agent used, and may be from about 0.01 mg / kg (of the subject's body weight) / day to about 50 mg / kg / day, preferably from about 1 mg / kg / day to about 25 mg / kg / day, once or several times daily, to obtain the desired therapeutic effect. In a preferred embodiment, the amount of the cancer immunotherapy is about 2.5 mg / kg (of the subject's body weight) / day or 7.5 mg / m, preferably administered once a week, more preferably administered parenterally, even more preferably administered intraperitoneally. 2In a preferred embodiment, the amount of the cancer immunotherapy is about 5 mg / kg (of subject's body weight) / day or 15 mg / m, preferably administered once a week, more preferably administered parenterally, and even more preferably administered intraperitoneally. 2 In another preferred embodiment, the amount of the cancer immunotherapy is about 10 mg / kg (of subject's body weight) / day or 30 mg / m, preferably administered once a week, more preferably administered parenterally, even more preferably administered intraperitoneally. 2 / day amount.

[0209] The pharmaceutical composition according to the present invention, which contains a first component (i) selected from a polypeptide comprising SEQ ID NO:1, its functionally equivalent variant, a fusion protein, a conjugate, a polynucleotide, a vector, or a cell according to the present invention, and a second component (ii) which is an immuno-oncology agent, can be presented as a single formulation (e.g. as a tablet or capsule containing a fixed amount of each of the components), or alternatively, as separate formulations which are subsequently combined for joint administration, sequential administration, separate administration. The composition of the present invention also encompasses the formulation as a kit of parts, where the components are formulated separately but packaged in the same container. The skilled person will understand that the formulation of the different components in the pharmaceutical composition according to the present invention may be similar, in other words formulated similarly (in tablets or pills), which allow their administration by the same route. When the different components of the present invention are formulated separately, the two components can be presented in blisters. Each blister contains the drugs that have to be consumed during a day. If the drug has to be administered several times a day, the drug corresponding to each administration can be placed in a different section of the blister, preferably with the time at which the drug should be administered being recorded in each section of the blister. Alternatively, the components of the composition of the invention can be formulated separately, such that the different components are administered separately. Thus, a first component can be formulated as a tablet or capsule for oral administration and a second component for intravenous administration, or vice versa. The ratio between the components that are part of the combination or pharmaceutical composition according to the invention can be adjusted by the skilled person depending on the antineoplastic drug used for each particular pathology as well as the desired indication. Thus, the present invention contemplates compositions in which the ratio between the amount of component (i) and the amount of component (ii) can range from 50:1 to 1:50, in particular from 20:1 to 1:20, 1:10 to 10:1, or from 5:1 to 1:5. In a more particular embodiment, the ratio between the amounts ranges from 1:1 to 1:5, preferably from 1:1 to 1:3.In a more preferred embodiment, the ratio is in the range of 1:1 to 1:1.5, preferably 1:1.3 to 1:1.4, more preferably 1:1.34. In another preferred embodiment, the ratio is in the range of 1:1 to 1:2.8, preferably 1:2.6 to 1:2.7, more preferably 1:2.67. In another particular embodiment, the ratio between the amounts is in the range of 30:1 to 5:1, preferably 30:1 to 8:1, more preferably 25:1 to 15:1, more preferably 20:1 to 10:1. In one embodiment, the ratio is 20:1. In another embodiment, the ratio is 10:1. Preferably, these ratios are w / w ratios.

[0210] The components of the pharmaceutical composition or combination of the present invention can be administered simultaneously. "Simultaneous administration" includes the coadministration of two therapeutic agents, regardless of the relative frequency or timing of administration of each agent. Thus, simultaneous administration includes the coadministration of two therapeutic agents at the same timing and frequency of administration. In addition, simultaneous administration also refers to the coadministration of two therapeutic agents, where one agent is administered more frequently than the other agent. In addition, simultaneous administration also refers to the coadministration of two therapeutic agents, where one agent is administered only once between the administration of the other agent.

[0211] In one embodiment, component (i) is administered intranasally. In another embodiment, component (i) is administered intravenously. In another embodiment, component (i) is administered parenterally, in particular intraperitoneally.

[0212] In a preferred embodiment, component (i) of the combination or pharmaceutical composition of the present invention is administered intranasally, while the cancer immunological agent is administered parenterally, particularly intraperitoneally or intravenously.For intranasal administration, the preferred dose of component (i) of the combination or composition of the present invention, preferably the preferred dose of the polypeptide or its functionally equivalent variant, fusion protein or conjugate, is in the range of 0.01mg / kg to 250mg / kg, more preferably between 0.1mg / kg and about 100mg / kg per day, which can be administered in a single dose or multiple doses.For intraperitoneal administration, the preferred dose of the cancer immunological agent is between 0.01mg / kg to 150mg / kg, more preferably between 0.1mg / kg to 100mg / kg.

[0213] In another embodiment, component (i) of the combination or pharmaceutical composition of the present invention is administered intravenously, while the cancer immunological agent is administered parenterally, in particular intraperitoneally or intravenously.

[0214] The pharmaceutical composition of the present invention may also contain one or more additional compounds for the prevention and / or treatment of pathologies in which uncontrolled cell division is present, such as cancer. The additional compounds, such as antitumor agents, may form part of the pharmaceutical composition as separate entities. In a preferred embodiment, the combination or pharmaceutical composition of the present invention comprises one or more antitumor agents selected from the group consisting of cytotoxic agents, angiogenesis inhibitors, antimetastatic agents, and antiproliferative agents.

[0215] The pharmaceutical composition of the present invention also contains one or more additional pharma- ceutically acceptable excipients. A "pharma-ceutically acceptable excipient" is understood as a therapeutically inactive substance that is intended to be used to incorporate an active ingredient and that is acceptable to the patient from a pharmacological / toxicological standpoint, and to the pharmacist who manufactures it from a physical / chemical standpoint in terms of composition, formulation, stability, patient acceptability, and bioavailability. The excipient may be a carrier. As used herein, "carrier" refers to any substance that serves to improve the delivery and efficacy of the active ingredient within the pharmaceutical composition. In a preferred embodiment, the carrier does not allow the direct delivery of components (i) and / or (ii) into the cytoplasm of the cell, i.e., the carrier cannot fuse with the plasma membrane of the target cell. Examples of pharma-ceutically acceptable carriers include one or more of water, saline, phosphate buffered saline, dextrose, glycerol, ethanol, and the like, and combinations thereof. In many cases, it will be preferable to include isotonic agents, such as sugars, polyalcohols, such as mannitol, sorbitol, or sodium chloride in the combination or composition. Pharmaceutically acceptable carriers may further include minor amounts of auxiliary substances, such as wetting and emulsifying agents, preservatives and buffers, which enhance the shelf life or effectiveness of the components that form part of the combination or composition of the present invention. Examples of suitable carriers are well known in the literature (see, for example, Remington's Pharmaceutical Sciences, 19th ed., Mack Publishing Company, Easton, PA, 1995). Examples of carriers are a series of sugars, such as lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, and maltitol; a series of starches, such as corn starch, wheat starch, rice starch, and potato starch; a series of celluloses, such as cellulose, methylcellulose, sodium carboxymethylcellulose, and hydroxylpropylmethylcellulose; and a series of fillers, such as gelatin and polyvinylpyrrolidone.Optionally, disintegrating agents may be added, such as cross-linked polyvinyl pyrrolidone, agar, alginic acid, or sodium alginate.

[0216] The number and nature of the pharmaceutically acceptable excipients vary according to the desired dosage form. Pharmaceutically acceptable excipients are known to those skilled in the art (Fauliy Trillo C. (1993) "Tratado de Farmacia Galenica", Luzan 5, SA Ediciones, Madrid). The composition can be prepared by conventional methods known in the state of the art (Remington: The Science and Practice of Pharmacy, 20th edition (2003) Genaro AR, ed., Lippincott Williams & Wilkins, Philadelphia, US).

[0217] In the case of pharmaceutical compositions containing agents that are nucleic acid molecules, the nucleic acid molecules can be present in any of a variety of delivery systems known to those skilled in the art, including nucleic acid and bacterial, viral, and mammalian expression systems, such as recombinant expression constructs as provided herein. Techniques for incorporating DNA into such expression systems are well known to those skilled in the art. The DNA can be "naked", for example, as described in Ulmer et al., Science 259:1745-49, 1993 and reviewed by Cohen, Science 259:1691-1692, 1993. The uptake of naked DNA can be increased by coating the DNA on biodegradable beads, which are efficiently transported into cells.

[0218] Nucleic acid molecules can be delivered into cells by any of several methods described in the art (e.g., Akhtar et al., Trends Cell Bio. 2:139 (1992); Delivery Strategies for Antisense Oligonucleotide Therapeutics, ed. Akhtar, 1995, Maurer et al., Mol. Membr. Biol. 16:129-40 (1999); Hofland and Huang, Handb. Exp. Pharmacol. 137:165-92 (1999); Lee et al., ACS Symp. Ser. 752:184-92 (2000); US6,395,713;WO 94 / 02595); Selbo et al., Int. J. Cancer 87:853-59 (2000); Selbo et al., Tumour Biol. 23:103-12 (2000)). (2002); US2001 / 0007666, and US2003 / 077829). Such delivery methods known to those skilled in the art include, but are not limited to, encapsulation in liposomes, by iontophoresis, or by incorporation into other vehicles such as biodegradable polymers; hydrogels; cyclodextrins (see, e.g., Gonzalez et al., Bioconjug. Chem. 10: 1068-74 (1999); Wang et al., International Application Publication Nos. WO 03 / 47518 and WO 03 / 46185); poly(lactic-co-glycolic) acid (PLGA) and PLCA microspheres (also useful for the delivery of peptides and polypeptides and other agents) (see, e.g., US 6,447,796; US 2002 / 130430); biodegradable nanocapsules and bioadhesive microspheres, or by proteinaceous vectors (International Application Publication No. WO 00 / 53722).In another embodiment, the nucleic acid molecule may be formulated or complexed with polyethyleneimine and its derivatives, such as polyethyleneimine-polyethylene glycol-N-acetylgalactosamine (PEI-PEG-GAL) or polyethyleneimine-polyethylene glycol-tri-N-acetylgalactosamine (PEI-PEG-triGAL) derivatives (see also, e.g., US 2003 / 0077829).

[0219] In certain embodiments, when the compound according to the present invention comprises a nucleic acid, the pharmaceutical composition may be formulated as a composition intended for use in gene therapy; by way of example and not by way of limitation, the pharmaceutical composition may contain a viral or non-viral vector containing a suitable polynucleotide or gene construct. By way of example and not by way of limitation, the vector may be viral, e.g., based on retrovirus, adenovirus, etc., or non-viral, e.g., based on ADN-liposome, ADN-polymer, ADN-polymer-liposome complex, etc. [see "Nonviral Vectors for Gene Therapy", edited by Huang, Hung and Wagner, Academic Press (1999)]. The vector containing the corresponding polynucleotide or gene construct may be directly administered to a subject by conventional methods. Alternatively, the vector may be used to transform, transfect, or infect cells, e.g., mammalian cells, e.g., human, ex vivo, which will then be implanted into a human or animal body to obtain the desired therapeutic effect. For administration to humans or animals, the cells will be formulated in a suitable vehicle that will not adversely affect cell viability.

[0220] The combination or pharmaceutical composition of the present invention can be administered by any type of suitable route, such as oral, topical, inhalation or parenteral, including the necessary pharma- ceutically acceptable excipients for the formulation of the desired dosage form. Other routes of administration can be rectal, intracisternal or intravaginal. The preferred route of administration of the combination or pharmaceutical composition is the intravenous route.

