Combination therapy for the treatment of cancer with omomyc and antibodies that bind pd-1 or ctla-4

Abstract

The present invention relates to a combination of a tumor immune agent and Omomyc, a functionally equivalent variant thereof, a conjugate comprising Omomyc or said functionally equivalent variant, a polynucleotide encoding said polypeptide, a vector comprising said polynucleotide, and a cell capable of secreting said polypeptide or conjugate. The invention also relates to a pharmaceutical composition comprising a combination of the invention and its medical use, in particular its use in the treatment of cancer.

Description

[0001] This application is a divisional application of the patent application 202080021965.2 (based on PCT application No. PCT / EP2020 / 057492) titled "Combination Therapies for the Treatment of Cancer Using Omomyc and Antibodies that Bind PD-1 or CTLA-4" filed by the applicant on March 18, 2020. Technical Field

[0002] The present invention relates to the field of cancer, and more particularly, to combinations comprising a polypeptide and a tumor immunotherapeutic agent and their medical use, more particularly their use in the prevention and / or treatment of cancer. Background Art

[0003] An ideal cancer drug should target non-redundant functions that are continuously required to maintain the tumor, but are not essential for the maintenance and function of any normal tissue. Thus, the most common logic has been to target gene products that are specifically mutated in cancer, on the basis that these mutant molecules may be "drivers" of cancer and may be less important for normal tissues. For these reasons, much attention has been focused on cataloging recurrent lesions in specific cancer types. Unfortunately, this approach has several problems. First, most human solid cancers undergo genomic instability events and exhibit mutational noise that can obscure "driver" mutations and their accompanying effector pathways. Second, cancer is the end result of a process involving transitions through multiple evolutionary bottlenecks. Each bottleneck requires a specific type of mutation, the function of which is no longer necessary for tumor maintenance thereafter and thus is no longer a good therapeutic target after that point in tumor evolution.

[0004] Myc is a basic helix-loop-helix leucine zipper (b-HLH-LZ) protein involved in growth control and cancer, which functions in a network of structurally related proteins Max, Mad, and Mnt. The Myc / Max dimer initiates gene transcription and induces cell proliferation or apoptosis. The Mad / Max and Mnt / Max complexes act as repressors, causing cell growth arrest and differentiation. All dimers recognize the same DNA consensus site, namely the CACGTG E-box.

[0005] Myc is tightly regulated in normal cells, with higher levels in proliferating cells and lower levels in non-proliferating cells. Abnormally high and / or dysregulated Myc activity is causally linked to most cancers and is generally associated with aggressive, poorly differentiated, and angiogenic tumors. Dysregulation of Myc expression is attributed to overexpression through gene amplification, loss of transcriptional control, impaired degradation, or increased stability. This results in abnormal proliferation, increased survival, altered metabolism, angiogenesis, and inflammation, all of which represent hallmarks of cancer. Multiple studies have confirmed the key role of Myc in regulating both intracellular and extracellular aspects of tumorigenesis, suggesting that targeting Myc function has therapeutic value.

[0006] It is known that the downregulation of Myc by BET bromodomain inhibitors leads to the regression of multiple tumor types. Although this approach shows great potential, it has some limitations, such as toxicity and a large number of off-target effects. Many small molecules that disrupt Myc / Max interactions show low specificity in cells.

[0007] However, Myc inhibitors have not yet entered clinical use, and their design poses various caveats: first, Myc is a nuclear transcription factor and thus more difficult to reach than membrane or cytoplasmic molecules; second, Myc does not have an enzymatic "active site" that can be targeted; third, the Myc family includes three different proteins, c-Myc, N-Myc, and L-Myc, which are functionally redundant in some cases, so all of these proteins need to be inhibited simultaneously. In addition, there are concerns that the inhibition of Myc may cause severe side effects by inhibiting the proliferation of normal tissues. For all these reasons, manufacturing Myc inhibitor drugs is challenging.

[0008] 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, retaining the ability to bind its natural partner Max and form homodimers with itself as well as heterodimers with wild-type c-Myc, N-Myc, and L-Myc.

[0009] Due to these properties, Omomyc can block Myc-dependent gene transactivation function in vitro and in vivo by invalidating the ability of Myc to bind its DNA recognition binding sites (E-boxes). At the same time, Omomyc strongly enhances Myc-induced apoptosis in a Myc expression level-dependent manner, thereby enhancing the transcriptional inhibitory activity of Myc. Omomyc thus blocks the binding of Myc to the promoter E-box and the transactivation of target genes, while retaining the Miz-1-dependent binding to the promoter and transcriptional repression. In the presence of Omomyc, the Myc interactome is redirected towards repression, and its activity is transformed from an oncogene into a tumor suppressor gene.

[0010] In EP2801370A1, it has been demonstrated that the Omomyc peptide itself is able to efficiently cross the cell membrane and translocate into the nucleus, where it exerts its tumor-inhibiting effect.

[0011] However, there is still a need in the art to develop new and improved therapeutic methods for treating cancer. Summary of the Invention

[0012] In a first aspect, the present invention relates to a combination comprising:

[0013] i) a first component selected from the group consisting of:

[0014] a) a polypeptide comprising the sequence SEQ ID NO: 1 or a functionally equivalent variant thereof,

[0015] b) a conjugate comprising: a polypeptide comprising the sequence SEQ ID NO: 1 or a functionally equivalent variant thereof; and a chemical moiety that promotes cellular uptake of the polypeptide or the functionally equivalent variant thereof,

[0016] c) a polynucleotide encoding the polypeptide of a) or the conjugate of b),

[0017] d) a vector comprising the polynucleotide according to c), and

[0018] e) a cell capable of secreting the polypeptide according to a) or the conjugate according to b) into the culture medium;

[0019] and

[0020] ii) a second component, which is a tumor immunotherapeutic agent.

[0021] In a second aspect, the present invention relates to a pharmaceutical composition comprising a pharmaceutically effective amount of the combination according to the present invention and a pharmaceutically acceptable excipient.

[0022] In a third aspect, the present invention relates to a combination according to the present invention or a pharmaceutical composition according to the present invention, which is used for medicine.

[0023] In a fourth aspect, the present invention relates to a combination according to the present invention or a pharmaceutical composition according to the present invention, which is used for preventing and / or treating cancer. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 : Intranasal administration of Omomyc recruits T cells to the tumor site. Mice bearing KRas G12D -driven NSCLC were treated intranasally with Omomyc four times a week for 4 weeks. (A) As early as 1 week after the start of treatment, administration of Omomyc induced recruitment of T cells to the tumor site, and the T cells remained there throughout the treatment. *p < 0.05; **p < 0.01. (B) FACS analysis showed that Omomyc induced recruitment of CD4 T cells to the tumor, especially activated CD4 T cells, which showed higher levels of PD-1 and PD-1 Tim-3 molecules. Omomyc also induced the expansion of regulatory T cells (Tregs).

[0025] Figure 2 : Systemic administration of Omomyc recruits T cells to the tumor site. The Kras / p53 mutant NSCLC MuH-163 cell line was subcutaneously inoculated into syngeneic mice. The mice were treated systemically with Omomyc for 3 weeks. Compared with its vehicle control, Omomyc recruited more CD3 + T cells to the tumor site (A), and there was a significant increase in CD4 and CD8 T cells expressing both PD-1 and Tim-3 molecules (B). **p < 0.01.

[0026] Figure 3 : The combination of Omomyc and anti-PD-1 recruits CD4+PD-1+Tim-3- T cells to the tumor. Mice bearing KRas G12D -driven NSCLC were treated intranasally with Omomyc four times a week and intraperitoneally with anti-PD-1 (250 μg) once a week for 4 weeks. The combination of Omomyc and anti-PD-1 induced recruitment of CD4 + PD-1 + Tim-3 - T cells to the tumor site.

[0027] Figure 4 : The combination of Omomyc and anti-PD-1 induces the production of IFN-γ. Treatment with the combination of Omomyc and anti-PD-1 induced the production of IFN-γ by CD4 (A) and CD8 (B) T cells in the tumor.

[0028] Figure 5 : The combination of Omomyc and anti-PD-1 antibody synergistically increases the proportion of healthy lungs and recruits T cells to the tumor site. Mice bearing KRas G12D -driven NSCLC were treated intranasally with Omomyc four times a week and intraperitoneally with anti-PD-1 antibody once a week. (A) Compared with vehicle and single treatments, animals treated with the combination of Omomyc and anti-PD-1 showed an increased proportion of healthy lungs. (B) Representative transverse plane CT images obtained from each experimental group at the start and end of treatment. Dark regions correspond to healthy lungs, and gray regions correspond to affected lungs. (C) FACS analysis showed that combination administration of Omomyc and anti-PD-1 induced T cell recruitment to the tumor site, particularly CD4 T cells and Th1 / Th17 cells. *p < 0.05; **p < 0.01; ***p < 0.0001.

[0029] Figure 6 : The combination of Omomyc and anti-CTLA-4 antibody synergistically reduces tumor growth and recruits anti-tumor T cells to the tumor site. Mice bearing KRas G12D -driven NSCLC were treated intranasally with Omomyc four times a week and intraperitoneally with anti-CTLA-4 antibody once a week. (A) Compared with vehicle and single treatments, animals treated with the combination of Omomyc and anti-CTLA-4 showed reduced tumor growth. The mean tumor growth of each treatment group is shown in the table. (B) FACS analysis showed that combination administration of Omomyc and anti-CTLA-4 induced T cell recruitment to the tumor site, particularly CD4 T cells and both CD4 PD-1 + T cells and CD8 PD-1 + T cells. *p < 0.05; **p < 0.01; ***p < 0.0001.

[0030] Figure 7 : The sequential combination of Omomyc and anti-PD-1 antibody synergistically recruits anti-tumor T cells to the tumor site. Every four days, mice bearing KRas G12D -driven NSCLC were treated intravenously with Omomyc for 10 days, and then mice bearing KRas G12D -driven NSCLC were treated intraperitoneally with anti-PD-1 antibody once a week. FACS analysis showed that treatment with Omomyc followed by anti-PD-1 induced T cell recruitment to the tumor site, particularly CD4 T cells expressing both PD-1 and Tim-3 molecules and Th1 / Th17 T cells expressing PD-1. *p < 0.05; **p < 0.01.

[0031] Figure 8: The combination of Omomyc and anti-PD-1 antibody synergistically recruits T cells to the tumor site. Mice bearing KRas G12D / p53-driven NSCLC were treated once a week with Omomyc (intravenously) and anti-PD-1 (intraperitoneally) concomitantly. (A) IHC staining showed that the combined treatment with Omomyc and anti-PD-1 significantly recruited T cells to the tumor site. (B) FACS analysis showed that treatment with Omomyc and anti-PD-1 induced the recruitment of all immune cells to the tumor site. *p < 0.05; **p < 0.01.

[0032] Figure 9 : High expression of CD3, CD4, IL-17, and IFN-γ is associated with higher survival rates. Representative Kaplan-Meier curves of NSCLC patients considering CD3, CD4, IL-17, and IFN-γ expression are shown. The table below the figure shows the survival rates at the upper quartile. The graph was plotted using the Kaplan-Meier Plotter at http: / / kmplot.com / analysis / index.php?p=background. Detailed Description of the Invention

[0033] The present invention relates to providing new therapeutic combinations for the prevention and treatment of cancer.

[0034] Unless otherwise defined, all technical terms used herein have the same meaning as commonly understood by one of ordinary skill in the technical field to which the present invention belongs.

[0035] All embodiments disclosed for one aspect of the present invention are applicable to other aspects.

[0036] Combinations and pharmaceutical compositions of the invention

[0037] The definitions provided herein and in each other aspect of the present invention equally apply to the entire invention.

[0038] The authors of the present invention have demonstrated that intranasal and systemic administration of Omomyc can recruit T cells to the tumor site ( Figure 1 and 2 ). Therefore, the combination of Omomyc and tumor immunomodulators can be used to treat cancer. In addition, it has been found that the combination of Omomyc and tumor immunomodulators has a synergistic effect in the treatment of cancer. For example, compared with the vehicle and anti-PD-1 treatment groups alone, the combination of Omomyc and anti-PD-1 therapy significantly increased the recruitment of CD4 + T cells expressing PD-1 but not Tim-3 to the tumor site ( Figure 3)。In addition, the combination of Omomyc and anti-PD-1 therapy significantly induced interferon-γ (IFN-γ) production by CD4 + helper cells and CD8+ cytotoxic intratumoral T cells compared to their vehicle controls ( Figure 4 ), a fact not observed in either the Omomyc or anti-PD-1 treated groups. When subjects with lung cancer were treated, the recruitment of T cells to the tumor site translated into a synergistic increase in the proportion of healthy lung ( Figure 5 ). This synergistic effect was maintained regardless of the route of administration, dose, and regimen ( Figure 7 and Figure 8 ). The combination of Omomyc and anti-CTLA-4 therapy was also found to synergistically reduce tumor growth and recruit anti-tumor T cells to the tumor site ( Figure 6 ).

[0039] Accordingly, in a first aspect, the present invention relates to a combination comprising:

[0040] i) a first component selected from the group consisting of:

[0041] a) a polypeptide comprising the sequence SEQ ID NO: 1 or a functionally equivalent variant thereof,

[0042] b) a conjugate comprising: a polypeptide comprising the sequence SEQ ID NO: 1 or a functionally equivalent variant thereof; and a chemical moiety that facilitates cellular uptake of the polypeptide or the functionally equivalent variant thereof, and

[0043] c) a polynucleotide encoding the polypeptide of a) or the conjugate of b),

[0044] d) a vector comprising the polynucleotide according to c), and

[0045] e) a cell capable of secreting the polypeptide according to a) or the conjugate according to b) into the culture medium;

[0046] and

[0047] ii) a second component which is a tumor immunotherapeutic agent.

[0048] According to the present invention, the expression "combination" represents various combinations of compounds (i) and (ii), such as a composition formulated as a single preparation, a combined mixture consisting of separate preparations of each component (e.g., a "tank-mix" that can be combined and used as a combined preparation), and the combined use of individual active ingredients in a sequential manner (i.e., one after another within a relatively short time, such as a few hours or days) or in a simultaneous administration manner. In the present invention, compound (i) refers to a therapeutically effective amount of a polypeptide comprising SEQ ID NO:1 or a functionally equivalent variant thereof; or a conjugate comprising a polypeptide comprising SEQ ID NO:1 or a functionally equivalent variant thereof and a chemical moiety that promotes cellular uptake of the polypeptide or the functionally equivalent variant thereof; or a polynucleotide encoding the polypeptide or the conjugate; or a vector comprising the polynucleotide; or a cell capable of secreting the polypeptide or the conjugate into the culture medium. In the present invention, compound (ii) refers to a therapeutically effective amount of a tumor immunomodulator. Preferably, the order of application of compounds (i) and (ii) is not essential for carrying out the present invention.

[0049] The combination may be a kit-of-parts, wherein each component is formulated and packaged separately.

[0050] The combination of compounds (i) and (ii) can be formulated for simultaneous, separate or sequential administration. In particular, if the administration is not simultaneous, the compounds are administered within a time close to each other. In addition, the compounds are administered in the same or different dosage forms or by the same or different routes of administration. For example, one compound can be administered orally, while 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.

[0051] The combination of the two compounds (i) and (ii) can be administered in the following ways:

[0052] - As a combination, which is a component of the same pharmaceutical preparation, and the two compounds are always administered simultaneously;

[0053] - As a combination of two units, each unit having one of the substances that allows for simultaneous, sequential or separate administration.

[0054] In a specific embodiment, compound (i) of the combination of the present invention can be administered independently of compound (ii), but at the same time, i.e., administered as two units.

[0055] In another specific embodiment, first the compound (i) of the combination of the present invention is administered, and then the compound (ii) is administered, i.e., the compound (ii) is administered separately or sequentially.

[0056] In yet another specific embodiment, first the compound (ii) of the combination of the present invention is administered first, and then the compound (i) is administered, i.e., the compound (i) is administered separately or sequentially as defined. If administered separately, the compounds (i) and (ii) of the combination of the present invention can be administered at intervals of time from each other, such as at intervals of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23 or 24 hours from each other. In another embodiment, the compounds (i) and (ii) of the combination of the present invention can be administered at intervals of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23 or 24 days from each other, preferably at intervals of 1 day from each other, more preferably at intervals of 10 days from each other. In a preferred embodiment, the compound (ii) is administered 10 days after the first administration of the compound (i). In one embodiment, the administration of the first compound is stopped before the start of the administration of the second compound.

[0057] In another aspect, the present invention relates to a combination or pharmaceutical composition comprising a synergistically effective amount of a first component according to the first aspect of the present invention and a tumor immunizing agent.

[0058] In a preferred embodiment, the compound (i) of the present invention is a polypeptide comprising the sequence SEQ ID NO: 1 or a functionally equivalent variant thereof.

[0059] The terms "polypeptide" and "peptide" are used interchangeably herein and refer to a polymer of amino acids of any length. The polypeptides of the present invention can comprise modified amino acids and can be interrupted by non-amino acids. In a preferred embodiment, the polypeptide is formed only of amino acids. Preferably, the polypeptide forming item (i) of the combination has a length of 80 to 500 amino acids, more preferably 80 to 300 amino acids, more preferably 80 to 250 amino acids, more preferably 80 to 150 amino acids, even more preferably 80 to 130 amino acids, preferably 90 to 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 90 to 98 amino acids, preferably 90 to 95 amino acids, more preferably 91 amino acids.

[0060] The term "amino acid" refers to natural and synthetic amino acids, as well as amino acid analogs and mimetics that act in a manner similar to natural amino acids. In addition, the term "amino acid" includes D-amino acids and L-amino acids (stereoisomers). Preferably, the amino acid is an L-amino acid.

[0061] The term "natural amino acid" or "naturally occurring amino acid" includes the 20 naturally occurring amino acids; those amino acids that are typically post-translationally modified in vivo, including, for example, hydroxyproline, phosphoserine, and phosphothreonine; and other less common amino acids, including but not limited to 2-aminoadipic acid, hydroxylysine, isodesmosine, norvaline, norleucine, and ornithine.

[0062] As used herein, the term "unnatural amino acid" or "synthetic amino acid" refers to a carboxylic acid or its derivative that has an amine group substituted at position "a" and is structurally related to a natural amino acid. Exemplary non-limiting examples of modified or less common amino acids include: 2-aminoadipic acid, 3-aminoadipic acid, β-alanine, 2-aminobutyric acid, 4-aminobutyric acid, 6-aminohexanoic acid, 2-aminoheptanoic acid, 2-aminoisobutyric acid, 3-aminoisobutyric acid, 2-aminoheptanedioic acid, 2,4-diaminobutyric acid, desmosine, 2,2'-diaminopimelic acid, 2,3-diaminopropionic acid, N-ethylglycine, N-ethylasparagine, hydroxylysine, alio hydroxy lysine, 3-hydroxyproline, 4-hydroxyproline, isodesmosine, alloisoleucine, N-methylglycine, N-methylisoleucine, 6-N-methyllysine, N-methylvaline, norvaline, norleucine, ornithine, and the like.

[0063] The polypeptides of the present invention may also contain non-amino acid moieties, such as hydrophobic moieties (various straight-chain, branched-chain, cyclic, polycyclic, or heterocyclic hydrocarbons and hydrocarbon derivatives) linked to the peptide; various protecting groups attached to the ends of the compound to reduce degradation. Suitable protecting functional groups are described in Chapter 5 and Chapter 7 of "Protecting Groups in Organic Synthesis" by Green and Wuts, John Wiley and Sons, 1991.

[0064] Chemical (non-amino acid) groups may be included in the polypeptide to improve various physiological properties, such as reducing degradation or clearance, reducing rejection by various cellular pumps, improving various modes of administration, increasing specificity, enhancing affinity, increasing stability, bioavailability, solubility, reducing toxicity, and the like.

[0065] "Mimetics" include molecules that have a chemical structure mimicking a peptide structure and retain the functional properties of the peptide structure. Methods for designing peptide analogs, derivatives, and mimetics are known in the art.

[0066] In one embodiment, the polypeptide of the present invention is a polypeptide consisting of the sequence SEQ ID NO: 1 or a polypeptide consisting of a functionally equivalent variant of SEQ ID NO: 1, preferably a polypeptide consisting of the sequence SEQ ID NO: 1. SEQ ID NO: 1 corresponds to:

[0067] TEENVKRRTHNVLERQRRNELKRSFFALRDQIPELENNEKAPKVVILKKATAYILSVQAETQKLISEIDLLRKQNEQLKHKLEQLRNSCA (SEQ ID NO: 1).

[0068] The polypeptide of the sequence SEQ ID NO: 1 corresponds to the Omomyc protein sequence. As used herein, the term "Omomyc" refers to a polypeptide consisting of a mutant form of the bHLHZip domain of Myc with E61T, E68I, R74Q, and R75N mutations (where the numbering of the mutation positions is relative to the sequence of the Myc region corresponding to amino acids 365 - 454 of the polypeptide defined by the accession number NP_002458 in the NCBI database (released on March 15, 2015)). The c-Myc sequence (SEQ ID NO: 2) provided in the NCBI database with the accession number NP_002458 is shown below, where the Omomyc-derived region is underlined:

[0069]

[0070] Omomyc also contains the M2 domain of c-Myc, which has the sequence RQRRNELKRSF (SEQ ID NO: 3) (see Dang and Lee, Mol. Cell. Biol., 1988, 8: 4048 - 4054) (the above double-underlined part), and corresponds to the nuclear localization signal.

[0071] Omomyc is characterized by its enhanced dimerization ability towards 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, provided that the mutations leading to tumor suppressor activity are retained. Thus, Omomyc that can be used in the present invention can be derived from any mammal, including but not limited to domestic and farm animals (cattle, horses, pigs, sheep, goats, dogs, cats, or rodents), primates, and humans. Preferably, the Omomyc protein is derived from the human Myc protein (accession number NP_002458, released on March 12, 2019).

[0072] As used herein, the term "Myc" refers to a family of transcription factors that includes c-Myc, N-Myc, and L-Myc. Myc proteins initiate the expression of many genes by binding to the consensus sequence CACGTG (enhancer box sequence or E-box and recruiting histone acetyltransferase or HAT). However, Myc can also act as a transcriptional repressor. By binding to the Miz-1 transcription factor and displacing the p300 co-activator, it can inhibit the expression of Miz-1 target genes. Myc also plays a direct role in controlling DNA replication.

