Compounds for the treatment of autoimmune diseases

Abstract

This invention relates to polypeptides containing Omomyc, functionally equivalent variants thereof, conjugates containing Omomyc or said functionally equivalent variants, polynucleotides encoding said polypeptides, carriers containing said polynucleotides, and cells capable of secreting said polypeptides or conjugates for the prevention and / or treatment of autoimmune diseases. The invention also relates to combinations and pharmaceutical compositions of said compounds and at least one other compound for the treatment of autoimmune diseases, and their medical uses.

Description

Technical Field

[0001] This invention relates to the field of autoimmune diseases, and more particularly, to a compound and its use in the prevention and / or treatment of autoimmune diseases. Background Technology

[0002] Abnormal regulation of the immune response is associated with a variety of human diseases because the inappropriate initiation of immune responses to various self- and foreign antigens plays a causal role in a large number of pathologies, including autoimmune disorders, asthma, allergic reactions, graft-versus-host disease, transplant rejection, and various other immune disorders.

[0003] Autoimmune diseases are a group of diseases caused by damage to the immune system, which leads to a reaction against the body's own tissues.

[0004] Changes in cellular recognition mechanisms that enable the body to distinguish between "self" and "non-self"—that is, to differentiate between elements belonging to the body and elements from outside the body—lead to the production of antibodies and the differentiation of specific T cells and B cells. These cells can target a single organ (organ-specific disease) or trigger systemic diseases, thereby impairing the function of the entire individual.

[0005] In particular, multiple sclerosis (MS) is a chronic, immune-mediated disease affecting the central nervous system (CNS), characterized by the destruction of myelin sheaths, axonal damage, and neuronal loss, leading to neurological dysfunction. The prevalence of this disease is increasing; in fact, up to 2.8 million people worldwide have been diagnosed with MS. Indeed, it is considered the leading non-traumatic cause of disability in young adults.

[0006] Most studies on the pathogenesis of MS have been conducted in animal models of experimental autoimmune encephalomyelitis (EAE), which can be induced by injecting myelin or peptides into susceptible rodent strains. These animals subsequently develop monophasic inflammatory CNS disease or various forms of chronic or relapsing-remitting EAE, and CD4+ T cells, monocytes / activated microglia and other cell populations, as well as humoral factors, are found in their CNS lesions (Martin R et al. Immunological aspects of demyelinating diseases. Ann Rev Immunol. 1992, 10:153-187).

[0007] The etiology of MS remains unclear; however, several genetic, environmental, and lifestyle factors are associated with disease risk. The most accepted hypothesis of pathogenesis posits that peripheral CNS autoreactive T cells are activated upon encountering CNS-derived antigens or antigens from pathogens highly homologous to CNS antigens. These CNS autoreactive T cells then circulate back into the CNS and are reactivated by resident antigen-presenting cells (APCs), which present CNS antigens to the autoreactive T cells, triggering a series of immune events that ultimately lead to neurodegeneration and neurological dysfunction. Because MS is an immune-mediated disease, current treatments focus on downregulating the peripheral immune response by suppressing and modulating it (immunosuppressants or immunomodulators).

[0008] Generally, treatment for autoimmune diseases depends on the type and severity of the condition. Nonsteroidal anti-inflammatory drugs (NSAIDs), steroids, and immunosuppressants are commonly used. Intravenous immunoglobulins may also be used occasionally. For example, in the case of multiple sclerosis, interferon (IFN)-β, glatiramer acetate, cyclophosphamide, or mitoxantrone are commonly used. Attempts to provide new treatments for autoimmune diseases include using proteins or small organic compounds as immunomodulators.

[0009] However, while reported treatments often improve symptoms, they typically do not cure the disease, and in some cases, the side effects can be just as harmful as the disease itself. In particular, genotoxic side effects, such as clastogenicity or aneugenicity, which are often associated with treatments that interfere with epigenetic regulation, are especially significant due to their severity and potential lethality.

