EA042570B1 - METHODS AND COMPOSITIONS FOR CANCER TREATMENT - Google Patents

Description

The field of technology to which the invention belongs

The invention relates to the field of oncological diseases and, in particular, to polypeptides, compositions and their use in medicine, in particular for the prevention and/or treatment of cancer.

State of the art

The ideal antitumor drug would target an essential function that is permanently required for the maintenance of a tumor, but which is not essential for the maintenance and function of any normal tissue. Therefore, the most general logic is to target gene products that specifically mutate in cancer, on the assumption that these mutant molecules will be likely drivers of cancer and perhaps of lesser importance in normal tissues. For these reasons, much attention has been paid to compiling a list of persistently recurring pathological changes in certain types of cancer. Unfortunately, this approach has several problems. First, most human solid tumors go through episodes of genomic instability and exhibit mutational noise that can mask driver mutations and their respective effector pathways. Second, cancers represent the end result of a process that involves transitions through multiple evolutionary critical sites. Each critical site may require a certain type of mutation, which then becomes unnecessary for tumor maintenance, and hence is not a good therapeutic target beyond this point in tumor development.

Myc is a protein containing the basic helix-loop-helix motif (b-HLH-LZ), a leucine zipper involved in the control of cancer growth and development, which functions in complex with structurally related proteins Max, Mad and Mnt. Myc/Max dimers activate gene transcription and induce cell proliferation or apoptosis. The Mad/Max and Mnt/Max complexes function as repressors and cause growth arrest and cell differentiation. All dimers recognize the same DNA consensus site, the CACGTG E-box.

Myc is highly regulated in normal cells, where levels are higher in proliferating cells and lower in non-proliferating cells. Aberrantly high and/or unregulated Myc activity is causally associated with most malignant tumors and is often associated with aggressive, poorly differentiated and angiogenic tumors. Dysregulation of Myc expression occurs through overexpression as a result of gene amplification, loss of transcriptional control, impaired degradation, or increased stabilization. This results in aberrant proliferation, increased survival, metabolic changes, angiogenesis, and inflammation, which are all major hallmarks of malignant tumors. The results of numerous studies have proven the critical role of Myc in the regulation of intracellular and extracellular aspects of oncogenesis, suggesting that targeting its function will be of therapeutic value.

Downregulation of Myc by the bromodomain inhibitor BET is known to result in regression of numerous tumor types. Although this approach has good potential, it has some limitations such as toxicity and numerous non-target effects. Many small molecules that interfere with the Myc/Max interaction have shown low specificity in cellulo.

However, the Myc inhibitor has not yet become clinically available, and its development is associated with various problems: firstly, Myc is a nuclear transcription factor, therefore, it is less available than membrane or cytoplasmic molecules; second, Myc lacks an enzymatic active site to target; thirdly, the Myc family includes three different proteins, c-, N-, and L-Myc, which under certain conditions are functionally duplicating each other, so they all require simultaneous inhibition. In addition, there were concerns that Myc inhibition would cause serious side effects by inhibiting the proliferation of normal tissues. For all these reasons, the development of a drug based on the Myc inhibitor is a difficult task.

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

Through these properties, Omomyc is able to prevent the transactivation function of Myc-dependent genes both in vitro and in vivo by eliminating the ability of Myc to bind to its DNA recognition binding site, the E-box. At the same time, Omomyc effectively enhances Myc-induced apoptosis in a Myc expression level dependent manner and thus enhances Myc transrepression activity. Thus, Omomyc prevents Myc binding to promoter E-boxes and transactivation of target genes, while maintaining Miz-1-dependent promoter binding and transrepression. In the presence of Omomyc interactom Moose is directed to

- 1 042570 repression, and its activity switches from pro-oncogenic to tumor suppressor.

In EP 2801370 A1, it was shown that the Omomyc peptide itself is able to efficiently pass through the cell membrane and translocate to the nucleus, where it exerts its tumor suppressive effect.

However, there is still a need in the art to develop new and improved therapeutic approaches for the treatment of cancer.

The essence of the invention

In a first aspect, the invention provides a polypeptide comprising a polypeptide of SEQ ID NO: 1 wherein the X residue at position 89 of SEQ ID NO: 1 is not a cysteine, or a functionally equivalent variant of said polypeptide wherein the X residue at position 89 of SEQ ID NO: :1 is not a cysteine.

In a second aspect, the invention relates to a conjugate comprising:

a) a polypeptide or a functionally equivalent variant of said polypeptide of the invention, and

b) a chemical group that promotes uptake by the cell of a polypeptide or a functionally equivalent variant of said polypeptide.

In a third aspect, the invention provides a polynucleotide encoding a polypeptide of the invention or a conjugate of the invention.

In a fourth aspect, the invention relates to a vector containing a polynucleotide according to the invention.

In a fifth aspect, the invention provides a host cell comprising a polypeptide of the invention, a conjugate of the invention, a polynucleotide of the invention, or a vector of the invention.

In a sixth aspect, the invention relates to a pharmaceutical composition comprising a pharmaceutically effective amount of a polypeptide or a functionally equivalent variant of said polypeptide of the invention, a conjugate of the invention, a polynucleotide of the invention, a vector of the invention, or a host cell of the invention, and a pharmaceutically acceptable excipient.

In a seventh aspect, the invention relates to a polypeptide or a functionally equivalent variant of said polypeptide of the invention, a conjugate of the invention, a polynucleotide of the invention, a vector of the invention, a host cell of the invention, or a pharmaceutical composition of the invention for use in medicine.

In an eighth aspect, the invention relates to a polypeptide or a functionally equivalent variant of said polypeptide of the invention, a conjugate of the invention, a polynucleotide of the invention, a vector of the invention, a host cell of the invention, or a pharmaceutical composition of the invention for use in the prevention and/or treatment of cancer.

Brief description of the figures

Fig. 1. Cell viability analysis shows that OmoCS (carrying the C89S change) inhibits the growth of U87 malignant glioma cells (panel A) and A549 lung adenocarcinoma cells (panel B) more effectively than Omomyc (at a lower concentration). Lipofectamine (Lipo) has no effect. t-test was used to calculate statistical significance. **P<0.01, ***p<0.001.

Fig. 2. Cell density analysis shows that OmoCA (carrying the C98A change) inhibits the growth of A549 lung adenocarcinoma cells as effectively as OmoCS (carrying the C98S change) at a lower concentration. Lipofectamine (Lipo) has no effect.

Detailed description of the invention

The present inventors have found that a polypeptide of SEQ ID NO: 1 wherein the cysteine at position 89 is replaced by a serine, called OmoCS, is able to exert a tumor suppressive effect more effectively than Omomyc (Example 1). This tumor suppressive effect is maintained when the cysteine at position 89 of SEQ ID NO: 1 is replaced with other amino acids. Example 2 shows that the polypeptide of SEQ ID NO: 1, where the cysteine at position 89 is replaced by alanine, called OmoCA, has the same tumor suppressive effect as OmoCS.

The polypeptide of the invention.

Therefore, in a first aspect, the invention relates to a polypeptide comprising a polypeptide of SEQ ID NO: 1, where the X residue at position 89 in SEQ ID NO: 1 is not a cysteine, or a functionally equivalent variant of said polypeptide, where the X residue at position 89 in SEQ ID NO: 1 is not cysteine.

SEQ ID NO: 1 corresponds to:

TEENVKRRTHNVLERQRRNELKRSFFALRDQIPELENNEKAPKWILKKATAYILSVQA

ETQKLISEIDLLRKQNEQLKHKLEQLRNSXA (SEQ ID NO: 1)

The polypeptide of SEQ ID NO: 1 corresponds to the sequence of the Omomyc protein, but the X residue at position 89 is not a cysteine. Within the scope of the invention, the term Omomyc refers to a polypeptide that consists of a mutated variant of the bHLHZip domain of the Myc protein carrying the E61T, E68I, R74Q, and R75N mutations (where the numeration of the mutated positions is relative to the sequence of the Myc region corresponding to amino acids 365-454 of the polypeptide having the accession number NP 002458 in the NCBI database, accessed March 15, 2015). The c-Myc sequence listed in the NCBI database under accession number NP 002458 is shown below (SEQ ID NO: 5), where the region from which Omomyc originates is shown underlined:

MDFFRWENQ QPPATMPLNV SFTNRNYDLD YDSVQPYFYC DEEENFYQQQ

QQSELQPPAP

SEDIWKKFEL LPTPPLSPSR RSGLCSPSYV AVTPFSLRGD NDGGGGSFST ADQLEMVTEL

121 LGGDMVNQSF ICDPDDETFI KNIIIQDCMW SGFSAAAKLV SEKLASYQAA RKDSGSPNPA

181 RGHSVCSTSS LYLQDLSAAA SECIDPSWF PYPLNDSSSP KSCASQDSSA FSPSSDSLLS

241 STESSPQGSP EPLVLHEETP PTTSSDSEEE QEDEEEIDW SVEKRQAPGK RSESGSPSAG

301 GHSKPPHSPL VLKRCHVSTH QHNYAAPPST RKDYPAAKRV KLDSVRVLRQ ISNNRKCTSP

361 RSSDTEENVK RRTHNVLERQ RRNELKRSFF ALRDQIPELE NNEKAPKWI LKKATAYILS

421 VQAEEQKLIS EEDLLRKRRE QLKHKLEQLR NSCA (SEQ ID NO: 5)

As used herein, the term Myc refers to a family of transcription factors that includes c-Myc, N-Myc, and L-Myc. The Myc protein activates the expression of many genes through the binding of the CACGTG consensus sequence (enhancer box sequences or E-boxes) and the involvement of histone acetyltransferases or HATs. However, Myc can also function as a transcriptional repressor. By binding the transcription factor Miz-1 and displacing the p300 coactivator, it inhibits the expression of Miz-1 target genes. Myc also plays a direct role in controlling DNA replication.

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

In a preferred embodiment, the polypeptide comprising the polypeptide of SEQ ID NO: 1 comprises, consists of, or essentially consists of the sequence of SEQ ID NO: 3 shown below.

MTEENVKRRTHNVLERQRRNELKRSFFALRDQIPELENNEKAPKWILKKATAYILSVQ

AETQKLISEIDLLRKQNEQLKHKLEQLRNSXA (SEQ ID NO: 3)

In this context, consisting essentially of means that said molecule will not contain any additional sequences that would alter the activity of SEQ ID NO: 3.

The invention relates to a polypeptide that contains, consists of or consists essentially of SEQ ID NO: 1, where the residue X at position 89 in SEQ ID NO: 1 is not a cysteine. Said polypeptide may be derived from the bHLHZip domain of any Myc protein known in the art, provided that mutations that result in a tumor suppressor effect are retained. Thus, the polypeptide that can be used in the present invention can be derived from any species of mammals, including, but not limited to, domestic and farm animals (cows, horses, pigs, sheep, goats, dogs, cats or rodents), primates and humans . Preferably, the polypeptide of the invention is derived from the human Myc protein (accession number NP_002458, version March 15, 2015).