[0073] The Myc b-HLH-LZ or Myc basic region helix-loop-helix leucine zipper domain refers to the region that determines the dimerization of Myc with the Max protein and the binding to Myc target genes. This region corresponds to amino acids 365-454 of human Myc and is characterized by two α helices connected by a loop (Nair, S.K., & Burley, S.K., 2003, Cell, 112:193-205).

[0074] 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:

[0075] MTEENVKRRTHNVLERQRRNELKRSFFALRDQIPELENNEKAPKVVILKKATAYILSVQAETQKLISEIDLLRKQNEQLKHKLEQLRNSCA (SEQ ID NO:4)

[0076] As used herein, "consisting essentially of" means that the specified molecule will not contain any other sequences that would alter the activity of SEQ ID NO:4.

[0077] Preferably, the polypeptide consists of SEQ ID NO:4.

[0078] The term "functionally equivalent variant" refers to any polypeptide obtained from the polypeptide of SEQ ID NO: 1 by the insertion or addition of one or more amino acids and / or by the deletion of one or more amino acids and / or by the conservative substitution of one or more amino acids, and / or refers to any polypeptide obtained by chemical modification of the polypeptide of SEQ ID NO: 1 and which substantially retains the tumor suppressor activity of SEQ ID NO: 1. Preferably, the functionally equivalent variant refers to any polypeptide produced from the polypeptide of SEQ ID NO: 1 by the insertion or addition of one or more amino acids and / or by the deletion of one or more amino acids and / or by the conservative substitution of one or more amino acids and which substantially retains the tumor suppressor activity of SEQ ID NO: 1; more preferably, it refers to any polypeptide produced from the polypeptide of SEQ ID NO: 1 by the insertion or addition of one or more amino acids.

[0079] Those skilled in the art will understand that retention of tumor suppressor activity requires that the variant be able to dimerize with Myc and / or its exclusive partner p21 / p22Max and inhibit Myc activity, be able to translocate across the cell membrane and be able to translocate across the nuclear envelope. In some embodiments, compared to Omomyc, the functionally equivalent variants of the polypeptides of the present invention dimerize less homodimerically, or are not forced to form homodimers by forming disulfide bridges. In particular, in certain embodiments of the polypeptides of the present invention, less disulfide bridge formation occurs in the homodimeric form than in the polypeptide OmoMyc.

[0080] As used herein, "less homodimerization" relates to a lower ability to form exclusive homodimers of the polypeptides of the present invention even under reducing conditions. In a preferred embodiment, this ability is at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95% lower than the ability to form homodimers of Omomyc.

[0081] As used herein, reducing conditions relate to the presence of a reducing agent, which is a compound that donates electrons to another chemical substance in a redox chemical reaction. Exemplary but non-limiting instances of reducing agents are DTT (dithiothreitol), β-mercaptoethanol, or TCEP (tris(2-carboxyethyl)phosphine). The amount of homodimers may be the same in vitro, and the difference between the functionally equivalent variant and Omomyc only exists in cells in which a heterodimeric partner is present, where no disulfides are present such that it is possible to form more heterodimers.

[0082] Several assays can be used to determine peptide homodimerization, for example but not limited to, thermal denaturation monitored by circular dichroism, and thus dimerization can be quantitatively detected by folding and thermal stability.

[0083] Suitable functionally equivalent variants include polypeptides consisting essentially of the polypeptide of SEQ ID NO: 1. As used herein, "consisting essentially of" means that the specified molecule does not contain any other sequences that would alter the activity of SEQ ID NO: 1.

[0084] In a preferred embodiment, a functionally equivalent variant of SEQ ID NO: 1 is a polypeptide obtained by inserting or adding one or more amino acids relative to the polypeptide of SEQ ID NO: 1. In one embodiment, the functionally equivalent variant is obtained by inserting less than 10 amino acids, more preferably less than 5 amino acids, and more preferably by inserting one amino acid. In a preferred embodiment, it is obtained by inserting one amino acid, i.e., methionine.

[0085] In another embodiment, a functionally equivalent variant of SEQ ID NO: 1 is a polypeptide obtained by deleting one or more amino acids relative to the polypeptide of SEQ ID NO: 1. In one embodiment, the functionally equivalent variant is obtained by deleting less than 10 amino acids, more preferably less than 5 amino acids, and more preferably by deleting one amino acid.

[0086] Suitable functional variants of the targeting peptide are those that exhibit an amino acid sequence identity of about greater than 25% relative to the peptide of SEQ ID NO: 1, such as 25%, 30%, 40%, 50%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity. The degree of identity between two polypeptides is determined using computer algorithms and methods well known to those skilled in the art. Preferably, the identity between two amino acid sequences is determined by using the BLASTP algorithm as 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, sequence identity is determined over the entire length of the polypeptide of SEQ ID NO: 1, or over the entire length of the variant, or both.

[0087] Functionally equivalent variants of the polypeptides of the present invention may also include post-translational modifications, such as glycosylation, acetylation, isoprenylation, myristoylation, proteolytic processing, etc.

[0088] In another embodiment, suitable functional variants of the targeting peptide are those functional variants in which one or more positions in the polypeptide of the invention contain amino acids that are conservative substitutions of the amino acids present in the above-mentioned protein. "Conservative substitution of an amino acid" is the replacement of one amino acid with another amino acid having a similar structure and / or chemical property. For example, each of the following six groups contains amino acids that are conservative substitutions of 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 substitutions of amino acids is within the ability of those of ordinary skill in 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).

[0089] It should be understood that functional equivalent variants of Omomyc contain mutations at positions corresponding to the mutations E61T, E68I, R74Q, and R75N present in Omomyc derived from human c-Myc. The positions at which mutations must occur in the functional equivalent variants can be determined by multiple sequence alignment of different Myc sequences and can be determined by alignment with those positions corresponding to positions 61, 68, 74, and 75 in the Omomyc sequence derived from human c-Myc. In one embodiment, functional equivalent variants of Omomyc contain mutations at positions corresponding to the mutations E61T, E68I, R74Q, and R75N present in Omomyc derived from human c-Myc.

[0090] In another embodiment, functional equivalent variants of Omomyc contain mutations at positions corresponding to E61, E68, R74, and R75 in the Omomyc sequence, where E61 has been mutated to E61A or E61S; E68 has been mutated to E68L, E68M, or E68V; R74 has been mutated to R74N; and R75 has been mutated to R75Q.

[0091] Multiple sequence alignment is an extension of pairwise alignment to combine more than two sequences at once. Multiple alignment methods align all the sequences in a given query set. Preferred multiple sequence alignment programs (and their algorithms) are ClustalW, Clusal2W or ClustalWXXL (see Thompson et al. (1994) Nucleic Acids Res 22: 4673-4680). Once the sequences of c-Myc and variants from different organisms have been compared (aligned) as described herein, one of ordinary skill in the art can readily determine the positions within each sequence corresponding to positions E61T, E68I, R74Q and R75N present in Omomyc, and introduce in Omomyc variants mutations corresponding to the E61T, E68I, R74Q and R75N mutations that occur in Omomyc derived from human c-Myc.

[0092] Suitable assays for determining whether a polypeptide can be considered a functionally equivalent variant of Omomyc include, but are not limited to:

[0093] - Assays that measure the ability of the polypeptide to form a dimer complex with Max and Myc, such as assays based on the expression of a reporter gene described by Soucek et al. (Oncogene, 1998, 17: 2463-2472) and assays of PLA (Protein Ligation Assay) or immunoprecipitation.

[0094] - Assays that measure the ability of the polypeptide to bind to the Myc / Max recognition site (CACGTG site) within DNA, such as the electrophoretic mobility shift assay (EMSA) described by Soucek et al. (ibid.).

[0095] - Assays that measure the ability to inhibit Myc-induced transactivation, such as assays based on the expression of a reporter gene under the control of a Myc / Max-specific DNA binding site described by Soucek et al. (ibid.).

[0096] - Assays based on the ability of the polypeptide to inhibit the growth of cells expressing the myc oncogene, as described by Soucek et al. (ibid.).

[0097] - Assays that measure the ability of the polypeptide to enhance Myc-induced apoptosis, such as the assays described by Soucek et al. (Oncogene, 1998: 17, 2463–2472). In addition, any assay known in the art for assessing apoptosis can be used, such as Hoechst staining, propidium iodide (PI) or annexin V staining, trypan blue, DNA laddering / fragmentation and TUNEL.

[0098] In a preferred embodiment, a polypeptide is considered a functionally equivalent variant of Omomyc if it shows at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or 100% of the activity of native Omomyc in one or more of the above assays.

[0099] In a specific embodiment, a functionally equivalent variant of the polypeptide of SEQ ID NO: 1 comprises a polypeptide of SEQ ID NO: 1 in which the residue X at position 89 of SEQ ID NO: 1 is not cysteine. Preferably, the residue X at position 89 of SEQ ID NO: 1 is an aliphatic amino acid, or a thio amino acid, or a dicarboxylic amino acid or its amide, or an amino acid with two basic groups, or an aromatic amino acid, or a cyclic amino acid, or a hydroxylated amino acid. More preferably, the amino acid is selected from serine, threonine and alanine, preferably from serine and alanine.

[0100] Suitable functionally equivalent variants of SEQ ID NO: 1 having residue X instead of cysteine at position 89 of SEQ ID NO: 1 are disclosed in the table below.

[0101]

[0102]

[0103] Thus, in a preferred embodiment, the functionally equivalent variants of SEQ ID NO:1 are 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.

[0104] In addition, a functionally equivalent variant of Omomyc is also capable of transducing cells after contact of the variant with the cells. It should be understood that a functionally equivalent variant of Omomyc comprises a protein transduction domain present in native Omomyc or another functional protein transduction domain.

[0105] In a preferred embodiment, a polypeptide is considered a functionally equivalent variant of SEQ ID NO: 1 if it is able to transduce target cells with at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or 100% of the efficiency of SEQ ID NO: 1.

[0106] In addition, a functionally equivalent variant of SEQ ID NO: 1 is also capable of translocating to the nucleus of target tumor cells.

[0107] In a preferred embodiment, a polypeptide is considered a functional equivalent variant of SEQ ID NO: 1 if it can translocate to the nucleus of target tumor cells with an efficiency of at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% of SEQ ID NO: 1.

[0108] For the ability of a polypeptide to cross the cell membrane and translocate into the nucleus, suitable assays for determining whether a polypeptide is a functional equivalent variant of SEQ ID NO: 1 include double-labeling cells with a reagent specific for the polypeptide and a dye that specifically labels the nucleus (such as DAPI or Hoechst dyes). Detection of the polypeptides of the present invention can be performed by confocal microscopy or fluorescence microscopy.

[0109] In another preferred embodiment, the compound (i) of the present invention is a conjugate comprising: a polypeptide comprising the sequence SEQ ID NO: 1 or a functional equivalent variant thereof, and a chemical moiety that promotes cellular uptake of the polypeptide or its functional equivalent variant.

[0110] As used herein, the term "conjugate" refers to two or more compounds covalently linked together such that the function of each compound is retained in the conjugate.

[0111] The term "chemical moiety" refers to any compound containing at least one carbon atom. Examples of chemical moieties include, but are not limited to, any peptide chain rich in hydrophobic amino acids and hydrophobic chemical moieties.

[0112] In a preferred embodiment, the conjugate according to the present 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 promote cellular uptake of the polypeptide or the functional equivalent variant of the polypeptide.

[0113] In one embodiment, the chemical moiety that promotes cellular uptake of the polypeptide is a lipid or a fatty acid.

[0114] Fatty acids are generally molecules with a carbon chain that has an acidic moiety (e.g., a carboxylic acid) at the end of the chain. The carbon chain of a fatty acid can have any length, but preferably has a length of 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, and any range derivable therefrom. In certain embodiments, the length of the carbon chain in the chain portion of the fatty acid is 4 to 18 carbon atoms. In certain embodiments, the fatty acid carbon chain can contain an odd number of carbon atoms, however, in certain embodiments, an even number of carbon atoms in the chain may be preferred. Fatty acids that contain only single bonds in their carbon chains are called saturated, while fatty acids that contain at least one double bond in their chains are called unsaturated. Fatty acids can be branched, although in the preferred embodiments of the present invention, it is unbranched. 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, arachidonic acid.

[0115] In a preferred embodiment, the chemical moiety that promotes cellular uptake of the polypeptide comprising SEQ ID NO:1 or a functionally equivalent variant thereof is a cell-penetrating peptide sequence, in which case the conjugate will comprise a fusion protein that includes: a polypeptide comprising SEQ ID NO:1 or a functionally equivalent variant thereof and a cell-penetrating peptide sequence.

[0116] The term "fusion protein" refers to a protein produced by genetic engineering techniques, which consists of two or more functional domains derived from different proteins. Fusion proteins can be obtained in a conventional manner, for example, by gene expression of a nucleotide sequence encoding the fusion protein in a suitable cell. It should be understood that the cell-penetrating peptide refers to a cell-penetrating peptide different from the cell-penetrating peptide that forms part of the polypeptide comprising SEQ ID NO:1 or a functionally equivalent variant of SEQ ID NO:1.

[0117] The term "cell-penetrating peptide sequence" is used interchangeably in this specification with "CPP", "protein transduction domain" or "PTD". It refers to a peptide chain of variable length that directs the intracellular transport of a protein. The delivery process into cells usually occurs by endocytosis, but the peptide can also be internalized into cells by direct membrane translocation. The amino acid composition of CPPs generally contains a relatively high abundance of positively charged amino acids (such as lysine or arginine), or has a sequence containing an alternating pattern of polar / charged amino acids and non-polar hydrophobic amino acids.

[0118] Examples of CPPs that can be used in the present invention include, but are not limited to: the CPP present in the Drosophila Antennapedia protein (RQIKIWFQNRRMKWKK; SEQ ID NO:13); the CPP present in the Herpes simplex virus 1 (HSV-1) VP22 DNA-binding protein (DAATATRGRSAASRPTERPRAPARSASRPRRPVE; SEQ ID NO:14); the CPP of Bac-7 (RRIRPRPPRLPRPRPRPLPFPRPG; SEQ ID NO:15); the CPPs of the HIV-1 TAT protein 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); the CPP of the S413-PV peptide (ALWKTLLKKVLKAPKKKRKV; SEQ ID NO:19); the CPP of penetratin (RQIKWFQNRRMKWKK; SEQ ID NO:20); the CPP of SynB1 (RGGRLSYSRRRFSTSTGR; SEQ ID NO:21); the CPP of SynB3 (RRLSYSRRRF; SEQ ID NO:22); the CPP of PTD-4 (PIRRRKKLRRLK; SEQ ID NO:23); the CPP of PTD-5 (RRQRRTSKLMKR; SEQ ID NO:24); the CPP of FHV Coat-(35-49) (RRRRNRTRRNRRRVR; SEQ ID NO:25); the CPP of BMV Gag-(7-25) (KMTRAQRRAAARRNRWTAR; SEQ ID NO:26); the CPP of HTLV-II Rex-(4-16) (TRRQRTRRARRNR; SEQ ID NO:27); the CPP of D-Tat (GRKKRRQRRRPPQ; SEQ ID NO:28); CPP R9-Tat (GRRRRRRRRRPPQ; SEQ ID NO:29); the CPP of MAP (KLALKLALKLALALKLA; SEQ ID NO:30); the CPP of SBP (MGLGLHLLVLAAALQGAWSQPKKKRKV; SEQ ID NO:31); the CPP of FBP (GALFLGWLGAAGSTMGAWSQPKKKRKV; SEQ ID NO:32); the CPP of MPG (ac-GALFLGFLGAAGSTMGAWSQPKKKRKV-cya; SEQ ID NO:33);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); polyarginine sequence with structure RN (where N is from 4 to 17); GRKKRRQRRR sequence (SEQ ID NO:37); RRRRRRLR sequence (SEQ ID NO:38); RRQRRTSKLMKR 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).;

[0119] In a preferred embodiment, the cell-penetrating peptide is not the endogenous peptide contained in SEQ ID NO: 1.

[0120] In a preferred embodiment, the CPP is the CPP of the HIV-1 TAT protein, which consists of amino acids 49 - 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).

[0121] In some embodiments, the CPP is the CPP as described in WO2019 / 018898, the entire content of WO2019 / 018898 is incorporated herein by reference in its entirety.

[0122] In one embodiment, the cell-penetrating peptide sequence is fused at the N-terminus of the polypeptide of the invention or a functionally equivalent variant of said polypeptide. In another embodiment, the cell-penetrating peptide is fused at the C-terminus of the polypeptide of the invention or a functionally equivalent variant of said polypeptide.

[0123] In a preferred embodiment, in addition to the native cell-penetrating peptide present in the polypeptide of SEQ ID NO: 1 or a functionally equivalent variant of said polypeptide, the conjugate or fusion protein of the combination according to the invention further 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 other cell-penetrating peptides.

[0124] Suitable fusion proteins of the invention include the polypeptides Omomyc*TAT and Omomyc*LZArg as defined below:

[0125]

[0126] Thus, in a preferred embodiment, the fusion protein is a polypeptide selected from SEQ ID NO: 11 and SEQ ID NO: 12.

[0127] Suitable assays for determining whether the conjugate retains the cell membrane translocation ability of Omomyc include, but are not limited to, assays measuring the ability of the conjugate to transduce cells in culture. The assay is based on contacting the conjugate with cultured cells and detecting the presence of the conjugate in the intracellular location.

[0128] In another preferred embodiment, the conjugate of the combination of the invention further comprises an additional nuclear localization signal.

[0129] As used herein, the term "nuclear localization signal" (NLS) refers to an amino acid sequence of about 4 to 20 amino acid residues in length that is used to direct a protein to the nucleus. Nuclear localization sequences are rich in basic amino acids, and exemplary sequences are well known in the art (Gorlich D. (1998) EMBO J. 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).

[0130] 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).

[0131] In another preferred embodiment, the NLS can be the N-terminus or C-terminus of a conjugate or fusion protein comprising the polypeptide of SEQ ID NO:1 or a functionally equivalent variant thereof.

[0132] Those skilled in the art will understand that it can be expected that the conjugates of the present invention may also contain one or more flexible peptides that link the polypeptide comprising SEQ ID NO: 1 or a functionally equivalent variant thereof, the cell-penetrating peptide sequence, and / or the NLS. Thus, in one specific embodiment, the polypeptide comprising SEQ ID NO: 1 or a functionally equivalent variant thereof is directly linked to the cell-penetrating peptide sequence. In another specific embodiment, the polypeptide comprising SEQ ID NO: 1 or a functionally equivalent variant thereof is linked to the cell-penetrating peptide sequence via a flexible peptide. In one embodiment, the polypeptide comprising SEQ ID NO: 1 or a functional variant thereof is directly linked to the NLS. In another embodiment, the polypeptide comprising SEQ ID NO: 1 or a functionally equivalent variant thereof is linked to the NLS via a flexible peptide.

[0133] In one specific embodiment, the polypeptide of the conjugate according to the present invention is directly linked to the cell-penetrating peptide sequence and the NLS.

[0134] In one embodiment, the NLS is one of the NLSs that occur endogenously in the Myc sequence, such as the M1 peptide (PAAKRVKLD, SEQ ID NO: 56) or the M2 peptide (RQRRNELKRSF, SEQ ID NO: 57).

[0135] In another embodiment, the additional NLS refers to an NLS that is different from the endogenous NLS present in the polypeptide comprising SEQ ID NO: 1 or the functionally equivalent variant of SEQ ID NO: 1.

[0136] In a preferred embodiment, in addition to the endogenous NLS present in the polypeptide of the present invention or its functionally equivalent variant, the conjugate or fusion protein according to the present invention further 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, or at least 10 NLSs.

[0137] In another specific embodiment, the polypeptide of the conjugate for use according to the present invention is linked to the cell-penetrating peptide sequence via a first flexible peptide linker and linked to the NLS via a second flexible peptide linker.

[0138] As used herein, the terms "flexible peptide", "spacer peptide", or "linker peptide" refer to a peptide that covalently binds two proteins or moieties but is not part of either polypeptide, which allows one to move relative to the other without substantially and detrimentally affecting the function of the protein or moiety. Thus, the flexible linker does not affect the tumor-tracking activity of the polypeptide sequence, the cell-penetrating activity of the cell-penetrating peptide, or the nuclear localization ability of the NLS.

[0139] The flexible peptide comprises at least one amino acid, at least two amino acids, at least three amino acids, at least four amino acids, at least five amino acids, at least six amino acids, at least seven amino acids, at least eight amino acids, at least nine 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 one protein to move relative to another protein to increase protein solubility and / or improve its activity. Suitable linker regions include polyglycine regions, the GPRRRR sequence (SEQ ID NO: 58) which is a combination of glycine, proline and alanine residues.

[0140] In one specific embodiment, the conjugate according to the invention comprises a tag that binds to the C-terminal or N-terminal domain of the conjugate or of the polypeptide or fusion protein or its variant. The tag is typically a peptide or amino acid sequence that can be used for the isolation or purification of the fusion protein. Thus, the tag is capable of binding one or more ligands, such as one or more ligands of an affinity matrix (e.g., a chromatographic support or beads), with high affinity. Examples of the tag are histidine tags (His tags or HTs), such as a tag comprising 6 histidine residues (His6 or H6), which can bind with high affinity to a nickel (Ni 2+ ) column or a cobalt (Co 2+ ) column. The His tag has the desired property that it can bind its ligand under conditions that denature most proteins and disrupt most protein-protein interactions. Thus, it can be used to remove the bait protein labeled with H6 after the protein-protein interaction involving the bait is disrupted.

[0141] Other exemplary non-limiting instances of tags for separating or purifying conjugates or polypeptides containing SEQ ID NO: 1 or variants or fusion proteins thereof include Arg-tag, FLAG-tag (DYKDDDDK; SEQ ID NO: 59), Strep-tag (WSHPQFEK, SEQ ID NO: 60), epitopes recognizable by antibodies such as c-myc-tag (recognized by anti-c-myc antibody), HA tag (YPYDVPDYA, SEQ ID NO: 61), V5 tag (GKPIPNPLLGLDST, SEQ ID NO: 62), SBP-tag, S-tag, calmodulin-binding peptide, cellulose-binding domain, chitin-binding domain, glutathione S-transferase-tag, maltose-binding protein, NusA, TrxA, DsbA, Avi-tag, etc. (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), β-galactosidase, etc.