[0010] In summary, there is an urgent need to develop new treatments for autoimmune diseases that complement the existing plethora of therapies and preferably overcome the drawbacks associated with existing treatments, such as the use of high doses and high frequency of administration. Summary of the Invention

[0011] The authors of this invention have demonstrated that inhibiting Myc using the peptide Omomyc constitutes a potential therapeutic option for treating autoimmune diseases. They found that in spleen cells of EAE-affected mice, Omomyc reduced spleen cell proliferation and induced cell death in a dose-dependent manner. Figure 1The study also found that when administered via different routes, at different doses and intervals, treatment with Omomyc in EAE-affected mice delayed disease onset and improved the clinical course of the disease. Figure 2-4 Surprisingly, the beneficial effects of Omomyc are long-lasting, as only four doses are needed to achieve a therapeutic effect. Figure 4 ).

[0012] Therefore, in a first aspect, the present invention relates to a compound, hereinafter referred to as the compound of the present invention, selected from the group consisting of: (a) A polypeptide containing the sequence SEQ ID NO: 1 or a functionally equivalent variant thereof, (b) A conjugate comprising a polypeptide comprising the sequence SEQ ID NO: 1 or a functionally equivalent variant thereof and a chemical moiety that promotes cellular uptake of said polypeptide or a functionally equivalent variant thereof. (c) A polynucleotide encoding the polypeptide of (a) or the conjugate of (b). (d) A vector containing the polynucleotide according to (c), and (e) Cells capable of secreting the polypeptide according to (a) or the conjugate according to (b) into a culture medium. Used for the prevention and / or treatment of autoimmune diseases in the subject.

[0013] In a second aspect, the present invention relates to a combination comprising: (i) Choose compounds from the following groups: (a) A polypeptide containing the sequence SEQ ID NO: 1 or a functionally equivalent variant thereof, (b) A conjugate comprising a polypeptide comprising the sequence SEQ ID NO: 1 or a functionally equivalent variant thereof, and a chemical moiety that promotes cellular uptake of said polypeptide or a functionally equivalent variant thereof. (c) A polynucleotide encoding the polypeptide of (a) or the conjugate of (b). (d) A vector comprising the polynucleotide according to (c), and (e) Cells capable of secreting the polypeptide according to (a) or the conjugate according to (b) into a culture medium; and (ii) An agent for the treatment of autoimmune diseases, preferably selected from the group consisting of: copaxone, teriflunomide, fumarate, natalizumab, fingolimod, ocrelizumab, poesimod, siponimod, ozanimod, and tocilizumab.

[0014] In a third aspect, the present invention relates to a pharmaceutical composition comprising a pharmaceutically effective amount of the combination of the second aspect of the present invention and a pharmaceutically acceptable excipient.

[0015] In a fourth aspect, the present invention relates to a combination of the second aspect of the invention or a pharmaceutical composition of the third aspect of the invention for medical use.

[0016] In a fifth aspect, the present invention relates to a combination of the second aspect of the invention or a pharmaceutical composition of the third aspect of the invention for the prevention and / or treatment of autoimmune diseases. Attached Figure Description

[0017] Figure 1 . for Experimental autoimmune encephalomyelitis (EAE) mice of Phytohemagglutinin (PHA) and Myelin oligodendrocyte glycoprotein (MOG) stimulated spleen cells , Omomyc reduces spleen cells in a dose-dependent manner. It proliferates and induces cell death. EAE was induced in C57BL6 / J mice. When the mice had developed neurological dysfunction, spleen cells were isolated and cultured in the presence of PHA or 35-55 MOG peptide, while simultaneously increasing the concentration of Omomyc. This was achieved through incorporation... 3 H-thymidine was used to measure proliferation, and flow cytometry was used to measure cell death.

[0018] Figure 2 . Omomyc treatment delayed the onset of the disease and improved its clinical course. Experimental autoimmune encephalomyelitis (EAE) was induced in C57BL6 / J mice, and clinical progress was tracked daily. Omomyc was administered daily via intraperitoneal (ip) injection starting from day 1 post-immunization. SEM, standard error of the mean; AUC, area under the curve; dpi, day post-immunization.

[0019] Figure 3 . Omomyc treatment significantly improved the clinical course of the disease, while animals exhibited milder clinical signs. (Maximum score decreased). Experimental autoimmune encephalomyelitis (EAE) was induced in C57BL6 / J mice, and clinical progress was tracked daily. Omomyc was administered intravenously (iv) at a dose of 75 mg / kg three times a week, starting from day 1 post-immunization.