In a preferred embodiment, the invention relates to a polypeptide consisting of a polypeptide of SEQ ID NO: 1 wherein the X residue at position 89 of SEQ ID NO: 1 is not a cysteine, or a functionally equivalent variant of said polypeptide.

According to the present invention, the residue at position 89 in SEQ ID NO: 1 may be any amino acid other than cysteine.

Within the scope of the invention, the term cysteine refers to an amino acid with the formula HO2CH(NH ₂ )CH ₂ SH. It is encoded by the codons UGU and UGC. The term also includes unnatural cysteine

Cysteine can form disulfide bonds in the homodimeric form of Omomyc, hence mutating it to any other amino acid will result in the inability to form disulfide bonds and should result in the same potency properties as OmoCS. Therefore, the residue at position 89 in SEQ ID NO: 1 may represent any natural, non-natural or synthetic amino acid other than cysteine, and in particular any amino acid that cannot be cross-linked to another monomer of the polypeptide of the invention to form a homodimer.

The term amino acid or amino acid residue refers to natural and synthetic

- 3 042570 amino acids, as well as amino acid analogs and amino acid mimetics that function similarly to natural amino acids. Amino acids may be designated here by the well-known three-letter symbols or by the single-letter symbols recommended by the IUPAC-IUB Commission on Biochemical Nomenclature.

The term amino acid includes naturally occurring amino acids (Ala, Arg, Asn, Asp, Gln, Glu, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr, Val), unusual natural amino acids and unnatural (synthetic) amino acids. The amino acids are preferably in the L-configuration, but amino acids in the D-configuration or mixtures of amino acids in the D- and L-configurations are also contemplated.

The term natural amino acids includes aliphatic amino acids (glycine, alanine, valine, leucine and isoleucine), hydroxylated amino acids (serine and threonine), sulfated amino acids (methionine), dicarboxylic amino acids and their amides (aspartic acid, asparagine, glutamic acid and glutamine), amino acids , having two main groups (lysine, arginine and histidine), aromatic amino acids (phenylalanine, tyrosine and tryptophan) and cyclic amino acids (proline). In an embodiment, the X residue at position 89 of SEQ ID NO: 1 is an aliphatic amino acid. In yet another embodiment, the X residue at position 89 of SEQ ID NO: 1 is a sulfated amino acid. In yet another embodiment, X at position 89 in SEQ ID NO: 1 is a dicarboxylic amino acid or amides thereof. In yet another embodiment, the X residue at position 89 in SEQ ID NO: 1 is an amino acid having two main groups. In yet another embodiment, the X residue at position 89 of SEQ ID NO: 1 is an aromatic amino acid. In yet another embodiment, the X residue at position 89 in SEQ ID NO: 1 is a cyclic amino acid. In a preferred embodiment, the X residue at position 89 in SEQ ID NO: 1 is a hydroxylated amino acid, preferably serine. In a preferred embodiment, the X residue at position 89 in SEQ ID NO: 1 is an amino acid selected from serine, threonine and alanine, preferably selected from serine and alanine.

In the context of the invention, the term non-natural amino acid refers to a carboxylic acid or derivative thereof which is substituted with an amino group and which is structurally related to a naturally occurring amino acid. Illustrative non-limiting examples of modified or unusual amino acids include 2-aminoadipic acid, 3-aminoadipic acid, beta-alanine, 2-aminobutyric acid, 4-aminobutyric acid, 6-aminocaproic acid, 2-aminoheptanoic acid, 2-aminoisobutyric acid, 3-aminoisobutyric acid, 2-amino pimelic acid, 2,4-diaminobutyric acid, desmosine, 2,2'-diaminopimelic acid, 2,3-diaminopropionic acid, N-ethylglycine, N-ethylasparagine, hydroxylysine or hydroxyproline, 3-hydroxyproline, 4-hydroxyproline , isodesmosine, alloisoleucine, N-methylglycine, N-methylisoleucine, 6-N-methyllysine, N-methylvaline, norvaline, norleucine, ornithine, etc.

Illustrative non-limiting examples of unusual amino acids are hydroxylysine and hydroxyproline, thyroxine, N-methylarginine, and N-acetyl lysine.

In a preferred embodiment, the X residue at position 89 is any other amino acid that cannot be cross-linked to another monomer of the polypeptide of the invention to form a homodimer pair.

In a more preferred embodiment of the polypeptide of the invention, the residue at position 89 of SEQ ID NO: 1 is the amino acid serine.

Within the scope of the invention, the term serine refers to 2-amino-3-hydroxypropanoic acid encoded in humans by the codons UCU, UCC, UCA, UCG, AGU and AGC. The term also includes modified serines such as phosphorylated or sulfated serine, as an illustrative non-limiting example N-benzoyl-(2R,3S)-3-phenylisoserine, D-cycloserine, L-isoserine, phenylserine.

In a preferred embodiment, the polypeptide of the invention contains the sequence shown in SEQ ID NO: 2:

TEENVKRRTHNVLERQRRNELKRSFFALRDQIPELENNEKAPKVVILKKATAYILSVQA

ETQKLISEIDLLRKQNEQLKHKLEQLRNSSA (SEQ ID NO: 2).

In yet another preferred embodiment, the polypeptide of the invention consists or consists essentially of SEQ ID NO: 4:

MTEENVKRRTHNVLERQRRNELKRSFFALRDQIPELENNEKAPKWILKKATAYILSVQ

AETQKLISEIDLLRKQNEQLKHKLEQLRNSSA (SEQ ID NO: 4).

In a more preferred embodiment of the polypeptide of the invention, the residue at position 89 of SEQ ID NO: 1 is the amino acid alanine.

Within the scope of the invention, the term alanine refers to 2-aminopropanoic acid encoded in humans by the codons GCU, GCC, GCA and GCG. The term also includes modified alanines such as N-acetyl-L-alanine.

In a preferred embodiment, the polypeptide of the invention comprises the sequence shown in SEQ ID NO: 63:

TEENVKRRTHNVLERQRRNELKRSFFALRDQIPELENNEKAPKWILKKATAYILSVQA

ETQKLISEIDLLRKQNEQLKHKLEQLRNSAA (SEQ ID NO: 63).

In yet another preferred embodiment, the polypeptide of the invention consists or consists essentially of SEQ ID NO: 64:

MTEENVKRRTHNVLERQRRNELKRSFFALRDQIPELENNEKAPKVVILKKATAYILSVQ

AETQKLISEIDLLRKQNEQLKHKLEQLRNSAA (SEQ ID NO: 64).

The term functionally equivalent variant, when referred to in SEQ ID NO: 1, wherein the X residue at position 89 in SEQ ID NO: 1 is not a cysteine, refers to any polypeptide that is derived from the deletion, insertion, or addition of one or more amino acids relative to a polypeptide of SEQ ID NO: 1, or which is derived from a chemical modification of a polypeptide of SEQ ID NO: 1, and which substantially retains the tumor suppressor activity of SEQ ID NO: 1, wherein the X residue at position 8 9 of SEQ ID NO: 1 is not is a cysteine, preferably which substantially retains the tumor suppressor activity of OmoCS. Those skilled in the art will appreciate that maintaining tumor suppressor activity requires that the variant be able to dimerize with Myc and/or its obligate partner p21/p22Max and inhibit Myc activity after nuclear entry, to be able to translocate across the cell membrane, and to be able to translocate across the nuclear envelope. In some embodiments, a functionally equivalent polypeptide variant of the invention homodimerizes less than Omomyc, or is not forced to become homodimers via disulfide bridge formation. In particular, the formation of a disulfide bridge in the homodimeric form of the polypeptide of the invention is weaker than in the OmoMyc polypeptide.

Within the scope of the invention, the expression weaker homodimerization refers to a lower ability to form obligate homodimers of a polypeptide of the invention even under reducing conditions. In a preferred embodiment, the 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 in Omomyc.

Within the scope of the invention, the expression reducing conditions refers to the presence of a reducing agent, a compound that donates an electron to other chemical compounds in a redox chemical reaction. Illustrative non-limiting examples of reducing agents are DTT (dithiothreitol), b-mercaptoethanol or TCEP (tris-(2carboxyethyl)phosphine). It is possible that the number of homodimers is the same under in vitro conditions, and that the difference between the polypeptide of the invention and Omomyc only occurs in cells in the presence of heterodimerization partners, where the absence of disulfide allows for potentially higher heterodimer formation.

Several assays can be used to determine the homodimerization of a peptide, and as an illustrative non-limiting example, thermal denaturation as determined by circular dichroism can be mentioned, hence dimerization can be detected by folding and thermal stability quantification.

Suitable functionally equivalent variants include polypeptides consisting essentially of the polypeptide of SEQ ID NO: 1 wherein the X residue at position 89 of SEQ ID NO: 1 is not a cysteine.

In this context, the expression consisting essentially of means that the specified molecule will not contain any additional sequences that could change the activity of SEQ ID NO: 1, where the X residue at position 89 in SEQ ID NO: 1 is not a cysteine.

Suitable functional variants of the targeting peptide are those that have a degree of identity to the peptide of SEQ ID NO: 1 (preferably to OmoCS, SEQ ID NO: 4) at a level of greater than 25% amino acid sequence identity, e.g., 25, 30, 40, 50, 60, 65, 70, 75, 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99%. The degree of identity between two polypeptides is determined using computer algorithms and methods that are widely known to those skilled in the art. The identity between two amino acid sequences is preferably determined using the BLASTP algorithm as previously described [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.

Functionally equivalent polypeptide variants of the invention may also include post-translational modifications such as glycosylation, acetylation, isoprenylation, myristoylation, proteolytic processing, and the like.

Alternatively, suitable functional variants of the targeting peptide are those in which one or more positions in the polypeptide of the invention contains an amino acid that is a conservative substitution for an amino acid present in the protein above. Conservative amino acid substitutions result from the substitution of one amino acid for another having similar structural and/or chemical properties. For example, each of the following six groups includes amino acids that are conservative substitutions for each other: 1) alanine (A), serine (S), threonine (T); 2) aspartic acid (D), glutamic acid (E); 3) asparagine (N), glutamine (Q); 4) arginine (R), lysine (K); 5) isoleucine (I), leucine (L), methionine (M), valine (V) and 6) phenylalanine (F), tyrosine (Y), tryptophan (W). The choice of such conservative amino acid substitutions is within the skill of the art and is described, for example, by Dordo et al. (J. Mol. Biol., 1999, 217:721-739) and Taylor et. al. (J. Theor. Biol., 1986, 119:205-218).

In a preferred embodiment, the entire sequence of the functionally equivalent variant of SEQ ID NO: 1 does not contain the amino acid cysteine. It will be appreciated that functionally equivalent variants of SEQ ID NO: 1 wherein the X residue at position 89 of SEQ ID NO: 1 is not a cysteine contain mutations at positions corresponding to the E61T, E68I, R74Q, and R75N mutations found in Omomyc derived from human s-mus. The position at which these mutations should occur in a functionally equivalent manner can be determined by multiple sequence alignments of different Myc sequences and identified by aligning those positions corresponding to positions 61, 68, 74 and 75 in the Omomyc sequence derived from human c-Myc.