[0142] If desired, the tag can be used for the separation or purification of the fusion protein.

[0143] In another preferred embodiment, the compound (i) of the present invention is a polynucleotide encoding the above polypeptide or fusion protein. In a preferred embodiment, the compound (i) of the present invention is a polynucleotide encoding a polypeptide containing the sequence SEQ ID NO: 1 or a functionally equivalent variant thereof. In another embodiment, the compound (i) of the present invention is a polynucleotide encoding a conjugate comprising a polypeptide containing the sequence SEQ ID NO: 1 or a functionally equivalent variant thereof and a chemical moiety that promotes cellular uptake of the polypeptide or its functionally equivalent variant; more preferably, it is a polynucleotide encoding a fusion protein that is a fusion between a polypeptide containing the sequence SEQ ID NO: 1 or a functionally equivalent variant thereof and a cell-penetrating peptide sequence.

[0144] The terms "polynucleotide", "nucleic acid", and "nucleic acid molecule" are used interchangeably and refer to nucleotides in polymeric form of any length. Polynucleotides may include deoxyribonucleotides, ribonucleotides, and / or their analogs. Nucleotides can have any three-dimensional structure and can perform any known or unknown function. The term "polynucleotide" includes, 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. In addition to natural nucleic acid molecules, the nucleic acid molecules of the present invention may also include modified nucleic acid molecules. As used herein, mRNA refers to RNA that can be translated in a cell.

[0145] In a preferred embodiment, the polynucleotide of the present invention is mRNA.

[0146] mRNA can be chemically synthesized, obtained by in vitro transcription, or synthesized in vivo in a target cell. The nucleotide sequence forming the polynucleotide encoding the conjugate or fusion protein of the present invention is in the same correct reading frame for its expression.

[0147] In a preferred embodiment, component (i) of the combination of the present invention is mRNA encoding a polypeptide consisting of SEQ ID NO: 1, or a polypeptide consisting of a functionally equivalent variant of SEQ ID NO: 1, or a polypeptide consisting of SEQ ID NO: 4.

[0148] In another embodiment, component (i) of the combination of the present invention is a vector comprising the polynucleotide of the present invention.

[0149] As used herein, the term "vector" refers to a nucleic acid sequence as described below, which contains essential sequences such that after transcription and translation of the sequence in a cell, a polypeptide encoded by the polynucleotide of the present invention is produced. The sequence is operably linked to other segments that allow it to replicate autonomously in a host cell of interest. Preferably, the vector is an expression vector, which is defined as a vector that, in addition to the region for autonomous replication in a host cell, also contains a region operably linked to the nucleic acid of the present invention and is capable of enhancing the expression of the product of the nucleic acid according to the present invention. The vectors of the present invention can be obtained by techniques widely known in the art.

[0150] 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, such as producer cells. Suitable vectors containing the polynucleotides of the present invention are vectors derived from: expression vectors in prokaryotes (e.g., pUC18, pUC19, pBluescript and its derivatives, mp18, mp19, pBR322, pMB9, ColE1, pCR1, RP4), phage and "shuttle" vectors (e.g., pSA3 and pAT28), expression vectors in yeast (e.g., vectors of the 2-micron plasmid type, integrating plasmids, YEP vectors, centromeric plasmids and the like), expression vectors in insect cells (e.g., vectors of the pAC series and pVL series), expression vectors in plants (e.g., pIBI, pEarleyGate, pAVA, pCAMBIA, pGSA, pGWB, pMDC, pMY, pORE and the like), and expression vectors in higher eukaryotic cells based on viral vectors (adenoviruses, adeno-associated viruses and retroviruses and especially lentiviruses), as well as non-viral vectors, such as pSilencer 4.1-CMV (Ambion), pcDNA3, pcDNA3.1 / hyg, pHCMV / Zeo, pCR3.1, pEFl / His, plND / 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 polynucleotides of the present invention are contained in a vector selected from the group consisting of pEGFP, or the pBabe retroviral vector and pTRIPZ, or the pSLIK lentiviral vector.

[0151] The vectors of the present invention can be used to transform, transfect or infect cells that can be transformed, transfected or infected by the vectors. The cells can be prokaryotic or eukaryotic.

[0152] The vector preferably comprises the polynucleotide of the present invention, which is operably linked to a sequence that regulates the expression of the polynucleotide of the present invention. The regulatory sequences used in the present invention can be nuclear promoters, or enhancer sequences and / or other regulatory sequences that increase the expression of heterologous nucleic acid sequences. In principle, any promoter can be used in the present invention as long as the promoter is compatible with the cells of the polynucleotide to be expressed. Thus, promoters suitable for carrying out the present invention include, but are not limited to, constitutive promoters, such as derivatives of eukaryotic viral genomes (e.g., polyomavirus, adenovirus, SV40, CMV, avian sarcoma virus, hepatitis B virus), metallothionein gene promoters, herpes simplex virus thymidine kinase gene promoters, LTR regions of retroviruses, immunoglobulin gene promoters, actin gene promoters, EF-1α gene promoters, and inducible promoters in which protein expression depends on the addition of a molecular or exogenous signal (e.g., tetracycline system, NFκB / UV light system, Cre / Lox system, and heat shock gene promoters), the regulatable RNA polymerase II activators and tissue-specific activators described in WO / 2006 / 135436.

[0153] In another embodiment, component (i) of the combination of the present invention is a cell capable of secreting the polypeptide of the present invention or the conjugate of the present invention, preferably the polypeptide of the present invention or the fusion protein of the present invention, into the culture medium.

[0154] 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, intimal cells, primary cultures of isolated cells from different organs (preferably cells isolated from islets of Langerhans), hepatocytes, leukocytes (including monocytes), mesenchymal stem cells, umbilical cord or adult (skin, lung, kidney, and liver), osteoclasts, chondrocytes, and other connective tissue cells. Established cell lines such as Jurkat T cells, NIH-3T3, CHO, Cos, VERO, BHK, HeLa, COS, MDCK, 293, 3T3 cells, C2C12 myoblasts, and W138 cells are also suitable. Those skilled in the art will understand that cells capable of secreting the polypeptide of the present invention into the culture medium can be found to form microparticles or microcapsules, thereby enabling these cells to have a longer lifespan in patients. Materials suitable for forming the microparticle objects of the present invention include any biocompatible polymer material that allows continuous secretion of therapeutic products and serves as a support for the cells. Thus, the biocompatible polymer material can be, for example, a thermoplastic polymer or a hydrogel polymer. Among thermoplastic polymers, there are acrylic acid, acrylamide, 2-aminoethyl methacrylate, poly(tetrafluoroethylene-co-hexafluoropropylene), (7-cumaroxy)ethyl methacrylate, N-isopropylacrylamide, polyacrylic acid, polyacrylamide, polyamidoamine, poly(amino)-p-xylene, poly(chloroethyl vinyl ether), polycaprolactone, poly(caprolactone carbonate-co-trimethylene), poly(urea carbonate)urethane, poly(carbonate)urethane, polyethylene, polyethylene and acrylamide copolymer, polyethylene glycol, polyethylene glycol methacrylate, poly(ethylene terephthalate), poly(4-hydroxybutyl acrylate), poly(2-hydroxyethyl methacrylate), poly(N-2-hydroxypropyl methacrylate), poly(lactic-co-glycolic acid), poly(L-lactic acid), poly(γ-methyl, L-glutamate), poly(methyl methacrylate), poly(propylene fumarate), poly(propylene oxide), polypyrrole, polystyrene, poly(tetrafluoroethylene), polyurethane, polyvinyl alcohol, ultra-high molecular weight polyethylene, 6-(p-vinylbenzamide)-hexanoic acid, N-p-vinylbenzyl-D-maltamide, and copolymers containing more than one of the above polymers. Among hydrogel-type polymers, there are natural materials such as alginate, agarose, collagen, starch, hyaluronic acid, bovine serum albumin, cellulose and its derivatives, pectin, chondroitin sulfate, fibrin, and silk fibroin, and synthetic hydrogels (e.g., and ).

[0155] Compound (ii) of the combination of the present invention is a tumor immunotherapeutic agent.

[0156] As used herein, the term "tumor immunizing agent" refers to an agent that is effective to enhance, stimulate, and / or upregulate the immune response of a subject. In some embodiments, the administration of the tumor immunizing agent in combination with compound (i) of the present invention has a synergistic effect on the treatment of cancer.

[0157] The tumor immunizing agent can be, for example, a small molecule drug, an antibody, or a biomolecule, or a small molecule. Examples of biological tumor immunizing 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 humanized or human.

[0158] In some embodiments, the tumor immunizing agent is a cytokine.

[0159] "Cytokine" is understood to mean peptides of different sizes and molecular weights that are synthesized by cells of the immune system for the purpose of regulating the immune response, and they can be hormones, growth factors, necrosis factors, chemokines, etc. They can be of natural origin or derived from recombinant cell cultures and biological activity equivalents of natural 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 the immune response. Their binding to antibodies produces immunocytokines. In some embodiments, the cytokine is recombinant human interleukin 15 (rhIL-15), recombinant human interleukin 12 (rhIL-12) (e.g., NM-IL-12 (Neumedicines, Inc.), or heterodimeric IL-15 (hetIL-15, Novartis / Admune), a fusion complex consisting of a synthetic form of endogenous IL-15 complexed with the soluble IL-15 binding protein IL-15 receptor α chain (IL15:sIL-15RA).

[0160] In another embodiment, the cytokine is selected from the group consisting of: 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, IFN-γ, 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 / TNFSFll / RANKL, and any combination thereof.

[0161] In a preferred embodiment, the tumor immunizing agent is not a cytokine. Thus, in a preferred embodiment, cytokines are excluded from the scope of the present invention. Preferably, the cytokines excluded by the present invention are TNF factor α, INF-γ, GM-GSF factor, and IL-2.

[0162] In another preferred embodiment, cytokines are excluded from the scope of the present invention only when the combined component (i) is component (i)(a) or (i)(b). Thus, in one embodiment, if the combined component (i) is a polypeptide comprising the sequence SEQ ID NO: 1 or a functionally equivalent variant thereof, or a conjugate comprising a polypeptide comprising the sequence 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, then the tumor immunizing agent is not a cytokine, preferably not a cytokine selected from the group consisting of TNF factor α, INF-γ, GM-GSF factor, and IL-2.

[0163] In some embodiments, the tumor immunizing agent is (i) an agonist of a stimulatory (including co-stimulatory) receptor or (ii) an antagonist of an inhibitory (including co-inhibitory) signal on T cells, both of which result in amplification of an antigen-specific T cell response.

[0164] Certain stimulatory and inhibitory molecules are members of the immunoglobulin superfamily (IgSF). An important family of membrane-bound ligands that bind to co-stimulatory or co-inhibitory receptors is the B7 family, including 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.

[0165] Another family of membrane-bound ligands that bind to co-stimulatory or co-inhibitory receptors is the family of TNF molecules that bind to members of the homologous TNF receptor family, which includes 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, OPG, RANK, RANKL, TWEAKR / Fn14, TWEAK, BAFFR, EDAR, XEDAR, TACI, APRIL, BCMA, LTβR, LIGHT, DcR3, HVEM, VEGI / TL1A, TRAMP / DR3, EDAR, EDA1, XEDAR, EDA2, TNFR1, lymphotoxin α / TNFβ, TNFR2, TNFα, LTβR, lymphotoxin α1β2, FAS, FASL, RELT, DR6, TROY, NGFR.

[0166] In some embodiments, the combination of the compound (i) of the present invention and a tumor immunotherapeutic agent can stimulate a T cell response. In some embodiments, the tumor immunotherapeutic 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; (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.

[0167] In some embodiments, the tumor immunizing 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 tumor immunizing agent is an antagonist of KIR, such as lirilumab.

[0168] In some embodiments, the tumor immunizing 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).

[0169] In some embodiments, the tumor immunizing agent is selected from agonists that ligate positive co-stimulatory receptors, blockers that attenuate signal transduction through inhibitory receptors, antagonists, and one or more agents that systemically increase the frequency of anti-tumor T cells, agents that overcome different immunosuppressive pathways in the tumor microenvironment (e.g., blocking inhibitory receptor engagement (e.g., PD-L1 / PD-1 interaction), depleting or inhibiting Tregs (e.g., using anti-CD25 monoclonal antibodies (e.g., daclizumab) or depletion by in vitro anti-CD25 beads), inhibiting metabolic enzymes such as IDO, or reversing / preventing T cell anergy or exhaustion), and agents that trigger innate immune activation and / or inflammation at the tumor site.

[0170] As used herein, the term "cytotoxic T lymphocyte-associated protein 4" (abbreviated "CTLA-4" and also known as cluster of differentiation 152 (CD152)) refers to a protein receptor that functions as an immune checkpoint. CTLA-4 is a member of the immunoglobulin superfamily and is expressed by activated T cells and transmits inhibitory signals to T cells. CTLA-4 is homologous to the T cell co-stimulatory protein CD28, and both molecules bind to CD80 and CD86 (also known as B7-1 and B7-2, respectively) on antigen-presenting cells. Compared to CD28, CTLA-4 binds CD80 and CD86 with greater affinity and avidity, enabling it to out-compete CD28 for binding to its ligands. CTLA-4 transmits inhibitory signals to T cells, while CD28 transmits stimulatory signals. CTLA-4 is also present in regulatory T cells (Tregs) and contributes to their inhibitory function. Activation of T cells through the T cell receptor and CD28 results in increased CTLA-4 expression. The CTLA-4 protein is encoded by the human CTLA-4 gene (database accession: ENSG00000163599). Typically, following T cell activation, CTLA-4 is upregulated on the plasma membrane where it downregulates T cell function through multiple mechanisms, including by out-competing CD28 for binding to its ligand B7 and by inducing T cell cycle arrest to prevent co-stimulation (Postow et al. (2015) J. Clinical oncology, Vol. 33, pp. 1974-1983; Pardoll, D. et al. (2012), Nature Reviews Cancer 12, 252-264).

[0171] In some embodiments, the cancer immunotherapeutic is a CTLA-4 antagonist. As used herein, the term "CTLA-4 antagonist" refers to, but is not limited to, any compound or agent or biomolecule that blocks the binding of CTLA-4 to its ligands B7-1 and / or B7-2. In the context of the present invention, it should be 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.

[0172] Non-limiting examples of CTLA-4 antagonist compounds currently being considered for clinical use in the treatment of cancer include antagonistic antibodies against CTLA-4.

[0173] In some embodiments, the CTLA-4 antagonist is an antagonistic CTLA-4 antibody. In some embodiments, the antagonistic CTLA-4 antibody is YERVOY (ipilimumab) or tremelimumab.

[0174] Other non-limiting examples of CTLA-4 antagonists include immunoadhesins (also known as fusion proteins), which are compounds capable of specifically binding to CTLA-4 and blocking its binding to B7-1 and / or B7-2.

[0175] As used herein, the term "programmed death 1 (PD-1)" receptor refers to an immunosuppressive receptor belonging to the CD28 family. In humans, PD-1 is encoded by the PDCD1 gene. PD-1 is mainly expressed on previously activated T cells in vivo and binds to two ligands, PD-L1 and PD-L2. As used herein, the term "PD-1" includes human PD-1 (hPD-1), variants, isoforms, and species homologs of hPD-1, and analogs having at least one epitope in common with hPD-1. The complete hPD-1 sequence can be found under GENBANK accession number 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, pp. 72-79; Gianchecchi et al. (2013), Autoimmun. Rev. 12:1091-1100).

[0176] In some embodiments, the cancer immunotherapeutic agent is a PD-1 antagonist. As used herein, the term "PD-1 antagonist" refers to, but is not limited to, any compound or agent or biomolecule (such as an antibody) that blocks the binding of PD-L1 expressed on cancer cells to PD-1 expressed on immune cells (T cells, B cells, or NKT cells), and / or any compound or agent or biomolecule (such as an antibody) that blocks the binding of PD-L2 expressed on cancer cells to PD-1 expressed on immune cells. In the context of the present invention, it should be understood that when a subject (such as 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, and 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.

[0177] Non-limiting examples of PD-1 antagonists are antibodies against PD-1 (also referred to as PD-1 antibodies or anti-PD-1 antibodies), such as PD-1 monoclonal antibodies (mAbs) or antigen-binding fragments thereof, which specifically bind to PD-1 and preferably specifically bind to human PD-1. The mAb can be a human antibody, a humanized antibody or a chimeric antibody and can include human constant regions. Non-limiting examples of PD-1 antagonist compounds include PD-1 antibodies such as nivolumab ( Bristol-Myers Squibb), pembrolizumab ( Merck), BGB-A317, and other antibodies such as PDR001 (Novartis). Other non-limiting examples of PD-1 antagonists include pidilizumab (CureTech), AMP-224 (GlaxoSmithKline), AMP-514 (GlaxoSmithKline), PDR001 (Novartis) and cemiplimab (Regeneron and Sanofi). Other PD-1 antagonists also include any anti-PD-1 antibodies described in US8008449, US7521051 and US8354509.

[0178] Other non-limiting examples of PD-1 antagonists include immunoadhesins (also referred to as fusion proteins), which are compounds capable of specifically binding to PD-1 and blocking its binding to PD-L1. Examples of immunoadhesion 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, which is a recombinant B7-DC Fc fusion protein composed of the extracellular domain of the PD-1 ligand programmed cell death ligand 2 (PD-L2, B7-DC) and the Fc region of human immunoglobulin (Ig) G1.

[0179] As used herein, the term "antibody" (e.g., PD-1 antibody and CTLA-4 antibody) refers to any form of antibody and its fragments that exhibit the desired biological activity or binding activity (e.g., as described above, blocking the binding of PD-1 to its ligand or blocking the binding of CTLA-4 to its ligand). Thus, it is used in the broadest sense and specifically covers but is not limited to monoclonal antibodies (including full-length monoclonal antibodies) and their fragments, polyclonal antibodies and their fragments, multispecific antibodies (e.g., bispecific antibodies) and their fragments, humanized antibodies, fully human antibodies, and their fragments, chimeric antibodies and their fragments, and camelized single-domain antibodies and their fragments.

[0180] In some embodiments, the tumor immunotherapeutic is an antibody or an 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 an antagonistic PD-1 antibody. In some embodiments, the antagonistic PD-1 antibody is OPDIVO (nivolumab), KEYTRUDA (pembrolizumab), or MEDI-0680 (AMP-514; WO2012 / 145493). In some embodiments, the tumor immunotherapeutic can be pidilizumab (CT-011). In some embodiments, the tumor immunotherapeutic is a recombinant protein composed of the extracellular domain of PD-L2 (B7-DC) fused to the Fc portion of IgG1, designated AMP-224.

[0181] In some embodiments, the tumor immunotherapeutic is a PD-L1 antagonist. In some embodiments, the PD-L1 antagonist is an antagonistic PD-L1 antibody. In some embodiments, the PD-L1 antibody is MPDL3280A (RG7446; WO2010 / 077634), durvalumab (MEDI4736), BMS-936559 (WO2007 / 005874), and MSB0010718C (WO2013 / 79174).

[0182] In some embodiments, the tumor immunotherapeutic is a LAG-3 antagonist. In some embodiments, the LAG-3 antagonist is an antagonistic LAG-3 antibody. In some embodiments, the LAG3 antibody is BMS-986016 (WO10 / 19570, WO14 / 08218) or IMP-731 or IMP-321 (WO08 / 132601, WO009 / 44273).

[0183] In some embodiments, the tumor immunotherapeutic agent is a CD137 (4-1BB) agonist. In some embodiments, the CD137 (4-1BB) agonist is an agonistic CD137 antibody. In some embodiments, the CD137 antibody is urelumab or PF-05082566 (WO12 / 32433).

[0184] In some embodiments, the tumor immunotherapeutic agent is a GITR agonist. In some embodiments, the GITR agonist is an agonistic GITR antibody. In some embodiments, the GITR antibody is BMS-986153, BMS-986156, TRX-518 (WO006 / 105021, WO009 / 009116), or MK-4166 (WO11 / 028683).

[0185] In some embodiments, the tumor immunotherapeutic 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); an enzyme that degrades kynurenine (Kynase, Kyn Therapeutics); and NLG-919 (WO09 / 73620, WO009 / 1156652, WO11 / 56652, WO12 / 142237).

[0186] In some embodiments, the tumor immunotherapeutic agent is an OX40 agonist. In some embodiments, the OX40 agonist is an agonistic OX40 antibody. In some embodiments, the OX40 antibody is MEDI-6383 or MEDI-6469.

[0187] In some embodiments, the tumor immunotherapeutic agent is an OX40L antagonist. In some embodiments, the OX40L antagonist is an antagonistic OX40L antibody. In some embodiments, the OX40L antagonist is RG-7888 (WO06 / 029879).

[0188] In some embodiments, the tumor immunizing agent is a CD40 agonist. In some embodiments, the CD40 agonist is an agonistic CD40 antibody. In some embodiments, the tumor immunizing agent is a CD40 antagonist. In some embodiments, the CD40 antagonist is an antagonistic CD40 antibody. In some embodiments, the CD40 antibody is lucatumumab or dacetuzumab.

[0189] In some embodiments, the tumor immunizing agent is a CD27 agonist. In some embodiments, the CD27 agonist is an agonistic CD27 antibody. In some embodiments, the CD27 antibody is varlilumab.

[0190] In some embodiments, the tumor immunizing agent is MGA271 (against B7H3) (WO11 / 109400).

[0191] In some embodiments, the tumor immunizing agent is abagovomab, adecatumumab, afutuzumab, alemtuzumab, anatumomab mafenatox, apolizumab, atezolimab, avelumab, blinatumomab, BMS-936559, catumaxomab, durvalumab, epacadostat, epratuzumab, indoximod, inotuzumab ozogamicin, intelumumab, ipilimumab, isatuximab, lambrolizumab, MED14736, MPDL3280A, nivolumab, obinutuzumab, ocaratuzumab, ofatumumab, olatatumab, pembrolizumab, pidilizumab, rituximab, ticilimumab, samalizumab or tremelimumab.

[0192] In some embodiments, the tumor immunizing agent is an immune stimulant. For example, antibodies that block the PD-1 and PD-L1 inhibitory axes can release activated tumor-reactive T cells and have been shown in clinical trials to induce durable anti-tumor responses in an increasing number of tumor histologies, including certain tumor types that have not traditionally been considered sensitive to immunotherapy. The anti-PD-1 antibody nivolumab( Bristol-Myers Squibb, also known as ONO-4538, MDX1106, and BMS-936558) has shown potential to improve overall survival in patients with RCC who experienced disease progression during or after prior anti-angiogenic therapy.