[0221] "Oral route" is understood as a pharmaceutical composition that is taken into the organism after swallowing. In certain embodiments, the pharmaceutical composition of the present invention can be in a dosage form suitable for administration by oral route, whether solid or liquid. The dosage form suitable for administration by oral route can be a tablet, capsule, syrup, or solution, and can contain any conventional excipient known in the art, such as a binder, such as syrup, acacia, gelatin, sorbitol, or polyvinylpyrrolidone; a filler, such as lactose, sugar, corn starch, calcium phosphate, sorbitol, or glycine; a lubricant for compression, such as magnesium stearate; a disintegrant, such as starch, polyvinylpyrrolidone, sodium starch glycolate, or microcrystalline cellulose; or a pharma-ceutically acceptable wetting agent, such as sodium lauryl sulfate. The solid oral composition can be prepared by conventional processes of mixing, filling, or compression. Repeated mixing operations can be used to completely distribute the active agent in those compositions that use a large amount of filler. Such operations are conventional in the art. Tablets can, for example, be prepared by wet or dry granulation and, optionally, by coating them with enteric coatings according to processes known in normal pharmaceutical practice.

[0222] On the other hand, "topical route" is understood as administration by a non-systemic route and includes application of the pharmaceutical compositions of the present invention externally, onto the epidermis, in the oral cavity, and by instillation of the compositions into the ears, eyes, and nose, such that they do not enter the bloodstream to a great extent. Dosage forms for topical or transdermal administration of the compounds of the present invention include ointments, pastes, creams, lotions, gels, powders, solutions, sprays, inhalants, or patches.

[0223] Ophthalmic formulations, ear drops, and eye drops are also contemplated as being within the scope of the invention. Additionally, the present invention contemplates the use of transdermal patches, which have the added advantage of providing controlled delivery of a compound to the body. Such dosage forms can be made by dissolving or dispersing the compound in a suitable medium. Absorption enhancers can also be used to increase the flux of the compound across the skin. The rate can be controlled by providing a rate controlling membrane or by dispersing the compound in a polymer matrix or gel.

[0224] In certain embodiments, the combination or pharmaceutical composition is administered systemically.

[0225] "Systemic route" is understood to mean administration by oral, intravenous, intraperitoneal and intramuscular routes. The amount of components (i) and (ii) required for a therapeutic or prophylactic effect will, of course, vary depending on the compound selected, the nature and severity of the disease being treated, and the patient.

[0226] In another embodiment, the combination or pharmaceutical composition is administered intranasally. In a preferred embodiment, intranasal administration is performed by drops or by intranasal inhalation.

[0227] "Inhalation" is understood to mean administration by the intranasal route and administration by oral inhalation. Suitable dosage forms for such administration, such as formulations in aerosols or metered dose inhalers, can be prepared by conventional techniques. In some embodiments, the administration route is the intranasal route.

[0228] As used herein, the term "parenteral" includes administration by intravenous, intraperitoneal, intramuscular, or subcutaneous routes. Subcutaneous, intramuscular, and intravenous dosage forms of parenteral administration are generally preferred.

[0229] In one embodiment, the combination or pharmaceutical composition of the present invention can be adapted for parenteral administration, such as, for example, sterile solutions, suspensions, or lyophilized products in suitable dosage unit forms. Suitable combination or pharmaceutical compositions for injection use include sterile aqueous solutions (if soluble in water), or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions. For administration by intravenous route, some suitable carriers include phosphate buffered saline (PBS). In all cases, the combination or composition must be sterile and fluid to the extent that it can be easily injected. It must be stable under preparation and storage conditions, and must be preserved against the contaminating action of microorganisms, such as bacteria and molds. The carrier can be, for example, a solvent or dispersion medium containing water, ethanol, a pharmaceutically acceptable polyol, such as glycerol, propylene glycol, liquid polyethylene glycol, and the like, and suitable mixtures thereof. Suitable fluidity can be maintained, for example, by using a coating such as lecithin, by maintaining the required particle size in the case of dispersions, and by using surfactants. Prevention of microbial contamination can be achieved by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, ascorbic acid, thiomersal, and the like. In most cases, it will be preferable to include isotonic agents, for example, sugars, polyalcohols, for example, mannitol, sorbitol, or sodium chloride in the composition. Prolonged absorption of the injectable composition can be brought about by including agents that delay absorption, for example, aluminum monostearate and gelatin.

[0230] The injectable sterile solution can be prepared by adding the required amount of active compound to a suitable solvent with one or combination of the above-mentioned raw materials, and then sterilizing by filtration through a sterile membrane as required.Generally, the dispersion is prepared by adding the active compound to a sterile vehicle that contains a basic dispersion medium and the rest of the required raw materials listed above.For the preparation of sterile powder for the preparation of injectable sterile solution, the preferred preparation process is vacuum drying and freeze-drying, which forms a powder with active raw materials and any desired additional raw materials from the previously filtered sterile solution.

[0231] The combination or pharmaceutical composition of the invention may be suitably administered, for example, by pulse infusion with declining doses of the composition. Preferably, the dose is administered by injection, more preferably intravenous or subcutaneous injection, depending in part on whether the administration is acute or continuous. In a preferred embodiment, the PD-1 antagonist is administered by infusion.

[0232] Alternatively, as described above, the different components in the composition are administered separately.

[0233] Thus, in one embodiment, component (i) of the combination or composition of the invention, preferably a polypeptide or a functionally equivalent variant or conjugate, is administered intranasally, while the cancer immunoagent is administered systemically.

[0234] In another preferred embodiment, component (i) of the combination or composition, preferably a polypeptide or a functionally equivalent variant, or a conjugate of the composition, is administered intranasally or by inhalation.

[0235] The dosage form of the composition intended for intranasal and pulmonary administration is preferably liquid, suspension or solid.Suspension is a liquid preparation containing solid particles dispersed in a liquid vehicle.The dosage form is preferably metered.For example, metered drops / spray means that the dispenser containing the drops / spray delivers the drops / spray containing a metered dose (predetermined amount) of the composition for use according to the present invention.

[0236] One of the preferred dosage forms related to intranasal administration route includes nasal drops.Drops are mostly deposited in the posterior part of the nose, and therefore are rapidly removed into the nasal pharynx.The concern with drops is often how to precisely control the dosage of the drug, which is particularly important for the administration of the composition.

[0237] Another intranasal dosage form that can administer the pharmaceutical composition of the present invention is nasal spray.Nasal spray typically contains the conjugate dissolved or suspended in a solution or mixture of excipients (e.g., preservatives, viscosity modifiers, emulsifiers, buffers) in a non-pressurized dispenser.Nasal spray has several advantages, such as compactness of delivery device, convenience, ease of use, and accuracy of delivering dosages of 25pL to 200pL.Nasal spray is deposited on the rear part of the nose and is slowly removed into the nasal pharynx by mucociliary clearance.Nasal spray, as used herein, can be a liquid or a suspension.

[0238] Another intranasal dosage form is nasal aerosol.Nasal aerosol differs from nasal spray in the way of dispensing the composition, in aerosol, compound is dispensed through valve by overpressure and release.In spray, compound is dispensed by being forced to release by micropump bucket, while the pressure in vial is the same as atmospheric pressure.Aerosol has the same advantages as spray.

[0239] Alternatively, the compositions herein may be preferably administered via nasal emulsions, ointments, gels, pastes, or creams, which are highly viscous solutions or suspensions that are applied to the nasal mucosa.

[0240] Due to the limited amount of composition that can be effectively delivered to the nasal mucosa, liquid intranasal dosage forms usually have a higher concentration than the corresponding intravenous dosage forms.When a substance is poorly soluble or insoluble in liquid form, powdered medicine can be used to administer the composition of the present invention.Another advantage of powdered medicine is that they do not require preservatives and usually have higher stability compared to liquid formulations.The main limitation of intranasal powdered medicine application is related to its irritating effect on the nasal mucosa.

[0241] One of the dosage forms in the context of pulmonary administration is inhalation aerosol. Inhalation aerosols are usually packaged under pressure and contain the composition according to the invention, which are released into the respiratory tract, particularly the lungs, upon actuation of a valve system. The released aerosol is a colloid (suspension) or liquid droplets (solution) of solid particles in air or another gas. Thus, the aerosol can be a solution aerosol or a suspension aerosol. The liquid droplets or solid particles preferably have a diameter of less than 100 pm, more preferably less than 10 pm, and most preferably less than 1 pm.

[0242] One of the dosage forms related to pulmonary administration is an inhalation spray. Inhalation sprays are typically aqueous-based and do not contain any propellant. They deliver the conjugate to the lungs by oral inhalation.

[0243] Nebulized inhalation solutions and suspensions can also be used to deliver the conjugates by the pulmonary route. Nebulized inhalation solutions and suspensions are typically water-based formulations containing the compositions according to the invention. Nebulized inhalation solutions and suspensions deliver the compositions to the lungs by oral inhalation for systemic effect, and are used with nebulizers.

[0244] Dry powder inhalation is an alternative to aerosol inhalation. The composition is usually contained in a capsule or in an inhaler for manual loading. Dry powder is typically delivered to the lungs by oral inhalation with an inhaler. Dry powder, as used herein, can be formulated purely. A neat formulation contains only drug or pseudo-drug, for example, as a spray-dried powder. Dry powder, as used herein, can also be formulated with a carrier such as lactose.

[0245] Pulmonary dosage forms are preferably metered, ie, delivered to the lungs in predetermined amounts.

[0246] Devices for intranasal delivery in the context of the present invention include spray pump systems, pipettes for delivering drops, metered-dose spray pumps, nasal pressurized metered-dose inhalers, powder spray systems, breath-actuated powder inhalers, and nasal powder insufflators. The intranasal delivery devices can be filled with single or multiple doses of the intranasal formulation.

[0247] Using the intrapulmonary route, the conjugates can be administered by a metered dose inhaler (MDI), which provides a fine mist of conjugates with an aerodynamic particle size generally less than 5 pm.

[0248] Dry powders can be used as an alternative method for delivering compositions into the lungs. Dry powder inhalers present the powder as a single or multi-dose powder.

[0249] Another device for pulmonary delivery is a nebulizer, such as an ultrasonic nebulizer and an air-jet nebulizer. In an ultrasonic nebulizer, ultrasonic waves are created in the ultrasonic nebulizer chamber by ceramic piezoelectric crystals that vibrate when electrically excited. This generates an aerosol cloud at the solution surface. The aerosol generated by an air-jet nebulizer is generated when compressed air is forced through an outlet hole. Liquid can be drawn from a vertical nozzle to mix with the atomized air jet using a baffle to promote the formation of an aerosol cloud (Bernoulli effect).

[0250] In one embodiment, each component of the combination or pharmaceutical composition of the invention is, for example, The composition is prepared with a carrier that protects the components, especially component (i), from rapid elimination from the body, such as controlled release formulations, including implants and microencapsulated delivery systems. Biodegradable biocompatible polymers can be used, such as vinyl acetate ethylene, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and polylactic acid. The process for preparing the formulation will be apparent to those skilled in the art. Materials can also be obtained commercially from Alza Corporation and Nova Pharmaceuticals, Inc.

[0251] The sustained release composition also includes preparations of crystals suspended in suitable formulations that can keep the crystals in suspension. These preparations can produce sustained release effects when they are injected subcutaneously or intraperitoneally. Other compositions also include components (i) and / or (ii) entrapped in liposomes. Liposomes containing such compounds are prepared by known methods, for example, Epstein et al., Proc. Natl. Acad. Sci. USA, (1985) 82:3688-3692; Hwang et al., Proc. Natl. Acad. Sci. USA, (1980) 77:4030-4034; EP52,322; EP36,676; EP88,046; EP143,949, etc. In a preferred embodiment, components (i) and / or (ii) are contained in liposomes, preferably both components are contained in liposomes, more preferably in the same liposome.