Multiple sequence alignment is an extension of pairwise alignment to include more than two sequences at the same time. Multiple alignment methods align all sequences in a given set of queries. The preferred multiple sequence alignment program (and algorithm) is ClustalW, Clustal2W, or ClustalW XXL (see Thompson et al. (1994) Nucleic Acids Res. 22:4673-4680). After comparing (aligning) c-Myc sequences from different organisms and variant as described here, one skilled in the art can easily identify the positions in each of the sequences corresponding to the positions E61T, E68I, R74Q and R75N found in Omomyc and enter into the variant SEQ ID NO: 1 wherein the X residue at position 89 of SEQ ID NO: 1 is not a cysteine, mutations corresponding to the E61T, E68I, R74Q and R75N mutations found in human c-Myc derived Omomyc.

Suitable assays to determine whether a polypeptide can be considered a functionally equivalent variant of SEQ ID NO: 1 where the X residue at position 89 of SEQ ID NO: 1 is not a cysteine include, without limitation:

assays that assess the ability of a polypeptide to form dimeric complexes with Max and Myc, such as assays based on reporter gene expression, as described in Soucek et al. (Oncogene, 1998, 17:2463-2472), as well as PLA (Protein Close Ligation Assay) or co-immunoprecipitation;

assays that evaluate the ability of a polypeptide to bind to the DNA Myc/Max recognition site (CACGTG site), such as the electrophoretic mobility shift assay (EMSA) described by Soucek et al. (see above);

assays that assess the ability to repress Myc-induced transactivation, such as the assay based on reporter gene expression under the control of Myc/Max-specific DNA binding sites, as described by Soucek et al. (see above);

assays based on the ability of a gene product or polypeptide to inhibit the growth of cells expressing the myc oncogene as described by Soucek et al. (see above);

assays that evaluate the ability of a polypeptide to enhance myc-induced apoptosis, such as those described by Soucek et al. (Oncogene, 1998:17, 2463-2472). In addition, any assay commonly known in the art can be used to evaluate cell apoptosis, such as Hoechst stain, propidium iodide (PI) or annexin V stain, trypan blue, ladder/fragmentation DNA detection, and TUNEL.

In a preferred embodiment, the functionally equivalent variant of SEQ ID NO: 1 wherein the X residue at position 89 in SEQ ID NO: 1 is not a cysteine includes those sequences that have one or more, preferably all, of the following characteristics: the ability to dimerize with Myc and inhibit its activity, cell membrane translocation, nuclear translocation, inability to form homodimers or reduced ability to form homodimers compared to Omomyc, cell viability in the in vitro assay as described in Example 1 is lower than Omomyc at 12.5 nM mRNA encoding a polypeptide of the invention. In a preferred embodiment, a functionally equivalent variant of the invention corresponds to a sequence that mediates cell viability in an in vitro assay,

- 6 042570 as described in example 1, lower than Omomyc, in the amount of 12.5 nm mRNA encoding the polypeptide according to the invention.

In a preferred embodiment, a polypeptide is considered to be functionally equivalent to a variant of SEQ ID NO: 1 where the X residue at position 89 in SEQ ID NO: 1 is not a cysteine if it exhibits an activity in one or more of the above assays that is at least 10, 20, 30, 40, 50, 60, 70, 80, 90 or 100% of the activity of SEQ ID NO: 1, where the X residue at position 89 in SEQ ID NO: 1 is not cysteine (preferably from the activity of OmoCS).

In addition, functionally equivalent variants of SEQ ID NO: 1 wherein the X residue at position 89 of SEQ ID NO: 1 is not a cysteine are also capable of transducing cells after the variant contacts said cell.

In a preferred embodiment, a polypeptide is considered to be functionally equivalent to a variant of SEQ ID NO: I where the X residue at position 89 in SEQ ID NO: 1 is not a cysteine if it is capable of transducing the target cell by at least 10, 20, 30, 40 , 50, 60, 70, 80, 90, or 100% as effective as SEQ ID NO: 1, where the X residue at position 8 9 in SEQ ID NO: 1 is not a cysteine (preferably as OmoCS, SEQ ID NO: 4) .

In addition, functionally equivalent variants of SEQ ID NO: 1 wherein the X residue at position 89 of SEQ ID NO: 1 is not a cysteine are also capable of translocating to the nucleus of the target tumor cell.

In a preferred embodiment, a polypeptide is considered to be functionally equivalent to a variant of SEQ ID NO: 1 where the X residue at position 89 in SEQ ID NO: 1 is not a cysteine if it is capable of translocating to the nucleus of the target tumor cells by at least 10, 20, 30, 40, 50, 60, 70, 80, 90 or 100% as effective as SEQ ID NO: 1, where the X residue at position 89 in SEQ ID NO: 1 is not a cysteine (preferably like OmoCS, SEQ ID NO : 4).

Suitable assays for determining whether a polypeptide is a functionally equivalent variant of SEQ ID NO: 1 where the X residue at position 89 in SEQ ID NO: 1 is not a cysteine, in terms of its ability to translocate across the cell membrane and into the nucleus, include double labeling cells with a polypeptide-specific reagent and a dye that specifically stains the cell nucleus (eg, DAPI or Hoechst stain). In a preferred embodiment, the detection of the polypeptide according to the invention is carried out using confocal microscopy or fluorescence microscopy.

The polypeptide of SEQ ID NO: 1 of the invention also contains an M2 c-Myc domain having the sequence RQRRNELKRSF (SEQ ID NO: 55) (see Dang and Lee, Mol. Cell Biol., 1988, 8:4048-4054), which corresponds to the signal of nuclear localization.

In yet another preferred embodiment, a functionally equivalent variant of SEQ ID NO: 1, where the X residue at position 89 in SEQ ID NO: 1 is not a cysteine, comprises the sequence of SEQ ID NO: 55.

In the context of the invention, the term nuclear localization signal refers to an amino acid sequence of about 4-20 amino acid residues in length that serves to direct a protein to the nucleus. Typically, the nuclear localization sequence is rich in basic amino acids, and typical sequences are well known in the art (Gorlich D. (1998) EMBO, 5.17:2721-7).

In some embodiments, the NLS is selected from the group consisting of SV40 large T antigen NLS (PKKKRKV, SEQ ID NO: 6); NLS of nucleoplasmin (KRPAATKKAGQAKKKK, SEQ ID NO: 7); NLS CBP80 (RRRHSDENDGGQPHKRRK, SEQ ID NO: 8); NLS protein Rev HIV-1 (RQARRNRRRWE, SEQ ID NO: 9); HTLV-I Rex (MPKTRRRPRRSQRKRPPT, SEQ ID NO: 10); NLS hnRNP A (NQSSNFGPMKGGNFGGRSSGPYGGGGQYFKPRNQGGY, SEQ ID NO: 11); NLS rpL23a (VHSHKKKKIRTSPTFTTPKTLRLRRQPKYPRKSAPRRNKLDHY, SEQ ID NO: 12). In one embodiment of the invention, the nuclear localization signal contains the motif K (K/R) X (K/R) (SEQ ID NO: 13).

In addition, functionally equivalent variants of SEQ ID NO: 1 wherein the X residue at position 89 of SEQ ID NO: 1 is not a cysteine are also capable of reaching the nuclei of transduced cells after the variant contacts said cell.

It is to be understood that functionally equivalent variants of SEQ ID NO: 1 where the X residue at position 89 of SEQ ID NO: 1 is not a cysteine contain the NLS found in SEQ ID NO: 1 where the X residue at position 89 of SEQ ID NO: : 1 is not a cysteine, or other functional NLS. In yet another embodiment, the polypeptide of the invention does not contain the native NLS found in SEQ ID NO: 1, but contains another functional NLS replacing said NLS found in SEQ ID NO: 1, or in any other part of the polypeptide of the invention.

The conjugate of the invention.

In another aspect, the invention relates to a conjugate comprising:

a) a polypeptide or functionally equivalent variant of said polypeptide according to the invention;

b) a chemical group that promotes cellular uptake of the polypeptide or functionally

- 7 042570 equivalent variant of the specified polypeptide.

Within the scope of the invention, the term conjugate refers to two or more compounds that are covalently linked to each other such that the conjugate retains the function of each compound.

In preferred embodiments, the conjugates of the invention comprise 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 at least 9, at least 10, or more chemical groups that promote cellular uptake of a polypeptide or a functionally equivalent variant of said polypeptide.

In one embodiment, the chemical group that promotes cellular uptake of the polypeptide is a lipid or fatty acid.

In general, a fatty acid is a molecule containing a carbon chain with an acidic group (eg, carboxylic acid) at the end of the chain. The fatty acid carbon chain may be of any length, however it is preferred that the carbon chain length be 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 value in the specified range. In some embodiments, the carbon chain length is from 4 to 18 carbon atoms in the chain portion of the fatty acid. In some embodiments, the fatty acid carbon chain may contain an odd number of carbon atoms, however, in some embodiments, an even number of carbon atoms in the chain may be preferred. A fatty acid containing only single bonds in its carbon chain is called saturated, while a fatty acid containing at least one double bond in its carbon chain is called unsaturated. The fatty acid may be branched, although in 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.

In a preferred embodiment, the chemical group that promotes cell uptake of the polypeptide is a cell-penetrating peptide sequence, in which case the conjugate is a fusion protein comprising a polypeptide of the invention or a functionally equivalent variant of said polypeptide and a cell-penetrating peptide sequence.

The term fusion protein refers to proteins obtained by genetic engineering, which consist of two or more functional domains derived from different proteins. The fusion protein can be obtained by conventional means, for example, by gene expression of the nucleotide sequence encoding said fusion protein in a suitable cell. It is understood that a cell-penetrating peptide refers to a cell-penetrating peptide that is different from a cell-penetrating peptide that forms a fragment of the polypeptide of SEQ ID NO: 1 or a functionally equivalent variant of said polypeptide.