[0020] Figure 4 . The beneficial effects of Omomyc are long-lasting. Experimental autoimmune encephalomyelitis (EAE) was induced in C57BL6 / J mice, and clinical progress was monitored daily. Starting from day 1 post-immunization, four doses of 75 mg / kg Omomyc were administered intraperitoneally every other day. Only four doses of Omomyc delayed disease onset and improved the clinical course of the disease. Animals treated with Omomyc did not show any weight loss compared to their counterparts treated with a vehicle, while the vehicle-treated counterparts showed significant weight loss after the onset of clinical signs. Detailed Implementation

[0021] This invention relates to therapeutic compounds for the prevention and / or treatment of autoimmune diseases in subjects.

[0022] Unless otherwise defined, all technical terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains.

[0023] Therapeutic uses of the present invention In a first aspect, the present invention relates to a compound selected from the group consisting of: (a) A polypeptide containing the sequence SEQ ID NO: 1 or a functionally equivalent variant thereof, (b) A conjugate comprising a polypeptide comprising the sequence SEQ ID NO: 1 or a functionally equivalent variant thereof and a chemical moiety that promotes cellular uptake of said polypeptide or a functionally equivalent variant thereof. (c) A polynucleotide encoding the polypeptide of (a) or the conjugate of (b). (d) A vector comprising the polynucleotide according to (c), and (e) Cells capable of secreting the polypeptide according to (a) or the conjugate according to (b) into a culture medium. Used for the prevention and / or treatment of autoimmune diseases in the subject.

[0024] Alternatively, the present invention relates to the use of a compound selected from the group consisting of in the preparation of a medicament for the prevention and / or treatment of an autoimmune disease in a subject: (a) A polypeptide containing the sequence SEQ ID NO: 1 or a functionally equivalent variant thereof, (b) A conjugate comprising a polypeptide comprising the sequence SEQ ID NO: 1 or a functionally equivalent variant thereof and a chemical moiety that promotes cellular uptake of said polypeptide or a functionally equivalent variant thereof. (c) A polynucleotide encoding the polypeptide of (a) or the conjugate of (b). (d) A vector comprising the polynucleotide according to (c), and (e) Cells capable of secreting the polypeptide according to (a) or the conjugate according to (b) into a culture medium.

[0025] Alternatively, the present invention relates to a method for preventing and / or treating autoimmune diseases, comprising administering to a subject in need a therapeutically effective amount of a compound selected from the group consisting of: (a) A polypeptide containing the sequence SEQ ID NO: 1 or a functionally equivalent variant thereof, (b) A conjugate comprising a polypeptide comprising the sequence SEQ ID NO: 1 or a functionally equivalent variant thereof and a chemical moiety that promotes cellular uptake of said polypeptide or a functionally equivalent variant thereof. (c) A polynucleotide encoding the polypeptide of (a) or the conjugate of (b). (d) A vector comprising the polynucleotide according to (c), and Cells capable of secreting the polypeptide according to a) or the conjugate according to b) into a culture medium. In a preferred embodiment, the compound used according to the invention is a polypeptide comprising the sequence SEQ ID NO: 1, hereinafter referred to as the polypeptide of the invention or a functionally equivalent variant thereof.

[0026] In a more preferred embodiment, the compound used according to the invention is a polypeptide comprising the sequence SEQ ID NO: 1.

[0027] The terms "polypeptide" and "peptide" are used interchangeably herein to refer to polymers of amino acids of any length. The polypeptides of the present invention may contain modified amino acids, and may be broken down by non-amino acid components. In a preferred embodiment, the polypeptide is formed entirely of amino acids. Preferably, the polypeptide forming item (a) has a length of 80 to 500 amino acids, more preferably 80 to 300 amino acids, more preferably 80 to 250 amino acids, more preferably 80 to 150 amino acids, even more preferably 80 to 130 amino acids, preferably 90 to 130 amino acids, preferably no more than 125 amino acids, and more preferably no more than 100 amino acids. In a preferred embodiment, the polypeptide has a length of 90 to 98 amino acids, preferably 90 to 95 amino acids, and more preferably 91 amino acids.