[0252] Despite the fact that Omomyc, its functionally equivalent variants, conjugates and fusion proteins of the present invention can cross biological membranes, it is possible to formulate Omomyc, any of its functionally equivalent variants, conjugates, polynucleotides, vectors, or cells in nanoparticles. The nanoparticles can contribute to preserving the integrity of the components in biological fluids until they reach the target organ. Moreover, in the case of compositions containing component (ii) or other antitumor drugs, encapsulation of the composition can reduce secondary effects caused by the antitumor drug. In addition, nanoparticles can be modified to include moieties that allow the nanoparticles to target the desired organ. In this way, component (i) of the combination or composition of the present invention is delivered near the target organ, facilitating the access of component (i) to the interior of cells where biological activity is required.

[0253] Thus, in another embodiment, component (i) of the combination or composition of the invention is provided to form part of a nanoparticle, hi another embodiment, both components of the combination or composition of the invention are provided to form part of a nanoparticle, preferably both components are provided in the same nanoparticle.

[0254] As used herein, the term "nanoparticle" refers to any material having a dimension in the range of 1-1,000 nm. In some embodiments, the nanoparticles have a dimension in the range of 2-200 nm, preferably in the range of 2-150 nm, and even more preferably in the range of 2-100 nm. Nanoparticles that can be used in the present invention include nanoscale materials such as, for example, lipid-based nanoparticles, superparamagnetic microparticles, nanoshells, semiconductor nanocrystals, quantum dots, polymer-based nanoparticles, silicon-based nanoparticles, silica-based nanoparticles, metal-based nanoparticles, fullerenes, and nanotubes. Molecules can be incorporated into the nanoparticle matrix or adsorbed to its surface, preferably, the molecules can be incorporated into the nanoparticles.

[0255] In a preferred embodiment, the nanoparticle is a liposome.

[0256] Targeted delivery can be achieved by adding ligands without sacrificing the nanoparticle's ability to deliver its contents. It is envisioned that this allows delivery to specific cells, tissues, and organs. The target specificity of ligand-based delivery systems is based on the distribution of ligand receptors to different cell types. Targeting ligands can be non-covalently or covalently associated with nanoparticles, and can be conjugated to nanoparticles by various methods described herein.

[0257] Examples of proteins or peptides that can be used to target nanoparticles include transferrin, lactoferrin, TGF-β, nerve growth factor, albumin, HIV Tat peptide, RGD peptide, and insulin and others.

[0258] It will be understood that the formulation of the invention in nanoparticles is not or is not merely intended to facilitate access of components (i) and / or (ii) to the interior of a cell, but is intended to protect components (i) and / or (ii) from degradation and / or to facilitate targeting of the nanoparticles to the organ of interest.

[0259] In one embodiment, the nanoparticles can be made of biodegradable polymers, such as poly(butyl cyanoacrylate) (PBCA). Examples of elemental nanoparticles include carbon nanoparticles and iron oxide nanoparticles, which can be coated with oleic acid (OA)-Pluronic®. In this approach, drugs (e.g., hydrophobic or water-insoluble drugs) are loaded into the nanoparticles. Other nanoparticles are made of silica.

[0260] The nanoparticles can be formed from any useful polymer. Examples of polymers include biodegradable polymers such as poly(butyl cyanoacrylate), poly(lactide), poly(glycolide), poly-s-caprolactone, poly(butylene succinate), poly(ethylene succinate), and poly(p-dioxanone); poly(ethylene glycol); poly-2-hydroxyethyl methacrylate (poly(HEMA)); copolymers such as poly(lactide-co-glycolide), poly(lactide)-poly(ethylene glycol), poly(poly(ethylene glycol) cyanoacrylate-cohexadecyl cyanoacrylate, and poly[HEMA-co-methacrylic acid]; proteins such as fibrinogen, collagen, gelatin, and elastin; and polysaccharides such as amylopectin, amylose, and chitosan.

[0261] Other nanoparticles include solid lipid nanoparticles (SLN). Examples of lipid molecules for solid lipid nanoparticles include stearic acid and modified stearic acid, such as PEG2000 stearate; soy lecithin; and emulsifying wax. Solid lipid nanoparticles can optionally include other components, such as surfactants, such as Epicuron® 200, poloxamer 188 (Pluronic® F68), Brij72, Brij78, polysorbate 80 (Tween80); and salts, such as sodium taurocholate. Drugs can be introduced into solid lipid nanoparticles by several methods described for liposomes, which can further include high pressure homogenization and dispersion of microemulsion.

[0262] Nanoparticles can also include nanometer-sized micelles. Micelles can be formed from any polymer described herein. Exemplary polymers for forming micelles include block copolymers, such as poly(ethylene glycol) and poly(ε-caprolactone) (e.g., aPEO-bPCL block copolymers including polymers of ε-caprolactone and α-methoxy-ω-hydroxy-poly(ethylene glycol)).

[0263] In certain embodiments, the properties of the nanoparticles are modified by coating them with a surfactant. Any biocompatible surfactant, such as a polysorbate surfactant, may be used. For example, polysorbates 20, 40, 60, and 80 (Tween 80), Epicuron® 200, poloxamer surfactants such as 188 (Pluronic® F68), poloxamer 908 and 1508, and Brij surfactants such as Brij 72 and Brij 78 may be used.

[0264] The nanoparticles can optionally be modified to include hydrophilic polymeric groups (e.g., poly(ethylene glycol) or poly(propylene glycol)), for example, by covalently attaching the hydrophilic polymeric groups to the surface or by using a polymer bearing such hydrophilic polymeric groups (e.g., poly[methoxypoly(ethylene glycol)cyanoacrylate-co-hexadecyl cyanoacrylate]). The nanoparticles can also optionally be crosslinked, which can be particularly useful in the case of protein-based nanoparticles.

[0265] In another embodiment, the pharmaceutical composition of the present invention is a nanoemulsion. "Nanoemulsion," as used herein, refers to a colloidal dispersion of droplets (or particles) in which at least some of the droplets have diameters in the nanometer size range. The nanoemulsion is composed of omega-3, -6, or -9 fatty acid rich oil in an aqueous phase, and is thermodynamically stabilized by an amphiphilic surfactant that creates an interfacial film, and is typically produced using a high shear microfluidization process, with droplet diameters in the range of about 80-220 nm.

[0266] Therapeutic uses of the present invention In one aspect the present invention relates to a combination or a pharmaceutical composition of the present invention for use in medicine.

[0267] In a further aspect, the present invention relates to a combination or a pharmaceutical composition according to the invention for use in the prevention and / or treatment of cancer.

[0268] In another embodiment the present invention refers to a combination or a pharmaceutical composition according to the invention for the preparation of a medicament for the prevention and / or treatment of cancer.

[0269] In another embodiment, the present invention also refers to a method for the prevention and / or treatment of cancer, comprising the step of administering a therapeutically effective amount of the combination or pharmaceutical composition of the present invention to a subject in need thereof.

[0270] In another aspect, the present invention also refers to a method for the prevention and / or treatment of cancer by recruiting T cells to a tumor site, comprising administering to a subject in need thereof a therapeutically effective amount of a combination or pharmaceutical composition of the present invention. In one embodiment, the T cells recruited to the tumor site are activated CD4 T cells, more particularly CD4 + PD-1 + T cells, and more particularly CD4 + PD-1 + Tim-3 - In another embodiment, the T cells recruited to the tumor site are CD4 + PD-1 + Tim-3 + In another embodiment, the T cells recruited to the tumor site are CD8 T cells, more particularly CD8 + PD-1 + In another embodiment, the T cells recruited to the tumor site are CD3 + In another embodiment, the T cells recruited to the tumor site are CD3 + CD4 + In another embodiment, the T cells recruited to the tumor site are Th1 / Th17 cells, particularly Th1 / Th17PD-1 + Cells, more specifically CD4 + IFN + IL-17 + T cells and more specifically CD4 + PD-1 + IFN + IL-17 + In another embodiment, the cells recruited to the tumor site are CD45 T cells. + It is a cell.

[0271] In another embodiment, the present invention also refers to a method for the prevention and / or treatment of cancer by inducing the proliferation of T regulatory cells, comprising the step of administering a therapeutically effective amount of the combination or pharmaceutical composition of the present invention to a subject in need thereof.

[0272] In another embodiment, the present invention also refers to a method for the prevention and / or treatment of cancer by inducing the production of IFN-γ by intratumoral CD4+ and CD8+ cells, comprising the step of administering a therapeutically effective amount of a combination or pharmaceutical composition of the present invention to a subject in need thereof.

[0273] In a preferred embodiment, the prophylactic or therapeutic method according to the invention involves the direct use of a combination or pharmaceutical composition comprising a polypeptide comprising Omomyc, or a functionally equivalent variant, conjugate, or fusion protein thereof. Thus, in a preferred embodiment, the prophylactic or therapeutic method according to the invention does not involve the administration of a nucleic acid encoding a polypeptide comprising Omomyc or a functionally equivalent variant or fusion protein thereof, or a vector encoding this nucleic acid, or a cell comprising said nucleic acid.

[0274] "Prevention" is understood as the administration of the combination or composition of the present invention at the initiation or early stage of the disease or to prevent even its onset.

[0275] The term "treatment" is used to indicate the administration of the combination or composition of the present invention to control disease progression before or after clinical signs appear. Control of disease progression is understood as beneficial or desired clinical outcome, including, but not limited to, alleviation of symptoms, reduction of disease duration, stabilization of morbidity (particularly avoiding further damage), delay of disease progression, improvement of morbidity, and remission (both partial and complete). Control of disease progression also involves prolongation of survival compared to expected survival if no treatment is applied. In a preferred embodiment, control of disease progression is measured as healthy lung / thorax volume ratio. In another embodiment, control of disease progression is measured as reduction of tumor volume.

[0276] The term "cancer" refers to diseases characterized by uncontrolled cell division (or increased survival or resistance to apoptosis), the ability of cells to invade other adjacent tissues (invasion), or the spread through lymphatic and blood vessels to other areas of the body where the cells are not normally located (metastasis). Depending on whether the tumor can spread by invasion and metastasis, tumors are classified as either benign or malignant; benign tumors are tumors that cannot spread by invasion or metastasis, i.e., they only grow locally, while malignant tumors are tumors that can spread by invasion and metastasis. The method according to the present invention is useful for the treatment of localized malignant tumors.

[0277] In one embodiment, cancers include, but are not limited to, leukemias (e.g., acute leukemia, acute lymphocytic leukemia, acute myeloid leukemia, acute myeloblastic leukemia, acute promyelocytic leukemia, acute myelomonocytic leukemia, acute monocytic leukemia, acute erythroleukemia, chronic leukemia, chronic myelogenous leukemia, chronic lymphocytic leukemia), hairy cell leukemia, polycythemia vera, lymphomas (e.g., Hodgkin's disease or non-Hodgkin's disease), AIDS-related leukemia, Waldenstrom's macroglobulinemia, multiple myeloma, heavy chain disease, and solid tumors, such as sarcomas and carcinomas (e.g., fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma, osteogenic sarcoma, chordoma, angiosarcoma, endothelial sarcoma, lymphangiosarcoma, lymphangioendothelial sarcoma, synovium, mesothelioma, Ewing's tumor, leiomyosarcoma, rhabdomyosarcoma, Kaposi's sarcoma, colon carcinoma, pancreatic cancer, breast cancer, biliary tract cancer, esophageal cancer, ovarian cancer, prostate cancer, oral cancer, e.g., squamous cell carcinoma, basal cell carcinoma, adenocarcinoma, sweat gland carcinoma, sebaceous gland carcinoma, papillary carcinoma, papillary adenocarcinoma, cystadenocarcinoma, medullary carcinoma, bronchogenic carcinoma, renal cell carcinoma, hepatocellular carcinoma, bile duct carcinoma, teratoma, choriocarcinoma, seminoma, embryonal carcinoma, Wilms' tumor, cervical cancer, uterine cancer, testicular cancer, lung cancer, small cell lung carcinoma, Bladder carcinoma, epithelial carcinoma, intraepithelial neoplasms such as Bowen's disease and Paget's disease, glioma, glioma, astrocytoma, glioblastoma multiforme (GBM, also known as glioblastoma), medulloblastoma, craniopharyngioma, ependymoma, pinealoma, hemangioblastoma, acoustic neuroma, oligodendroglioma, neurofibroma, neurofibrosarcoma, meningioma, melanoma, neuroblastoma, and retinal germinomas.