[0193] In some embodiments, the immunomodulatory therapeutic agent specifically induces apoptosis of tumor cells. Approved immunomodulatory therapeutic agents that can be used in the present invention include pomalidomide( Celgene); and lenalidomide( Celgene); ingenol mebutate( LEO Pharma).

[0194] In some embodiments, the tumor immunizing agent is a cancer vaccine. In some embodiments, the cancer vaccine is selected from sipuleucel-T( Dendreon / Valeant Pharmaceuticals) and talimogene laherparepvec( BioVex / Amgen, formerly known as T-VEC). In some embodiments, the tumor immunizing agent is selected from oncolytic virus therapies, such as pexastimogene devacirepvec (PexaVec / JX-594, SillaJen / formerly Jennerex Biotherapeutics), pelareorep( Oncolytics Biotech), enadenotucirev (NG-348, PsiOxus, formerly known as ColoAd1), ONCOS-102 (Targovax / formerly Oncos), vaccinia viruses engineered to express β-galactosidase (β-gal) / β-glucuronidase and / or β-gal / human sodium iodide symporter (hNIS) such as GL-ONC1 (GLV-1h68 / GLV-1h153, Genelux GmbH), and adenoviruses engineered to express GM-CSF such as CG0070 (Cold Genesys).

[0195] In some embodiments, the tumor immunizing agent is selected from JX-929 (SillaJen / formerly Jennerex Biotherapeutics), TG01 and TG02 (Targovax / formerly Oncos), TILT-123 (TILT Biotherapeutics), and VSV-GP (Vira Therapeutics).

[0196] In some embodiments, the tumor immunizing agent is a T cell engineered to express a chimeric antigen receptor or CAR. T cells engineered to express such a chimeric antigen receptor are referred to as CAR-T cells. CARs have been constructed that consist of a binding domain that can be derived from a natural ligand, a single-chain variable fragment (scFv) derived from a monoclonal antibody specific for a cell surface antigen, and can be fused to an intracellular domain that functions as the end of the T cell receptor (TCR), such as the CD3-ζ (zeta) signaling domain of the TCR, which is capable of generating an activation signal in T lymphocytes. Once the antigen binds, such CARs are linked to the endogenous signaling pathways in effector cells and generate activation signals similar to those triggered by the TCR complex.

[0197] For example, in some embodiments, the CAR-T cells are those described in U.S. Patent 8,906,682 (June; incorporated herein by reference in its entirety), which discloses that CAR-T cells are engineered to include an extracellular domain having an antigen-binding domain (e.g., a domain that binds to CD19), fused to an intracellular signaling domain of the T cell antigen receptor complex ζ chain (e.g., CD3ζ). When expressed in T cells, the CAR is capable of redirecting antigen recognition based on antigen-binding specificity. In the case of CD19, the antigen is expressed on malignant B cells. Currently, over 200 clinical trials are underway using CAR-T in various indications. [https: / / clinicaltrials.gov / ct2 / results?term=chimeric+antigen+receptors&pg=1].

[0198] In some embodiments, the immunostimulant is an activator of retinoic acid receptor-related orphan receptor γ (RORγt). RORγt is a transcription factor that plays a key role in the differentiation and maintenance of the type 17 effector subsets of CD4 + (Th17) and CD8 + (Tc17) T cells and the differentiation of innate immune cell subsets expressing IL-17 such as NK cells. In some embodiments, the activator of RORγt is LYC-55716 (Lycera), which is currently being evaluated in a clinical trial for the treatment of solid tumors (NCT02929862).

[0199] In some embodiments, the immunostimulant is an agonist or activator of toll-like receptor (TLR). Suitable activators of TLR include agonists or activators of TLR9, such as SD-101 (Dynavax). Agonists or activators of TLR8 that can be used in the present invention include motolimod (VTX-2337, VentiRx Pharmaceuticals).

[0200] Other tumor immunotherapeutics that can be used in the present invention include: urelumab (BMS-663513, Bristol-Myers Squibb), anti-CD137 monoclonal antibody, varlilumab (CDX-1127, Celldex Therapeutics), anti-CD27 monoclonal antibody, BMS-986178 (Bristol-Myers Squibb), anti-OX40 monoclonal antibody, lirilumab (IPH2102 / BMS-986015, Innate Pharma, Bristol-Myers Squibb), anti-KIR monoclonal antibody, monalizumab (IPH2201, Innate Pharma, AstraZeneca), anti-NKG2A monoclonal antibody, andecaliximab (GS-5745, Gilead Sciences), anti-MMP9 antibody, MK-4166 (Merck & Co.), anti-GITR monoclonal antibody.

[0201] In some embodiments, the immunostimulant is selected from elotuzumab, mifamurtide, an agonist or activator of a toll-like receptor, and an activator of RORγt.

[0202] In some embodiments, the tumor immunotherapeutic is selected from those described by Jerry L. Adams et al., “Big opportunities for small molecules in immuno-oncology,” Cancer Therapy 2015, Vol. 14, pp. 603-622, the entire content of which is incorporated herein by reference in its entirety. In some embodiments, the tumor immunotherapeutic is selected from the examples described in Table 1 of Jerry L. Adams et al. In some embodiments, the tumor immunotherapeutic is a small molecule targeting a tumor immunotherapy target selected from those listed in Table 2 of Jerry L. Adams et al. In some embodiments, the tumor immunotherapeutic is a small molecule agent selected from those listed in Table 2 of Jerry L. Adams et al.

[0203] In some embodiments, the tumor immunotherapeutic agent is selected from the small molecule tumor immunotherapeutic agents described in Peter L. Toogood, “Small molecule immuno-oncology therapeutic agents,” Bioorganic & Medicinal Chemistry Letters 2018, Vol. 28, pp. 319-329, the entire content of which is incorporated herein by reference. In some embodiments, the tumor immunotherapeutic agent is an agent that targets the pathway described by Peter L. Toogood.

[0204] In some embodiments, the tumor immunotherapeutic agent is selected from those described in Sandra L. Ross et al., “Bispecific T cell engager antibody constructs can mediate bystander tumor cell killing”, PLoS ONE 12(8):e0183390, the entire content of which is incorporated herein by reference. In some embodiments, the tumor immunotherapeutic agent is a bispecific T cell engager antibody construct. In some embodiments, the bispecific T cell engager antibody construct is a CD19 / CD3 bispecific antibody construct. In some embodiments, the bispecific T cell engager antibody construct is an EGFR / CD3 bispecific antibody construct. In some embodiments, the bispecific T cell engager antibody construct activates T cells. In some embodiments, the bispecific T cell engager antibody construct activates T cells, which release cytokines, inducing upregulation of intercellular adhesion molecule 1 (ICAM-1) and FAS on bystander cells. In some embodiments, the bispecific T cell engager antibody construct activates T cells, which results in the lysis of induced bystander cells. In some embodiments, the bystander cells are solid tumors. In some embodiments, the lysed bystander cells are associated with -Adjacent to activated T cells. In some embodiments, the bystander cells comprise tumor-associated antigen (TAA)-negative cancer cells. In some embodiments, the bystander cells include EGFR-negative cancer cells. In some embodiments, the tumor immunizing agent is an antibody that blocks the PD-L1 / PD1 axis and / or CTLA4. In some embodiments, the tumor immunizing agent is ex-vivo expanded tumor-infiltrating T cells. In some embodiments, the tumor immunizing agent is a bispecific antibody construct or chimeric antigen receptor (CAR) that directly links T cells to tumor-associated surface antigens (TAA).

[0205] In another embodiment, the tumor immunizing agent of the combination of the present invention is an antagonist of a protein that inhibits T cell activation or an immune checkpoint inhibitor.

[0206] As used herein, the term "checkpoint inhibitor" refers to an agent that can be used to prevent cancer cells from evading the patient's immune system. One of the main mechanisms of antitumor immunity disruption is "T cell exhaustion", which is caused by long-term exposure to antigens that lead to upregulation of inhibitory receptors. These inhibitory receptors act as immune checkpoints to prevent uncontrolled immune responses.

[0207] 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 3 (Tim-3), lymphocyte activation gene-3 (Lag-3, CD223), etc. are generally referred to as checkpoint regulators. They act as molecular "gatekeepers" that enable extracellular information to indicate whether the cell cycle process and other intracellular signaling processes should continue.

[0208] In some embodiments, the immune checkpoint inhibitor is an antibody against PD-1. PD-1 binds to the programmed cell death 1 receptor (PD-1) to prevent this receptor from binding to the inhibitory ligand PDL-1, thereby overriding the tumor's ability to suppress the host's anti-tumor immune response.

[0209] On the one hand, checkpoint inhibitors are biotherapeutic agents or small molecules. On the other hand, checkpoint inhibitors are monoclonal antibodies, humanized antibodies, fully human antibodies, fusion proteins, or combinations thereof. In other aspects, checkpoint inhibitors inhibit checkpoint proteins selected from the following: 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 ligands, or combinations thereof. In other aspects, checkpoint inhibitors interact with ligands of checkpoint proteins selected from the following: 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 ligands, or combinations thereof. In one aspect, checkpoint inhibitors are immunostimulants, T cell growth factors, interleukins, antibodies, vaccines, or combinations thereof. In another aspect, the interleukin is IL-7 or IL-15. In a specific aspect, the interleukin is glycosylated IL-7. In another aspect, the vaccine is a dendritic cell (DC) vaccine.

[0210] Checkpoint inhibitors include any agent that blocks or inhibits an inhibitory pathway of the immune system in a statistically significant manner. Such inhibitors can include small molecule inhibitors, or can include antibodies or antigen-binding fragments thereof that bind and block or inhibit immune checkpoint receptors, or antibodies that bind and block or inhibit immune checkpoint receptor ligands. Exemplary checkpoint molecules that can be targeted to block or inhibit include, but are not limited to, CTLA-4, PDL1, PDL2, PD1, B7-H3, B7-H4, BTLA, HVEM, GAL9, LAG3, TIM3, VISTA, KIR, 2B4 (which belongs to the CD2 molecule family and is expressed on all NK, γδ, and memory CD8+(αβ) T cells), CD160 (also known as 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 the following antibodies, or antigen-binding fragments thereof, other binding proteins, biotherapeutic agents, or small molecules that bind to one or more of CTLA-4, PDL1, PDL2, PD1, BTLA, HVEM, TIM3, GAL9, LAG3, VISTA, KIR, 2B4, CD160, and CGEN-15049 and block or inhibit their activity. Exemplary immune checkpoint inhibitors include Tremelimumab (a CTLA-4 blocking antibody), anti-OX40, a PD-L1 monoclonal antibody (anti-B7-Hl; MEDI4736), MK-3475 (a PD-1 blocker), Nivolumab (an anti-PD1 antibody), CT-011 (an anti-PD1 antibody), a BY55 monoclonal antibody, AMP224 (an anti-PDL1 antibody), BMS-936559 (an anti-PDL1 antibody), MPLDL3280A (an anti-PDL1 antibody), MSB0010718C (an anti-PDL1 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.

[0211] In certain embodiments, the immune checkpoint inhibitor is selected from PD-1 antagonists, PD-L1 antagonists, and CTLA-4 antagonists. In some embodiments, the checkpoint inhibitor is selected from the group consisting of nivolumab ipilimumab and pembrolizumab A group consisting thereof. In some embodiments, the checkpoint inhibitor is selected from nivolumab (an anti-PD-1 antibody, Bristol-Myers Squibb); pembrolizumab (an anti-PD-1 antibody, Merck); ipilimumab (an anti-CTLA-4 antibody, Bristol-Myers Squibb); durvalumab (an anti-PD-L1 antibody, AstraZeneca); and atezolizumab (an anti-PD-L1 antibody, Genentech).

[0212] 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, lirlumab, IPH2101, pembrolizumab and tremelimumab.

[0213] In some embodiments, the immune checkpoint inhibitor is REGN2810 (Regeneron), which is an anti-PD-1 antibody (NCT03132636) tested in patients with basal cell carcinoma (NCT03132636), NSCLC (NCT03088540), cutaneous squamous cell carcinoma (NCT02760498), lymphoma (NCT02651662), and melanoma (NCT03002376); pidilizumab (CureTech), also known as CT-011, which is an antibody that binds to PD-1 in clinical trials for diffuse large B-cell lymphoma and multiple myeloma; avelumab ( Pfizer / Merck KGaA), also known as MSB0010718C, which is 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), which is 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, which has been studied in clinical trials for various indications, including: mesothelioma, colorectal cancer, renal cancer, breast cancer, lung cancer and non-small cell lung cancer, pancreatic ductal adenocarcinoma, pancreatic cancer, germ cell carcinoma, head and neck squamous cell carcinoma, hepatocellular carcinoma, prostate cancer, endometrial cancer, liver metastases, liver cancer, large B-cell lymphoma, ovarian cancer, cervical cancer, metastatic anaplastic thyroid cancer, urothelial cancer, fallopian tube cancer, multiple myeloma, bladder cancer, soft tissue sarcoma, and melanoma. AGEN-1884 (Agenus) is an anti-CTLA4 antibody, which is undergoing a Phase I clinical trial for advanced solid tumors (NCT02694822).

[0214] 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, which is being studied in solid tumors (NCT02817633). LY3321367 (Eli Lilly) is an anti-TIM-3 antibody, which is being studied in solid tumors (NCT03099109). MBG453 (Novartis) is an anti-TIM-3 antibody, which is being studied in advanced malignancies (NCT02608268).

[0215] In some embodiments, the checkpoint inhibitor is an inhibitor of the T cell immunoreceptor with Ig and ITIM domains, or an inhibitor of TIGIT (an immunoreceptor on certain T cells and NK cells). TIGIT inhibitors that can be used in the present invention include BMS-986207 (Bristol-Myers Squibb), which is an anti-TIGIT monoclonal antibody (NCT02913313); OMP-313M32 (Oncomed); and an anti-TIGIT monoclonal antibody (NCT03119428).

[0216] 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, REGN3767, and IMP321. BMS-986016 (Bristol-Myers Squibb) is an anti-LAG-3 antibody that is being studied in glioblastoma and gliosarcoma (NCT02658981). REGN3767 (Regeneron) is also an anti-LAG-3 antibody that is being studied in malignancies (NCT03005782). IMP321 (Immutep S.A.) is a LAG-3-Ig fusion protein that is being studied in melanoma (NCT02676869), adenocarcinoma (NCT02614833), and metastatic breast cancer (NCT00349934).

[0217] Checkpoint inhibitors that can be used in the present invention include OX40 agonists. OX40 agonists being studied in clinical trials include the following: the agonistic anti-OX40 antibody PF-04518600 / PF-8600 (Pfizer) in metastatic renal cell carcinoma (NCT03092856) and advanced cancers and tumors (NCT02554812; NCT05082566); the agonistic anti-OX40 antibody GSK3174998 (Merck) in a phase I cancer trial (NCT02528357); the agonistic anti-OX40 antibody MEDI0562 (Medimmune / AstraZeneca) in advanced solid tumors (NCT02318394 and NCT02705482); the agonistic anti-OX40 antibody MEDI6469 (Medimmune / AstraZeneca) in patients with colorectal cancer (NCT02559024), breast cancer (NCT01862900), head and neck cancer (NCT02274155), and metastatic prostate cancer (NCT01303705); and the agonistic anti-OX40 antibody BMS-986178 (Bristol-Myers Squibb) in advanced cancers (NCT02737475).

[0218] Checkpoint inhibitors that can be used in the present invention include CD137 (also known as 4-1BB) agonists. CD137 agonists being studied in clinical trials include: the agonistic anti-CD137 antibody utomilumab (PF-05082566, Pfizer) in diffuse large B-cell lymphoma (NCT02951156) and advanced cancers and tumors (NCT02554812 and NCT05082566); the agonistic anti-CD137 antibody urelumab (BMS-663513, Bristol-Myers Squibb) in melanoma and skin cancer (NCT02652455) and glioblastoma and gliosarcoma (NCT02658981).

[0219] Checkpoint inhibitors that can be used in the present invention include CD27 agonists. CD27 agonists being studied in clinical trials include: the agonistic anti-CD27 antibody varlilumab (CDX-1127, Celldex Therapeutics) in squamous cell head and neck cancer, ovarian cancer, colorectal cancer, renal cell cancer, and glioblastoma (NCT02335918); lymphoma (NCT01460134); and glioma and astrocytoma (NCT02924038).

[0220] Checkpoint inhibitors that can be used in the present invention include glucocorticoid-induced tumor necrosis factor receptor (GITR) agonists. GITR agonists being studied in clinical trials include: the agonistic anti-GITR antibody TRX518 (Leap Therapeutics) in malignant melanoma and other malignant solid tumors (NCT01239134 and NCT02628574); the agonistic anti-GITR antibody GWN323 (Novartis) in solid tumors and lymphoma (NCT 02740270); the agonistic anti-GITR antibody INCAGN01876 (Incyte / Agenus) in advanced cancers (NCT02697591 and NCT03126110); the agonistic anti-GITR antibody MK-4166 (Merck) in solid tumors (NCT02132754) and the agonistic hexameric GITR-ligand molecule MEDI1873 (Medimmune / AstraZeneca) with a human IgG1 Fc domain in advanced solid tumors (NCT02583165).

[0221] Checkpoint inhibitors that can be used in the present invention include inducible T cell co-stimulator (ICOS, also known as CD278) agonists. ICOS agonists being studied in clinical trials include: the agonistic anti-ICOS antibody MEDI-570 (Medimmune) in lymphoma (NCT02520791); the agonistic anti-ICOS antibody GSK3359609 (Merck) in Phase 1 (NCT02723955); the agonistic anti-ICOS antibody JTX-2011 (Jounce Therapeutics) in Phase 1 (NCT02904226).

[0222] Checkpoint inhibitors that can be used in the present invention include killer immunoglobulin-like receptor (KIR) inhibitors. KIR inhibitors being studied in clinical trials include: the anti-KIR antibody lirilumab (IPH2102 / BMS-986015, Innate Pharma / Bristol-Myers Squibb) in leukemia (NCT01687387, NCT02399917, NCT02481297, NCT02599649), multiple myeloma (NCT02252263), and lymphoma (NCT01592370); IPH2101 (1-7F9, Innate Pharma) in myeloma (NCT01222286 and NCT01217203); the anti-KIR antibody IPH4102 (Innate Pharma) that binds to three domains of the long cytoplasmic tail region (KIR3DL2) in lymphoma (NCT02593045).

[0223] Checkpoint inhibitors that can be used in the present invention include CD47 inhibitors that interact between CD47 and signal regulatory protein alpha (SIRPa). CD47 / SIRPa inhibitors being studied in clinical trials include: the antagonistic variant ALX-148 (Alexo Therapeutics) of (SIRPa) that binds to CD47 and blocks CD47 / SIRPa-mediated signal transduction in Phase I (NCT03013218); the soluble recombinant fusion protein TTI-621 (SIRPa-Fc, Trillium Therapeutics) in Phase I of clinical trials (NCT02890368 and NCT02663518), which is generated by linking the N-terminal CD47-binding domain of SIRPa to the Fc domain of human IgG1, acts by binding to human CD47 and blocking its delivery of the "don't eat" signal to macrophages; the anti-CD47 antibody CC-90002 (Celgene) in leukemia (NCT02641002); and Hu5F9-G4 (Forty Seven, Inc.) in colorectal tumors and solid tumors (NCT02953782), acute myeloid leukemia (NCT02678338), and lymphoma (NCT02953509). In a preferred embodiment, the checkpoint inhibitor is a CD47 inhibitor.

[0224] Checkpoint inhibitors that can be used in the present invention include CD73 inhibitors. CD73 inhibitors being studied in clinical trials include: the anti-CD73 antibody MEDI9447 (Medimmune) in solid tumors (NCT02503774); and the anti-CD73 antibody BMS-986179 (Bristol-Myers Squibb) in solid tumors (NCT02754141).

[0225] Checkpoint inhibitors that can be used in the present invention include agonists of stimulators of interferon genes protein (STING, also known as transmembrane protein 173 or TMEM173). STING agonists being studied in clinical trials include: the agonistic synthetic cyclic dinucleotide MK-1454 (Merck) in lymphoma (NCT03010176); and the agonistic synthetic cyclic dinucleotide ADU-S100 (MIW815, Aduro Biotech / Novartis) in Phase 1 (NCT02675439 and NCT03172936).

[0226] Checkpoint inhibitors that can be used in the present invention include CSF1R inhibitors. CSF1R inhibitors being studied in clinical trials include: the CSF1R small molecule inhibitor pexidartinib (PLX3397, Plexxikon) in colorectal cancer, pancreatic cancer, metastatic and advanced cancers (NCT02777710) and melanoma, non-small cell lung cancer, squamous cell head and neck cancer, gastrointestinal stromal tumor (GIST), and ovarian cancer (NCT02452424); and the anti-CSF-1R antibody IMC-CS4 (LY3022855, Lilly) in pancreatic adenocarcinoma (NCT03153410), melanoma (NCT03101254), and solid tumors (NCT02718911); and the orally available CSF1R inhibitor BLZ945 (4-[2((1R,2R)-2-hydroxycyclohexylamino)-benzothiazol-6-yloxy]-pyridine-2-carboxylic acid methylamide, Novartis) in advanced solid tumors (NCT02829723).

[0227] Checkpoint inhibitors that can be used in the present invention include NKG2A receptor inhibitors. NKG2A receptor inhibitors being studied in clinical trials include: the anti-NKG2A antibody monalizumab (IPH2201, Innate Pharma) in head and neck tumors (NCT02643550) and chronic lymphocytic leukemia (NCT02557516).

[0228] In some embodiments, the immune checkpoint inhibitor is selected from nivolumab, pembrolizumab, ipilimumab, avelumab, durvalumab, atezolizumab, or pidilizumab.

[0229] In a preferred embodiment, the antagonist of the protein that inhibits T cell activation is selected from anti-PD-1 and anti-CTLA-4.

[0230] In a preferred embodiment, the tumor immunizing agent is a CTLA-4 antagonist, preferably a CTLA-4 antibody, more preferably ipilimumab or tremelimumab.

[0231] In a more preferred embodiment, the antagonist of the protein that inhibits T cell activation is anti-PD-1. In a preferred embodiment, the anti-PD-1 is an antibody or an antigen-binding portion thereof, and the preferred antibodies are 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 consisting of the extracellular domain of PD-L2 (B7-DC) fused to the Fc portion of IgG1, which is referred to as AMP-224.