[0028] The term "amino acid" refers to naturally occurring amino acids and synthetic amino acids, as well as amino acid analogs and amino acid mimics that function similarly to naturally occurring amino acids. Furthermore, the term "amino acid" includes both D-amino acids and L-amino acids (stereoisomers). Preferably, the amino acid is an L-amino acid.

[0029] The term “natural amino acid” or “naturally occurring amino acid” includes 20 naturally occurring amino acids; those that are typically modified post-translationally in vivo, including, for example, hydroxyproline, phosphoserine, and phosphothreonine; and other unusual amino acids, including but not limited to, 2-aminoadipic acid, hydroxylysine, isodesmosine, valine, leucine, and ornithine.

[0030] As used herein, the term "non-natural amino acid" or "synthetic amino acid" refers to a carboxylic acid or a derivative thereof that is structurally related to a natural amino acid by replacing an amino group at position "a". Exemplary non-limiting examples of modified or uncommon amino acids include 2-aminoadipic acid, 3-aminoadipic acid, β-alanine, 2-aminobutyric acid, 4-aminobutyric acid, 6-aminohexanoic acid, 2-aminoheptanoic acid, 2-aminoisobutyric acid, 3-aminoisobutyric acid, 2-aminopimelic acid, 2,4-diaminobutyric acid, desmodium, 2,2'-diaminopimelic acid, 2,3-diaminopropionic acid, N-ethylglycine, N-ethylasparagine, hydroxylysine, allohydroxylysine, 3-hydroxyproline, 4-hydroxyproline, isodesmodium, alloisoleucine, N-methylglycine, N-methylisoleucine, 6-N-methyllysine, N-methylvaline, valine, leucine, ornithine, etc.

[0031] The polypeptides used according to the present invention may also contain non-amino acid moieties, such as, for example, hydrophobic moieties attached to the peptide (various linear, branched, cyclic, polycyclic or heterocyclic hydrocarbons and hydrocarbon derivatives); and various protecting groups attached to the ends of the compound to reduce degradation. Suitable protecting functional groups are described in Green and Wuts, "Protecting Groups in Organic Synthesis", John Wiley and Sons, Chapters 5 and 7, 1991.

[0032] Chemical (non-amino acid) groups present in peptides can be included to improve various physiological properties, such as reducing degradation or clearance rates; reducing the repulsive forces of various cell pumps; improving various drug delivery methods; increasing specificity; increasing affinity; improving stability, bioavailability, solubility; and reducing toxicity.

[0033] "Mimetic" refers to molecules that mimic the chemical structure of a peptide while retaining its functional properties. Methods for designing peptide analogs, derivatives, and mimics are known in the art.

[0034] In embodiments, the polypeptide used according to the present invention is a polypeptide consisting of the sequence SEQ ID NO: 1 or a polypeptide consisting of a functionally equivalent variant of SEQ ID NO: 1, preferably a polypeptide consisting of the sequence SEQ ID NO: 1.

[0035] SEQ ID NO: 1 corresponds to TEENVKRRTHNVLERQRRNELKRSFFALRDQIPELENNEKAPKVVILKKATAYILSVQAETQKLISEIDLLRKQNEQLKHKLEQLRNSCA (SEQ ID NO: 1).

[0036] The polypeptide sequence SEQ ID NO: 1 corresponds to the Omomyc protein sequence. As used herein, the term "Omomyc" refers to a polypeptide consisting of a mutant version of the bHLHZip domain of Myc carrying E61T, E68I, R74Q, and R75N ​​mutations (where the mutation positions are numbered according to the Myc region sequence corresponding to amino acids 365-454 of the polypeptide as defined in NCBI accession number NP_002458 published on March 15, 2015). The c-Myc sequence provided under NCBI accession number NP_002458 is shown below (SEQ ID NO: 2), where the region from which Omomyc originates is underlined:

[0037] Omomyc also contains the M2 domain of c-Myc, with the sequence RQRRNELKRSF (SEQ ID NO: 3) (see Dang and Lee, Mol.Cell. Biol., 1988, 8:4048-4054) (double underlined above), and it corresponds to the nuclear localization signal.