[0278] In some embodiments, the cancer is a glioma, astrocytoma, glioblastoma multiforme (GBM, also known as glioblastoma), medulloblastoma, craniopharyngioma, ependymoma, pinealoma, hemangioblastoma, acoustic neuroma, oligodendroglioma, neurofibroma, neurofibrosarcoma, meningioma, melanoma, neuroblastoma, or retinal germinomas.

[0279] In some embodiments, the cancer is an acoustic neuroma, astrocytoma, (e.g., grade I-pilocytic astrocytoma, grade II-low grade astrocytoma, grade III-anaplastic astrocytoma, or grade IV-glioblastoma (GBM)), chordoma, CNS lymphoma, craniopharyngioma, brain stem glioma, ependymoma, mixed glioma, optic nerve glioma, subependymoma, medulloblastoma, meningioma, metastatic brain tumor, oligodendroglioma, pituitary tumor, primitive neuroectodermal (PNET) tumor, or neurofibroma. In some embodiments, the cancer is of a type more commonly found in children than adults, such as, for example, brain stem glioma, craniopharyngioma, ependymoma, juvenile pilocytic astrocytoma (JPA), medulloblastoma, optic nerve glioma, pineal tumor, primitive neuroectodermal tumor (PNET), or rhabdoid tumor. In some embodiments, the patient is an adult. In some embodiments, the patient is a child or a pediatric patient.

[0280] In another embodiment, the cancer includes, but is not limited to, mesothelioma, hepatobiliary (liver and bile duct), bone cancer, pancreatic cancer, skin cancer, cancer of the head and neck, cutaneous or intraocular melanoma, ovarian cancer, colon cancer, rectal cancer, cancer of the anal region, stomach cancer, gastrointestinal cancer (stomach, colorectum, and duodenum), uterine cancer, fallopian tube cancer, endometrial carcinoma, cervical carcinoma, vaginal cancer, vulvar cancer, Hodgkin's disease, esophageal cancer, small intestine cancer, cancer of the endocrine system, thyroid cancer, cancer of the parathyroid gland, cancer of the adrenal gland, soft tissue sarcoma, cancer of the urethra, cancer of the penis, prostate cancer, testicular cancer, chronic or acute leukemia, chronic myeloid leukemia, lymphocytic lymphoma, bladder cancer, cancer of the kidney or ureter, renal cell carcinoma, renal pelvis cancer, non-Hodgkin's lymphoma, spinal axis tumor, brain stem glioma, pituitary adenoma, adrenal cortical carcinoma, gallbladder cancer, multiple myeloma, cholangiocarcinoma, fibrosarcoma, neuroblastoma, retinal germinomas, or a combination of one or more of the foregoing cancers.

[0281] In some embodiments, the cancer is selected from hepatocellular carcinoma, ovarian cancer, epithelial ovarian cancer, or fallopian tube cancer; papillary serous cystadenocarcinoma or uterine papillary serous carcinoma (UPSC); prostate cancer; testicular cancer; gallbladder cancer; hepatic cholangiocarcinoma; synovial sarcoma of soft tissue and bone; rhabdomyosarcoma; osteosarcoma; chondrosarcoma; Ewing's sarcoma; thyroid anaplastic carcinoma; adrenal cortical adenoma; pancreatic cancer; pancreatic ductal carcinoma or adenocarcinoma; gastrointestinal / gastric (GIST) cancer; lymphoma; squamous cell carcinoma of the head and neck (SCCHN); salivary gland cancer; glioma or brain tumor; neurofibromatosis-1 associated malignant peripheral nerve sheath tumor (MPNST); Waldenstrom's macroglobulinemia; or medulloblastoma.

[0282] In some embodiments, the cancer is selected from hepatocellular carcinoma (HCC), hepatoblastoma, colon cancer, rectal cancer, ovarian cancer, epithelial ovarian cancer, fallopian tube cancer, papillary serous cystadenocarcinoma, uterine papillary serous carcinoma (UPSC), hepatic cholangiocarcinoma, synovial sarcoma of soft tissue and bone, rhabdomyosarcoma, osteosarcoma, thyroid anaplastic carcinoma, adrenal cortical adenoma, pancreatic cancer, pancreatic ductal carcinoma, pancreatic adenocarcinoma, glioma, neurofibromatosis-1 associated malignant peripheral nerve sheath tumor (MPNST), Waldenstrom's macroglobulinemia, or medulloblastoma.

[0283] In some embodiments, the cancer is a solid tumor, such as a sarcoma, carcinoma, or lymphoma. A solid tumor generally comprises a mass of abnormal tissue that typically does not contain cysts or liquid areas. In some embodiments, the cancer is a renal cell carcinoma, or renal cancer; hepatocellular carcinoma (HCC) or hepatoblastoma, or liver cancer; melanoma; breast cancer; colorectal cancer, or colorectal cancer; colon cancer; rectal cancer; anal cancer; lung cancer, such as non-small cell lung cancer (NSCLC) or small cell lung cancer (SCLC); ovarian cancer, such as epithelial ovarian cancer, ovarian carcinoma, or fallopian tube cancer; papillary serous cystadenocarcinoma or uterine papillary serous carcinoma (UPSC); prostate cancer; testicular cancer; biliary selected from: bladder cancer; hepatic cholangiocarcinoma; synovial sarcoma of soft tissue and bone; rhabdomyosarcoma; osteosarcoma; chondrosarcoma; Ewing's sarcoma; anaplastic thyroid carcinoma; adrenal cortical carcinoma; pancreatic cancer; pancreatic ductal carcinoma or adenocarcinoma; gastrointestinal / gastric (GIST) cancer; lymphoma; squamous cell carcinoma of the head and neck (SCCHN); salivary gland carcinoma; glioma, or brain tumor; neurofibromatosis-1 associated malignant peripheral nerve sheath tumor (MPNST); Waldenstrom's macroglobulinemia; or medulloblastoma.

[0284] In some embodiments, the cancer is selected from hepatocellular carcinoma (HCC), hepatoblastoma, colon cancer, rectal cancer, ovarian cancer, epithelial ovarian cancer, ovarian carcinoma, fallopian tube carcinoma, papillary serous cystadenocarcinoma, uterine papillary serous carcinoma (UPSC), hepatic cholangiocarcinoma, synovial sarcoma of soft tissue and bone, rhabdomyosarcoma, osteosarcoma, thyroid anaplastic carcinoma, adrenal cortical carcinoma, pancreatic cancer, pancreatic ductal carcinoma, pancreatic adenocarcinoma, glioma, neurofibromatosis-1 associated malignant peripheral nerve sheath tumor (MPNST), Waldenstrom's macroglobulinemia, or medulloblastoma.

[0285] In some embodiments, the cancer is hepatocellular carcinoma (HCC). In some embodiments, the cancer is hepatoblastoma. In some embodiments, the cancer is colorectal cancer. In some embodiments, the cancer is rectal cancer. In some embodiments, the cancer is ovarian cancer or ovarian carcinoma. In some embodiments, the cancer is epithelial ovarian cancer. In some embodiments, the cancer is fallopian tube cancer. In some embodiments, the cancer is papillary serous cystadenocarcinoma. In some embodiments, the cancer is uterine papillary serous carcinoma (UPSC). In some embodiments, the cancer is hepatocholangiocarcinoma. In some embodiments, the cancer is synovial sarcoma of soft tissue and bone. In some embodiments, the cancer is rhabdomyosarcoma. In some embodiments, the cancer is osteosarcoma. In some embodiments, the cancer is anaplastic thyroid carcinoma. In some embodiments, the cancer is adrenocortical carcinoma. In some embodiments, the cancer is pancreatic cancer or pancreatic ductal carcinoma. In some embodiments, the cancer is pancreatic adenocarcinoma. In some embodiments, the cancer is a glioma. In some embodiments, the cancer is a malignant peripheral nerve sheath tumor (MPNST). In some embodiments, the cancer is a neurofibromatosis-1 associated MPNST. In some embodiments, the cancer is Waldenstrom's macroglobulinemia.

[0286] In some embodiments, the cancer is medulloblastoma. In some embodiments, the cancer is a virus-associated cancer, such as human immunodeficiency virus (HIV)-associated solid tumor, human papillomavirus (HPV)-16 positive incurable solid tumor, and adult T-cell leukemia (https: / / clinicaltrials.gov / ct2 / show / study / ), a highly aggressive form of CD4+ T-cell leukemia caused by human T-cell leukemia virus type I (HTLV-I) and characterized by clonal integration of HTLV-I in leukemic cells. NCT02631746); as well as virus-associated tumors in gastric cancer, nasopharyngeal carcinoma, cervical cancer, vaginal cancer, vulvar cancer, squamous cell carcinoma of the head and neck, and Merkel cell carcinoma (see https: / / clinicaltrials.gov / ct2 / show / study / NCT02488759; see also https: / / clinicaltrials.gov / ct2 / show / study / NCT0240886; https: / / clinicaltrials.gov / ct2 / show / NCT02426892).

[0287] Other cancers will be known to those of skill in the art.

[0288] In some embodiments, the cancer is melanoma cancer.In some embodiments, the cancer is breast cancer.

[0289] In another embodiment, the cancer is glioblastoma.

[0290] "Glioblastoma," also known as glioblastoma and grade IV astrocytoma, is the most common and most aggressive cancer that begins in the brain.

[0291] In a preferred embodiment, the cancer is lung cancer.

[0292] The term "lung cancer" or "lung tumor" refers to a physiological condition in mammals that is characterized by unregulated cell proliferation in the tissue of the lung. The term "lung cancer" is intended to mean any cancer of the lung, and includes non-small cell lung cancer and small cell lung cancer. In one embodiment, the lung cancer is non-small cell lung cancer (NSCLC). In another embodiment, the lung cancer is small cell lung cancer (SCLC).

[0293] The term non-small cell lung cancer (NSCLC), as used herein, refers to a group of heterogeneous diseases that have been classified together because their prognosis and management are roughly the same, as well as, for example, according to the World Health Organization / International Society for Lung Cancer (Travis WD et al. Histological typing of lung and pleural tumours. 3rd ed. Berlin: Springer-Verlag, 1999) histological classification as follows: (i) Squamous cell carcinoma (SCC), which accounts for 30% to 40% of NSCLC, begins in the larger respiratory tract but grows more slowly, which means that the size of these tumors varies at the time of diagnosis. (ii) adenocarcinoma, the most common subtype of NSCLC, accounting for 50% to 60% of NSCLC, develops near the gas-exchanging surfaces of the lung, as well as the subtype bronchoalveolar carcinoma, which may have different responses to treatment. (iii) Large cell carcinoma, a fast-growing form that grows near the surface of the lung. It is primarily a diagnosis of exclusion, and when more investigations have been completed, it is usually reclassified as squamous cell carcinoma or adenocarcinoma. (iv) Adenosquamous carcinoma, a type of cancer that contains two types of cells: squamous cells (thin, flat cells that line certain organs) and adenoid cells. (v) Carcinomas with pleomorphic, sarcomatoid, or sarcomatous elements: This is a rare group of tumors that reflects histologic heterogeneity and continuity in epithelial and mesenchymal differentiation. (vi) Carcinoid tumors, slow-growing neuroendocrine lung tumors that arise in cells capable of releasing hormones in response to stimulation by the nervous system. (vii) Salivary-type carcinoma, which begins in the salivary gland cells located in the large airways of the lungs. (viii) Unclassified carcinoma, which includes cancers that do not fall into any of the lung cancer categories listed above. Examples include:

[0294] In certain embodiments, the NSCLC is selected from squamous cell carcinoma of the lung, large cell carcinoma of the lung, and adenocarcinoma of the lung.