The term cell-penetrating peptide sequence is used herein interchangeably with CPP, protein transduction domain, or PTD. The term refers to a peptide chain of various lengths that directs the transport of a protein into the cell. The process of delivery into the cell usually occurs by endocytosis, but the peptide can also be internalized into the cell by direct translocation across membranes. CPPs typically have an amino acid composition that either includes a high relative content of positively charged amino acids such as lysine or arginine, or has sequences that alternately contain polar/charged amino acids and nonpolar hydrophobic amino acids. Examples of CPPs that can be used in the present invention include, without limitation:

CPP found in Drosophila antennapedia protein (RQIKIWFQNRRMKWKK; SEQ ID NO: 14),

CPP found in herpes simplex virus 1 (HSV-1) DNA binding protein VP22 (DAATATRGRSAASRPTERPRAPARSASRPRRPVE; SEQ ID NO: 15),

CPP Vas-7 (RRIRPRPPRLPRPRPRPLPFPRPG; SEQ ID NO: 16),

HIV-1 TAT protein CPP consisting of amino acids 49-57 (RKKRQRR; SEQ ID NO:17), amino acids 48-60 (GRKKRRQRRRTPQ; SEQ ID NO: 18), amino acids 47-57 (YGRKKRRQRRR; SEQ ID NO: 19) ,

CPP peptide S413-PV (ALWKTLLKKVLKAPKKKRKV; SEQ ID NO: 20),

CPP penetratin (RQIKWFQNRRMKWKK; SEQ ID NO: 21),

CPP SynB1 (RGGRLSYSRRRFSSTGR; SEQ ID NO: 22),

CPP SynB3 (RRLSYSRRRF; SEQ ID NO: 23),

CPP PTD-4 (pIRRRKKLRRLK; SEQ ID NO: 24),

CPP PTD-5 (RRQRRTSKLMKR; SEQ ID NO: 25),

CPP FHV Coat-(35-49) (RRRRRNRTRRNRRRVR; SEQ ID NO: 26),

CPP BMV Gag-(7-25) (KMTRAQRRAAARRNRWTAR; SEQ ID NO: 27),

CPP HTLV-II Rex-(4-16) (TRRQRTRRARRNNR; SEQ ID NO: 28),

CPP D-Tat (GRKKRRQRRRPPQ; SEQ ID NO: 29),

CPP R9-Tat (GRRRRRRRRRPPQ; SEQ ID NO: 30),

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CPP MAP (KLALKLALKLALALKLA; SEQ ID NO: 31),

CPP SBP (MGLGLHLLVLAAALQGAWSQPKKKRKV; SEQ ID NO: 32),

CPP FBP (GALFLGWLGAAGSTMGAWSQPKKKRKV; SEQ ID NO: 33),

CPP MPG (ac-GALFLGFLGAAGSTMGAWSQPKKKRKV-cya; SEQ ID NO: 34),

CPP MPG (ENLS) (ac-GALFLGFLGAAGSTMGAWSQPKSKRKV-cya; SEQ ID NO: 35),

CPP Pep-1 (ac-KETWWETWWTEWSQPKKKRKV-cya; SEQ ID NO: 36),

Pep-2 CPP (ac-KETWFETWFTEWSQPKKKRKV-cya; SEQ ID NO: 37), a polyarginine sequence having the structure _RN (where N is in the range of 4 to 17), a GRKKRRQRRR sequence (SEQ ID NO: 38), a RRRRRRLR sequence ( SEQ ID NO: 39), RRQRRTSKLMKR sequence (SEQ ID NO: 40), transportan (GWTLNSAGYLLGKINLKALAALAKKIL; SEQ ID NO: 41),

KALAWEAKLAKALAKALAKHLAKALAKALKCEA (SEQ ID NO: 42),

RQIKIWFQNRRMKWKK (SEQ ID NO: 43), sequence YGRKKRRQRRR (SEQ ID NO: 44), sequence RKKRRQRR (SEQ ID NO: 45), sequence YARAAARQARA (SEQ ID NO: 46), sequence THRLPRRRRRR (SEQ ID NO: 47), sequence GGRRARRRRRR (SEQ ID NO: 48).

In a preferred embodiment, said cell-penetrating peptide is non-endogenous and is part of SEQ ID NO: 1.

In a preferred embodiment, the CPP is the CPP of the HIV-1 TAT protein of amino acids 49-57 (RKKRRQRRR, SEQ ID NO: 49). In yet another preferred embodiment, the CPP is the sequence GRKKRRQRRR (SEQ ID NO: 50) or RRRRRRRR (SEQ ID NO: 51).

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

In preferred embodiments, the conjugates or fusion proteins of the invention comprise, in addition to the intrinsic cell-penetrating peptide found in the polypeptide of SEQ ID NO: 1 or a functionally equivalent variant of said polypeptide, at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10 or more additional cell-penetrating peptides.

Suitable fusion proteins of the invention include the OmoCS*TAT and OmoCS*LZArg polypeptides as defined below:

Name SEQID NO: Subsequence OmoCS*TAT 52 MTEENVKRRTHNVLERQRRNELKRSFFALRDQIPELENNEKAPKWILKKATA YILSVQAETQKLISEIDLLRKQNEQLKHKLEQLRNSSAGRKKRRQRRR OmoCS*LZArg 53 MTEENVKRRTHNVLERQRRNELKRSFFALRDQIPELENNEKAPKWILKKATA YILSVQAETQKLISEIDLLRKQNEQLKHKLEQLRNSSARRRRRRRR

In yet another preferred embodiment, the conjugates or fusion proteins of the invention comprise a polypeptide of the invention, or a functionally equivalent variant thereof, and further comprise an N-terminal or C-terminal nuclear localization signal.

One of skill in the art will appreciate that it may be desirable for the fusion protein to further comprise one or more flexible peptides that connect a polypeptide of the invention, or a functionally equivalent variant of said polypeptide, a cell-penetrating peptide sequence, and/or an NLS. Thus, in a particular embodiment, a polypeptide of the invention is directly linked to a cell-penetrating peptide sequence. In yet another specific embodiment, a polypeptide of the invention is linked to a cell-penetrating peptide sequence via a flexible peptide. In one embodiment, the polypeptide of the invention is directly linked to NLS. In yet another embodiment, the polypeptide of the invention is linked to the NLS via a flexible peptide.

In a particular embodiment, a polypeptide of the invention is directly linked to a cell-penetrating peptide sequence and to an NLS.

In one embodiment, the NLS is one of the NLS that appears endogenously in the Myc sequence, such as an M1 peptide (PAAKRVKLD, SEQ ID NO: 54) or an M2 peptide (RQRRNELKRSF, SEQ ID NO: 55).

- 9 042570

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

In preferred embodiments, the conjugates or fusion proteins of the invention comprise, in addition to the endogenous NLS found in the polypeptide of the invention or its functional equivalent, 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 NLS.

In yet another specific embodiment, the polypeptide of the invention is linked to a cell-penetrating peptide sequence via a first flexible peptide linker and to NLS via a second flexible peptide linker.

As used herein, the terms flexible peptide, spacer peptide, or linker peptide refer to a peptide that covalently links two proteins or fragments, but which is not part of either polypeptide, allowing movement of one relative to the other without significantly adversely affecting the function of the protein or fragment. Thus, the flexible linker does not adversely affect the tumor-suppressive activity of the polypeptide sequence, the cell-penetrating activity of the cell-penetrating peptide, or the ability of NLS to localize to the nucleus.

Flexible peptide contains 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 implementation of the flexible peptide will provide movement of one protein relative to another, in order to increase the solubility of the protein and/or increase its activity. Suitable linker regions include a polyglycine region, the sequence GPRRRR (SEQ ID NO: 56) combinations of glycine, proline and alanine residues.

In some embodiments, a fusion protein of the invention may contain an additional chemical group including, but not limited to, fluorescent groups, biotin, polyethylene glycol (PEG), amino acid analogs, unnatural amino acids, phosphate groups, glycosyl groups, radioisotope labels, and pharmaceutical molecules. In still other embodiments, the heterologous polypeptide may contain one or more reactive groups including, but not limited to, a ketone, an aldehyde, Cys residues, and Lys residues.

In a particular embodiment, the conjugates or fusion proteins of the invention comprise a tag linked to the conjugate or to the C-terminal or N-terminal domain of said fusion protein or variant of said polypeptide. Said label typically represents a peptide or amino acid sequence that can be used to isolate or purify said fusion protein. Thus, said label is capable of binding to one or more ligands, such as, for example, one or more ligands of an affinity matrix such as a high affinity chromatographic support or bead. An example of such a label is a histidine label (His-tag or HT), for example a label containing 6 histidine residues (His6 or H6), which can bind on a nickel (Ni2+) or cobalt (Co2+) column with high affinity. A His tag has the necessary property that it can bind to its ligands under conditions that denature most proteins and disrupt most protein-protein interactions. Thus, it can be used to remove the H6-labeled bait protein, followed by the disruption of protein-protein interactions involving the bait protein.

Additional illustrative non-limiting examples of labels useful for isolating or purifying a fusion protein include an Arg tag, a FLAG tag (DYKDDDDK; SEQ ID NO: 57), a Strep tag (WSHPQFEK; SEQ ID NO: 58), an epitope that can be recognized antibody such as c-myc tag (recognized by anti-c-myc antibody), HA tag (YPYDVPDYA; SEQ ID NO: 59), V5 tag (GKPIPNPLLGLDST; SEQ ID NO: 60), 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), an amino acid sequence such as AHGHRP (SEQ ID NO: 61) or PIHDHDHPHLVIHSGMTCXXC (SEQ ID NO: 62), β-galactosidase, and the like .

If desired, the label can be used to isolate or purify said fusion protein.

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Polynucleotide, vector and host cell of the invention.

In another aspect, the invention provides a polynucleotide encoding a polypeptide of the invention or a conjugate of the invention.

The terms polynucleotide, nucleic acid, and nucleic acid molecule are used interchangeably to refer to polymeric forms of nucleotides of any length. Polynucleotides may contain deoxyribonucleotides, ribonucleotides and/or their analogs. Nucleotides may have any three-dimensional structure and may perform any function, currently known or unknown. The term polynucleotide includes, for example, single-stranded, double-stranded and triple helical molecules, gene or gene region, exons, introns, mRNA, tRNA, rRNA, ribozymes, cDNA, recombinant polynucleotides, branched polynucleotides, plasmids, vectors, isolated DNA of any sequence, isolated RNA any sequence, nucleic acid probes and primers. In addition to the native nucleic acid molecule, the nucleic acid molecule of the present invention may also include modified nucleic acid molecules.

Within the scope of the invention, the term mRNA refers to RNA that can be translated in a cell. In a preferred embodiment, the polynucleotide of the invention is an mRNA. The mRNA may be chemically synthesized, may be produced by in vitro transcription, or may be synthesized in vivo in the target cell. The nucleotide sequences that form the polynucleotide encoding the conjugate or fusion protein of the invention are in the same correct reading frame for their expression.

In another aspect, the invention relates to a vector containing a polynucleotide according to the invention.

Within the scope of the invention, the term vector refers to a nucleic acid sequence containing the necessary sequences so that, after transcription and translation of said sequences, the polypeptide encoded by the polynucleotide of the invention is produced in a cell. Said sequence is operably linked to additional segments that allow its autonomous replication in the host cell of interest. Preferably, the vector is an expression vector, which is defined as a vector which, in addition to regions of autonomous replication in the host cell, contains regions operably linked to a nucleic acid of the invention and which is capable of enhancing the expression of nucleic acid products of the invention. Vectors according to the invention can be obtained using methods widely known in this field.