[0038] Omomyc is characterized by its enhanced dimerization ability with all three oncogenic Myc proteins (c-Myc, N-Myc, and L-Myc). Omomyc can be derived from the bHLHZip domain of any Myc protein known in the art, provided that the mutation leading to the Myc repressive effect is retained. Therefore, the Omomyc used in this invention can be derived from any mammalian species, including but not limited to livestock and farm animals (cattle, horses, pigs, sheep, goats, dogs, cats, or rodents), primates, and humans. Preferably, the Omomyc protein is derived from a human Myc protein (accession number NP_002458, published March 12, 2019).

[0039] As used herein, the term "Myc" refers to the family of transcription factors, including c-Myc, N-Myc, and L-Myc. Myc proteins activate the expression of many genes by binding to the concordant sequence CACGTG (an enhancer cassette sequence or E-box and recruiting histone acetyltransferases or HAT). However, Myc can also act as a transcriptional repressor. By binding to the Miz-1 transcription factor and replacing the p300 coactivator, it represses the expression of Miz-1 target genes. Myc also plays a direct role in the control of DNA replication.

[0040] The Myc b-HLH-LZ or Myc basic region helix-loop-helix leucine zipper domain refers to the region that determines the dimerization of Myc with Max proteins and their binding to Myc target genes. This region corresponds to amino acid 365-454 of human Myc and is characterized by two α-helices linked by loops (Nair, SK, & Burley, SK, 2003, Cell, 112: 193–205).

[0041] In a preferred embodiment, the polypeptide of the present invention is a polypeptide comprising or substantially comprising the following SEQ ID NO: 4.

[0042] MTEENVKRRTHNVLERQRRNELKRSFFALRDQIPELENNEKAPKVVILKKATAYILSVQAETQKLISEIDLLRKQNEQLKHKLEQLRNSCA (SEQ ID NO: 4).

[0043] In this document, “consistently composed of” means that a particular molecule does not contain any additional sequences that would alter the activity of SEQ ID NO: 4.

[0044] Preferably, the polypeptide consists of SEQ ID NO: 4.

[0045] The term "functionally equivalent variant" refers to any polypeptide derived by inserting or adding one or more amino acids and / or deleting one or more amino acids and / or conservatively substituting one or more amino acids relative to the polypeptide of SEQ ID NO: 1, and / or any polypeptide derived by chemically modifying the polypeptide of SEQ ID NO: 1, and which substantially retains the inhibitory activity of SEQ ID NO: 1. Preferably, a functionally equivalent variant refers to any polypeptide derived by inserting or adding one or more amino acids and / or deleting one or more amino acids and / or conservatively substituting one or more amino acids relative to the polypeptide of SEQ ID NO: 1, and which substantially retains the inhibitory activity of SEQ ID NO: 1; more preferably, any polypeptide derived by inserting or adding one or more amino acids relative to the polypeptide of SEQ ID NO: 1.

[0046] Those skilled in the art will understand that retention of inhibitory activity requires the variant to dimerize with Myc and / or its dedicated chaperone molecules p21 / p22Max and inhibit Myc activity, which is capable of translocation across the cell membrane and across the nuclear envelope. In some embodiments, functionally equivalent variants of the peptides of the present invention exhibit less homodimerization than Omomyc, or are not forced to become homodimers due to disulfide bond formation. In particular, disulfide bond formation in the homodimeric form of certain embodiments of the peptides of the present invention is less than that in the peptide Omomyc.

[0047] As used herein, "less homodimerization" means that the peptides of the present invention have a lower ability to form specific homodimers even under reducing conditions. In a preferred embodiment, this ability is at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% lower than the ability of Omomyc to form homodimers.

[0048] As used herein, reduction conditions involve the presence of a reducing agent, which is a compound that donates electrons to other chemicals in a redox chemical reaction. Exemplary, non-limiting examples of reducing agents are DTT (dithiothreitol), β-mercaptoethanol, or TCEP (tris(2-carboxyethyl)phosphine). The amount of homodimer may be the same in vitro, and the difference between functionally equivalent variants and Omomyc exists only in the cells of heterodimerized partner molecules, where the absence of disulfide bonds may result in a higher formation rate of heterodimers.

[0049] Several assays can be used to determine the homodimerization of peptides, such as exemplary non-limiting examples of monitoring thermal denaturation by circular dichroism, thus dimerization can be detected by quantifying folding and thermal stability.