[0295] The term small cell lung cancer (SCLC), as used herein, refers to the proliferation of small cells with distinctive and strict morphological characteristics, including dense neurosecretory granules that confer an associated endocrine / paraneoplastic syndrome to the tumor. Most cases arise in the larger airways (primary and secondary bronchi). These cancers grow rapidly and spread early during the course of the disease.

[0296] In an even more preferred embodiment, the lung cancer is an adenocarcinoma, more preferably a KRas-driven lung adenocarcinoma, preferably a cancer associated with a mutation in the KRAS gene. In one embodiment, the mutation in the KRAS gene is a mutation in the glycine at position 12, the glycine at position 13, or the glutamine at position 61. In a more preferred embodiment, the mutation is selected from the group consisting of a G12S mutation, a G12V mutation, a G12D mutation, a G13D mutation, a G12C mutation, a G12R mutation, a G12F mutation, a G12I mutation, a G13C mutation, a G13R mutation, or a Q61L mutation. In a preferred embodiment, the mutation is a G12D mutation. In another embodiment, the lung cancer is a KRas-driven lung adenocarcinoma. GD12 / p53-driven lung cancer, preferably KRas GD12 / p53-driven NSCLC.

[0297] In certain embodiments, the cancer is a primary tumor. The term "primary tumor," as used herein, refers to a tumor that originates in a location or organ and is present in that location but has not metastasized to another location.

[0298] In another embodiment, the cancer is cancer metastasis. In the context of the present invention, "metastasis" is understood as the spread of cancer from the organ in which it originates to another organ. It generally occurs through the blood or lymphatic system. When cancer cells spread and form new tumors, the latter are called secondary or metastatic tumors. The cancer cells that form secondary tumors are similar to the cancer cells of the original tumor. For example, when breast cancer spreads (metastasizes) to the lungs, secondary tumors are formed by malignant breast cancer cells. The lung disease is metastatic breast cancer, not lung cancer. The inventors have also observed that the combination or composition of the present invention can reduce cell proliferation, regardless of whether the cancer shows an increase in Myc protein expression or activity. In a preferred embodiment, the cancer to be prevented or treated is a Myc-induced cancer.

[0299] In some embodiments, the cancer is a solid tumor.

[0300] All combinations of the compounds of the present invention and types of cancer are encompassed by the present invention.

[0301] In some embodiments, the combination or composition of the present invention causes tumor growth to stop. In some embodiments, the combination or composition of the present invention causes a reduction in tumor size (e.g., volume or mass) of at least 5%, 10%, 25%, 50%, 75%, 90%, or 99% relative to the size of the tumor before treatment. In some embodiments, the combination or composition of the present invention causes a reduction in tumor mass in a patient of at least 5%, 10%, 25%, 50%, 75%, 90%, or 99% relative to the tumor mass before treatment.

[0302] "Subject" as used herein includes any animal that has cancer or shows symptoms of cancer or is at risk of having cancer or showing symptoms of cancer. Suitable subjects (patients) include laboratory animals (such as mice, rats, rabbits, or guinea pigs), farm animals, and livestock animals or pets (such as cats or dogs). Non-human primates, preferably human patients, are included. Preferably, the subject is a mammal, most preferably a human.

[0303] The combination or composition for use in the prevention and / or treatment of cancer may be administered using any amount and any route of administration effective for treating or reducing the severity of cancer. The exact amount required will vary from subject to subject, depending on the species, age, and general condition of the subject, the severity of the disease or condition, the specific drug, the mode of administration, and the like. The compounds of the present invention are preferably formulated in unit dosage form for ease of administration and uniformity of dosage. The expression "unit dosage form" as used herein means a single physically separate and separate dose of drug appropriate for the patient to be treated. However, the total daily usage of the compounds and compositions of the present invention will be determined by the attending physician within the scope of sound medical judgment. The specific effective dosage level for any particular patient or organism will depend on a variety of factors, such as the disorder being treated and the severity of the disorder; the activity of the particular compound employed; the particular composition employed, the patient's age, weight, general health, sex, and diet; the timing of administration, route of administration, and rate of excretion of the particular compound employed; the duration of treatment; drugs used in combination with or concomitantly with the particular compound employed, as well as factors well known in the medical arts.

[0304] In a preferred embodiment, component (i) of the present invention, preferably a polypeptide or a functionally equivalent variant or conjugate thereof, interacts synergistically with the cancer immunoagent of the combination or composition in the treatment of cancer (to achieve a therapeutic effect).

[0305] In particular, in a more preferred embodiment, the combination or pharmaceutical composition for use in the prevention and / or treatment of cancer is a combination or pharmaceutical composition in which the polypeptide or its functionally equivalent variant, or conjugate, is in an amount that interacts synergistically with a cancer immunotherapy agent in the treatment of cancer.

[0306] The terms "synergy" or "synergistically interacting" are used interchangeably. Synergy is an effect that exceeds the additive effect that would be expected by adding up the actual effects of the individual agents in vitro. In vivo, synergy is a physiological effect, particularly a therapeutic effect, that exceeds the additive effect that would be expected by adding up the actual effects of the individual agents in vivo.

[0307] Thus, when two drugs are administered, they together provide a measurable physiological effect, particularly a therapeutic effect, when the actual effect of the combined drugs exceeds that expected by adding up the actual therapeutic effects of the individual drugs. In particular, a synergistic effect is provided when a first drug alone provides some measurable effect, a second drug alone provides some measurable effect, and the two drugs together provide a measurable effect that exceeds the effect provided by the sum of both individual drugs. More specifically, a synergistic effect is provided when a first drug alone provides no measurable effect, a second drug alone provides some measurable effect, and the two drugs together provide a measurable effect that exceeds the effect provided by the second drug alone. Even more specifically, a synergistic effect is provided when neither a first drug alone nor a second drug alone provides any measurable effect, but the two drugs together provide a measurable effect. Because components (i) and (ii) act synergistically, the amount of components (i) and / or (ii) in the combinations or compositions of the invention may be less than that required in monotherapy using only one of them as a therapeutic agent. Preferably, in these combinations or compositions, a dosage of between 0.01 and 1.000 μg / kg body weight / day of one or the other therapeutic agent may be administered.

[0308] The amount of the therapeutic agent present in the combination or composition may be equal to or less than the amount normally administered in a composition containing that therapeutic agent as the active agent. Preferably, the amount of the therapeutic agent in the composition will be in the range of about 50% to 100% of the amount normally present in a composition containing that agent as the only therapeutically active agent. In some embodiments, a therapeutic agent is administered at a dosage of about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, or about 95% of the amount that the agent is normally administered. As used herein, the phrase "normally administered" refers to the amount that an FDA approved therapeutic agent is approved for administration per the FDA label insert.

[0309] The combinations or compositions of the invention may also be used in combination with known therapeutic processes, for example in combination with chemotherapy, radiotherapy, immunotherapy, phototherapy, surgical intervention, hormones, or combinations thereof.

[0310] In a preferred embodiment, said combination or pharmaceutical composition for use in the treatment and / or prevention of cancer is selected from the group consisting of lung cancer, preferably NSCLC, more preferably KRas-driven cancer, even more preferably KRAS G12DFor the treatment of cancer, the first component, preferably a polypeptide comprising the sequence of SEQ ID NO: 1, more preferably a polypeptide consisting of SEQ ID NO: 4, is administered intranasally, and the second component, preferably an antagonist of a protein inhibiting T cell activation, more preferably an anti-PD-1 or anti-CTLA-4, even more preferably an anti-PD-1 antibody or an anti-CTLA-4 antibody, is administered systemically, preferably parenterally, even more preferably intraperitoneally. In a preferred embodiment, the first component is administered four times a week. In a preferred embodiment, the second component is administered once a week. In a more preferred embodiment, the first component is administered four times a week and the second component is administered once a week. In an embodiment, the first and second components are administered consecutively, i.e., the administration of the first component is stopped before starting the administration of the second component. In a preferred embodiment, the treatment continues for at least four weeks. In a preferred embodiment, the first and second components are administered on different days. In a preferred embodiment, the first component is administered on days 1, 2, 4 and 5, and the second component is administered on day 3. In a preferred embodiment of the combined administration, the first and second components are administered on different days, preferably the first component is administered on days 1, 2, 4 and 5, and the second component is administered on day 3. In a preferred embodiment, the ratio between components (i) and (ii) may range from 50:1 to 1:50, in particular from 20:1 to 1:20, from 1:10 to 10:1, or from 5:1 to 1:5, preferably from 1:1 to 1:5, more preferably from 1:1 to 1:3. In a more preferred embodiment, the ratio may range from 1:1 to 1:1.5, preferably from 1:1.3 to 1:1.4, more preferably from 1:1.34. In another preferred embodiment, the ratio ranges from 1:1 to 1:2.8, preferably from 1:2.6 to 1:2.7, more preferably 1:2.67. These ratios are preferably w / w ratios.

[0311] In a preferred embodiment, said combination or pharmaceutical composition for use in the treatment and / or prevention of cancer is selected from the group consisting of lung cancer, preferably NSCLC, more preferably KRas-driven cancer, even more preferably KRAS G12D Cancer driven, preferably KRAS G12D / p53-driven cancer, in which the first component, preferably a polypeptide comprising the sequence of SEQ ID NO:1, more preferably a polypeptide consisting of SEQ ID NO:4, is administered intravenously; and the second component, preferably an antagonist of a protein inhibiting T-cell activation, more preferably an anti-PD-1 or anti-CTLA-4, even more preferably an anti-PD-1 antibody or an anti-CTLA-4 antibody, even more preferably an anti-PD-1 antibody, is administered systemically, preferably parenterally, even more preferably intraperitoneally. In a preferred embodiment, the first component is administered twice weekly. In a preferred embodiment, the second component is administered once weekly. In a more preferred embodiment, the first component is administered twice weekly and the second component is administered once weekly. In an embodiment, the first and second components are administered sequentially, i.e., administration of the first component is stopped before starting administration of the second component. In another embodiment, the first and second components are administered simultaneously, preferably once weekly. In a preferred embodiment, the treatment is continued for at least 3 weeks, preferably at least 4 weeks. In a preferred embodiment, the first and second components are administered on different days. In a preferred embodiment, the first component is administered on the second and fifth days, and the second component is administered on the third day. In a preferred embodiment of combined administration, the first and second components are administered on different days, preferably the first component is administered on the second and fifth days, and the second component is administered on the third day. In a more preferred embodiment, the first component is administered for a period of time, preferably at least 5 days, at least 10 days, at least 15 days, more preferably 10 days, before administering the second component. In an embodiment, administration of the first component is stopped before starting administration of the second compound. In a preferred embodiment, the ratio between component (i) and component (ii) may range from 50:1 to 1:50, in particular 20:1 to 1:20, 1:10 to 10:1, or 5:1 to 1:5. In another particular embodiment, the ratio between the amounts ranges from 30:1 to 5:1, preferably from 30:1 to 8:1, more preferably from 25:1 to 15:1, more preferably from 20:1 to 10:1. In one embodiment, the ratio is 20:1.In another embodiment, the ratio is 10: 1. These ratios are preferably w / w ratios.

[0312] All embodiments of the combination of the present invention are also applicable to the treatment method of the present invention.