Examples of vectors include, but are not limited to, viral vectors, naked DNA or RNA expression vectors, plasmid, cosmid or phage vectors, DNA or RNA expression vectors coupled with cationic condensing agents, DNA or RNA expression vectors encapsulated in liposomes, and some eukaryotic cells such as producer cells. Suitable vectors containing a polynucleotide of the invention are those derived from prokaryotic expression vectors such as pUC18, pUC19, pBluescript and their derivatives, mp18, mp19, pBR322, pMB9, ColE1, pCR1, RP4, phages and shuttle vectors such as pSA3 and pAT28, yeast expression vectors such as 2-micron plasmid type vectors, integrative plasmids, YEP vectors, centromeric plasmids and the like, insect cell expression vectors such as the pAC series and pVL series vectors, plant expression vectors such as vectors of the pIBI, pEarleyGate, pAVA, pCAMBIA, pGSA, pGWB, pMDC, pMY, pORE series and the like, and expression vectors in higher eukaryotic cells based on viral vectors (adenoviruses, adeno-associated viruses, as well as retroviruses and, in particular, lentiviruses) , as well as non-viral vectors such as pSilencer 4.1-CMV (Ambion), pcDNA3, pcDNA3.1/hyg, pHCMV/Zeo, pCR3.1, pEFl/His, pIND/GS, pRc/HCMV2, pSV40/Zeo2, pTRACER- HCMV, pUB6/V5-His, pVAXl, pZeoSV2, pCI, pSVL, pKSV-10, pBPV-1, pML2d and pTDT1. In a preferred embodiment, the polynucleotide of the invention is in a vector selected from the group consisting of pEGFP or pBabe retroviral vectors and pTRIPZ or pSLIK lentiviral vectors.

The vector of the invention can be used to transform, transfect or infect cells that can be transformed, transfected or infected with said vector. These cells may be prokaryotic or eukaryotic cells.

The vector preferably contains a polynucleotide of the invention operably linked to sequences that regulate the expression of the polynucleotide of the invention. Regulatory sequences for use in the present invention may be nuclear promoters or, alternatively, enhancer sequences and/or other regulatory sequences that increase the expression of a heterologous nucleic acid sequence. In principle, any promoter can be used in the present invention, provided that said promoter is compatible with the cells in which the polynucleotide is to be expressed. Thus, promoters suitable for the practice of the present invention include, but are not limited to, constitutive promoters such as those derived from the genomes of eukaryotic viruses such as polyomavirus,

- 11 042570 adenovirus, SV40, CMV, avian sarcoma virus, hepatitis B virus, metallothionein gene promoter, herpes simplex virus thymidine kinase gene promoter, retrovirus LTR regions, immunoglobulin gene promoter, actin gene promoter, EF-1alpha gene promoter, and inducible promoters when the expression of proteins depends on the addition of a molecule or an exogenous signal, such as tetracycline systems, the NFKB/UV light system, the Cre/Lox system and the heat shock protein gene promoter, the regulated RNA polymerase II promoters described in WO 2006/135436, and tissue-specific promoters.

In another aspect, the invention provides a host cell comprising a polypeptide of the invention, a conjugate of the invention, a polynucleotide of the invention, or a vector of the invention.

Cells suitable for the present invention include, but are not limited to, mammalian, plant, insect, fungal and bacterial cells. Bacterial cells include, but are not limited to, Gram-positive bacteria cells such as strains of the genera Bacillus, Streptomyces, and Staphylococcus, and Gram-negative bacteria cells, such as cells of the Escherichia and Pseudomonas genera. Fungal cells preferably include yeast cells such as Saccharomyces, Pichia pastoris and Hansenula polymorpha. Insect cells include, without limitation, Drosophila cells and Sf9 cells. Plant cells include, but are not limited to, cells from agricultural crops such as cereals, medicinal or ornamental plants, or bulbs. Suitable mammalian cells for the present invention include epithelial cell lines (pigs, etc.), osteosarcoma cell lines (human, etc.), neuroblastoma cell lines (human, etc.), epithelial carcinomas (human, etc.). etc.), glial cells (mouse, etc.), liver cell lines (monkeys, etc.), CHO (Chinese Hamster Ovary) cells, COS cells, BHK cells, HeLa cells, 911, AT1080, A549, 293 or PER.C6, human NTERA-2 ECC cells, mESC D3 cells, non-human embryonic stem cells, NIH3T3, 293N, REH and MCF-7 cells, and hMSCs.

All terms and embodiments previously described apply equally to this aspect of the invention.

Pharmaceutical compositions according to the invention.

In yet another aspect, the invention relates to a pharmaceutical composition comprising a pharmaceutically effective amount of a polypeptide or a functionally equivalent variant of said polypeptide of the invention, a conjugate of the invention, a polynucleotide of the invention, a vector of the invention, or a host cell of the invention, and a pharmaceutically acceptable excipient.

In the context of the present invention, the term "pharmaceutical composition" refers to a formulation that is adapted to administer a predetermined dose of one or more therapeutically useful agents to a cell, group of cells, organ, tissue, or animal where uncontrolled cell division occurs, as in cancer.

Within the scope of the invention, the expression pharmaceutically effective amount is understood as the amount capable of providing a therapeutic effect, and which can be determined by a person skilled in the art using commonly used means. The amount of a polypeptide of the invention or a functionally equivalent variant thereof, conjugate, polynucleotide, vector or host cell of the invention, or anticancer compound that may be combined in pharmaceutical compositions of the invention will vary depending on the subject and the particular route of administration. One of skill in the art will appreciate that dosages can also be determined using Goodman and Goldman's, The Pharmacological Basis of Therapeutics, Ninth Edition (1996), Appendix II, p. 1707-1711 and Goodman and Goldman's, The Pharmacological Basis of Therapeutics, Tenth Edition (2001), Appendix II, p. 475-493.

The appropriate dosage of the active compound or compounds in a pharmaceutical composition will depend on the type of cancer being treated, the severity and course of the disease, whether the composition is administered prophylactically or therapeutically, prior therapy, the patient's medical history and response to the peptide or polypeptide, and the judgment of the treating physician. doctor. An amount of a polypeptide of the invention or a functionally equivalent variant thereof, conjugate, polynucleotide, vector or host cell of the invention, respectively, is administered to a patient once or over a series of treatments. Depending on the type and severity of the disease, a suitable dosage level is usually from about 0.01 to 500 mg per 1 kg of patient body weight per day, which can be administered in single or multiple doses. Preferably, the dosage level will be from about 0.1 to about 250 mg/kg per day; more preferably from about 0.5 to about 100 mg/kg per day. A suitable dosage level may be from about 0.01 to 250 mg/kg per day, from about 0.05 to 100 mg/kg per day, or from about 0.1 to 50 mg/kg per day. Within this range, the dosage may be 0.05 to 0.5, 0.5 to 5, or 5 to 50 mg/kg per day. For oral administration, the compositions are preferably provided in the form of tablets containing from 1.0 to 1000 mg of the active ingredient, in particular 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 mg of the active ingredient for symptomatic dose adjustment for the patient being treated. The compounds may be administered on a schedule of 1 to 4 times per day, preferably 1 or 2 times per day.

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The pharmaceutical compositions of the invention also contain one or more additional pharmaceutically acceptable excipients. Pharmaceutically acceptable excipient means a therapeutically inactive substance which is used to incorporate an active ingredient and which is acceptable to the patient from a pharmacological/toxicological point of view and to the pharmaceutical chemist who manufactures it, from a physical/chemical point of view, taking into account composition, formulation, stability, patient acceptability and bioavailability. An excipient or carrier also includes any substance that serves to enhance the delivery and efficacy of the active compound in the pharmaceutical composition. Examples of pharmaceutically acceptable carriers include one or more of water, saline, phosphate buffered saline, dextrose, glycerol, ethanol, and the like, as well as combinations thereof. In many cases, it will be preferable to include isotonic agents, for example sugars, polyhydric alcohols such as mannitol, sorbitol, or sodium chloride, in the composition. Pharmaceutically acceptable carriers may further include minor amounts of excipients, such as wetting or emulsifying agents, preservatives or buffers, which increase the shelf life or potency of the fusion protein or compositions forming part of the pharmaceutical compositions. Examples of suitable carriers are well known in the literature (see, for example, Remington's Pharmaceutical Sciences, ^19th ed., Mack Publishing Company, Easton, PA, 1995). Examples of carriers, without limitation, is the group of saccharides such as lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol and maltitol; a group of starches such as corn starch, wheat starch, rice starch and potato starch; a group of celluloses such as cellulose, methylcellulose, sodium carboxymethylcellulose and hydroxylpropylmethylcellulose; and a group of excipients such as gelatin and polyvinylpyrrolidone. In some cases, a disintegrant may be added, such as cross-linked polyvinylpyrrolidone, agar, alginic acid, or sodium alginate.

The amount and nature of pharmaceutically acceptable excipients depend on the desired dosage form. Pharmaceutically acceptable excipients are known to those skilled in the art (Fauli y Trillo C. (1993) Tratado de Farmacia Galenica, Luzan 5, SA Ediciones, Madrid). These compositions can be prepared using conventional methods known in the art (Remington: The Science and Practice of Pharmacy, ^20th edition (2003) Genaro AR, ed., Lippincott Williams & Wilkins, Philadelphia, US).

For pharmaceutical compositions containing an agent that is a nucleic acid molecule, the nucleic acid molecule may be present in any of a variety of delivery systems known to those skilled in the art, including the nucleic acid and bacterial, viral, and mammalian expression systems such as as the recombinant expression constructs provided herein. Methods for incorporating DNA into such expression systems are well known to those skilled in the art. 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. Naked DNA uptake can be increased by applying DNA to biodegradable beads that are efficiently transported into cells.

Nucleic acid molecules can be delivered into a cell according to any of several methods described in the art (see, for example, 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 Application Publication WO 94/02595); Selbo et al., Int. J. Cancer 87:853-59 (2000); Selbo et al., Tumor Biol. 23:103-12 (2002); U.S. Patent Application Publication Nos. 2001/0007666 and 2003/077829). Such delivery methods known to those skilled in the art include, but are not limited to, encapsulation in liposomes, iontophoresis, or incorporation into other carriers such as biodegradable polymers; hydrogels; cyclodextrins (see, for example, Gonzalez et al., Bioconjug. Chem. 10:1068-74 (1999); Wang et al., International Publication Nos. WO 03/47518 and WO 03/46185); microspheres based on poly(lactic glycolic) acid (PLGA) and PLCA (also suitable for delivery of peptides, polypeptides and other substances) (see, for example, US patent No. 6447796; US patent application publication No. 2002/130430); biodegradable nanocapsules; and bioadhesive microspheres or protein-like vectors (International Publication No. WO 00/53722). In yet another embodiment, nucleic acid molecules for use in modifying (suppressing or enhancing) the immune response in immune cells and for treating an immunological disease or disorder can also be formulated or complexed with polyethyleneimine and its derivatives, such as polyethyleneimine-polyethylene glycol-N- derivatives. acetylgalactosamine (PEI-PEG-GAL) or polyethyleneimine polyethylene glycol-tri-N-acetylgalactosamine (PEI-PEG-triGAL) (see also, for example, US Patent Application Publication No. 2003/0077829).

In a preferred embodiment, the pharmaceutical composition or pharmaceutical combination of the invention further comprises, together or separately, an antineoplastic agent.