[0232] In one embodiment, the combination of the present invention is a conjugate between component (i) and component (ii) of the combination of the present invention, particularly a conjugate between a polypeptide comprising SEQ ID No: 1 or a functionally equivalent variant thereof and a tumor immunotherapeutic agent.

[0233] In some embodiments, the conjugation between component (i) and (ii) is via an uncleavable linker. In some embodiments, the conjugation between component (i) and (ii) is via a cleavable linker. Exemplary uncleavable and cleavable linkers are described in US8088387, US8142784, WO2013075048, US6630579, US8512707, US9120854, US9023351, US20160095938, US9446146, WO2005009369, US5773001, US6214345, US10111954, US8153768, US7829531, US20160082119, WO2018218004, US8568728, WO2015057699, US20170182181, US9198979, the contents of each of which are incorporated herein by reference in their entirety.

[0234] In another aspect, the present invention relates to a pharmaceutical composition comprising a pharmaceutically effective amount of the combination of the present invention and a pharmaceutically acceptable excipient.

[0235] As used in the present invention, the expression "pharmaceutical composition" refers to a preparation suitable for administering a predetermined dose of one or several therapeutically useful agents to cells, cell populations, organs, tissues, or animals (such as cancer) in which cell division is uncontrolled.

[0236] The pharmaceutical composition of the present invention comprises a pharmaceutically effective amount of the combination of the present invention and a pharmaceutically active carrier. The pharmaceutical composition of the present invention includes a polypeptide comprising the sequence SEQ ID NO: 1, a functional equivalent variant thereof, a conjugate according to the present invention, a polynucleotide encoding the polypeptide or conjugate, a vector comprising the polynucleotide, or a cell capable of secreting the polypeptide or conjugate into the culture medium, and a tumor immunizing agent. Suitable functional equivalent variants of the polypeptide of SEQ ID NO: 1, suitable conjugates, fusion proteins, polynucleotides, vectors or cells for use in the pharmaceutical composition of the present invention are as defined above.

[0237] As used herein, the expression "pharmaceutically effective amount" should be understood as an amount capable of providing a therapeutic effect, and it can be determined by those skilled in the art by conventional means. The amount of the Omomyc polypeptide, its functional equivalent variant, conjugate, fusion protein, polynucleotide, vector, cell or tumor immunizing agent that can be combined in the pharmaceutical composition according to the present invention will vary depending on the subject to which it is administered and the particular mode of administration. Those skilled in the art will understand that the dosage can also be determined under the guidance of Goodman and Gilman's "The Pharmacological Basis of Therapeutics", 9th Edition (1996), Appendix II, pages 1707-1711 and Goodman and Gilman's "The Pharmacological Basis of Therapeutics", 10th Edition (2001), Appendix II, pages 475-493.

[0238] The appropriate dosage of one or more active ingredients in the pharmaceutical composition will depend on the type of cancer to be treated, the severity and course of the disease, whether the composition is administered for prophylactic or therapeutic purposes, previous therapies, the patient's clinical history and response to the peptide or polypeptide, and the diagnosis of the attending physician.

[0239] The amount of the polypeptide comprising the sequence SEQ ID NO: 1, its functional equivalent variant, fusion protein, conjugate, polynucleotide, vector or cell is suitable for administration to a patient either once or by a series of treatments. Depending on the type and severity of the disease, suitable dosage levels are generally from about 0.01 mg / kg patient body weight / day to 500 mg / kg patient body weight / day, which can be administered as a single dose or multiple doses. Preferably, the dosage level is from about 0.1 mg / kg / day to about 250 mg / kg / day, more preferably from about 0.5 mg / kg / day to about 100 mg / kg / day.

[0240] In a preferred embodiment, the amount of the first component is about 3.75 mg / kg of subject body weight per day, preferably administered four times a week, preferably by intranasal administration. In a preferred embodiment, the amount of the first component is about 8 mg / m 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, preferably administered four times a week, preferably by intranasal administration.

[0241] In a preferred embodiment, the amount of the first component is about 50 mg / kg of subject body weight per day, preferably administered twice a week, preferably by intravenous administration. In a preferred embodiment, the amount of the first component is about 100 mg / m 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, preferably administered twice a week, preferably by intravenous injection.

[0242] Suitable dose levels can be about 0.01 mg / kg / day to 250 mg / kg / day, about 0.05 mg / kg / day to 100 mg / kg / day, or about 0.1 mg / kg / day to 50 mg / kg / day. Within this range, the dose can be 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 tablets containing 1.0 to 1000 milligrams of the active ingredient, particularly 1.0, 5.0, 10.0, 15.0, 20.0, 25.0, 50.0, 75.0, 100.0, 150.0, 200.0, 250.0, 300.0, 400.0, 500.0, 600.0, 750.0, 800.0, 900.0, and 1000.0 milligrams of the active ingredient, for symptomatic adjustment of the dose for the patient to be treated. The compound can be administered according to a regimen of one to four times a day, preferably once or twice a day.

[0243] In one embodiment, 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, or seven times a week. In one embodiment, 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. The two compounds can be administered simultaneously or sequentially. When the compounds are administered sequentially, administration of the first compound is stopped before starting administration of the second compound.

[0244] The duration of treatment can be at least one week, at least two weeks, at least three weeks, at least four weeks, at least five weeks, at least six weeks, at least seven weeks, at least eight weeks, at least nine weeks, at least ten weeks, or longer. Preferably, the duration of treatment is at least four weeks. In another embodiment, the duration of treatment is at least three weeks.

[0245] The amount of the tumor immunizing agent depends on the specific agent used and can be from about 0.01 mg / kg of subject body weight per day to about 50 mg / kg of subject body weight per day, preferably from about 1 mg / kg of subject body weight per day to about 25 mg / kg of subject body weight per day, once or multiple times a day to achieve the desired therapeutic effect. In one preferred embodiment, the amount of the tumor immunizing agent is about 2.5 mg / kg of subject body weight per day or 7.5 mg / m 2 / day, preferably administered once a week, more preferably parenterally, even more preferably intraperitoneally. In one preferred embodiment, the amount of the tumor immunizing agent is about 5 mg / kg of subject body weight per day or 15 mg / m 2 / day, preferably administered once a week, more preferably parenterally, even more preferably intraperitoneally. In another preferred embodiment, the amount of the tumor immunizing agent is about 10 mg / kg of subject body weight per day, or 30 mg / m 2 / day, preferably administered once a week, more preferably parenterally, even more preferably intraperitoneally.

[0246] A pharmaceutical composition according to the present invention comprises a first component (i), said first component being selected from a polypeptide comprising SEQ ID NO: 1 according to the present invention, a functionally equivalent variant thereof, a fusion protein, a conjugate, a polynucleotide, a vector or a cell, and a second component (ii) as a tumor immunotherapeutic agent, which may be in the form of a single formulation (e.g., in the form of a tablet or capsule containing a quantified amount of each component), or on the other hand may be in the form of separate formulations, which may subsequently be combined for co-administration, sequential administration or separate administration. The compositions of the present invention also include formulations as part of a kit, wherein the components are formulated separately but packaged in the same container. Those skilled in the art will understand that the formulations of the different components in the pharmaceutical compositions according to the present invention may be similar, in other words, similarly formulated (in the form of tablets or pills), which allows them to be administered by the same route. In the case of separately formulating the different components of the present invention, the two components may be in the form of blisters. Each blister contains the drug that must be taken during the day. If the drug must be administered several times a day, the drugs corresponding to each administration may be placed in different parts of the blister, preferably recording the time of day when the drug should be administered in each part of the blister. Alternatively, the components of the composition of the present invention may be formulated differently such that the different components are administered differently. Thus, it is possible to formulate the first component as a tablet or capsule for oral administration and the second component as an intravenous administration, and vice versa. Those skilled in the art can adjust the ratio between the components as part of the combination or pharmaceutical composition according to the present invention based on the anti-tumor agent used in each specific case and the desired indication. Thus, the present invention contemplates such compositions wherein the ratio between the amounts of component (i) and component (ii) may be from 50:1 to 1:50, particularly from 20:1 to 1:20, from 1:10 to 10:1, or from 5:1 to 1:5. In a more specific 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 ranges from 1:1 to 1:1.5, preferably from 1:1.3 to 1:1.4, more preferably 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. In another specific embodiment, the ratio between the amounts is 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. Preferably, these ratios are weight / weight ratios.

[0247] The components of the pharmaceutical composition or the combinations of the present invention can be administered simultaneously. "Simultaneous administration" includes the co-administration of two therapeutic agents, regardless of the relative frequency or timing of administration of the individual therapeutic agents. Thus, simultaneous administration includes the co-administration of two therapeutic agents at the same time and at the same dosing frequency. Additionally, simultaneous administration refers to the co-administration of two therapeutic agents, where one therapeutic agent is administered more frequently than the other. Additionally, simultaneous administration refers to the co-administration of two therapeutic agents, where one treatment is administered only once during the administration of the other treatment.

[0248] In one embodiment, component (i) is administered intranasally, and in another embodiment, component (i) is administered intravenously. In another embodiment, component (ii) is administered parenterally, particularly intraperitoneally.

[0249] In a preferred embodiment, the combination of the present invention or component (i) of the pharmaceutical composition is administered intranasally, while the tumor immunizing agent is administered parenterally, particularly intraperitoneally or intravenously. For the preferred dose of the combination of the present invention or component (i) of the composition for intranasal administration, the preferred dose range of the polypeptide or its functionally equivalent variant, fusion protein, or conjugate is from 0.01 mg / kg / day to 250 mg / kg / day (which can be administered as a single dose or multiple doses), more preferably from 0.1 mg / kg / day to about 100 mg / kg / day. The preferred dose of the tumor immunizing agent for intraperitoneal administration is from 0.01 mg / kg to 150 mg / kg, more preferably from 0.1 mg / kg to 100 mg / kg.

[0250] In another embodiment, component (i) of the combination of the present invention or the pharmaceutical composition is administered intravenously, while the tumor immunizing agent is administered parenterally, particularly intraperitoneally or intravenously.

[0251] The pharmaceutical composition of the present invention may further comprise one or several additional compounds for the prevention and / or treatment of conditions where cell division is uncontrolled, such as cancer. The additional compound, such as an anti-tumor agent, may form part of the pharmaceutical composition as an independent entity. In a preferred embodiment, the combination of the present invention or the pharmaceutical composition comprises one or more anti-tumor agents selected from the group consisting of cytotoxic agents, anti-angiogenic agents, anti-metastatic agents, and anti-proliferative agents.

[0252] The pharmaceutical composition of the present invention further comprises one or several additional pharmaceutically acceptable excipients. "Pharmaceutically acceptable excipients" should be understood to mean substances that are therapeutically inactive for incorporation into the active ingredient and are acceptable to the patient from a pharmacological / toxicological perspective, and are acceptable to the pharmaceutical chemist manufacturing it in terms of composition, formulation, stability, patient acceptance, and bioavailability from a physical / chemical perspective. The excipient can be a carrier. As used herein, "carrier" refers to any substance used to improve the delivery and effectiveness of the active ingredient in the pharmaceutical composition. In a preferred embodiment, the carrier is not capable of directly delivering component (i) and / or (ii) to the cytoplasm of the cell, i.e., the carrier cannot fuse with the plasma membrane of the target cell. Examples of pharmaceutically acceptable carriers include one or more of water, saline, phosphate buffered saline, dextrose, glycerol, ethanol, etc. and combinations thereof. In many cases, an isotonic agent, such as a sugar, a polyol (e.g., mannitol, sorbitol) or sodium chloride, is preferably included in the combination or composition. The pharmaceutically acceptable carrier may also contain small amounts of auxiliary substances, such as wetting agents or emulsifiers, preservatives or buffers, which extend the shelf life or effectiveness of the components forming 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 Edition, Mack Publishing Company, Easton, PA, 1995). Examples of carriers are, without limitation, 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 hydroxypropylmethylcellulose; and a series of fillers, such as gelatin and polyvinylpyrrolidone. In some cases, a disintegrant, such as cross-linked polyvinylpyrrolidone, agar, alginic acid, or sodium alginate, can be added.

[0253] The amount and nature of the pharmaceutically acceptable excipients depend on the desired dosage form. Pharmaceutically acceptable excipients are known to those skilled in the art (Faulíy Trillo C. (1993) “Tratado de Farmacia Galénica”, Luzán 5, S.A. Ediciones, Madrid). The compositions can be prepared by conventional methods known in the art (“Remington: The Science and Practice of Pharmacy”, 20th edition (2003) Genaro A.R., ed., Lippincott Williams & Wilkins, Philadelphia, US).

[0254] For pharmaceutical compositions containing a reagent that is a nucleic acid molecule, the nucleic acid molecule can be present in any of a variety of delivery systems known to those of ordinary skill in the art, including nucleic acids, bacteria, viruses, and mammalian expression systems, such as the recombinant expression constructs provided herein. Techniques for incorporating DNA into such expression systems are well known to those of ordinary skill in the art. The DNA can also be “naked”, as described, for example, in Ulmer et al., Science 259:1745-49, 1993, and reviewed by Cohen, Science 259:1691-1692, 1993. Uptake of naked DNA is increased by coating the DNA onto biodegradable beads that are efficiently transported into cells.

[0255] Nucleic acid molecules can be delivered into cells according to any of several methods described in the art (see, 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); U.S. Patent No. 6,395,713; International Patent Application Publication No. WO 94 / 02595); Selbo et al., Int. J. Cancer 87:853-59 (2000); Selbo et al., Tumour Biol. 23:103-12 (2002); U.S. Patent Application Publication No. 2001 / 0007666, and No. 2003 / 077829). Such delivery methods known to those of skill in the art include, but are not limited to: encapsulation in liposomes, by iontophoresis, or by incorporation into other carriers such as biodegradable polymers, hydrogels, cyclodextrins (see, e.g., Gonzalez et al., Bioconjug. Chem. 10:1068-74 (1999); Wang et al., International Patent Publication No. WO03 / 47518 and WO03 / 46185); poly(lactic-co-glycolic) acid (PLGA) and PLCA microspheres (also useful for delivering peptides and polypeptides as well as other substances) (see, e.g., U.S. Patent No. 6,447,796; U.S. Patent Application Publication No. 2002 / 130430); biodegradable nanocapsules; and bioadhesive microspheres, or by protein carriers (International Application Publication No. WO00 / 53722). In another embodiment, the nucleic acid molecule can also 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) (also see, e.g., U.S. Patent Application Publication No. 2003 / 0077829).

[0256] In a specific embodiment, when the compounds according to the invention contain nucleic acids, the pharmaceutical composition can be formulated as a composition intended for gene therapy; by way of example and not limitation, the pharmaceutical composition can contain viral or non-viral vectors, which contain suitable polynucleotides or gene constructs. By way of example and not limitation, the vectors can be viral (e.g., retrovirus-based, adenovirus-based, etc.), or non-viral (e.g., ADN-liposomes, ADN-polymers, ADN-polymer-liposome complexes, etc.) [see “Nonviral Vectors for Gene Therapy”, edited by Huang, Hung and Wagner, Academic Press (1999)]. The vectors containing the corresponding polynucleotides or gene constructs can be administered directly to a subject by conventional methods. Alternatively, the vectors can be used for ex vivo transformation, or transfection, or infection of cells, such as mammalian cells, including human, and subsequently implanted into a human or an animal to obtain the desired therapeutic effect. For administration to a human or an animal, the cells are formulated in a suitable culture medium that has no adverse effect on cell viability.

[0257] The combination or pharmaceutical composition of the present invention can be administered by any type of suitable route, such as by the oral route, topical route, by inhalation, or parenteral route, and thus pharmaceutically acceptable excipients required for formulating the desired dosage form will be included. 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.

[0258] The “oral route” should be understood as the pharmaceutical composition being incorporated into the organism after being swallowed. In a specific embodiment, the pharmaceutical composition of the present invention can be in a dosage form suitable for administration by the oral route, whether solid or liquid. Dosage forms suitable for administration by the oral route can be tablets, capsules, syrups or solutions, and can contain any conventional excipients known in the art, such as binders, such as syrups, gum arabic, gelatin, sorbitol or polyvinylpyrrolidone; fillers, such as lactose, sugar, corn starch, calcium phosphate, sorbitol or glycine; compression lubricants, such as magnesium stearate; disintegrants, such as starch, polyvinylpyrrolidone, sodium starch glycolate or microcrystalline cellulose; or pharmaceutically acceptable wetting agents, such as sodium lauryl sulfate. Solid oral compositions can be prepared by conventional mixing, filling or compression methods. Repeated mixing operations can be used to completely disperse the active agent in those compositions using a large amount of filler. Such operations are conventional in the art. Tablets can be prepared, for example, by wet or dry granulation and optionally coated according to methods known in conventional pharmaceutical practice, especially with enteric coatings.

[0259] On the other hand, "local route" should be understood as administration by non-systemic routes and includes applying the pharmaceutical composition of the present invention to the exterior of the epidermis, in the oral cavity, and dropping the composition into the ear, eye, and nose, and not significantly entering the bloodstream. Dosage forms for topical or transdermal administration of the compounds for use in the present invention include ointments, pastes, creams, lotions, gels, powders, solutions, sprays, inhalants, or patches.

[0260] Ophthalmic preparations, ear drops, and eye drops are also considered to be within the scope of the present invention. Additionally, the use of transdermal patches is contemplated in the present invention, which have the additional advantage of providing a controlled delivery of the compound to the body. Such dosage forms can be prepared by dissolving or dispersing the compound in a suitable medium. Absorption enhancers can also be used to increase the flux of the compound through the skin. The rate can be controlled by providing a rate-controlling membrane or by dispersing the compound in a polymer matrix or gel.

[0261] In one embodiment, the combination or pharmaceutical composition is administered systemically.

[0262] "Systemic administration" is understood to be administration by oral, intravenous, intraperitoneal, and intramuscular routes. The amounts of components (i) and (ii) required for a therapeutic or prophylactic effect will naturally vary depending on the selected compound, the nature and severity of the disease to be treated, and the patient.

[0263] In another embodiment, the combination or pharmaceutical composition is administered intranasally. In a preferred embodiment, intranasal administration is by instillation or nasal inhalation.

[0264] "Inhalation" should be understood as administration by intranasal and oral inhalation routes. Dosage forms suitable for such administration can be prepared by conventional techniques, such as those in aerosols or metered inhalers. In one embodiment, the administration route is the intranasal route.

[0265] As used herein, the term "parenteral" includes administration by intravenous, intraperitoneal, intramuscular, or subcutaneous routes. Subcutaneous, intramuscular, and intravenous dosage forms for parenteral administration are generally preferred.

[0266] In one embodiment, the combination or pharmaceutical composition of the present invention may be suitable for its parenteral administration, such as sterile solutions, suspensions or lyophilized products in suitable dosage unit forms. The combination or pharmaceutical composition suitable for its injectable use includes sterile aqueous solutions (when they are soluble in water), or dispersions, and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions. For its administration by the intravenous route, some suitable carriers include phosphate-buffered saline solution (PBS). In all cases, the combination or composition must be sterile and must be fluid to reach the point of easy injection. It must be stable under the conditions of preparation and storage and must be protected against the contamination of microorganisms such as bacteria and fungi. The carrier may be a solvent or a dispersion medium, which includes, for example, water, ethanol, pharmaceutically acceptable polyols (such as glycerol, propylene glycol, liquid polyethylene glycol) and their suitable mixtures. For example, the appropriate fluidity can be maintained by using coatings such as lecithin, by maintaining the particle size required in the dispersion case, and by using surfactants. The prevention of the action of microorganisms can be achieved by various antibacterial and antifungal agents, such as parabens, chlorobutanol, phenol, ascorbic acid, thimerosal, etc. In many cases, an isotonic agent, such as sugar, polyol (such as mannitol, sorbitol) or sodium chloride, is preferably included in the composition. The prolonged absorption of the injectable composition can be achieved by including an agent that delays absorption, such as aluminum monostearate and gelatin.

[0267] The sterile injectable solution can be prepared as needed by incorporating the required amount of the active compound into a suitable solvent alone or in combination with one or more of the above ingredients, and then sterilizing by filtration through a sterile membrane. Generally, the dispersion is prepared by incorporating the active compound into a sterile carrier that contains a basic dispersion medium and the remaining ingredients required from those listed previously. In the case of the sterile powder for the preparation of the sterile injectable solution, the preferred preparation methods are vacuum drying and lyophilization, which produce a powder with the active ingredient plus any required additional ingredients from the previously filtered sterile solution.

[0268] The combination or pharmaceutical composition of the present invention may be suitably administered by pulsed infusion, using, for example, a decreasing dose of the composition. Preferably, the dose is administered by injection, more preferably by intravenous injection or subcutaneous injection, partly depending on whether the administration is acute or chronic. In a preferred embodiment, the PD-1 antagonist is administered by infusion.

[0269] Alternatively, as described above, the different components of the composition are administered differently.

[0270] Thus, in one embodiment, component (i) of the combination or composition of the present invention, preferably a polypeptide or a functionally equivalent variant or conjugate, is administered intranasally, while the tumor immunizing agent is administered systemically.

[0271] In another preferred embodiment, component (i) of the combination or composition, preferably a polypeptide or a functionally equivalent variant thereof, or a conjugate of the composition, is administered intranasally or by inhalation.

[0272] The dosage forms of the composition for intranasal and pulmonary administration are preferably liquid, suspension or solid. A suspension is a liquid preparation containing solid particles dispersed in a liquid carrier. The dosage form is preferably metered. For example, a metered drop / spray refers to a drop / spray including a dispenser that delivers a composition according to the use of the present invention containing a metered dose (predetermined amount).

[0273] In the context of the intranasal administration route, a preferred dosage form includes nasal drops. Most of the drops are deposited at the back of the nose and thus quickly move into the nasopharynx. The problem with drops is usually how to precisely control the drug dose, which is particularly important for the administration of the composition.

[0274] Another intranasal dosage form in which the pharmaceutical composition of the present invention can be administered is a nasal spray. A nasal spray typically contains, in an unpressurized dispenser, a conjugate dissolved or suspended in a solution or a mixture of excipients (such as preservatives, viscosity regulators, emulsifiers, buffers). Nasal sprays have several advantages, including the simplicity of the delivery device, convenience, ease of use, and accuracy of the delivered dose from 25 pL to 200 pL. They are deposited at the front of the nose and slowly enter the nasopharynx by mucociliary clearance. The nasal sprays used herein can be liquid or suspension.