[0050] Suitable functionally equivalent variants include polypeptides that consist essentially of the polypeptide of SEQ ID NO: 1. In this context, "consistently of" means that the particular molecule does not contain any additional sequence that would alter the activity of SEQ ID NO: 1.

[0051] In a preferred embodiment, the functionally equivalent variant of SEQ ID NO: 1 is a polypeptide generated by inserting or adding one or more amino acids relative to the polypeptide of SEQ ID NO: 1. In embodiments, the functionally equivalent variant is generated by inserting fewer than 10 amino acids, more preferably fewer than 5 amino acids, and even more preferably by inserting one amino acid. In a preferred embodiment, it is generated by inserting one amino acid, namely methionine.

[0052] In another embodiment, the functionally equivalent variant of SEQ ID NO: 1 is a polypeptide generated by deleting one or more amino acids relative to the polypeptide of SEQ ID NO: 1. In this embodiment, the functionally equivalent variant is generated by deleting fewer than 10 amino acids, more preferably fewer than 5 amino acids, and more preferably by deleting one amino acid.

[0053] Suitable functional variants of the targeting peptide are those that show a degree of identity with the peptide of SEQ ID NO: 1 of approximately 25% amino acid sequence identity, such as 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 the two peptides is determined using computer algorithms and methods well known to those skilled in the art. The identity between the 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 the entire length of the variant, or both.

[0054] The functionally equivalent variants of the peptides of the present invention may also include post-translational modifications, such as glycosylation, acetylation, isopreneation, myristylation, proteolytic processing, etc.

[0055] In another embodiment, suitable functional variants of the targeting peptide are those in which one or more positions within the polypeptide used according to the invention contain an amino acid (which is a conserved substitution of an amino acid present in the aforementioned protein). "Conserved amino acid substitution" refers to the replacement of one amino acid with another amino acid having similar structure and / or chemical properties. For example, each of the following six groups of amino acids contains one mutually conserved substitution: 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). Such choices of conserved amino acid substitutions fall within the skill of those skilled in the art, as 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).

[0056] It should be understood that, in a preferred embodiment, the functionally equivalent variant of Omomyc contains mutations at positions E61, E68, R74, and R75 found in Omomyc derived from human c-Myc, and preferably at positions E61T, E68I, R74Q, and R75N ​​found in Omomyc derived from human c-Myc. The positions where these mutations must occur in the functionally equivalent variant can be determined by multiple sequence alignment of different Myc sequences and identified by comparing those positions 61, 68, 74, and 75 within the Omomyc sequence derived from human c-Myc. In this embodiment, the functionally equivalent variant of Omomyc contains mutations at positions E61T, E68I, R74Q, and R75N ​​found in Omomyc derived from human c-Myc.

[0057] In another embodiment, the functionally equivalent variant of Omomyc contains mutations at positions E61, E68, R74, and R75 within the sequence corresponding to Omomyc, wherein E61 has been mutated to E61A or E61S; E68 has been mutated to E68L, E68M, or E68V; R74 has been mutated to R74N; and R75 has been mutated to R75Q.

[0058] Multiple sequence alignment is an extension of paired alignment, allowing the merging of two or more sequences at once. Multiple alignment methods align all sequences in a given query set. Preferred multiple sequence alignment procedures (and their algorithms) are ClustalW, Clusal2W, or ClustalW XXL (see Thompson et al. (1994) Nucleic Acids Res 22:4673-4680). Once c-Myc sequences and variants from different organisms are compared (aligned) as described herein, those skilled in the art can easily identify positions in each sequence corresponding to E61T, E68I, R74Q, and R75N ​​found in Omomyc, and introduce mutations in Omomyc variants corresponding to the E61T, E68I, R74Q, and R75N ​​mutations found in Omomyc derived from human c-Myc.

[0059] Suitable assays for determining whether a peptide can be considered a functionally equivalent variant of Omomyc include, but are not limited to: - Assays that measure the ability of peptides to form dimer complexes with Max and Myc, such as assays based on reporter gene expression, as described by Soucek et al. (Oncogene, 1998, 17: 2463-2472), as well as PLA (protein linkage assay) or coimmunoprecipitation.