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Within the scope of the invention, the term antineoplastic agent is understood as the specified biological or chemical compound with which tumors are treated or prevented from forming. In a preferred embodiment, said anticancer agent is selected from the group consisting of a cytotoxic agent, an antiangiogenic agent, an antimetastatic agent, and an antiproliferative agent.

In the context of the present invention, the term cytotoxic agent refers to an agent that is capable of inducing cell death and has the ability to slow down growth, stop growth or destroy cells, and in particular rapidly proliferating cells, and even more specifically tumor cells. Cell death can be caused by any mechanism, such as, for example, apoptosis, although it is not limited to this cause, and also by inhibition of metabolism, interference with the organization of the cytoskeleton, or chemical modification of DNA. The term cytotoxic agent includes any chemotherapeutic agent, including small organic molecules, peptides, oligonucleotides, and the like; toxins; enzymes; cytokines; radioisotopes or radiotherapeutic agents.

An anti-angiogenic agent is understood as a chemical or biological substance that inhibits or reduces the formation of new blood vessels, i. angiogenesis.

Anti-angiogenic agents that can be used with the polypeptide of the first aspect of the invention or with the fusion protein of the second aspect of the invention include, without limitation, an anti-angiogenic agent selected from the group of paclitaxel, 2-methoxyestradiol, primomastat, batimastat, BAY 12-9566, carboxyamidotriazole, CC -1088, dextromethorphan acetic acid, dimethylxanthenone acetic acid, endostatin, IM-862, marimastat, penicillamine, PTK787/ZK 222584, RPI.4610, squalamine lactate, SU5416, thalidomide, combrestatin, tamoxifen, COL-3, neovastat, BMS-275291 , SU6668, anti-VEGF antibodies, Medi-522 (Vitaxin II), CAI, intereukin-12, IM862, amiloride, angiostatin, angiostatin KI-3, angiostatin KI-5, captopril, DL-alpha-difluoromethylornithine, DL-alpha -difluoromethylornithine HCl, endostatin, fumagillin, herbimycin A, 4-hydroxyphenylretinamide, juglone, laminin, laminin hexapeptide, laminin pentapeptide, lavendustin A, medroxyprogesterone, minocycline, placental inhibitor ribonuclease, suramin, thrombospondin, antibodies directed against pro-angiogenic factors (eg Avastin, Erbitux, Vectibix, Herceptin); small molecule tyrosine kinase inhibitors, pro-angiogenic growth factors (eg Tarceva, Nexavar, Sutent, Iressa); mTOR inhibitors (eg Torisel); α-, β- and γ-interferon, IL-12, matrix metalloproteinase inhibitors (eg COL3, marimastat, batimastat); ZD6474, SUI1248, Vitaxin; PDGFR inhibitors (eg Gleevec); NM3 and 2-ME2; cyclopeptides such as cilengitide.

An antimetastatic agent is understood as a chemical or biological substance that inhibits or reduces metastasis, i.e. further spread, mainly by the lymphatic or circulatory route, of cancer-causing cells and the growth of new tumors at the sites of said metastases.

An anti-proliferative agent is understood as a chemical or biological substance that is capable of preventing or inhibiting the formation or growth of tumors. Antiproliferative agents include, but are not limited to:

(i) antimetabolites such as folate antimetabolites (aminopterin, denopterin, methotrexate, edatrexate, trimetrexate, nolatrexed, lometrexol, pemetrexed, raltitrexed, pyrithrexim, pteropterin, leucovorin, 10-propargyl-5,8-dideazafolate (PDDF, CB3717)) , purine analogs (cladribine, clofarabine, fludarabine, mercaptopurine, pentostatin, thioguanine) and pyrimidine analogs (capecitabine, cytarabine or ara-C, decitabine, fluorouracil, 5-fluorouracil, doxifluridine, floxuridine, and gemcitabine);

ii) natural products such as antitumor antibiotics and mitosis inhibitors such as vinca alkaloids such as vindesine, vincristine, vinblastine, vinorelbine; taxanes such as paclitaxel (Taxol™), docetaxel (Taxotere™); colchicine (NSC 757), thiocolchicine (NSC 361792), colchicine derivatives (eg NSC 33410) and allocolchicine (NSC 406042); halichondrin B (NSC 609395); dolastatin 10 (NSC 376128); maytansine (NsC 153858); rhizoxin (NSC 332598); epothilone A, epothilone B; discodermolide; estramustine; nocodazole;

(iii) hormones and their antagonists such as tamoxifen, toremifene, anastrozole, arzoxifene, lasofoxifene, raloxifene, nafoxidine, fulvestrant, aminoglutethimide, testolactone, atamestan, exemestane, fadrozole, formestane, letrozole, goserelin, leuprorelin or leuprolide, buserelin, histrelin, megestrol and fluoxymesterone;

(iv) biological agents such as viral vectors, interferon alpha and interleukins;

(v) platinum-based compounds such as carboplatin, [cis-diammine dichloroplatinum, (CDDP)], oxaliplatin, iproplatin, nedaplatin, triplatin tetranitrate, tetraplatin, satraplatin (JM216), JM118 [cis-ammindichloroplatinum(P)), JM149 [ cis-ammindichloro(cyclohexylamine)trans-dihydroxoplatinum(IV)], JM335 [trans-ammindichlorodihydroxoplatinum(IV)], transplatinum, ZD0473,

- 14 042570 cis, trans, cis-Pt (NH ₃ ) (C ₆ H ₁₁ NH ₂ ) (OOCC ₃ H ₇ ) ₂ Cl, malanate-2-diaminocyclohexanoplatinum (II),

5-sulfosalicylate-trans-(1,2-diaminocyclohexane)platinum(P) (SSP), poly-[(trans-1,2-diaminocyclohexane)platinum]carboxyamylose (POLY-PLAT) and 4-hydroxysulfonylphenylacetate (trans-1, 2diaminocyclohexane) platinum (II) (SAP) and the like; and (vi) DNA alkylating drugs such as nitrogen mustards, nitrosoureas, ethyleneimine derivatives, alkylsulfonates, and triazenes, including, but not limited to, cyclophosphamide (Cytoxan™), busulfan, improsulfan, piposulfan, pipobroman, melphalan (L-sarcolysin) , chlorambucil, mechlorethamine or mustine, uramustine or uracil mustard, novembiquine, fenesterol, trofosfamide, ifosfamide, carmustine (BCNU), lomustine (CCNU), chlorzotocin, fotemustine, nimustine, ranimnustine, semustine (methyl-CCNU), streptozocin, thiotepa, triethylenemelamine , triethylenethiophosphoramide, procarbazine, altretamine, dacarbazine, mitozolomide, and temozolomide.

In the case of pharmaceutical compositions or combinations of the invention which contain an antineoplastic agent, the composition may be presented as a single formulation (for example, as a tablet or capsule containing a fixed amount of each of the components) or, alternatively, may be presented as separate formulations, which are then combined for joint, sequential or separate administration. The compositions or combinations of the invention also include a kit formulation in which the components are formulated separately but packaged in the same container. Those skilled in the art will recognize that the formulation of the various components in the case of the second pharmaceutical composition of the invention may be analogous, in other words formulated similarly (in tablets or pills), allowing them to be administered by the same route. In the case where the various components of the invention are formulated separately, the two components may be presented in a blister pack. Each blister contains medicines to be taken throughout the day. If the drugs are to be taken several times a day, the drugs corresponding to each administration can be placed in different sections of the blister, preferably indicating in each section of the blister the time of day when they should be taken. Alternatively, the components of the composition of the invention may be formulated in different ways so that the different components are administered differently. Thus, it is possible for the first component to be formulated as a tablet or capsule for oral administration and the second component to be formulated for intravenous administration, or vice versa. The ratio of the components that make up the compositions used in the second pharmaceutical composition according to the invention can be adjusted by a person skilled in the art depending on the antitumor agent used in each particular case, as well as on the target indication. Thus, the invention provides compositions in which the ratio of the two components can be from 50:1 to 1:50, in particular from 20:1 to 1:20, from 1:10 to 10:1 or from 5: to 1: 5.

Pharmaceutical compositions or combinations of the invention may be administered by any suitable route, such as the oral route, topical route, inhalation or parenteral route, provided that pharmaceutically acceptable excipients necessary to prepare the desired dosage form are included. The preferred route of administration of these pharmaceutical compositions is intravenously.

The oral route is understood as the route in which the pharmaceutical composition enters the body after it is swallowed. In a particular embodiment, the pharmaceutical composition of the invention may be in a dosage form suitable for oral administration, whether solid or liquid. Dosage forms suitable for their oral administration may be tablets, capsules, syrups or solutions and may contain any conventional excipient known in the art such as binders such as syrup, gum, gelatin, sorbitol or polyvinylpyrrolidone; fillers, for example lactose, sugar, corn starch, calcium phosphate, sorbitol or glycine; tabletting lubricants such as magnesium stearate; disintegrating agents, for example starch, polyvinylpyrrolidone, sodium starch glycolate or microcrystalline cellulose; or pharmaceutically acceptable wetting agents such as sodium lauryl sulfate. Solid compositions for oral administration can be prepared using conventional mixing, filling or compression processes. Repetitive mixing operations can be used to completely distribute the active agent in those compositions that use large amounts of excipients. These operations are generally accepted in this field. Tablets can be prepared, for example, by wet or dry granulation and optionally by coating according to methods known in conventional pharmaceutical practice, in particular by enteric coating.

On the other hand, the topical route is understood as non-systemic administration and includes applying the pharmaceutical composition of the invention externally to the epidermis, into the oral cavity and instilling said composition into the ears, eyes and nose, and in which it does not enter the bloodstream to a large extent. The systemic route is understood as administration by the oral route, intravenous route, intraperitoneal route and intramuscular route. Inhalation is understood to mean intranasal administration and oral inhalation. Dosage forms suitable for said administration, such as an aerosol formulation or a metered dose inhaler, can be prepared by conventional techniques. In one embodiment, the route of administration is the intranasal route.

Within the scope of the invention, the term parenteral includes administration by intravenous route, intraperitoneal route, intramuscular route or subcutaneous route. Dosage forms for subcutaneous, intramuscular and intravenous administration with parenteral administration are generally preferred.

In one embodiment, the pharmaceutical compositions of the invention may be adapted for parenteral administration, such as sterile solutions, suspensions, or lyophilized products in a suitable single dosage form. Pharmaceutical compositions suitable for its injectable use include sterile aqueous solutions (when soluble in water) or dispersions and sterile powders for the preparation of sterile injectable solutions or extempore dispersions. For intravenous administration, some suitable carriers include phosphate buffered saline (PBS). In all cases, the composition must be sterile and must be fluid to the extent that it can be easily administered. It must be stable under the conditions of preparation and storage and must be protected from contamination by microorganisms such as bacteria and fungi. The carrier may be a solvent or dispersion medium which includes, for example, water, ethanol, a pharmaceutically acceptable polyol such as glycerol, propylene glycol, liquid polyethylene glycol, and suitable mixtures thereof. The desired fluidity can be achieved, for example, by using a coating such as lecithin, by providing the required particle size in the case of a dispersion, and by using surfactants. Prevention of the action of microorganisms can be achieved using various antibacterial and antifungal agents, such as parabens, chlorobutanol, phenol, ascorbic acid, thiomersal, and the like. In many cases it is preferable to include isotonic agents, for example sugars, polyhydric alcohols such as mannitol or sorbitol, and sodium chloride, in the composition. Prolonged absorption of injectable compositions can be achieved by the inclusion in the composition of an agent that delays absorption, such as aluminum monostearate and gelatin.