[0275] Another intranasal dosage form is a nasal aerosol. The composition dispensing method of a nasal aerosol is different from that of a nasal spray: in an aerosol, the compound is dispensed due to excessive pressure and released through a valve. In a spray, the compound is dispensed by being pushed by a micropump barrel, and the pressure in the vial is similar to atmospheric pressure. Aerosols have advantages similar to those of sprays.

[0276] Alternatively, the composition according to the present invention can preferably be administered by a nasal emulsion, ointment, gel, paste or cream. These are high-viscosity solutions or suspensions applied to the nasal mucosa.

[0277] Due to the limited volume of the composition that can be effectively delivered to the nasal mucosa, liquid intranasal dosage forms, such as the corresponding intravenous dosage forms, usually have a higher concentration. When a substance becomes poorly soluble or unstable in liquid form, powders can be used to administer the composition of the present invention. Other advantages of powders are that they do not require preservatives and generally have higher stability compared to liquid preparations. The main limitation of intranasal powders is their irritating effect on the nasal mucosa.

[0278] One dosage form in the context of pulmonary administration is the inhaled aerosol. Inhaled aerosols are usually packaged under pressure and contain a composition according to the invention which is released into the respiratory tract, in particular the lungs, when the valve system is actuated. The released aerosol is a colloidal suspension of fine solid particles (suspensions) or droplets (solutions) in air or other gases. Thus, the aerosol can be a solution or a suspension aerosol. The diameter of the droplets or solid particles is preferably less than 100 pm, more preferably less than 10 pm and most preferably less than 1 pm.

[0279] Another dosage form for pulmonary administration is the inhaled spray. Inhaled sprays are usually water-based and do not contain any propellants. The conjugate is delivered to the lungs by oral inhalation.

[0280] Nebulized inhalation solutions and suspensions can also be used to deliver the conjugate via the pulmonary route. Nebulized inhalation solutions and suspensions are usually water-based formulations containing a composition according to the invention. Nebulized inhalation solutions and suspensions deliver the composition to the lungs by oral inhalation to produce a systemic effect and are used with a nebulizer.

[0281] Dry powder inhalation is an alternative to aerosol inhalation. The composition usually comprises in a capsule for manual loading or in an inhaler. The dry powder is usually delivered to the lungs by oral inhalation via an inhaler. The dry powder used here can be formulated neat. The neat formulation contains only the drug or almost only the drug, such as the dry powder as a spry. The dry powder used herein can also be formulated with a carrier such as lactose.

[0282] Pulmonary dosage forms are preferably metered, i.e., delivered to the lungs in a predetermined amount.

[0283] In the context of the present invention, devices for nasal delivery include spray pump systems, pipettes for delivering drops, metered spray pumps, nasal pressurized metered inhalers, powder spraying systems, breath-actuated powder inhalers and nasal powder insufflators. Nasal delivery devices can be filled with single-dose or multi-dose nasal preparations.

[0284] Using the pulmonary route, the conjugate can be administered with a metered-dose inhaler. A metered-dose inhaler (MDI) can provide a fine mist of the conjugate, the aerodynamic particle size of which is usually less than 5 pm.

[0285] A dry powder inhaler can alternatively be used to deliver the composition to the lungs. The dry powder inhaler makes the powder into a single-dose or multi-dose powder.

[0286] Another device for pulmonary delivery is an ultrasonic nebulizer and a jet nebulizer. In an ultrasonic nebulizer, ultrasonic waves are formed in an ultrasonic nebulizer chamber by a ceramic piezotransistor that vibrates upon electrical excitation. This generates an aerosol cloud at the surface of the solution. An aerosol is produced by a jet nebulizer when compressed air is forced through an orifice. Liquid can be withdrawn from a vertical nozzle (Bernoulli effect) to mix with an air jet stream that is atomized using a baffle to facilitate the formation of an aerosol cloud.

[0287] In one embodiment, each component of the combination or pharmaceutical composition of the present invention is prepared with a carrier that protects the component, particularly component (i), from rapid clearance from the body (e.g., a controlled release formulation), including implants and microencapsulated drug delivery systems. Biodegradable biocompatible polymers such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and polylactic acid can be used. The methods for preparing such formulations are known to those skilled in the art. Such materials are also commercially available from Alza Corporation and Nova Pharmaceuticals, Inc.

[0288] Sustained release compositions also include the preparation of crystals suspended in a suitable formulation that can keep the crystals suspended. When these formulations are injected by subcutaneous or intraperitoneal routes, a sustained release effect can be produced. Other compositions also include component (i) and / or (ii) entrapped in liposomes. Liposomes containing such components are prepared by known methods, such as Epstein et al., Proc. Natl. Acad. Sci. USA, (1985) 82:3688 - 3692; Hwang et al., Proc. Natl. Acad. Sci. USA, (1980) 77:4030 - 4034; EP 52,322; EP 36,676; EP 88,046; EP143,949. In a preferred embodiment, component (i) and / or (ii) is contained within the liposome, preferably both components are contained within the liposome, more preferably within the same liposome.

[0289] Although the Omomyc of the present invention, its functionally equivalent variants, conjugates, and fusion proteins are capable of translocating across biological membranes, it is possible to formulate any one of Omomyc, its functionally equivalent variants, conjugates, polynucleotides, vectors, or cells in nanoparticles. Nanoparticles can help maintain the integrity of the components in biological fluids until they reach the target organ. In addition, in the case of a composition comprising component (ii) or other anti-tumor agents, encapsulation of the composition can reduce secondary reactions caused by the anti-tumor agent. Additionally, the nanoparticles can also be modified to include moieties that allow the nanoparticles to target organs of interest. In this way, component (i) of the combination or composition of the present invention will be delivered near the target organ, thereby facilitating the entry of component (i) into the interior of the cells that require its biological activity.

[0290] Accordingly, in another embodiment, it is provided that component (i) of the combination or composition of the present invention forms part of a nanoparticle. In another embodiment, it is provided that both components of the combination or composition of the present invention form part of a nanoparticle, preferably with both components provided inside the same nanoparticle.

[0291] As used herein, the term "nanoparticle" refers to any material having a size range from 1 nm to 1000 nm. In some embodiments, the nanoparticles have a size range from 2 nm to 200 nm, preferably from 2 nm to 150 nm, and even more preferably from 2 nm to 100 nm. Nanoparticles that can be used in the present invention include the following nanoparticles: such as lipid-based nanoparticles, superparamagnetic nanoparticles, nanoshells, semiconductor nanocrystals, quantum dots, polymer-based nanoparticles, silicon-based nanoparticles, silica-based nanoparticles, metal-based nanoparticles, fullerenes, and nanotubes. Molecules can be encapsulated in the nanoparticle matrix or can be adsorbed on its surface, preferably with the molecules encapsulated in the nanoparticles.

[0292] In a preferred embodiment, the nanoparticle is a liposome.

[0293] Targeted delivery can be achieved by adding ligands without compromising the ability of the nanoparticles to deliver their contents. It is expected that this will enable delivery to specific cells, tissues, and organs. The targeting specificity of ligand-based delivery systems is based on the distribution of ligand receptors on different cell types. The targeting ligand can be non-covalently or covalently bound to the nanoparticle and can be conjugated to the nanoparticle by a variety of methods discussed herein.

[0294] 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, among others.

[0295] It will be understood that the inventive formulation in the nanoparticles is not intended or not solely intended to facilitate the entry of component (i) and / or (ii) into the interior of the cell, but also to protect component (i) and / or (ii) from degradation and / or to facilitate the targeting of the nanoparticles to the target organ.

[0296] In one example, the nanoparticles can be made of a biodegradable polymer such as poly(butyl cyanoacrylate) (PBCA). Examples of elemental nanoparticles include carbon nanoparticles and iron oxide nanoparticles, which can then be coated with oleic acid (OA)-Pluronic®. In this method, a drug (e.g., a hydrophobic or water-insoluble drug) is loaded into the nanoparticles. Other nanoparticles are made of silica.

[0297] 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(butylene adipate), 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-co-hexadecyl cyanoacrylate), and poly[HEMA-co-methacrylic acid]; proteins such as fibrinogen, collagen, gelatin, and elastin; and polysaccharides such as amylopectin, amylose, and chitosan.

[0298] Other nanoparticles include solid lipid nanoparticles (SLN). Examples of lipid molecules for solid lipid nanoparticles include stearic acid and modified stearic acids such as stearic acid-PEG 2000; soy lecithin; and emulsifying wax. The solid lipid nanoparticles can optionally contain other components including surfactants such as Epicuron® 200, poloxamer 188 (Pluronic® F68), Brij 72, Brij 78, polysorbate 80 (Tween 80); and salts such as sodium taurocholate. The reagent can be introduced into the solid lipid nanoparticles by many of the methods discussed for liposomes, which methods can also include high-pressure homogenization and dispersion of microemulsions.

[0299] The nanoparticles can also include nanosized micelles. The 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., the PEO-b-PCL block copolymer including ε-caprolactone and α-methoxy-ω-hydroxy-poly(ethylene glycol)).

[0300] In certain embodiments, the properties of the nanoparticles are altered by coating with surfactants. Any biocompatible surfactant can be used, for example, polysorbate surfactants such as polysorbate 20, polysorbate 40, polysorbate 60, and polysorbate 80 (Tween 80); Epicuron(R) 200; poloxamer surfactants such as 188 (Pluronic(R) F68), poloxamer 908, and 1508; and Brij surfactants such as Brij 72 and Brij 78.

[0301] The nanoparticles can optionally be modified to include hydrophilic polymer groups (e.g., poly(ethylene glycol) or poly(propylene glycol)), for example, by covalently attaching the hydrophilic polymer groups to the surface, or by using a polymer that includes such hydrophilic polymer groups (e.g., poly[methoxypoly(ethylene glycol) cyanoacrylate - co - hexadecyl cyanoacrylate]). The nanoparticles can optionally be crosslinked, which can be particularly useful for protein - based nanoparticles.

[0302] In another embodiment, the pharmaceutical composition of the present invention is a nanoemulsion. As used herein, "nanoemulsion" refers to a colloidal dispersion of droplets (or particles), at least some of which have diameters in the nanometer size range. The nanoemulsion consists of an oil rich in omega - 3, omega - 6, or omega - 9 fatty acids in an aqueous phase and is thermodynamically stabilized by an amphiphilic surfactant that forms an interfacial film, produced using a high - shear microfluidization process, and typically has a droplet diameter range of about 80 nm to 220 nm.

[0303] Therapeutic uses of the invention

[0304] In one aspect, the present invention relates to the combination or pharmaceutical composition of the present invention for use in medicine.

[0305] In another aspect, the present invention relates to the combination or pharmaceutical composition of the present invention for use in the prevention and / or treatment of cancer.

[0306] In another aspect, the present invention relates to the combination or pharmaceutical composition of the present invention for use in the preparation of a medicament for the prevention and / or treatment of cancer.

[0307] In yet another aspect, the present invention further relates to a method for the prevention and / or treatment of cancer, the method comprising administering to a subject in need thereof a therapeutically effective amount of the combination or pharmaceutical composition of the present invention.

[0308] On the other hand, the present invention also relates to a method for preventing and / or treating cancer by recruiting T cells to the tumor site, the method comprising administering to a subject in need thereof a therapeutically effective amount of the 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 specifically CD4 + PD-1 + T cells, even more specifically CD4 + PD-1 + Tim-3 - T cells. In another embodiment, the T cells recruited to the tumor site are CD4 + PD-1+Tim-3 + T cells. In another embodiment, the T cells recruited to the tumor site are CD8 T cells, more specifically CD8 + PD-1 + T cells. In another embodiment, the T cells recruited to the tumor site are CD3 + T cells. In another embodiment, the T cells recruited to the tumor site are CD3 + CD4 + T cells. In another embodiment, the T cells recruited to the tumor site are Th1 / Th17 cells, specifically Th1 / Th17PD-1 + cells, more specifically CD4 + IFN + IL-17 + T cells, even more specifically CD4 + PD-1 + IFN + IL-17 + T cells. In another embodiment, the cells recruited to the tumor site are CD45 + cells.

[0309] On the other hand, the present invention also relates to a method for preventing and / or treating cancer by inducing the expansion of regulatory T cells, the method comprising administering to a subject in need thereof a therapeutically effective amount of the combination or pharmaceutical composition of the present invention.

[0310] On the other hand, the present invention also relates to a method for preventing and / or treating cancer by inducing IFN-γ production by intratumoral CD4+ and CD8+ cells, the method comprising administering to a subject in need thereof a therapeutically effective amount of the combination or pharmaceutical composition of the present invention.

[0311] In a preferred embodiment, the prophylactic or therapeutic method according to the invention involves the direct use of a combination or composition comprising a polypeptide comprising Omomyc, a functionally equivalent variant thereof, a conjugate, or a fusion protein. Thus, in a preferred embodiment, the prophylactic or therapeutic method according to the invention does not involve administering a nucleic acid encoding a polypeptide comprising Omomyc, a functionally equivalent variant thereof, or a fusion protein, nor does it involve administering a vector encoding said nucleic acid or a cell comprising said nucleic acid.

[0312] "Prophylaxis" should be understood as administering the combination or composition of the invention at the initial or early stage of a disease, or also preventing its onset.

[0313] The term "treatment" is used to denote administering the combination or composition of the invention before or after the appearance of clinical symptoms to control the progression of a disease. Control of disease progression is understood as a beneficial or desired clinical outcome, including but not limited to alleviation of symptoms, reduction in the duration of the disease, stabilization of the pathological condition (in particular avoiding additional damage), postponement of disease progression, improvement of the pathological condition, and remission (partial and complete). Control of disease progression also involves an extension of the survival period compared to the expected survival without application of the treatment. In a preferred embodiment, control of disease progression is measured by a healthy lung / chest volume ratio. In another embodiment, control of disease progression is measured as a reduction in tumor volume.

[0314] The term "cancer" refers to a disease characterized by uncontrolled cell division (or with increased survival or apoptosis resistance), the ability of said cells to invade other adjacent tissues (invasion), or spread to other regions of the human body where cells are not normally located via lymphatic vessels and blood vessels (metastasis). Tumors are classified as benign or malignant depending on whether they can spread by invasion and metastasis: a benign tumor is a tumor that cannot spread by invasion or metastasis, i.e., it grows only locally; while a malignant tumor is a tumor that can spread by invasion and metastasis. The method according to the invention can be used to treat local and malignant tumors.

[0315] In one embodiment, the cancer includes, but is not limited to, leukemia (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, lymphoma (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, osteosarcoma, chordoma, angiosarcoma, endotheliosarcoma, lymphangiosarcoma, lymphangioendotheliosarcoma, synovioma, mesothelioma, Ewing's tumor, leiomyosarcoma, rhabdomyosarcoma, Kaposi's sarcoma, colon cancer, pancreatic cancer, breast cancer, biliary cancer, esophageal cancer, ovarian cancer, prostate cancer, oral cancer including squamous cell carcinoma, basal cell carcinoma, adenocarcinoma, sweat gland carcinoma, sebaceous gland carcinoma, papillary carcinoma, papillary adenocarcinoma, cystadenocarcinoma, medullary carcinoma, bronchial carcinoma, renal cell carcinoma, liver cancer, cholangiocarcinoma, teratoma, choriocarcinoma, seminoma, embryonal carcinoma, Wilm's tumor, cervical cancer, uterine cancer, testicular cancer, lung cancer, small cell lung cancer, bladder cancer, epithelial carcinoma, intraepithelial neoplasia including Bowen's disease and Paget's disease, glioma, glioblastoma, astrocytoma, glioblastoma multiforme (GBM, also known as malignant glioblastoma), medulloblastoma, craniopharyngioma, ependymoma, pinealoma, hemangioblastoma, acoustic neuroma, oligodendroglioma, schwannoma, neurofibrosarcoma, meningioma, melanoma, neuroblastoma, and retinoblastoma.

[0316] In some embodiments, the cancer is glioma, glioblastoma, astrocytoma, glioblastoma multiforme (GBM, also known as malignant glioblastoma), medulloblastoma, craniopharyngioma, ependymoma, pinealoma, hemangioblastoma, acoustic neuroma, oligodendroglioma, schwannoma, neurofibrosarcoma, meningioma, melanoma, neuroblastoma, and retinoblastoma.

[0317] In certain embodiments, the cancer is acoustic neuroma, astrocytoma (e.g., grade I - pilocytic astrocytoma, grade II - low - grade astrocytoma, grade III - anaplastic astrocytoma, or grade IV - glioblastoma multiforme (GBM)), chordoma, CNS lymphoma, craniopharyngioma, brainstem glioma, ependymoma, mixed glioma, optic nerve glioma, subependymoma, medulloblastoma, meningioma, metastatic brain tumor, oligodendroglioma, pituitary tumor, primitive neuroectodermal (PNET) tumor, or schwannoma. In some embodiments, the cancer is a type that is more common in children than in adults, such as brainstem glioma, craniopharyngioma, ependymoma, juvenile pilocytic astrocytoma (JPA), medulloblastoma, optic nerve glioma, pinealoma, primitive neuroectodermal tumor (PNET), or rhabdoid tumor. In some embodiments, the patient is an adult. In some embodiments, the patient is a child or pediatric patient.

[0318] In another embodiment, the cancer includes but is not limited to mesothelioma, hepatobiliary cancer (hepatic bile duct), bone cancer, pancreatic cancer, skin cancer, head and neck cancer, cutaneous or intraocular melanoma, ovarian cancer, colon cancer, rectal cancer, anal cancer, gastric cancer, gastrointestinal cancer (stomach, colorectal, and duodenum), uterine cancer, fallopian tube cancer, endometrial cancer, cervical cancer, vaginal cancer, vulvar cancer, Hodgkin's disease, esophageal cancer, small intestine cancer, endocrine system cancer, thyroid cancer, parathyroid cancer, adrenal cancer, soft tissue sarcoma, urethral cancer, penile cancer, prostate cancer, testicular cancer, chronic or acute leukemia, chronic myeloid leukemia, lymphocytic lymphoma, bladder cancer, kidney or ureteral cancer, renal cell cancer, renal pelvic cancer, non - Hodgkin's lymphoma, spinal cord tumor, brainstem glioma, pituitary adenoma, adrenocortical carcinoma, gallbladder cancer, multiple myeloma, cholangiocarcinoma, fibrosarcoma, neuroblastoma, retinoblastoma, or a combination of one or more of the above cancers.

[0319] In some embodiments, the cancer is selected from: hepatocellular carcinoma, ovarian cancer, ovarian epithelial cancer or fallopian tube cancer; papillary serous cystadenocarcinoma or uterine papillary serous carcinoma (UPSC); prostate cancer; testicular cancer; gallbladder cancer; hepatocholangiocarcinoma; soft tissue and bone synovial sarcoma; rhabdomyosarcoma; osteosarcoma; chondrosarcoma; Ewing's sarcoma; anaplastic thyroid cancer; adrenocortical adenoma; pancreatic cancer; pancreatic ductal carcinoma or pancreatic adenocarcinoma; gastrointestinal / gastric stromal tumor (GIST); lymphoma; head and neck squamous cell carcinoma (SCCHN); salivary gland cancer; glioma or brain cancer; neurofibromatosis - 1 - associated malignant peripheral nerve sheath tumor (MPNST); Waldenström macroglobulinemia; or medulloblastoma.

[0320] In some embodiments, the cancer is selected from: hepatocellular carcinoma (HCC), hepatoblastoma, colon cancer, rectal cancer, ovarian cancer, ovarian epithelial cancer, fallopian tube cancer, papillary serous cystadenocarcinoma, uterine papillary serous carcinoma (UPSC), cholangiocarcinoma, soft tissue and bone synovial sarcoma, rhabdomyosarcoma, osteosarcoma, anaplastic thyroid cancer, adrenocortical adenoma, pancreatic cancer, pancreatic ductal carcinoma, pancreatic adenocarcinoma, glioma, neurofibromatosis-1 associated malignant peripheral nerve sheath tumor (MPNST), Waldenström macroglobulinemia, or medulloblastoma.

[0321] In some embodiments, the cancer is a solid tumor, such as a sarcoma, carcinoma, or lymphoma. Solid tumors typically comprise an abnormal mass of tissue and generally do not include cysts or fluid areas. In some embodiments, the cancer is selected from: renal cell carcinoma or kidney cancer; hepatocellular carcinoma (HCC), or hepatoblastoma, or liver cancer; melanoma; breast cancer; colorectal cancer, or colorectal carcinoma; colon cancer; rectal cancer; anal cancer; lung cancer, such as non-small cell lung cancer (NSCLC) or small cell lung cancer (SCLC); ovarian cancer, ovarian epithelial cancer or fallopian tube cancer; papillary serous cystadenocarcinoma or uterine papillary serous carcinoma (UPSC); prostate cancer; testicular cancer; gallbladder cancer; cholangiocarcinoma; soft tissue and bone synovial sarcoma; rhabdomyosarcoma; osteosarcoma; chondrosarcoma; Ewing's sarcoma; anaplastic thyroid cancer; adrenocortical tumor; pancreatic cancer; pancreatic ductal carcinoma or pancreatic adenocarcinoma; gastrointestinal / gastric cancer (GIST); lymphoma; head and neck squamous cell carcinoma (SCCHN); salivary gland cancer; glioma or brain cancer; neurofibromatosis-1 associated malignant peripheral nerve sheath tumor (MPNST); Waldenström macroglobulinemia; or medulloblastoma.

[0322] In some embodiments, the cancer is selected from: hepatocellular carcinoma (HCC), hepatoblastoma, colon cancer, rectal cancer, ovarian cancer, ovarian epithelial cancer, ovarian tumor, fallopian tube cancer, papillary serous cystadenocarcinoma, uterine papillary serous carcinoma (UPSC), cholangiocarcinoma, soft tissue and bone synovial sarcoma, rhabdomyosarcoma, osteosarcoma, anaplastic thyroid cancer, adrenocortical tumor, pancreatic cancer, pancreatic ductal carcinoma, pancreatic adenocarcinoma, glioma, neurofibromatosis-1 associated malignant peripheral nerve sheath tumor (MPNST), Waldenström macroglobulinemia, or medulloblastoma.