[0060] - Assays that measure the ability of a peptide to bind to the Myc / Max recognition site (CACGTG site) within DNA, such as the electrophoretic mobility shift assay (EMSA) described by Soucek et al. (above). - Assays for measuring the ability to repress Myc-induced transcriptional activation, such as the assays described by Soucek et al. (above) based on control of the reported gene expression at a specific DNA binding site of Myc / Max.

[0061] - Assays based on the ability of peptides to inhibit the growth of cells expressing Myc, as described by Soucek et al. (above).

[0062] - Assays for measuring the ability of peptides to enhance Myc-induced apoptosis, such as those described in Soucek et al. (Oncogene, 1998: 17, 2463-2472). Alternatively, any assay commonly known in the art for assessing cell apoptosis may be used, such as Hurst staining, propidium iodide (PI) or annexin V staining, trypan blue, DNA ladder / fragmentation, and TUNEL assays.

[0063] - A assay for measuring the ability of peptides to reduce proliferation and induce cell death in spleen cells derived from EAE-infected mice, as described in the examples of this invention.

[0064] - An assay for measuring the ability of peptides to delay disease onset and improve the clinical course of EAE disease, as described in the examples of the present invention.

[0065] In a preferred embodiment, if the polypeptide exhibits at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% of the activity of natural Omomyc in one or more of the above assays, the polypeptide is considered to be a functionally equivalent variant of Omomyc.

[0066] In a particular embodiment, a functionally equivalent variant of the polypeptide of SEQ ID NO: 1 includes the polypeptide of SEQ ID NO: 1, wherein residue X at position 89 of SEQ ID NO: 1 is not cysteine. Preferably, residue X at position 89 of SEQ ID NO: 1 is an aliphatic amino acid, or a sulfurized amino acid, or a dicarboxylic acid amino acid or their amide, or an amino acid having two basic groups, or an aromatic amino acid, or a cyclic amino acid, or a hydroxylated amino acid. More preferably, the amino acid is selected from serine, threonine, and alanine, and more preferably from serine and alanine.

[0067] The table below discloses suitable functionally equivalent variants of SEQ ID NO:1 where residue X at position 89 of SEQ ID NO:1 is not cysteine.

[0068]

[0069] Therefore, in a preferred embodiment, the functionally equivalent variant of the polypeptide of SEQ ID NO: 1 is selected from the group consisting of: SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9 and SEQ ID NO: 10.

[0070] Furthermore, functionally equivalent variants of Omomyc are also capable of transducing cells upon contact with the cells. It should be understood that these functionally equivalent variants of Omomyc contain either the protein transduction domain found in natural Omomyc or another functional protein transduction domain.

[0071] In a preferred embodiment, if the polypeptide can transduce target cells with an efficiency of at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% of SEQ ID NO: 1, then the polypeptide is considered a functionally equivalent variant of SEQ ID NO: 1.

[0072] In addition, the functionally equivalent variant of SEQ ID NO: 1 can also translocate to the nucleus of the target cell.

[0073] In a preferred embodiment, if the polypeptide can translocate to the nucleus of the target cell with an efficiency of at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% of SEQ ID NO: 1, then the polypeptide is considered a functionally equivalent variant of SEQ ID NO: 1.

[0074] A suitable assay for determining whether a polypeptide is a functionally equivalent variant of SEQ ID NO: 1 (in terms of its ability to cross the cell membrane and translocate to the cell nucleus) comprises double labeling the cells with a reagent specific to the polypeptide and a dye that specifically labels the cell nucleus (such as DAPI or Hearst dye). Detection of the polypeptide according to the invention can be performed by confocal microscopy or by fluorescence microscopy.

[0075] In another preferred embodiment, the compound used according to the invention is (b) a conjugate containing a polypeptide comprising the sequence SEQ ID NO: 1 or a functionally equivalent variant thereof and a chemical moiety that promotes cellular uptake of the polypeptide or a functionally equivalent variant thereof.

[0076] As used in this article, the term "conjugate" refers to two or more compounds covalently linked together such that the function of each compound is retained in the conjugate.

[0077] The term "chemical moiety" refers to any chemical compound containing at least one carbon atom. Examples of chemical moieties include, but are not limited to, any peptide chain rich in hydrophobic amino acids and hydrophobic chemical moieties.