[0313] Articles of Manufacture and Kits The disclosure also provides an article of manufacture comprising any one of the combinations or pharmaceutical compositions disclosed herein in one or more containers. In some embodiments, the article of manufacture includes, for example, a pamphlet, printed instructions, label, or package insert directed to a user (e.g., a distributor or end user) for combining and / or using the compositions of the article of manufacture for the prevention and / or treatment of cancer.

[0314] In some embodiments, the article of manufacture includes, for example, a bottle, a vial, a cartridge, a box, a syringe, an injector, or any combination thereof. In some embodiments, the label indicates the use or administration of the combination or pharmaceutical composition in the article of manufacture according to the methods disclosed herein. In some aspects, the label indicates, for example, a dosing regimen for use, a dosing regimen for treating, preventing, or ameliorating cancer, etc.

[0315] The contents of all cited references (including references, patents, patent applications, and websites) that may be cited throughout this application are expressly incorporated by reference in their entirety for any purpose, as are the references cited therein.

[0316] All terms used herein shall be understood in their ordinary meanings known in the art unless otherwise specified. Other more specific definitions for certain terms used in this application are detailed below and are intended to apply uniformly throughout the description and claims unless another clearly specified definition provides a broader definition. Throughout the description and claims, the word "comprise" and variations of the word are not intended to exclude other technical features, additives, ingredients, or steps. Moreover, the word "comprise" includes the case of "consisting of. Further objects, advantages, and features of the present invention will be apparent to those skilled in the art upon perusal of the description or may be learned by the practice of the present invention. Moreover, the present invention covers all possible combinations of the specific embodiments described herein.

[0317] In this specification and the appended claims, the singular forms "a", "an" and "the" include plural referents unless the context clearly negates. The terms "a" (or "an"), as well as "one or more" and "at least one" can be used interchangeably herein. Moreover, "and / or" as used herein is understood as a specific disclosure of each of the two specified features or components with or without the other. Thus, when the term "and / or" is used herein in phrases such as "A and / or B", it is intended to include "A and B", "A or B", "A" (only), and "B" (only). Similarly, when the term "and / or" is used in phrases such as "A, B, and / or C", it is intended to include each of the following aspects: A, B, and C; A, B, or C; A, or C; A or B; B or C; A and C; A and B; B and C; A (only); B (only); and C (a only). The term "about", when used in connection with a numerical value throughout this specification and claims, indicates an interval of precision, familiarity, and acceptability to those of ordinary skill in the art. Typically, such intervals of precision are ±15%. Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by those of ordinary skill in the art to which this disclosure pertains. Units, prefixes, and symbols are designated in their International System of Units (SI) accepted form. Numerical ranges include the numerical values ​​that define the range. Unless otherwise specified, amino acid sequences are written from left to right in the amino to carboxy direction. The headings provided herein are not limitations of the various aspects of the disclosure, which may be obtained by reference to the specification as a whole. Thus, the terms defined immediately below are more fully defined by reference to the specification.

[0318] The present invention is illustrated by the following examples, which are considered merely as illustrative and not as limiting the scope of the invention. EXAMPLES

[0319] Generation and purification of Omomyc The Omomyc peptide sequence of SEQ ID NO: 4, containing a methionine at the N-terminus, was reverse transcribed, codon-optimized for expression in Escherichia coli (E. coli), cloned in the pET3a expression vector (Novagen), and purified from the BL21(DE3) arabinose-inducible (Invitrogen®) strain using a protocol adapted from the Max° purification protocol described in J.-F. Naud et al. 2003. J Mol Biol, 326:1577-1595; F.-O. and Mcduff et al. 2009. J Mol Recognit, 22:261-269. The resulting purified construct was the polypeptide of SEQ ID NO: 4. The identity of each purified construct was confirmed by mass spectrometry and Western blot analysis. Omomyc was purified by cation exchange chromatography, and purity was confirmed by mass spectrometry, SDS-PAGE, and ultraviolet spectroscopy. For in vivo administration, an additional purification step was performed to remove endotoxin using the ToxinEraser™ Endotoxin Removal Kit (Genscript). Endotoxin concentrations were quantified using the Pierce® LAL Chromogenic Endotoxin Quantification Kit (Thermo Scientific). Buffer exchange was performed in an Amicon Ultra-15 (Merck Millipore) with a 3 kDa exclusion limit.

[0320] Intranasal Omomyc treatment specifically increased the recruitment of T lymphocytes to tumor sites KRas LSL-G12D / +Mice were genotyped by Transnetyx and lung tumor generation in both males and females was performed as described above (Jackson, EL, et al., Genes Dev, 2001. 15(24): p. 3243-8). Animals were maintained on a mixed C57BL / 6JxFVBN background. A minimum of 5 mice per time point and condition were randomized and treatment began 14-16 weeks after Adeno-Cre infection when mice showed tumors detectable by microCT. Animals were anesthetized by inhalation of isoflurane (AbbVie Farmaceutica SLU) and treated intranasally with either Omomyc polypeptide (2.4 mg / kg) or vehicle (10 mM sodium acetate, pH 6.5) in a total volume of 30 μL (1101100) four times per week for either 1 or 4 weeks.

[0321] At the end point, mice were euthanized, and lungs were excised, perfused through the trachea with 4% PFA, fixed overnight, transferred to 70% ethanol, embedded in paraffin, and cut into 4 μm sections. For CD3 immunofluorescence, antigen retrieval was performed by heating in a 400W microwave for 20 min in 0.01M citrate buffer, pH 6.0. After blocking for 1 h in 3% BSA and 0.05% Tween20, slides were incubated overnight at 4°C with anti-CD3 (Dako A0452) diluted 1 / 100 in Dako Ready-to-use diluent (Dako S2022). After washing with PBS, slides were incubated with goat anti-rabbit IgG (H+L)-AlexaFluor® 488 conjugate (Thermo Fisher Scientific A-11008), stained with DAPI (Life Technologies D1306) diluted 1 / 10000, washed with PBS, and mounted with Fluorescence Mounting Medium (Dako S3023). Images were taken using a Nikon C2+ confocal microscope and NIS-elements software. Five photographs of representative tumors were taken per mouse, and the CD3 per area was + Cell averages are shown.

[0322] Immunostaining with anti-CD3 revealed that Omomyc treatment specifically increased the recruitment of T lymphocytes to the tumor site as early as one week after treatment initiation, and that T cells remained there throughout all treatments (Figure 1A), demonstrating a possible immune contribution as part of the mechanism of action of the Omomyc polypeptide.

[0323] Intranasal Omomyc treatment recruits activated CD4 T cells to tumor sites The experimental model and Omomyc treatment are the same as described above.

[0324] At the end point, mice were euthanized, and lungs were excised, separated using the Mouse Tumor Dissociation Kit (Miltenyi), and stained with conjugated antibodies to analyze immune cell content by flow cytometry. Prior to staining, dead cells were stained with Fixable Viability Stain 510 (BD Biosciences 564406) according to the manufacturer's instructions. Non-specific binders were then blocked by incubation with anti-CD16 / 32 antibody for 10 minutes at room temperature. For surface staining, cells were incubated with the antibody for 20 minutes at 4°C in the dark. The antibodies used are listed in Table 1. For FoxP3 intracellular staining, FoxP3 Transcription Buffer Set (eBioscience 00-5523-00) was used according to the manufacturer's instructions. Cells were acquired using a CytoFlex cytometer (Beckman Coulter) and data were analyzed using CytoExpert 2.0 software (Beckman Coulter).

[0325] Figure 1B shows FACS analysis showing that Omomyc induces the recruitment of CD4 T cells to tumors and activates them. Indeed, these cells show higher levels of PD-1 and both PD-1 and Tim-3 molecules, suggesting that Omomyc induces anti-tumor immune responses. In addition, Omomyc also induces the expression of T regulatory cells (Tregs).

[0326] Systemic administration of Omomyc recruits T cells to tumor sites 1x10 for studies with Kras / p53 syngeneic models 6 MuH-163 cells were inoculated subcutaneously into the dorsal flank of 7-week-old female C57BL / 6 (JANVIER LABS). Tumors were established and grew to approximately 100 mm 3After reaching a tumor volume of 1000 mg / kg, mice were randomized into two groups and treated intravenously once a week with vehicle (PBS, pH 7.0) or Omomyc (32 mg / kg). After 3 weeks of treatment, mice were euthanized, tumors were excised, and cut into two halves. One half of the tumor was then fixed overnight with 4% PFA, transferred to 70% ethanol, embedded in paraffin, and cut into 4 μm sections. For CD3 immunofluorescence, antigen retrieval was performed by heating in a 400W microwave for 20 minutes in 0.01M citrate buffer, pH 6.0. After blocking for 1 hour in 3% BSA and 0.05% Tween 20, slides were incubated overnight at 4°C with anti-CD3 (Dako A0452) diluted 1 / 100 in Dako Ready-to-use diluent (Dako S2022). After washing with PBS, slides were incubated with goat anti-rabbit IgG (H+L)-AlexaFluor® 488 conjugate (Thermo Fisher Scientific A-11008), stained with DAPI (Life Technologies D1306) diluted 1 / 10000, washed with PBS, and mounted with Fluorescence Mounting Medium (Dako S3023). Images were taken using a Nikon Tie fluorescence microscope and NIS-elements software. Four photographs per mouse of representative areas of the tumor were taken, and CD3 per field was counted. + Cell averages are shown.

[0327] For flow cytometry analysis, the other half of the tumor was dissociated using Mouse Tumor Dissociation Kit (Miltenyi) and stained with conjugated antibodies to analyze immune cell content by flow cytometry. Prior to staining, dead cells were stained with Fixable Viability Stain 510 (BD Biosciences 564406) according to the manufacturer's instructions. Non-specific binders were then blocked by incubation with anti-CD16 / 32 antibody for 10 minutes at room temperature. For surface staining, cells were incubated with the antibody for 20 minutes at 4°C in the dark. The antibodies used are listed in Table 1. Cells were acquired using a CytoFlex cytometer (Beckman Coulter) and data were analyzed using CytoExpert 2.0 software (Beckman Coulter).

[0328] Omomyc administration induced T cell recruitment to the tumor site (Figure 2A). Omomyc recruited more CD8 T cells to the tumor, and there was a significant increase in CD4 and CD8 T cells expressing both PD-1 and Tim-3 molecules (Figure 2B).

[0329] Omomyc in combination with anti-PD-1 targets CD4 + PD-1+Tim-3 - Mobilizing T cells KRas LSL-G12D / +Mice were genotyped by Transnetyx, and generation of lung tumors in both males and females was performed as described previously (Jackson, EL, et al., Genes Dev, 2001. 15(24): p. 3243-8). Animals were maintained in a pure C57BL / 6 background. Conditions were randomized with a minimum of 5 mice per time point, and treatment was initiated 14-16 weeks after mice showed tumors detectable by microCT following Adeno-Cre infection. Mice were randomized into 4 groups: vehicle + isotype murine IgG2a,k, Omomyc + isotype murine IgG2a,k, vehicle + anti-PD-1, and Omomyc + anti-PD-1. For Omomyc treatment, animals were anesthetized with inhaled isoflurane (AbbVie Farmaceutica SLU) and treated intranasally with either Omomyc polypeptide (2.4 mg / kg) or vehicle (PBS, pH=7) in a total volume of 30 μL four times per week (1101100). Anti-PD-1 (BioXCell BE0146) or its isotype Rat IgG2a,k (BioXCell BE0089) was given intraperitoneally once a week (0010000) at a dose of 200 μg / mouse for four weeks.

[0330] At the end point, mice were euthanized, and lungs were excised, dissociated using the Mouse Tumor Dissociation Kit (Miltenyi), and stained with conjugated antibodies to analyze immune cell content by flow cytometry. Prior to staining, dead cells were stained with Fixable Viability Stain 510 (BD Biosciences 564406) according to the manufacturer's instructions. Non-specific interactions were blocked by incubation with anti-CD16 / 32 antibody for 10 minutes at room temperature. For surface staining, cells were incubated with the antibody for 20 minutes at 4°C in the dark. The antibodies used are listed in Table 1. Cells were acquired using a CytoFlex cytometer (Beckman Coulter) and data were analyzed using CytoExpert 2.0 software (Beckman Coulter).