[0323] In some embodiments, the cancer is hepatocellular carcinoma (HCC). In some embodiments, the cancer is hepatoblastoma. In some embodiments, the cancer is colon cancer. In some embodiments, the cancer is rectal cancer. In some embodiments, the cancer is ovarian cancer, or ovarian tumor. In some embodiments, the cancer is ovarian epithelial 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 cholangiocarcinoma. In some embodiments, the cancer is soft tissue and bone synovial sarcoma. In some embodiments, the cancer is rhabdomyosarcoma. In some embodiments, the cancer is osteosarcoma. In some embodiments, the cancer is anaplastic thyroid cancer. 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 glioma. In some embodiments, the cancer is malignant peripheral nerve sheath tumor (MPNST). In some embodiments, the cancer is neurofibromatosis-1 associated MPNST. In some embodiments, the cancer is Waldenström macroglobulinemia. In some embodiments, the cancer is medulloblastoma.

[0324] In some embodiments, the cancer is a virus-associated cancer, including human immunodeficiency virus-associated solid tumors, human papillomavirus (HPV)-16 positive incurable solid tumors, and adult T-cell leukemia, which is caused by human T-cell leukemia virus type I (HTLV-1) and is a highly aggressive form of CD4 + T-cell leukemia characterized by clonal integration of HTLV-1 in leukemic cells (see https: / / clinicaltrials.gov / ct2 / show / study / NCT02631746); and virus-associated tumors of gastric cancer, nasopharyngeal cancer, cervical cancer, vaginal cancer, vulvar cancer, head and neck squamous cell carcinoma, and Merkel cell carcinoma. (See https: / / clinicaltrials.gov / ct2 / show / study / NCT02488759; also see https: / / clinicaltrials.gov / ct2 / show / study / NCT0240886; https: / / clinicaltrials.gov / ct2 / show / NCT02426892).

[0325] Other cancers are known to those of ordinary skill in the art.

[0326] In some embodiments, the cancer is melanoma cancer. In some embodiments, the cancer is breast cancer.

[0327] In another embodiment, the cancer is glioblastoma.

[0328] "Glioblastoma", also known as glioblastoma multiforme and grade IV astrocytoma, is the most common and aggressive cancer that begins within the brain.

[0329] In a preferred embodiment, the cancer is lung cancer.

[0330] The term "lung cancer" or "lung tumor" refers to a physiological condition in mammals characterized by unregulated cell growth in lung tissue. The term lung cancer refers to any cancer of the lungs, including 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).

[0331] As used herein, the term non-small cell lung cancer (NSCLC) refers to a group of heterogeneous diseases because their prognosis and management methods are generally the same and, according to the histological classification of the World Health Organization / International Association for the Study of Lung Cancer (Travis WD et al., Histological typing of lung and pleural tumours, 3rd edition, Berlin: Springer-Verlag, 1999) includes:

[0332] (i) Squamous cell carcinoma (SCC), which accounts for 30% to 40% of NSCLC, begins in the larger airways but grows slowly, meaning that the size of these tumors can vary at the time of diagnosis.

[0333] (ii) Adenocarcinoma is the most common subtype of NSCLC, accounting for 50% to 60% of NSCLC. It begins near the gas exchange surface of the lung and includes the subtype bronchioloalveolar carcinoma, which may respond differently to treatment.

[0334] (iii) Large cell carcinoma is a fast-growing form that grows near the surface of the lung. It is mainly a diagnosis of exclusion and is usually reclassified as squamous cell carcinoma or adenocarcinoma when more studies are done.

[0335] (iv) Adenosquamous carcinoma is a cancer that contains two types of cells: squamous cells (thin flat cells lining certain organs) and gland-like cells.

[0336] (v) Cancers with pleomorphic, sarcomatoid or sarcomatous components. This is a group of rare tumors that reflects histological heterogeneity and the continuity of epithelial and mesenchymal differentiation.

[0337] (vi) Carcinoid tumors are slow-growing neuroendocrine lung tumors that originate from cells capable of releasing hormones in response to stimuli provided by the nervous system.

[0338] (vii) Cancers of salivary gland type originate in salivary gland cells located within the large airways of the lung.

[0339] (viii) Unclassified cancers include cancers that do not belong to any of the above categories of lung cancer.

[0340] In one specific embodiment, NSCLC is selected from squamous cell carcinoma of the lung, large cell carcinoma of the lung, and adenocarcinoma of the lung.

[0341] As used herein, the term small cell lung cancer (SCLC) refers to the proliferation of small cells with unique and strict morphological features, containing dense neurosecretory granules, which renders the tumor associated with endocrine / paraneoplastic syndromes. Most cases occur in the larger airways (main and lobar bronchi). These cancers grow rapidly and spread early in the disease.

[0342] In an even more preferred embodiment, the lung cancer is adenocarcinoma, more preferably KRas-driven lung adenocarcinoma, preferably a cancer associated with a KRAS gene mutation. In one embodiment, the mutation in the KRAS gene is a mutation at glycine at position 12, glycine at position 13, or glutamine at position 61. In a more preferred embodiment, the mutation is selected from the group consisting of G12S mutation, G12V mutation, G12D mutation, G13D mutation, G12C mutation, G12R mutation, G12F mutation, G12I mutation, G13C mutation, G13R mutation, or Q61L mutation. In a preferred embodiment, the mutation is G12D mutation. In another embodiment, the lung cancer is KRas GD12 / p53-driven lung cancer, preferably KRas GD12 / p53-driven NSCLC.

[0343] In one embodiment, the cancer is a primary tumor. As used herein, the term "primary tumor" refers to a tumor that originates at the location or organ where it is present and has not metastasized to that location from another location.

[0344] In another embodiment, the cancer is cancer metastasis. In the context of the present invention, "metastasis" is understood as the proliferation of cancer from the organ where it starts to a different organ. It typically occurs through the blood or lymphatic system. When cancer cells spread and form new tumors, the latter are referred to as secondary or metastatic tumors. The cancer cells that form the secondary tumors are similar to the cancer cells of the original tumor. For example, if breast cancer spreads (metastasizes) to the lungs, the secondary tumor is formed by malignant breast cancer cells. The disease in the lungs is metastatic breast cancer rather than lung cancer. The authors of the present invention have also observed that the combination or composition of the present invention is capable of reducing cell proliferation, regardless of whether the cancer exhibits increased expression or activity of the Myc protein. In a preferred embodiment, the cancer to be prevented or treated is Myc-induced cancer.

[0345] In one embodiment, the cancer is a solid tumor.

[0346] All combinations of the compounds of the present invention and cancer types are included in the present invention.

[0347] In some embodiments, the combination or composition of the present invention inhibits tumor growth. In some embodiments, relative to the size (e.g., volume or mass) of the tumor before treatment, the combination or composition of the present invention reduces the tumor size by at least 5%, 10%, 25%, 50%, 75%, 90% or 99%. In some embodiments, relative to the number of tumors in the patient before treatment, the combination or composition of the present invention reduces the number of tumors by at least 5%, 10%, 25%, 50%, 75%, 90% or 99%.

[0348] As used herein, "subject" includes any animal that has cancer, or exhibits symptoms of cancer, or is at risk of developing cancer or exhibiting symptoms of cancer. Suitable subjects (patients) include laboratory animals (e.g., mice, rats, rabbits or guinea pigs), farm animals and domestic or pet animals (e.g., cats or dogs). Non-human primates are included, preferably human patients. Preferably, the subject is a mammal, most preferably a human.

[0349] Combinations or compositions for the prevention and / or treatment of cancer can be administered in any amount and by any route of administration effective to treat or alleviate the severity of cancer. The exact amount required varies from subject to subject and depends on the species, age, and general condition of the subject, the severity of the disease or condition, the specific agent, the mode of its administration, etc. The compounds of the present invention are preferably formulated in unit dosage form for ease of administration and uniformity of dosage. As used herein, the expression "unit dosage form" refers to physically discrete units of the agent suitable for the patient to be treated. However, it should be understood that the total daily usage of the compounds and compositions of the present invention will be determined by the attending physician within the scope of reasonable medical judgment. The specific effective dosage level for any particular patient or organism will depend on a variety of factors, including the disorder being treated and the severity of the disorder; the activity of the specific compound used; the specific composition employed; the age, body weight, general health, sex, and diet of the patient; the time of administration, the route of administration, and the rate of excretion of the specific compound used; the duration of the treatment; drugs used in combination with or concurrently with the specific compound employed, and like factors well known in the medical arts.

[0350] 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 tumor immunizing agent of the combination or composition to treat cancer (to achieve a therapeutic effect).

[0351] In particular, in a more preferred embodiment, the combination or pharmaceutical composition for the prevention and / or treatment of cancer is a combination or pharmaceutical composition in which the amount of the polypeptide or a functionally equivalent variant or conjugate thereof interacts synergistically with the tumor immunizing agent in the treatment of cancer.

[0352] The terms "synergism" or "interact synergistically" are used interchangeably. Synergism is greater than the additive effect predicted by adding the actual effects of the individual agents in vitro. In vivo, synergism is a physiological effect, particularly a therapeutic effect, that is greater than the additive effect predicted by adding the actual effects of the individual agents in vivo.

[0353] Thus, if two agents are administered, they provide a measurable physiological effect, particularly a therapeutic effect, if the actual effect of the two agents together is greater than the effect expected by adding the actual therapeutic effects of the individual agents. Specifically, a synergistic effect is provided when the first agent alone provides some measurable effect, the second agent alone provides some measurable effect, and the measurable effect provided by the two agents together is greater than the sum of the effects provided by the two individual agents. More specifically, a synergistic effect is provided when the first agent alone does not provide a measurable effect, the second agent alone provides some measurable effect, and the measurable effect provided by the two agents together is greater than the effect provided by the second agent alone. Even more particularly, a synergistic effect is provided when neither the first agent alone nor the second agent alone provides any measurable effect, but the two agents together provide a measurable effect. Since components (i) and (ii) act synergistically, the amounts of components (i) and / or (ii) of the combination or composition of the present invention can be less than the amounts required for a single therapy using only one of them as a therapeutic agent. Preferably, in these combinations or compositions, the dosage of one or the other therapeutic agent can be from 0.01 μg / kg body weight / day to 1.000 μg / kg body weight / day.

[0354] The amount of the therapeutic agent present in the combination or composition can be no greater than the amount normally administered in a composition containing the therapeutic agent as the sole active agent. Preferably, the amount of the therapeutic agent in the composition of the present invention ranges from about 50% to 100% of the amount normally present in a composition containing the agent as the sole therapeutic active agent. In some embodiments, one therapeutic agent is administered at about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, or about 95% of its normal dosage amount. As used herein, the phrase "normal administration" means that the amount of a therapeutic agent approved by the FDA is the dosage approved according to each FDA label insert.

[0355] The combination or composition of the present invention can also be used in combination with known methods of treatment, such as chemotherapy, radiotherapy, immunotherapy, phototherapy, surgery, hormones, or combinations thereof.

[0356] In a preferred embodiment, the combination or pharmaceutical composition for treating and / or preventing cancer is for treating lung cancer, preferably NSCLC, more preferably Kras-driven cancer, even more preferably KRAS G12DA cancer driven by, wherein the first component preferably comprises the polypeptide of SEQ ID NO: 1, more preferably consists of the polypeptide of SEQ ID NO: 1, and is administered intranasally; and wherein the second component is preferably an antagonist of a protein that inhibits 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, and 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 one embodiment, the first component and the second component are administered sequentially, i.e., administration of the first component is stopped before starting administration of the second component. In a preferred embodiment, the treatment lasts for at least four weeks. In a preferred embodiment, the first component and the second component are administered on different days. In a preferred embodiment, the first component is administered on days 1, 2, 4, and 5, while the second component is administered on day 3. In a preferred embodiment of co-administration, the first component and the second component are administered on different days, preferably, the first component is administered on days 1, 2, 4, and 5, while the second component is administered on day 3. In a preferred embodiment, the ratio range of the amounts of component (i) and component (ii) can be from 50:1 to 1:50, specifically 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 range can be from 1:1 to 1:1.5, preferably from 1:1.3 to 1:1.4, more preferably 1:1.34. In another preferred embodiment, the ratio range is 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 weight / weight ratios.

[0357] In a preferred embodiment, the combination or pharmaceutical composition for treating and / or preventing cancer is used for treating lung cancer, preferably NSCLC, more preferably Kras-driven cancer, even more preferably KRAS G12D driven cancer, preferably KRAS G12D / p53-driven cancers, wherein the first component preferably comprises a polypeptide of SEQ ID NO: 1, more preferably consists of the polypeptide of SEQ ID NO: 1, and is administered intravenously; and wherein the second component is preferably an antagonist of a protein that inhibits 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, and is administered systemically, preferably parenterally, even more preferably intraperitoneally. In a preferred embodiment, the first component is administered twice a week. In a preferred embodiment, the second component is administered once a week. In a more preferred embodiment, the first component is administered twice a week and the second component is administered once a week. In one embodiment, the first component and the second component are administered sequentially, i.e., administration of the first component is stopped before starting administration of the second component. In another embodiment, the first component and the second component are administered simultaneously, preferably once a week. In a preferred embodiment, the treatment lasts for at least three weeks, preferably at least four weeks. In a preferred embodiment, the first component and the second component are administered on different days. In a preferred embodiment, the first component is administered on days 2 and 5, and the second component is administered on day 3. In a preferred embodiment of co-administration, the first component and the second component are administered on different days, preferably, the first component is administered on days 2 and 5, and the second component is administered on day 3. In a more preferred embodiment, the first component is administered during 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 one embodiment, administration of the first component is stopped before starting administration of the second component. In a preferred embodiment, the ratio of the amounts of component (i) and component (ii) can range from 50:1 to 1:50, specifically 20:1 to 1:20, 1:10 to 10:1, or 5:1 to 1:5. In another specific embodiment, the ratio of 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 weight / weight ratios.

[0358] All embodiments of the combination of the present invention are also applicable to the treatment method of the present invention.

[0359] Articles and kits

[0360] The present disclosure also provides an article of manufacture in one or more containers comprising any of the combinations or pharmaceutical compositions disclosed herein. In some embodiments, the article of manufacture includes, for example, a manual, printed instructions, label, or package insert that instructs a user (e.g., a distributor or end user) to combine and / or use the composition of the article of manufacture to prevent and / or treat cancer.

[0361] In some embodiments, the article of manufacture includes, for example, a bottle, vial, cartridge, box, syringe, injector, or any combination thereof. In some embodiments, the label refers to using or administering the combination or pharmaceutical composition in the article of manufacture according to the methods disclosed herein. In some aspects, the label recommends, for example, a usage regimen, a regimen for treating, preventing, or ameliorating cancer.

[0362] The content of all cited references (including references, patents, patent applications, and websites) that may be cited in this application, as well as the references cited therein, are hereby expressly incorporated by reference in their entirety for any purpose into this application.

[0363] Unless otherwise specified, all terms used herein shall be understood in their ordinary meaning as known in the art. Other more specific definitions of certain terms used in this application are set forth below and are intended to be applied uniformly throughout the specification and the scope of the invention for which a patent is sought, unless a more extensive definition is provided by an expressly listed definition. Throughout the specification and the scope of the invention for which a patent is sought, the word "comprising" and variations of the word are not intended to exclude other technical features, additives, components, or steps. Furthermore, the word "comprising" encompasses the case of "consisting of". By reading the specification, other objects, advantages, and features of the present invention will become apparent to those skilled in the art or can be learned by practicing the present invention. In addition, the present invention encompasses all possible combinations of the specific and particular embodiments described herein.

[0364] In this specification and the appended claims of the invention, the indefinite article terms in the singular form ("a", "an") and the definite article term ("the") include plural referents, unless the context clearly dictates otherwise. The indefinite article term "a" (or "an") and the terms "one or more" and "at least one" may be used interchangeably herein. In addition, the "and / or" used herein shall be regarded as clearly disclosing that each of the two specified features or components has or does not have the other. Thus, the term "and / or" used in a phrase herein (e.g., "A and / or B") is intended to include "A and B", "A or B", "A" (alone), and "B" (alone). Similarly, the term "and / or" used in a phrase (e.g., "A, B and / or C") is intended to cover 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 (alone); B (alone); and C (alone). The term "about" used in connection with a numerical value throughout the specification and the claims of the invention represents a range of precision familiar and acceptable to those skilled in the art. Generally, such a range of precision is ±15%. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the technical field to which this disclosure pertains. Units, prefixes, and symbols are expressed in their internationally accepted SI form. Numerical ranges include the numbers defining the range. Unless otherwise stated, amino acid sequences are written from left to right in the amino to carboxyl direction. The headings provided herein are not limitations on the various aspects or aspects of the disclosure, which can be obtained by reference to the entire specification as a whole. Thus, the terms defined directly below are more fully defined by reference to the entire specification.

[0365] The present invention will be described by the following examples, which should be considered illustrative only and not limiting of the scope of the invention.

[0366] Examples

[0367] Production and purification of Omomyc

[0368] Using an improved protocol of the Max° purification protocol described by 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 Omomyc peptide sequence SEQ ID NO: 4 containing methionine at the N-terminus was reverse transcribed, codons were optimized for expression in Escherichia coli E. coli, cloned into the pET3a expression vector (Novagen), and expressed by BL21(DE3) arabinose-inducible Bacterial strain purification. The purified construct obtained is the polypeptide of SEQ ID NO: 4. The identity of each purified construct is confirmed by mass spectrometry and Western blot analysis. Omomyc is purified by cation exchange chromatography and its purity is confirmed by mass spectrometry, SDS-PAGE and UV spectroscopy. For in vivo administration, an additional purification step using the ToxinEraser TM Endotoxin Removal Kit (Genscript) is performed to remove endotoxin. The Chromogenic Endotoxin Quantification Kit (Thermo Scientific) is used to quantify the endotoxin concentration. Buffer exchange is performed in Amicon Ultra-15 (Merck Millipore) with an exclusion limit of 3 kDa.

[0369] Intranasal treatment with Omomyc increases the specific recruitment of T lymphocytes to the tumor site KRas LSL-G12D / + mice are genotyped by Transnetyx and lung tumors are generated in males and females as previously described (Jackson, E.L. et al., Genes Dev, 2001. 15(24): pp. 3243-8). The animals are maintained on a mixed C57BL / 6J×FVBN background. At least 5 mice are randomly grouped at each time point and condition and treatment is initiated 14 to 16 weeks after Adeno-Cre infection once tumors detectable by micro-CT appear in these mice. During weeks one to four, the animals are anesthetized by inhalation of isoflurane (AbbVie Farmaceutica S.L.U.) and treated intranasally four times a week (1101100) with a total volume of 30 μL of Omomyc polypeptide (2.4 mg / kg) or vehicle (10 mM sodium acetate, pH 6.5).

[0370] At the endpoint, the mice are euthanized, the lungs are excised and perfused through the trachea with 4% PFA, fixed overnight, transferred to 70% ethanol, embedded in paraffin, and sectioned at 4 μm. For CD3 immunofluorescence, antigen retrieval is performed by heating in 0.01 M citrate buffer (pH 6.0) in a microwave at 400 W for 20 minutes. After blocking for 1 hour in 3% BSA plus 0.05% Tween 20, the sections are incubated overnight at 4°C with anti-CD3 (Dako A0452) diluted 1 / 100 with Dako ready-to-use diluent (Dako S2022). After washing with PBS, the sections are incubated with goat anti-rabbit IgG (H+L)– Incubated with the conjugate (Thermo Fisher Scientific A-11008), stained with 1 / 10,000 diluted DAPI (Life Technologies D1306), washed with PBS, and mounted with fluorescent mounting medium (Dako S3023). Images were acquired using a Nikon C2+ confocal microscope and NIS-elements software. Five representative tumors were photographed per mouse, and the mean number of CD3 + cells per area is shown.

[0371] Immunostaining with anti-CD3 revealed that Omomyc treatment increased the recruitment of T lymphocytes specifically to the tumor site as early as 1 week after the start of treatment, and T cells remained there throughout the treatment ( Figure 1 A), indicating that part of the mechanism of action of the Omomyc polypeptide may be an immune contribution.

[0372] Intranasal treatment with Omomyc recruits activated CD4 T cells to the tumor site

[0373] The experimental model and Omomyc treatment were the same as previously described.

[0374] At the end point, the mice were euthanized, the lungs were excised and dissociated using the Mouse Tumor Dissociation Kit (Miltenyi), and stained with conjugated antibodies to analyze the 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. Then, non-specific binding was blocked by incubating with anti-CD16 / 32 antibody for 10 minutes at room temperature. For surface staining, cells were incubated with the antibodies in the dark at 4°C for 20 minutes. Antibodies used are listed in Table 1. For intracellular staining of FoxP3, the 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).

[0375] Figure 1Panel B shows FACS analysis indicating that Omomyc induces CD4 T cell recruitment to the tumor and their activation. Indeed, these cells show higher levels of the PD-1 and PD-1Tim-3 molecules, indicating that Omomyc induces an anti-tumor immune response. Additionally, Omomyc also induces the expansion of T regulatory cells (Tregs).

[0376] Systemic administration of Omomyc recruits T cells to the tumor site

[0377] For the study using the Kras / p53 syngeneic model, 1×10 6 MuH-163 cells were subcutaneously inoculated into the dorsal side of 7-week-old female C57BL / 6 mice (JANVIER LABS). Once tumors were formed and reached an approximate volume of 100 mm 3 the mice were randomly divided into two groups and treated intravenously once a week with vehicle (PBS pH 7.0) or Omomyc (32 mg / kg). Three weeks after treatment, the mice were euthanized, the tumors were excised and cut into two parts. Then, half of the tumors were fixed overnight in 4% PFA, transferred to 70% ethanol, embedded in paraffin, and sectioned at 4 μm. For CD3 immunofluorescence, antigen retrieval was performed by heating in 0.01 M citrate buffer (pH 6.0) in a microwave at 400 W for 20 minutes. After blocking for 1 hour in 3% BSA plus 0.05% Tween20, the sections were incubated overnight at 4 °C with anti-CD3 (Dako A0452) diluted 1 / 100 with Dako ready-to-use diluent (Dako S2022). After washing with PBS, the sections were incubated with goat anti-rabbit IgG (H+L)– conjugate (Thermo Fisher Scientific A-11008), stained with DAPI (Life Technologies D1306) diluted 1 / 10000, washed with PBS, and mounted with fluorescent mounting medium (DakoS3023). Images were acquired using a mechanical Nikon Tie fluorescence microscope and NIS-elements software. Four representative areas of the tumor were photographed for each mouse, and the mean number of CD3 + cells per field was shown.