Injectable sterile solutions can be prepared by incorporating the active compound in the required amount in a suitable solvent with one or a combination of the above ingredients, if necessary, followed by sterile filtration through sterile membranes. Typically, dispersions are prepared by incorporating the active compound into a sterile vehicle containing a basic dispersion medium and the remaining necessary ingredients from among the above agents. In the case of sterile powders for the preparation of sterile injectable solutions, the preferred methods of preparation are vacuum drying and lyophilization, which provide the powder with the active ingredient plus any desired additional ingredient from its pre-filtered sterile solution.

The pharmaceutical compositions of the invention can suitably be administered by pulsed infusion, for example by reducing the doses of the composition. Preferably, the dose is administered by injection, more preferably by intravenous or subcutaneous injection, depending in part on whether the administration is short-term or long-term.

In one embodiment, the first or second pharmaceutical compositions of the invention are formulated with carriers that will protect said polypeptide from rapid elimination from the body, such as a controlled release formulation, including implants and microencapsulated administration systems. Biodegradable biocompatible polymers such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters and polylactic acid can be used. Methods for the preparation of these formulations will be understood by those skilled in the art. Materials are also available commercially from Alza Corporation and Nova Pharmaceuticals, Inc.

Sustained release formulations also include preparations of antibody crystals suspended in suitable formulations that can keep the crystals in suspension. These drugs, when administered by the subcutaneous or intraperitoneal route, can provide a sustained release effect. Other compositions also include antibodies included in liposomes. Liposomes containing such antibodies are prepared using known methods such as those described in 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 52322; EP 36676; EP 88046; EP 143949.

Despite the fact that a polypeptide of the invention and conjugates and fusion proteins comprising a polypeptide of the invention are capable of translocation across biological membranes, one skilled in the art will recognize that it may also be convenient to include conjugates or fusion proteins comprising polypeptides of the invention, into nanoparticles.

Within the scope of the invention, the term nanoparticle refers to any material having dimensions in the range of 1-1000 nm. In some embodiments, the nanoparticles have sizes in the range of 2-200 nm, preferably in the range of 2-150 nm, and even more preferably in the range of 2-100 nm.

- 16 042570

Nanoparticles can help maintain the integrity of the polypeptide in biological fluids until it reaches the target organ. In addition, in the case of compositions containing an antineoplastic agent, encapsulation of the composition can reduce the secondary effects caused by the antineoplastic agent. Finally, the nanoparticles can also be modified to include moieties that allow the nanoparticle to be targeted to the organ of interest.

Thus, in yet another embodiment, the pharmaceutical compositions of the invention comprise conjugates, fusion proteins, and compositions of the invention forming part of a nanoparticle.

Suitable nanoparticles that can be used in the context of the present invention include nanoscale materials such as lipid-based nanoparticle, superparamagnetic nanoparticle, nanoshell, semiconductor nanocrystal, quantum dot, polymer-based nanoparticle, silicon-based nanoparticle, silica-based nanoparticle, nanoparticle based on metal, fullerene and nanotube.

Targeted delivery can be achieved by adding ligands without compromising the ability of the nanoparticles to deliver their polypeptide payloads. It is assumed that this will provide delivery to certain 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 may be non-covalently or covalently associated with the nanoparticle and may be conjugated to the nanoparticles in various ways, as discussed here.

Examples of proteins or peptides that can be used for targeted delivery of nanoparticles include transferrin, lactoferrin, TGF-β, nerve growth factor, albumin, HIV Tat peptide, RGD peptide and insulin, among others.

It is understood that formulation of the product of the invention as a nanoparticle is not intended or intended not only to facilitate penetration of the product into the cell, but to protect the product from degradation and/or to facilitate targeted delivery of the nanoparticle to the organ of interest.

The pharmaceutical compositions of the invention are suitable for administration to any kind of mammal, preferably a human.

All terms and embodiments previously described apply equally to this aspect of the invention.

medical applications.

In yet another aspect, the invention relates to a polypeptide or a functionally equivalent variant of said polypeptide of the invention, a conjugate of the invention, a polynucleotide of the invention, a vector of the invention, a host cell of the invention, or a pharmaceutical composition of the invention for use in medicine.

In yet another aspect, the invention relates to a polypeptide or a functionally equivalent variant of said polypeptide of the invention, a conjugate of the invention, a polynucleotide of the invention, a vector of the invention, a host cell of the invention, or a pharmaceutical composition of the invention for use in the prevention and/or treatment of cancer.

Alternatively, the invention relates to a method for the prevention and/or treatment of cancer comprising administering to a patient a therapeutically effective amount of a polypeptide or a functionally equivalent variant of said polypeptide of the invention, a conjugate of the invention, a polynucleotide of the invention, a vector of the invention, a host cell of the invention, or a pharmaceutical composition of the invention. invention.

Alternatively, the invention relates to the use of a polypeptide or a functionally equivalent variant of said polypeptide of the invention, a conjugate of the invention, a polynucleotide of the invention, a vector of the invention, a host cell of the invention, or a pharmaceutical composition of the invention for the manufacture of a medicament for the prevention and/or treatment of cancer.

Prophylaxis is understood to mean the administration of a compound at the initial or early stage of a disease or also to prevent its occurrence.

The term treatment is used to refer to the administration of a compound to control the progression of a disease before or after the onset of clinical signs. Controlling the progression of a disease is understood as beneficial or desirable clinical outcomes, which include, but are not limited to, reduction in symptoms, reduction in the duration of the disease, stabilization of pathological conditions (in particular, prevention of additional disorders), delay in disease progression, suppression of the pathological condition, and remission (both partial, and complete). Controlling disease progression also includes prolonging survival compared to expected survival when no treatment is given.

Within the meaning of the invention, a patient includes any animal that has cancer or is exhibiting a symptom or cancer, or is at risk of developing cancer or exhibiting a symptom of cancer. Suitable patients include laboratory animals (such as mice, rats, rabbits, or

- 17 042570 guinea pig), farm animals and pets or companion animals (such as a cat or dog). Non-human primates are included, and preferably human patients.

The term cancer refers to a disease characterized by uncontrolled division of cells (or an increase in their survival or resistance to apoptosis), the ability of these cells to invade other nearby tissues (invasion), or spread to other areas of the body where cells are normally absent (metastasis) through the lymphatic and blood vessels. Depending on whether tumors can spread through invasion and metastasis, they are classified as benign or malignant: benign tumors are tumors that cannot spread through invasion or metastasis, i.e. they grow only locally; while malignant tumors are tumors that are able to spread through invasion and metastasis. The methods of the present invention are suitable for the treatment of local and malignant tumors.

As used herein, the term cancer includes, but is not limited to, the following types of cancer: breast cancer; biliary tract cancer; bladder cancer; brain cancer, including glioblastomas and medulloblastomas; cervical cancer; choriocarcinoma; colon cancer; endometrial cancer; esophageal carcinoma; stomach cancer; hematological neoplasms, including acute lymphocytic leukemia and myeloid leukemia; T-cell acute lymphoblastic leukemia/lymphoma; hairy cell leukemia; chronic myeloid leukemia, multiple myeloma; AIDS-associated leukemia and adult T-cell leukemia/lymphoma; intraepithelial neoplasias including Bowen's disease and Paget's disease; liver cancer; lungs' cancer; lymphomas, including Hodgkin's disease and lymphocytic lymphomas; neuroblastomas; oral cancer, including squamous cell carcinoma; ovarian cancer, including tumors arising from epithelial cells, stromal cells, germ cells and mesenchymal cells; pancreas cancer; prostate cancer; rectal cancer; sarcomas, including leiomyosarcoma, rhabdomyosarcoma, liposarcoma, fibrosarcoma, and osteosarcoma; skin cancer, including melanoma, Merkel cell carcinoma, Kaposi's sarcoma, basal cell carcinoma and squamous cell carcinoma; testicular cancer, including germ cell tumors such as seminoma, non-seminoma (teratomas, choriocarcinomas), stromal tumors and embryonic cell tumors; thyroid cancer, including thyroid adenocarcinoma and medullary carcinoma; and kidney cancer, including adenocarcinoma and Wilms tumor. Other types of cancer will be known to the person skilled in the art. In a preferred embodiment, the cancer being treated is lung cancer, preferably lung adenocarcinoma, more preferably KRas-mediated lung adenocarcinoma.

In a preferred embodiment, the cancer is a solid tumor.

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

In a preferred embodiment, the cancer is selected from the group consisting of glioblastoma and non-small cell lung cancer.

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

Within the scope of the invention, the term NSCLC or non-small cell lung cancer refers to a group of heterogeneous diseases grouped together because their prognosis and treatment are approximately the same 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 ed. Berlin: Springer-Verlag, 1999):

1. Squamous cell carcinoma (SCC), which accounts for 30 to 40% of NSCLC, starts in the large airways but grows more slowly, meaning that the size of these tumors depends on the diagnosis.

2. Adenocarcinoma is the most common subtype of NSCLC, accounting for 50 to 60% of NSCLC that begins near the gas exchange surface of the lung and which includes the subtype, bronchoalveolar carcinoma, which may have varying responses to treatment.

3. Large cell carcinoma is a fast growing form that grows near the surface of the lungs. It is primarily a diagnosis of exclusion, and when more tests are done, it is usually reclassified to either squamous cell carcinoma or adenocarcinoma.

4. Adenosquamous carcinoma is a type of cancer that includes two types of cells: squamous cells (thin, flat cells that line certain organs) and glandular cells.

5. Carcinomas with pleomorphic, sarcomatoid or sarcomatous elements. This is a group of rare tumors reflecting continuity in histological heterogeneity as well as epithelial and mesenchymal differentiation.

6. A carcinoid tumor is a slowly growing neuroendocrine tumor of the lung that begins in cells capable of releasing a hormone in response to a stimulus generated by the nervous system.

7. Salivary gland-type carcinomas begin in the cells of the salivary glands located inside

- 18 042570 large airways of the lung.

8. Unclassified carcinomas include cancers that do not fit into any of the above categories of lung cancer.

The invention also includes the following:

1. A polypeptide comprising a polypeptide of SEQ ID NO: 1 wherein the X residue at position 89 in SEQ ID NO: 1 is not a cysteine, or a functionally equivalent variant of said polypeptide.

2. A polypeptide according to claim 1, wherein said polypeptide consists of a polypeptide of SEQ ID NO: 1, wherein the X residue at position 89 of SEQ ID NO: 1 is not a cysteine, or consists of a functionally equivalent variant of said polypeptide.