[0331] The combination of Omomyc and anti-PD-1 therapy significantly increases the recruitment of CD4+ T cells expressing PD-1, but not Tim-3, to the tumor site compared to both vehicle and anti-PD-1 only treated groups (Figure 3). This finding indicates that the combination of Omomyc with anti-PD-1 provides a synergistic effect to promote anti-tumor immune responses. Recent findings demonstrate that PD-1 expression on tumor-infiltrating lymphocytes (TILs) precisely identifies the repertoire of clonally expanded tumor-reactive cells (Gros A et al., J Clin Invest (2014) 124(5): 2246-2259). In this view, it is clear that initially PD-1 expression is a marker of T cell activation and of high avidity TILs specific for tumor antigens, although ligation with its ligands (PD-L1 and PD-L2) leads to an inhibitory signal (reviewed in Simon S and Labarriere N.. OncoImmunology (2018). 7:1, e1364828).

[0332] Omomyc in combination with anti-PD-1 induces IFN-γ production The experimental model, Omomyc and anti-PD-1 treatment are the same as those described above.

[0333] At the end point, mice were euthanized, and lungs were excised and dissociated using the Mouse Tumor Dissociation Kit (Miltenyi) and stained with conjugated antibodies to analyze immune cell content by flow cytometry. Prior to staining, dead cells were stained with Fixable Viability Stain 510 (BD Biosciences 564406) according to the manufacturer's instructions. Non-specific interactions were blocked by incubation with anti-CD16 / 32 antibody for 10 minutes at room temperature. For surface staining, cells were incubated with the antibody for 20 minutes at 4°C in the dark. The antibodies used are listed in Table 1. For IFN-γ staining, harvested and dissociated tumor cells were stimulated with PMA and ionomicin (both from Sigma-Aldrich) in the presence of monensin and befeldrin A (both from BD Biosciences) for 12 hours. Cells were then harvested and stained for flow cytometry analysis. For intracellular staining of IFN-γ, BD Cytofix / Cytoperm buffer set was used according to the manufacturer's instructions (BD Biosciences 554722). Cells were acquired using a CytoFlex cytometer (Beckman Coulter) and data were analyzed using CytoExpert 2.0 software (Beckman Coulter).

[0334] These experiments demonstrated that combination therapy of Omomyc and anti-PD-1 significantly induced interferon-γ (IFN-γ) production by both CD4+ helper and CD8+ cytotoxic intratumoral T cells compared with their vehicle counterparts (Figure 4), which was indeed not observed in either the Omomyc- or anti-PD-1-treated groups.

[0335] In the last few years, a considerable amount of evidence has accumulated demonstrating a key role for IFN-γ in promoting tumor rejection and clearance. This cytokine is mainly produced by activated T cells and NK cells and exerts its antitumor effects by directly inducing anti-proliferative, pro-apoptotic, and necroptotic effects on tumor cells and by enhancing immunogenicity by inducing upregulation of major histocompatibility molecules (reviewed extensively in Castro F et al., Front. Immunol. (2018). 9:847 and in Ikeda H et al., Cytokine & Growth Factors Reviews (2002). 13 95-109). Moreover, this cytokine also affects the intratumoral microenvironment, impairing angiogenesis by inhibiting the proliferation and survival of endothelial cells surrounding the tumor, and consequently inducing ischemia at the tumor site, which is a key mechanism leading to tumor rejection (Beatty G and Paterson Y. J Immunol (2001) 166:2276-82, Kammertoens T et al., Nature (2017) 545:98-102 and Briesemeister D et al., Int J Cancer (2011) 128:371-8). Moreover, this cytokine is important for the trafficking of T and NK cells to the tumor site, so that the production of IFN-γ by Th1 CD4+ and CD8+ T cells enhances tumor clearance (Melero I et al., Cancer Discov (2014) 4:522-6). In addition, IFN-γ also plays a crucial role in activating macrophages and promoting their tumoricidal activity (Celada A et al., J Exp Med (1984) 160:55-74).Importantly, increased levels of IFN-γ are a predictive biomarker of response to both chemotherapy and radiation therapy, as well as anti-PD-1 and CLTA-4 immunotherapy (Karachaliou N et al., Ther Adv Med Oncol (2018) 10:1758834017749748 and Mo X et al., Cancer Res (2018) 78:436-50). Consistently, recent clinical trials already have promising results showing a link between IFN-γ producing effector T cells and tumor growth inhibition (Liakou CI et al., Proc Natl Acad Sci USA (2008) 105:14987-92, Peng W et al., Cancer Res (2012) 72:5209-18 and Overacre-Delgoffe AE et al., Cell (2017) 169:1130-41.e11).

[0336] [Table 3]

[0337] Combination of Omomyc with anti-PD-1 antibody synergistically increases the proportion of healthy lung and recruits T cells to tumor sites KRas by Transnetyx LSL-G12D / +Mice were genotyped and lung tumor generation in both males and females was performed as described above (Jackson, EL, et al., Analysis of lung tumor initiation and progression using conditional expression of oncogenic K-ras. Genes Dev, 2001. 15(24): p. 3243-8). Animals were maintained on a pure C57BL / 6 background. 14-16 weeks after Adeno-Cre infection, when mice showed tumors detectable by microCT, they were randomized into four groups treated for 4 weeks as follows: vehicle + isotype type Rat IgG2a,k, Omomyc + isotype type Rat IgG2a,k, vehicle + anti-PD-1, and Omomyc + anti-PD-1. For Omomyc treatment, animals were anesthetized by inhalation of isoflurane (AbbVie Farmaceutica SLU) and treated intranasally with either Omomyc polypeptide (3.75 mg / kg) or vehicle (PBS, pH=7) in a total volume of 30 μL four times per week (1101100). Anti-PD-1 (BioXCell BE0146) or its isotype Rat IgG2a,k (BioXCell BE0089) was given intraperitoneally at 5 mg / kg once per week (0010000) for four weeks.

[0338] Micro-CT studies were acquired with a Quantum FX imaging system (Perkin Elmer, 940 Winter Street, Waltham, MA, EEUU) and image reconstruction was based on the Feldkamp method. For thoracic volume, Quantum FX analysis software was used. First, the distance between the contralateral ribs was measured at the carina level (r2). The second measurement was defined as the maximum distance from the carina level to the diaphragmatic cupule. The final measurement was the thoracic height, defined as the distance between the sternum and the sublumbar musculature at the diaphragmatic cupule level (r1). These three values ​​were used to calculate the volume of the truncated cone using the following formula: Volume = height * π / 3 * (r13-r23) / (r1-r2)

[0339] Lung healthy tissue was calculated using the AMIDE software (Amide® Andreas Loening) using a thresholding method that selects the complete amount of voxels in the images with intensity values ​​that fall within the -950 / -350 gray range. This grayscale range was selected manually after examining different studies.

[0340] Finally, based on the notion that intact thoracic volume is maintained while healthy lung volume is gradually decreased with progression of pathology, we calculated the healthy lung / thoracic volume ratio. Animals treated with Omomyc in combination with anti-PD-1 showed an increased percentage of healthy lung compared to vehicle and treatment alone (Figures 5A and 5B).

[0341] At the end point, mice were euthanized, and lungs were excised and dissociated using the Mouse Tumor Dissociation Kit (Miltenyi) and stained with conjugated antibodies. Prior to staining, dead cells were stained with Fixable Viability Stain 510 (BD Biosciences 564406) according to the manufacturer's instructions. Non-specific cross-reaction was blocked by incubation with anti-CD16 / 32 antibody for 10 minutes at room temperature. For surface staining, cells were incubated with the antibody for 20 minutes at 4°C in the dark. The antibodies used are listed in Table 2.

[0342] [Table 4]

[0343] For IFN-γ and IL-17 staining, harvested and dissociated tumor cells were stimulated with PMA and ionomicin (both from Sigma-Aldrich) in the presence of monensin and befeldrin A (both from BD Biosciences). Cells were then harvested and stained for flow cytometry analysis. For intracellular staining of IFN-γ, BD Cytofix / Cytoperm buffer set (BD Biosciences 554722) was used according to the manufacturer's instructions. Cells were acquired using a CytoFlex cytometer (Beckman Coulter) and data were analyzed using CytoExpert 2.0 software (Beckman Coulter).

[0344] Figure 5C shows that Omomyc and anti-PD-1 administered in combination induce T cell recruitment to the tumor site, specifically CD4 T cells and Th1 / Th17c cells. Table 3 shows that the effect obtained is synergistic. A synergistic effect is considered when the increase in the immune cell population of interest is greater than the sum of the increases of the individual treatments.

[0345] [Table 5]

[0346] Combination of Omomyc with anti-CTLA-4 antibody synergistically reduces tumor growth and recruits anti-tumor T cells to tumor sites The experimental model, micro-CT scan, and FACS staining were the same as those described in FIG.

[0347] 14-16 weeks after Adeno-Cre infection, when mice showed tumors detectable by microCT, they were randomized into four groups that were treated for 4 weeks as follows: vehicle + isotype Syrian hamster IgG, Omomyc + isotype Syrian hamster IgG, vehicle + anti-CTLA-4, and Omomyc + anti-CTLA-4. For Omomyc treatment, animals were anesthetized by inhalation of isoflurane (AbbVie Farmaceutica SLU) and treated intranasally with either Omomyc polypeptide (3.75 mg / kg) or vehicle (PBS, pH=7) in a total volume of 30 μL four times a week (1101100). Anti-CTLA-4 (BioXCell BE0131) or its isotype Syrian hamster IgG (BioXCell BE0087) were given intraperitoneally at 10 mg / kg once a week (0010000) for 4 weeks.

[0348] Figure 6A shows that animals treated with Omomyc in combination with anti-CTLA-4 exhibit reduced tumor growth compared to vehicle and treatment alone. Figure 6B shows that Omomyc and anti-CTLA-4 administered in combination significantly reduced tumor growth, particularly in CD4 T cells, and in CD8 PD-1 T cells. + Table 4 shows that both of the treatments induce T cell recruitment to the tumor site. Table 4 shows that the effect obtained is synergistic. If the increase in the immune cell population of interest is greater than the sum of the increases of the individual treatments, it is considered synergistic.

[0349] [Table 6]

[0350] Sequential combination of intravenously administered Omomyc with anti-PD-1 antibody synergistically recruits anti-tumor T cells to tumor sites The experimental model, micro-CT scanning, and FACS staining were the same as those described for FIG.

[0351] 14-16 weeks after Adeno-Cre infection, when mice showed tumors detectable by microCT, they were randomized into four groups that were treated for 4 weeks as follows: vehicle, Omomyc, vehicle + anti-PD-1, and Omomyc + anti-PD-1. For Omomyc treatment, mice were treated intravenously with either Omomyc polypeptide (50 mg / kg) or vehicle (NaAc 24 mM + 150 mM sodium chloride) (0100100) twice a week for 10 days. The group that received the combination was treated with Omomyc twice a week for the first 10 days. After 10 days of Omomyc treatment, the combination group was discontinued from Omomyc treatment and started receiving 2.5 mg / kg anti-PD-1 (BioXCell BE0146) intraperitoneally once a week (0010000) until the end of the experiment. The Omomyc monotherapy group received Omomyc twice a week for the first 10 days, and then continued treatment but only once a week until the end of the study.