[0378] For flow cytometry analysis, the other half of the tumor was dissociated using the Mouse Tumor Dissociation Kit (Miltenyi) and stained with conjugated antibodies to analyze the 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. Then, non-specific binding was blocked by incubating with anti-CD16 / 32 antibody for 10 minutes at room temperature. For surface staining, the cells were incubated with the antibodies in the dark at 4 °C for 20 minutes. 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).

[0379] Administration of Omomyc induced the recruitment of T cells to the tumor site ( Figure 2 A). Omomyc recruited more CD8 T cells to the tumor site, and there was a significant increase in both CD4 and CD8 T cells expressing both PD-1 and Tim-3 molecules ( Figure 2 B).

[0380] The combination of Omomyc and anti-PD-1 recruits CD4 + PD-1 + Tim-3 - T cells into the tumor

[0381] KRas genotyping of mice was performed by Transnetyx LSL-G12D / + and lung tumors were generated in male and female mice as previously described (Jackson, E.L. et al., Genes Dev, 2001.15(24): pp. 3243-8). Animals were maintained on a pure C57BL / 6 background. At least 5 mice were randomly grouped at each time point and disease condition, and treatment was initiated 14-16 weeks after Ad-Cre infection once tumors detectable by micro-CT appeared in these mice. Mice were randomly divided into 4 groups: vehicle + isotype rat IgG2a,k, Omomyc + isotype rat IgG2a,k, vehicle + anti-PD-1, and Omomyc + anti-PD-1. For Omomyc treatment, animals were anesthetized by inhalation of isoflurane (AbbVie Farmaceutica S.L.U.) and treated intranasally four times a week (1101100) with a total volume of 30 μL of Omomyc polypeptide (2.4 mg / kg) or vehicle (PBS, pH = 7). Anti-PD-1 (BioXCell BE0146) at a dose of 200 μg / mouse or its isotype rat IgG2a,k (BioXCell BE0089) was administered intraperitoneally once a week (0010000) for four weeks.

[0382] At the end point, the mice were euthanized, and the lungs were excised and dissociated using the Mouse Tumor Dissociation Kit (Miltenyi), and stained with conjugated antibodies for analysis of the content of immune cells by flow cytometry. Before staining, dead cells were stained with Fixable Viability Stain 510 (BD Biosciences 564406) according to the manufacturer's instructions. Nonspecific interactions were blocked by incubating with anti-CD16 / 32 antibody for 10 minutes at room temperature. For surface staining, the cells were incubated with the antibodies in the dark at 4 °C for 20 minutes. 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).

[0383] Compared with both the vector and the anti-PD-1 treatment group alone, the combination of Omomyc and anti-PD-1 therapy significantly increased the recruitment of CD4 + T cells expressing PD-1 but not Tim-3 to the tumor site ( Figure 3 ). This finding indicates that the combination of Omomyc and anti-PD-1 synergistically promotes the anti-tumor immune response. Recent findings have shown that the expression of PD-1 on tumor-infiltrating lymphocytes (TILs) accurately identifies all components of clonally expanded tumor-reactive cells (Gros A et al., J Clin Invest (2014) 124(5):2246–2259). Along this line of thought, although inhibitory signals are generated upon ligation to its ligands (PD-L1 and PD-L2), it is now clear that PD-1 expression is a primary marker of T cell activation and high-affinity TILs specifically directed against tumor antigens (see Simon S and Labarriere N., OncoImmunology (2018). 7:1, e1364828).

[0384] The combination of Omomyc and anti-PD-1 induces the production of IFN-γ

[0385] The experimental model, Omomyc, and anti-PD-1 treatment were the same as previously described.

[0386] At the end point, the mice were euthanized, and the lungs were excised and dissociated using the Mouse Tumor Dissociation Kit (Miltenyi), and stained with conjugated antibodies for analysis of the 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. Nonspecific interactions were blocked by incubation with anti-CD16 / 32 antibody for 10 minutes at room temperature. For surface staining, the cells were incubated with the antibodies for 20 minutes at 4 °C in the dark. Antibodies used are listed in Table 1. For IFN-γ staining, harvested and isolated tumor cells were stimulated with PMA plus ionomycin (both from Sigma-Aldrich) in the presence of monensin and brefeldin A (both from BD Biosciences) for 12 hours. The cells were then harvested and stained for flow cytometry analysis. For intracellular staining of IFN-γ, the 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).

[0387] These experiments showed that the combination therapy of Omomyc plus anti-PD-1 significantly induced interferon-γ (IFN-γ) production by CD4 + helper cells and CD8+ cytotoxic intratumoral T cells compared to their vehicle controls, a fact not observed in either the Omomyc or anti-PD-1 treatment groups. Figure 4 )

[0388] In recent years, a large body 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 antiproliferative, proapoptotic, and necrotic effects on tumor cells and by enhancing immunogenicity through upregulation of major histocompatibility molecules (for more, see Castro F et al., Front. Immunol. (2018). 9:847 and Ikeda H et al., Cytokine & Growth Factors Reviews (2002) 13, 95–109). In addition, this cytokine also affects the tumor microenvironment, impairing angiogenesis by inhibiting the proliferation and survival of endothelial cells surrounding the tumor and thus inducing local ischemia at the tumor site, which is an important 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, IFN-γ produced by Th1 CD4 + and CD8 + T cells enhances tumor clearance because this cytokine is crucial for the trafficking of T and NK cells to the tumor site (Melero I et al., Cancer Discov (2014) 4:522–6). In addition, IFN-γ also plays a key role in activating macrophages and promoting their antitumor activity (Celada A et al., J Exp Med (1984) 160:55–74). Importantly, elevated levels of IFN-γ are predictive biomarkers in response to both chemotherapy and radiotherapy as well as anti-PD-1 and CLTA-4 immunotherapies (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 have had promising results showing an association between effector T cells producing IFN-γ and tumor growth inhibition (Liakou CI et al., Proc Natl Acad Sci U S A (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).

[0389]

[0390]

[0391] Table 1: Anti-mouse antibodies for flow cytometry analysis

[0392] The combination of Omomyc and anti-PD-1 antibody synergistically increases the proportion of healthy lungs and recruits T cells to the tumor site.

[0393] Genotyping of mice for KRas was performed by Transnetyx LSL-G12D / + and lung tumors were generated in males and females as previously described (Jackson, E.L. et al., Analysis of lung tumor initiation and progression using conditional expression of oncogenic K-ras. Genes Dev, 2001. 15(24): pp. 3243-8). Animals were maintained on a pure C57BL / 6 background. Fourteen to sixteen weeks after Ad-Cre infection, once tumors were detectable by micro-CT, the mice were randomly divided into 4 groups and treated for 4 weeks as follows: vehicle + isotype rat IgG2a,k, Omomyc + isotype rat IgG2a,k, vehicle + anti-PD-1, and Omomyc + anti-PD-1. For Omomyc treatment, animals were anesthetized by inhalation of isoflurane (AbbVie Farmaceutica S.L.U.) and treated intranasally four times a week (1101100) with a total volume of 30 μL of Omomyc polypeptide (3.75 mg / kg) or vehicle (PBS, pH = 7). Five mg / kg of anti-PD-1 (BioXCell BE0146) or its isotype rat IgG2a,k (BioXCell BE0089) was administered intraperitoneally once a week (0010000) for four weeks.

[0394] MicroCT studies were obtained using a Quantum FX imaging system (Perkin Elmer. 940 Winter St. Waltham, Massachusetts. USA), and image reconstruction was based on the Feldkamp method. For thoracic volume, Quantum FX analysis software was used. First, the distance between the lateral ribs (r2) was measured at the carina level. The second measurement was defined as the maximum distance from the carina level to the cup of the diaphragm (h). The last measurement was the thoracic height, which was defined as the distance between the sternum and the psoas minor at the level of the cup of the diaphragm (r1). Using these three values, the volume of a frustum was calculated using the following mathematical formula:

[0395] Volume = height * π / 3 * (r1³ - r2³) / (r1 - r2)

[0396] Using the AMIDE software ( Loening), the healthy lung tissue was calculated using the threshold method. This threshold selection intensity value includes all the voxel amounts in the image within the gray scale range of -950 / -350. After conducting different studies, this gray scale range was manually selected.

[0397] Finally, based on the intention of maintaining the complete thoracic volume, the ratio of healthy lung volume / thoracic volume was calculated, and the healthy lung volume gradually decreased with the progression of pathology. Compared with the vehicle alone and the treatment, the animals treated with the combination of Omomyc and anti-PD-1 showed an increased proportion of healthy lungs ( Figure 5 A and 5B).

[0398] At the end point, the mice were euthanized, and the lungs were excised and isolated using the Mouse Tumor Dissociation Kit (Miltenyi), and stained with conjugated antibodies to analyze the content of immune cells by flow cytometry. Before staining, the dead cells were stained with Fixable Viability Stain 510 (BD Biosciences 564406) according to the manufacturer's instructions. Non-specific cross-reactions were blocked by incubating with anti-CD16 / 32 antibody at room temperature for 10 minutes. For surface staining, the cells were incubated with the antibody in the dark at 4°C for 20 minutes. Table 2 lists the antibodies used.

[0399]

[0400] Table 2: Anti-mouse antibodies for flow cytometry analysis

[0401] For IFN-γ and IL-17 staining, the harvested and isolated tumor cells were stimulated with PMA plus ionomycin (both from Sigma-Aldrich) for 12 hours in the presence of monensin and brefeldin A (both from BD Biosciences). Then the cells were harvested and stained for flow cytometry analysis. For intracellular staining of IFN-γ, the BD Cytofix / Cytoperm buffer set (BD Biosciences 554722) was used according to the manufacturer's instructions. The cells were collected using a CytoFlex cytometer (Beckman Coulter), and the data were analyzed using CytoExpert 2.0 software (Beckman Coulter).

[0402] Figure 5 C shows that the combined administration of Omomyc and anti-PD-1 induces the recruitment of T cells to the tumor site, particularly CD4 T cells and Th1 / Th17 cells. Table 3 shows that the obtained effect is synergistic. A synergistic effect is considered when the increase in the immune cell population of interest is higher than the sum of the increases of the individual treatments.

[0403]

[0404] Table 3: Mean values of each immune cell population

[0405] The combination of Omomyc and anti-CTLA-4 antibody synergistically reduces tumor growth and recruits anti-tumor T cells to the tumor site

[0406] The experimental model, micro-CT scans, and FACS staining were the same as those shown in Figure 5 the same.

[0407] Fourteen to sixteen weeks after Adeno-Cre infection, once the mice developed tumors detectable by micro-CT, they were randomly divided into 4 groups and 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 the Omomyc treatment, the animals were anesthetized by inhalation of isoflurane (AbbVie Farmaceutica S.L.U.) and treated intranasally four times a week (1101100) with a total volume of 30 μL of Omomyc polypeptide (3.75 mg / kg) or vehicle (PBS, pH = 7). Anti-CTLA-4 (BioXCell BE0131) or its isotype Syrian hamster IgG (BioXCell BE0087) at 10 mg / kg was administered intraperitoneally once a week (0010000) for four weeks.

[0408] Figure 6 A shows that animals treated with the combination of Omomyc and anti-CTLA-4 showed reduced tumor growth compared to the vehicle and treatment alone. Figure 6 B shows that the combined administration of Omomyc and anti-CTLA-4 induces the recruitment of T cells to the tumor site, particularly CD4 T cells and both CD4 and CD8 PD-1 + T cells. Table 4 shows that the obtained effect is synergistic. A synergistic effect is considered when the increase in the immune cell population of interest is higher than the sum of the increases of the individual treatments.

[0409]

[0410] Table 4: Mean values for each immune cell population

[0411] Sequential intravenous administration of the combination of Omomyc and anti-PD-1 antibody synergistically recruits anti-tumor T cells to the tumor site site.

[0412] The experimental models, micro-CT scans, and FACS staining were the same as those shown in Figure 5 the same as those shown in

[0413] Fourteen to sixteen weeks after Adeno-Cre infection, once tumors detectable by micro-CT appeared in the mice, they were randomly divided into four groups and treated for 4 weeks as follows: vehicle, Omomyc, vehicle + anti-PD-1, and Omomyc + anti-PD-1. For the Omomyc treatment, animals were treated intravenously twice a week with Omomyc peptide (50 mg / kg) or with vehicle (NaAc 24 mM + 150 mM NaCl) (0100100) for 10 days. The groups receiving the combination were treated with Omomyc twice a week for the first 10 days. Ten days after the Omomyc treatment, the groups receiving the combination stopped the Omomyc treatment and started receiving 2.5 mg / kg of anti-PD-1 (BioXCell BE0146) intraperitoneally once a week (0010000) until the end of the experiment. The single-therapy group using only Omomyc received Omomyc twice a week for the first 10 days and then continued the treatment but only once a week until the end of the experiment.

[0414] Figure 7 Sequential treatment with Omomyc followed by anti-PD-1 was shown to induce the recruitment of T cells to the tumor site, particularly CD4 T cells expressing both PD-1 and Tim-3 molecules and Th1 / Th17 T cells expressing PD-1. Table 5 shows that the effects obtained were synergistic. Synergy was considered when the increase in the immune cell population of interest was higher than the sum of the increases with single therapies.

[0415]

[0416] Table 5: Mean values for each immune cell population

[0417] Intravenous administration of the combination of Omomyc and anti-PD-1 antibody synergistically recruits T cells to the tumor site

[0418] The highly invasive Kras / p53-mutated NSCLC MuH-163 cell line (1×10 6Cells were subcutaneously inoculated into syngeneic C57 / BL6 mice. Once tumors formed, the mice were randomly divided into 4 groups: vehicle, Omomyc, vehicle + anti-PD-1, and Omomyc + anti-PD-1. Omomyc treatment was administered intravenously at 50 mg / kg (0010000), along with intraperitoneal administration of the anti-PD-1 antibody at 5 mg / kg once a week for 3 weeks. Mice were monitored twice a week, and tumor growth was monitored by caliper measurement.

[0419] Tumors were collected at the endpoint. Half of them were fixed with 4% PFA and embedded in paraffin for IHC analysis, while the other half was digested using the Mouse Tumor Dissociation Kit (Miltenyi) and stained with conjugated antibodies to analyze the immune cell content by flow cytometry. As Figure 5 shown, FACS staining and analysis were performed.

[0420] For CD3 immunofluorescence, antigen retrieval was performed by heating in 0.01 M citrate buffer (pH 6.0) using a microwave set at 400 W for 20 minutes. After blocking in 3% BSA for 45 minutes and washing in PBS, the sections were incubated overnight at 4°C with anti-CD3 (Dako A0452) diluted 1 / 100 with Dako ready-to-use diluent (Dako S2022). After washing with PBS, the sections were incubated with goat anti-rabbit IgG (H+L)– conjugate (Thermo Fisher Scientific A-11008) diluted 1 / 200 and stained with DAPI (Life Technologies D1306) diluted 1 / 10000, washed once with water, and mounted with fluorescence mounting medium (Dako S3023). CD3 positivity was detected by capturing 5 representative fluorescence microscopy images at 20× magnification for each animal.

[0421] Figure 8 It is shown that combination therapy with Omomyc and anti-PD-1 significantly recruits T cells to the tumor site. Table 6 shows that the effect obtained is synergistic. Synergy is considered when the increase in the immune cell population of interest is higher than the sum of the increases with single therapies.

[0422]

[0423] Table 6: Mean values of each immune cell population

[0424] High expression of CD3, CD4, IL-17 and IFN-γ is associated with higher survival rates

[0425] The Kaplan-Meier plots were generated using the online software Kaplan-Meier Plotter (http: / / kmplot.com / analysis / index.php?p=background). For this purpose, the lung cancer patient database was selected. All histological types, all stages, and all grades of NSCLC were included in the analysis.

[0426] Figure 9 High expression of CD3, CD4, IL-17, and IFN-γ was shown to be associated with higher survival rates in NSCLC patients.

[0427] Discussion

[0428] Compared to the improvements shown by Omomyc alone (0.86) and anti-PD-1 (0.92) therapy alone, the combination of intranasal Omomyc and anti-PD-1 antibody synergistically increased the proportion of healthy lung in the total thoracic volume (mean 7.969) ( Figure 5 A and 5B). Additionally, the combined treatment significantly induced T cell recruitment to the tumor site, particularly CD4 T cells and Th1 / Th17 cells, which are known to exert effective anti-tumor effects (Chatterjee, S. et al., CD38-NAD(+) Axis Regulates Immunotherapeutic Anti-Tumor T Cell Response. Cell Metab, 2018. 27(1): p. 85-100e8) ( Figure 5 C).

[0429] Consistent with these results, the combination of intranasal Omomyc and anti-CTLA-4 also showed reduced tumor growth (1.11) compared to the vehicle (3.85) and the two single treatments (Omomyc: 2.3; α-CTLA-4: 3.0) ( Figure 6 A).

[0430] In addition to the direct effect on tumor growth, the treatment combination also synergistically induced T cell recruitment to the tumor site, particularly CD4 T cells and both CD4 and CD8 T cells expressing the PD-1 molecule (cells known to recognize 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): pp. 2246-59)) ( Figure 6B). In summary, intranasal administration of Omomyc in combination with anti-PD-1 or anti-CTLA-4 reduced tumor growth and synergistically recruited anti-tumor T cells to the tumor site.

[0431] In addition, using the same mouse model (Kras G12D -driven NSCLC), the inventors have demonstrated that sequential administration of intravenous Omomyc in combination with anti-PD-1 (first Omomyc and then the anti-PD-1 antibody) also synergistically induces T cell recruitment to the tumor site, particularly tumor-specific CD4 and Th1 / Th17 anti-tumor T cells that co-express the PD-1 and Tim-3 molecules ( Figure 7 ).

[0432] To verify this synergy in another model, the authors used Omomyc in combination with anti-PD-1 in another highly aggressive NSCLC model driven by mutations in both Kras and p53. Again, both drugs synergistically and significantly recruited more T cells to the tumor site ( Figure 8 A), and also recruited more total immune cells ( Figure 8 B).

[0433] In summary, the authors concluded that combining Omomyc treatment with anti-PD-1 and CTLA-4 therapies reduced tumor growth and synergistically recruited 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.

[0434] This immune cell recruitment has obvious therapeutic effects on NSCLC cancer patients, as an increased proportion of total CD3 T cells, CD4, and T cells secreting IFN-γ and IL-17 is associated with increased survival ( Figure 9 ). This evidence emphasizes the importance of discovering the above-described combinations of Omomyc with tumor immunotherapeutics. The same conclusion can be extrapolated to other types of cancer, where data based on immune signatures have been established, a strong immune cell component predicts a good response to chemotherapy in breast cancer, and high tumor-infiltrating lymphocytes (TILs) are associated with a higher response rate to neoadjuvant therapy. In liver metastases of colorectal cancer, CD8 +High infiltration of T cells predicts better response to chemotherapy and longer survival. In melanoma, the expression of immune signatures (i.e., high expression of Th1 cells and genes related to cytotoxicity) is associated with good clinical response to therapeutic vaccines using melanoma-associated antigen 3 (MAGEA3) (described by Fridman, W.H. et al., The immune contexture in cancer prognosis and treatment. Nat Rev Clin Oncol, 2017. 14(12): p. 717-734).

[0435] In the past few years, a large amount of evidence has accumulated, indicating that TILs play a crucial role in both tumor elimination and the efficacy of tumor immunotherapy. In fact, the main factor contributing to resistance to tumor immunotherapy is the lack of tumor T cell infiltration, which is characteristic of so-called "cold tumors". Treating such immunologically inert tumors with tumor immunomodulators poses a great challenge because they do not have any adaptive immune response against the tumor and cannot respond to this type of therapy (Bonaventura, P. et al., Cold Tumors: A Therapeutic Challenge for Immunotherapy. Front Immunol, 2019. 10: p. 168).

[0436] Patients who have never shown a clinical response or disease stability after PD-1 / PD-L1 blockade are said to have "primary resistance" to the treatment. In contrast, early data from clinical trials indicate that the presence of pre-existing tumor-infiltrating lymphocytes (TILs) in and around the tumor, as well as co-localized PD-1 and PD-L1 expression on T cells and tumor cells, predict treatment response to anti-PD-1 therapy (Nowicki, T.S., 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 line of evidence, the efficacy of Pembrolizumab (anti-PD-1) is associated with the presence of intratumoral T cells and PD-1 / PD-L1 expression, which are requirements for an effective anti-tumor response (Tumeh, P.C. et al., PD-1 blockade induces responses by inhibiting adaptive immune resistance. Nature, 2014. 515(7528): p. 568-71) and 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).

[0437] Taking into account all this evidence, the combination of Omomyc with tumor immunomodulators, which synergistically induces T cell infiltration and stimulation, will ultimately increase the clinical response rate to tumor immunotherapy.

Claims

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

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 or 2, wherein the chemical moiety that promotes cellular uptake of the polypeptide or the functionally equivalent variant thereof is a cell-penetrating peptide sequence, and wherein the cell-penetrating peptide sequence forms a fusion protein with the polypeptide or the functionally equivalent variant thereof.

4. The combination according to claim 3, wherein the cell-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 1-4, wherein the conjugate further comprises an additional nuclear localization signal.

6. The combination according to any one of claims 1-5, provided that the tumor immunomodulator is not a cytokine.

7. The combination according to any one of claims 1-6, wherein the tumor immunomodulator 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 the 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 the protein that inhibits T cell activation is anti-PD-1.

10. The combination according to any one of claims 8 or 9, wherein the antagonist is an antagonistic antibody.

11. The combination according to claim 10, wherein the antagonistic antibody is pembrolizumab.

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

1.

13. A pharmaceutical composition comprising a pharmaceutically effective amount of the combination according to any one of claims 1 to 12 and a pharmaceutically acceptable excipient.

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

15. The combination according to any one of claims 1 to 12 or the pharmaceutical composition according to claim 13, which is used for preventing and / or treating cancer.

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

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

18. The combination or pharmaceutical composition for the use according to claim 17, wherein the intranasal administration is carried out by instillation or nasal inhalation.

19. The combination or pharmaceutical composition for the 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. The combination or pharmaceutical composition for the use according to any one of claims 14 to 19, wherein the polypeptide or its functional equivalent variant, or the conjugate interacts synergistically with the tumor immunomodulator in the treatment of cancer.

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