3. A polypeptide according to claim 1 or 2, wherein the X residue at position 89 in SEQ ID NO: 1 is serine.

4. A polypeptide according to claim 3, consisting of SEQ ID NO: 4.

5. Conjugate containing:

a) a polypeptide or functionally equivalent variant of said polypeptide according to any one of claims 1-4, and

b) a chemical group that promotes cellular uptake of a polypeptide or a functionally equivalent variant of said polypeptide.

6. The conjugate of claim 5, wherein the chemical group that promotes cell uptake of a polypeptide or a functionally equivalent variant of said polypeptide is a cell-penetrating peptide sequence, and wherein said cell-penetrating peptide sequence and said polypeptide or a functionally equivalent variant of said polypeptide form a fusion protein.

7. The conjugate of claim 6 wherein the cell-penetrating peptide sequence is selected from the group consisting of GRKKRRQRRR (SEQ ID NO: 38) and RRRRRRLR (SEQ ID NO: 39).

8. A conjugate according to any one of claims 5 to 7, further comprising an additional nuclear localization signal, wherein in particular the nuclear localization signal is selected from the group consisting of PKKKRKV (SEQ ID NO: 6), PAAKRVKLD (SEQ ID NO: 54) and KRPAATKKAGQAKKKK (SEQ ID NO: 7).

9. A polynucleotide encoding a polypeptide according to any one of claims 1-4 or a conjugate according to any one of claims 5-8.

10. A vector comprising a polynucleotide according to claim 9.

11. A host cell comprising a polypeptide according to any one of claims 1 to 4, a conjugate according to any one of claims 5 to 8, a polynucleotide according to claim 9, or a vector according to claim 10.

12. A pharmaceutical composition comprising a pharmaceutically effective amount of a polypeptide or a functionally equivalent variant of said polypeptide according to any one of claims 1 to 4, a conjugate according to any one of claims 5 to 8, a polynucleotide according to claim 9, a vector according to claim 10, or a host cell 11, and a pharmaceutically acceptable excipient.

13. A polypeptide or a functionally equivalent variant of said polypeptide according to any one of claims 1 to 4, a conjugate according to any one of claims 5 to 8, a polynucleotide according to claim 9, a vector according to claim 10, a host cell according to claim 11, or a pharmaceutical composition according to claim 12 for use in medicine.

14. A polypeptide or a functionally equivalent variant according to any one of claims 1-4, a conjugate according to any one of claims 5-8, a polynucleotide according to claim 9, a vector according to claim 10, a host cell according to claim 11, or a pharmaceutical composition according to 12 for use in the prevention and/or treatment of cancer.

15. A polypeptide or functionally equivalent variant, conjugate, polynucleotide, vector, host cell, or pharmaceutical composition for use according to claim 14, wherein the cancer is selected from the group consisting of glioblastoma and non-small cell lung cancer.

All terms and embodiments previously described apply equally to this aspect of the invention.

The invention is described in detail below by means of the following examples, which are purely illustrative and do not limit the scope of the invention in any way.

Examples

Materials and methods.

Cell transfection with Omomyc and OmoCS mRNA.

mRNA for Omomyc and the OmoCS mutant was obtained from Trilink Biotechnologies at concentrations of 0.782 and 0.876 mg/ml, respectively (ARCA capped and fully substituted with modified 5-methyl-C and pseudo-U). The Omomyc or OmoCS DNA sequence was inserted into the vector downstream of the T7 RNA polymerase promoter and the 3' poly(T) tail. RNA modified with 5-methylcytidine-5'-triphosphate and pseudouridine-5'-triphosphate was obtained by in vitro transcription using T7 RNA polymerase. The RNA was also capped using the analog of the cap structure [3'-0-Me-m7G(5')ppp(5')G] RNA. Template vector DNA was digested using DNase and residual triphosphate was removed by treatment with phosphatase. The RNA product was then purified and assessed for integrity and quantity using agarose gel electrophoresis and a Nanodrop spectrophotometer, respectively. RNA was stored at -80°C.

- 19 042570

Lipofectamine MessengerMAX transfection reagent was obtained from Thermo Fisher Scientific. Cell lines A549 and U87 were seeded at 500 and 1000 cells per well, respectively, in 96-well plates. Cells were cultured in Dulbecco's Modified Eagle's Medium (DMEM) supplemented with 10% fetal bovine serum (FBS) and 1% L-glutamine (complete medium). After 24 h, cells were transfected with lipofectamine-mRNA complexes. 3 µg of mRNA for every 2 µl of Lipofectamine was separately diluted in serum-free medium and incubated for 10 min. The mRNA and lipofectamine dilutions were then mixed and incubated for 5 min to form complexes. The wells were washed twice with serum-free medium. Cells were treated with serial dilutions starting from 1 to 0.0625 µg of mRNA per well in 100 µl of complete serum-free medium. For each concentration, control cells were treated with Lipofectamine alone and triplicates were used for each test condition. After 4 hours of transfection, serum-free media and mRNA-lipofectamine complexes were removed from the wells and replaced with complete media. Cells were incubated for 3 days. Viability was then assessed using CellTiter-Blue from Promega. Absorbance relative to untreated wells was calculated for each test condition. Statistical significance was calculated using the t-test.

Transfection of OmoCS and OmoCA mRNA cells.

mRNA for Omomyc and the OmoCA mutant was obtained from Trilink Biotechnologies (ARCA capped and fully substituted with modified 5-methyl-C and pseudo-U). The mRNA concentrations for OmoCS and OmoCA were determined at 0.845 and 0.832 mg/ml, respectively, using a Nanodrop spectrophotometer. Lipofectamine MessengerMAX transfection reagent was obtained from Thermo Fisher Scientific. A549 cells were plated at 500 cells per well in a 96-well plate. Cells were cultured in Roswell Park Memorial Institute (RPMI) medium supplemented with 10% fetal bovine serum (FBS) and 1% L-glutamine (complete medium). After 24 h, cells were transfected with lipofectamine-mRNA complexes. 2 µg of mRNA for every 1.5 µl of Lipofectamine was separately diluted in serum-free medium and incubated for 10 min. The mRNA and lipofectamine dilutions were then mixed and incubated for 5 min to form complexes. The wells were washed twice with serum-free medium. Cells were treated with 1:2 serial dilutions starting with 1 μg of mRNA per well in 50 μl of complete serum-free medium (200 nM). For each concentration, cells were either untreated (untreated) or treated with Lipofectamine only (Lipo only) and used as control wells. Triplicates were used for each condition. After 4 hours of transfection, serum-free media and mRNA-lipofectamine complexes were removed from the wells and replaced with complete media. Cells were incubated for 3 days. At this point, cell density was assessed using crystal violet staining.

Absorbance relative to untreated wells was calculated for each concentration.

Example 1

Surprisingly, the OmoCS mutant showed higher cell growth inhibition compared to the original Omomyc sequence at low concentration (FIG. 1). This increased efficiency over the original Omomyc sequence can at least in part be explained by the hypothesis that the oxidized cysteine boundary of the Omomyc homodimer will prevent Omomyc from heterodimerizing to Myc or to Max, limiting Omomyc activity through simple competition for binding to the E-box. Instead, the OmoCS mutant will increase other biological activities of Omomyc, promoting the formation of heterodimeric populations.

Example 2

Surprisingly, the OmoCA mutant behaved exactly like OmoCS in this proliferation assay (FIG. 2).

Claims (17)

1. A polypeptide comprising a polypeptide of SEQ ID NO: 1, where the X residue at position 89 in SEQ ID NO: 1 is not a cysteine, or a functionally equivalent variant of the specified polypeptide, which is obtained by deletion, insertion, addition or conservative replacement of one or more than amino acids relative to the polypeptide of SEQ ID NO: 1, or which is obtained by chemical modification of the polypeptide of SEQ ID NO: 1, and which essentially retains the tumor suppressive activity of SEQ ID NO: 1, where the X residue is at position 89 in SEQ ID NO: 1 is not a cysteine. 2. The polypeptide according to claim 1, where the specified polypeptide consists of a polypeptide with SEQ ID NO: 1, where the X residue at position 89 in SEQ ID NO: 1 is not a cysteine or consists of a functionally equivalent variant of the specified polypeptide, where the X residue at position 89 in SEQ ID NO: 1 is not a cysteine. 3. A polypeptide according to claim 1, wherein said polypeptide consists of a polypeptide of SEQ ID NO: 3, wherein the X residue at position 90 of SEQ ID NO: 3 is not a cysteine. 4. A polypeptide according to any one of claims 1 to 3, wherein the X residue is serine. - 20 042570 5. A polypeptide according to claim 4, consisting of SEQ ID NO: 4. 6. A polypeptide according to any one of claims 1 to 3, wherein X is an alanine. 7. Conjugate containing: a) a polypeptide or functionally equivalent variant of said polypeptide according to any one of claims 1-6, and b) a chemical group that promotes uptake by the cell of a polypeptide or a functionally equivalent variant of said polypeptide. 8. The conjugate of claim 7, wherein the chemical group that promotes cell uptake of a polypeptide or a functionally equivalent variant of said polypeptide is a cell-penetrating peptide sequence, and wherein said cell-penetrating peptide sequence and said polypeptide or a functionally equivalent variant of said polypeptide form a fusion protein. 9. The conjugate of claim 8, wherein the cell-penetrating peptide sequence is selected from the group consisting of GRKKRRQRRR (SEQ ID NO: 38) and RRRRRRLR (SEQ ID NO: 39). 10. A conjugate according to any one of claims 7 to 9, further comprising an additional nuclear localization signal, wherein in particular the nuclear localization signal is selected from the group consisting of PKKKRKV (SEQ ID NO: 6), PAAKRVKLD (SEQ ID NO: 54) and KRPAATKKAGQAKKKK (SEQ ID NO: 7). 11. A polynucleotide encoding a polypeptide according to any one of claims 1-6. 12. A polynucleotide encoding a conjugate according to any one of claims 8-10. 13. A vector comprising a polynucleotide according to any one of claims 11 or 12. 14. A host cell comprising a polypeptide according to any one of claims 1 to 6, a conjugate according to any one of claims 7 to 10, a polynucleotide according to any one of claims 11 or 12, or a vector according to claim 13. 15. A pharmaceutical composition comprising a pharmaceutically effective amount of a polypeptide or a functionally equivalent variant of said polypeptide according to any one of claims 1 to 6, a conjugate according to any one of claims 7 to 10, a polynucleotide according to any one of claims 11 or 12, or a vector according to claim 13 and a pharmaceutically acceptable excipient. 16. The use of a polypeptide or a functionally equivalent variant according to any one of claims 1 to 6, a conjugate according to any one of claims 7 to 10, a polynucleotide according to any one of claims 11 or 12, a vector according to claim 13 or a pharmaceutical composition according to claim 15 for the prevention and/or treatment of cancer. 17. Use according to claim 16 wherein the cancer is selected from the group consisting of glioblastoma and non-small cell lung cancer.