[0352] Figure 7 shows that sequential treatment with Omomyc and then anti-PD-1 induced T cell recruitment to the tumor site, specifically of CD4 T cells expressing both PD-1 and Tim-3 molecules, and of Th1 / Th17 T cells expressing PD-1. Table 5 shows that the effect obtained is synergistic. If the increase in the immune cell population of interest is greater than the sum of the increases of the individual treatments, it is considered a synergistic effect.

[0353] [Table 7]

[0354] The combination of Omomyc administered intravenously with an anti-PD-1 antibody synergistically recruits T cells to tumor sites The highly aggressive Kras / p53 mutant NSCLC MuH-163 cell line was inoculated subcutaneously (1 × 10 6Once tumors were established, mice were randomized into four groups: vehicle, Omomyc, vehicle + anti-PD-1, and Omomyc + anti-PD-1. They received Omomyc treatment at 50 mg / kg (0010000) intravenously and anti-PD-1 antibody at 5 mg / kg intraperitoneally once a week for three weeks. Mice were monitored twice a week and tumor growth was measured with calipers.

[0355] At endpoint, tumors were harvested and half were fixed with 4% PFA and embedded in paraffin for IHC analysis, while the other half was digested using a Mouse Tumor Dissociation Kit (Miltenyi) and stained with conjugated antibodies to analyze immune cell content by flow cytometry. FACS staining and analysis were performed as described for FIG. 5.

[0356] For CD3 immunofluorescence, antigen retrieval was performed by heating in 0.01 M citrate buffer, pH 6.0, for 20 min using a microwave set at 400 W. After blocking in 3% BSA for 45 min and washing with PBS, slides were incubated overnight at 4° C. with anti-CD3 (Dako A0452) diluted 1 / 100 in Dako Ready-to-use diluent (Dako S2022). After PBS washing, slides were incubated with goat anti-rabbit IgG (H+L)-AlexaFluor® 488 conjugate (Thermo Fisher Scientific A-11008) diluted 1 / 200, stained with DAPI (Life Technologies D1306) diluted 1 / 10000, washed once with water, and mounted with Fluorescence Mounting Medium (Dako S3023). CD3 positivity was measured from five representative fluorescence microscopy images per animal taken at 20x magnification.

[0357] Figure 8 shows that co-treatment with Omomyc and anti-PD-1 significantly recruits T cells to the tumor site. Table 6 shows that the effect obtained is synergistic. A synergistic effect is considered when the increase in the immune cell population of interest is greater than the sum of the increases of the individual treatments.

[0358] [Table 8]

[0359] High expression of CD3, CD4, IL-17, and IFN-γ correlates with better survival Kaplan-Meier plots were performed using the Kaplan-Meier plotter (http: / / kmplot.com / analysis / index.php?p=background). To do so, a database of lung cancer patients was selected. All histological types, all stages, and all grades of NSCLC were included for the analysis.

[0360] FIG. 9 shows that high expression of CD3, CD4, IL-17, and IFN-γ correlates with higher survival in NSCLC patients.

[0361] conclusion The combination of anti-PD-1 antibody with intranasal Omomyc synergistically increased the percentage of healthy lung relative to total thoracic volume (average 7.969) compared to the improvement shown by Omomyc (0.86) and anti-PD-1 (0.92) therapy alone (Figure 5A and 5B). In addition, the simultaneously administered treatments significantly induced the recruitment of T cells to the tumor site, particularly CD4 T cells and Th1 / Th17c cells, which are known to exert potent antitumor effects (Chatterjee, S., et al., CD38-NAD(+)Axis Regulates Immunotherapeutic Anti-Tumor T Cell Response. Cell Metab, 2018. 27(1): p. 85-100 e8) (Figure 5C).

[0362] In line with these results, the combination of anti-CTLA-4 with intranasal Omomyc showed reduced tumor growth (1.11) compared to vehicle (3.85) and both treatments administered alone (Omomyc: 2.3; a-CTLA-4: 3.0) (Figure 6A).

[0363] In addition to this direct effect on tumor growth, the therapeutic combination also synergistically induced T cell recruitment to the tumor site, specifically CD4 T cells as well as both CD4 and CD8 T cells expressing the PD-1 molecule, cells known to identify tumor-specific T cells (Gros, A., et al., PD-1 identifies the patient-specific CD8(+) tumor-reactive repertoire infiltrating human tumors. J Clin Invest, 2014. 124(5): p. 2246-59) (Figure 6B). In summary, Omomyc administered intranasally in combination with anti-PD-1 or anti-CTLA-4 reduces tumor growth and synergistically recruits anti-tumor T cells to the tumor site.

[0364] Furthermore, the same mouse model (Kras G12D By using CD4+-driven NSCLC, we demonstrated that intravenous Omomyc in combination with sequentially administered anti-PD-1 (Omomyc administered first, followed by anti-PD-1 antibody) also conferred a synergistic effect and induced the recruitment of T cells to the tumor site, particularly tumor-specific CD4 and Th1 / Th17 anti-tumor T cells expressing both PD-1 and Tim-3 molecules (Figure 7).

[0365] To validate this synergy in another model, we combined Omomyc and anti-PD-1 in another highly aggressive model of NSCLC driven by mutations in both Kras and p53. Again, both drugs synergized, recruiting significantly more T cells to the tumor site (Figure 8A), as well as more overall immune cells (Figure 8B).

[0366] In summary, we conclude that Omomyc treatment in combination with both anti-PD-1 and CTLA-4 therapy can reduce tumor growth and synergistically recruit anti-tumor T cells to the tumor site. This therapeutic effect was observed using different administration routes, different Omomyc doses, and different doses of anti-PD-1 and CTLA-4.

[0367] This immune cell mobilization has clear therapeutic impacts for NSCLC cancer patients, since an increase in the percentage of CD3 T cells, CD4 cells, and total T cells secreting IFN-g and IL-17 correlates with increased survival (Figure 9). This evidence highlights the importance of the findings described above regarding the combination of Omomyc with cancer immunotherapy. If immune signature-based data demonstrate that a strong immune cell component predicts a good response to chemotherapy in breast cancer, such as high tumor-infiltrating lymphocytes (TILs) being associated with a higher response rate to neoadjuvant therapy, the same conclusions can be extrapolated to other types of cancer. In colorectal cancer liver metastases, CD8 + High infiltration of T cells predicts good response to chemotherapy and long-term survival. In melanoma, expression of immune signature (i.e., high expression of Th1 cells and cytotoxicity-related genes) correlates with good clinical response to therapeutic vaccine using melanoma-associated antigen 3 (MAGEA3) (reviewed in Fridman, WH, et al., The immune contexture in cancer prognosis and treatment. Nat Rev Clin Oncol, 2017. 14(12): p. 717-734).

[0368] In the last few years, a considerable amount of evidence has been accumulated that demonstrates the important role of TILs for both tumor eradication and for the efficacy of cancer immunotherapy. Indeed, a major factor involved in resistance to cancer immunotherapy is the lack of tumor T cell infiltration, which characterizes so-called "cold tumors". The treatment of these immune-inactive tumors with cancer immunotherapy presents great challenges, since they do not display any adaptive immune response against the tumor and are unresponsive to this type of therapy (Bonaventura, P., et al., Cold Tumors: A Therapeutic Challenge for Immunotherapy. Front Immunol, 2019. 10: p. 168).

[0369] Patients who do not show any clinical response or stabilized disease to PD-1 / PD-L1 blockade are considered to have "primary resistance" to the therapy. In contrast, early data from clinical trials have demonstrated that the presence of pre-existing TILs in and around the tumor, together with coexistent PD-1 and PD-L1 expression on T cells and tumor cells, respectively, predicts therapeutic response to anti-PD-1 therapy (Nowicki, TS, S. Hu-Lieskovan, and A. Ribas, Mechanisms of Resistance to PD-1 and PD-L1 Blockade. Cancer J, 2018. 24(1): p. 47-53). Along the same lines of evidence, the efficacy of pembrolizumab (anti-PD-1) correlates with the presence of intratumoral T cells and PD-1 / PD-L1 expression as prerequisites for a strong anti-tumor response (Tumeh, PC, et al., PD-1 blockade induces responses by inhibiting adaptive immune resistance. Nature, 2014. 515(7528): p. 568-71) as well as the efficacy of anti-PD-1 agents (Ribas, A., Tumor immunotherapy directed at PD-1. N Engl J Med, 2012. 366(26): p. 2517-9).

[0370] Considering all of this evidence, it is demonstrated that the combination of Omomyc with cancer immunotherapy synergistically induces T cell infiltration as well as stimulation that will ultimately lead to improved clinical response rates to cancer immunotherapy.

Claims

1. i) the group consisting of: a) a polypeptide comprising the sequence of SEQ ID NO:1 or a functionally equivalent variant thereof; b) a conjugate comprising a polypeptide comprising the sequence of SEQ ID NO:1 or a functionally equivalent variant thereof and a chemical moiety that promotes the cellular uptake of said polypeptide or a functionally equivalent variant thereof; c) a polynucleotide encoding the polypeptide of a) or the conjugate of b); d) a vector comprising the polynucleotide of c), and e) a cell capable of secreting the polypeptide of a) or the conjugate of b) into the culture medium. A first component selected from ii) a second component which is an immuno-oncology agent; and A combination comprising:

2. The combination according to claim 1, wherein the functionally equivalent variant of SEQ ID NO:1 is selected from the group consisting of SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, and SEQ ID NO:

10.

3. The combination according to any one of claims 1 to 2, wherein the chemical moiety that promotes cellular uptake of the polypeptide or its functionally equivalent variant is a cell membrane-permeable peptide sequence, and the cell membrane-permeable peptide sequence and the polypeptide or its functionally equivalent variant form a fusion protein.

4. The combination according to claim 3 , wherein the cell membrane penetrating peptide sequence is selected from the group consisting of GRKKRRQRRR (SEQ ID NO: 37) and RRRRRRLR (SEQ ID NO: 38).

5. The combination according to any one of claims 1 to 4, wherein the conjugate further comprises an additional nuclear localization signal.

6. The combination according to any one of claims 1 to 5, wherein the cancer immunological agent is not a cytokine.

7. The combination according to any one of claims 1 to 6, wherein the cancer immunotherapy agent is an antagonist of a protein that inhibits T cell activation or an immune checkpoint inhibitor.

8. The combination according to claim 7, wherein the antagonist of a protein that inhibits T cell activation is selected from anti-PD-1 and anti-CTLA-4.

9. The combination according to claim 8, wherein the antagonist of a protein that inhibits T cell activation is anti-PD-1.

10. 10. The combination according to claim 8 or 9, wherein the antagonist is an antagonist antibody.

11. The combination of claim 10, wherein the antagonist antibody is pembrolizumab.

12. A combination according to any one of claims 1 to 11, wherein the first component is a polypeptide comprising the sequence of SEQ ID NO:

1.

13. A pharmaceutical composition comprising a therapeutically effective amount of a combination according to any one of claims 1 to 12 and a pharma- ceutically acceptable excipient.

14. A combination according to any one of claims 1 to 12 or a pharmaceutical composition according to claim 13 for use in medicine.

15. A combination according to any one of claims 1 to 12 or a pharmaceutical composition according to claim 13 for use in the prevention and / or treatment of cancer.

16. 16. The combination or pharmaceutical composition for use according to claim 15, wherein the cancer is lung cancer.

17. The combination or pharmaceutical composition according to any one of claims 14 to 16, wherein said composition is administered systemically or intranasally.

18. 18. A combination or pharmaceutical composition for use according to claim 17, wherein said intranasal administration is performed by drops or by intranasal inhalation.

19. A combination or pharmaceutical composition for use according to any one of claims 14 to 16, wherein the first component is administered intranasally or intravenously and the second component is administered systemically.

20. A combination or pharmaceutical composition for use according to any one of claims 14 to 19, wherein said polypeptide or a functionally equivalent variant thereof, or said conjugate, interacts synergistically with said cancer immunotherapy agent in the treatment of cancer.

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