[0078] In a preferred embodiment, the conjugate according to the invention comprises at least one, at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, at least ten or more chemical moieties that promote cellular uptake of the polypeptide or a functionally equivalent variant of the polypeptide.

[0079] In one embodiment, the chemical component that promotes cellular uptake of the peptide is a lipid or fatty acid.

[0080] Fatty acids are typically molecules comprising carbon chains with an acidic portion (e.g., a carboxylic acid) at the end. The carbon chain of a fatty acid can be of any length; however, the length is preferably at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or more carbon atoms, and any range thereof. In some embodiments, the length of the carbon chain in the chain portion of the fatty acid is from 4 to 18 carbon atoms. In some embodiments, the fatty acid carbon chain can contain an odd number of carbon atoms; however, in some embodiments, an even number of carbon atoms in the chain is preferred. Fatty acids containing only single bonds in their carbon chain are called saturated fatty acids, while fatty acids containing at least one double bond in their chain are called unsaturated fatty acids. Fatty acids can be branched, although in a preferred embodiment of the invention, they are 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, and arachidonic acid.

[0081] In a preferred embodiment, the chemical component that promotes cellular uptake of the polypeptide comprising the sequence SEQ ID NO: 1 or a functionally equivalent variant thereof is a cell-penetrating peptide sequence. In this case, the conjugate will comprise a fusion protein containing the polypeptide comprising SEQ ID NO: 1 or a functionally equivalent variant thereof and a cell-penetrating peptide sequence.

[0082] The term "fusion protein" refers to a protein produced by genetic technology that consists of two or more functional domains derived from different proteins. Fusion proteins can be obtained by conventional methods, such as by gene expression of the nucleotide sequence encoding the fusion protein in suitable cells. It should be understood that a cell-penetrating peptide refers to a cell-penetrating peptide that is distinct from cell-penetrating peptides that form portions of the polypeptide comprising SEQ ID NO: 1 or functionally equivalent variants of SEQ ID NO: 1.

[0083] The term "cell-penetrating peptide sequence" is used interchangeably with "CPP," "protein transduction domain," or "PTD" in this invention. It refers to a variable-length peptide chain that directs protein transport within the cell. Delivery into the cell typically occurs via endocytosis, but peptides can also be internalized into the cell via direct membrane translocation. CPPs typically have an amino acid composition that either contains a relatively large abundance of positively charged amino acids (such as lysine or arginine) or has a sequence containing alternating patterns of polar / charged and nonpolar hydrophobic amino acids.

[0084] Examples of CPPs that can be used in this invention include, but are not limited to: the CPP found in Drosophila antennal foot protein (RQIKIWFQNRRMKWKK. SEQ ID NO: 13), the CPP found in the herpes simplex virus 1 (HSV-1) VP22 DNA-binding protein (DAATATRGRSAASRPTERPRAPARSASRPRRPVE, SEQ ID NO: 14), the CPP of Bac-7 (RRIRPRPPRLPRPRPRPLPFPRPG; SEQ ID NO: 15), the CPP of HIV-1 TAT protein composed of amino acids 49-57 (RKKRRQRRR, SEQ ID NO: 16), amino acids 48-60 (GRKKRRQRRRTPQ, SEQ ID NO: 17), amino acids 47-57 (YGRKKRRQRRR; SEQ ID NO: 18), and the CPP of S413-PV peptide (ALWKTLLKKVLKAPKKKRKV; SEQ ID NO: 18). 19), CPP of penetratin (RQIKWFQNRRMKWKK; SEQ ID NO: 20), CPP of SynB1 (RGGRLSYSRRRFSTSTGR; SEQ ID NO: 21), CPP of SynB3 (RRLSYSRRRF; SEQ ID NO: 22), CPP of PTD-4 (PIRRRKKLRRLK; SEQ ID NO: 23), CPP of PTD-5 (RRQRRTSKLMKR; SEQ ID NO: 24), CPP of FHV Coat-(35-49) (RRRRRNRTRRNRRRVR; SEQ ID NO: 25), CPP of BMV Gag-(7-25) (KMTRAQRRAAARRNRWTAR; SEQ ID NO: 26), HTLV-II CPP of Rex-(4-16) (TRRQRTRRARRRNR; SEQ ID NO: 27), CPP of D-Tat (GRKKRRQRRRPPQ; SEQ ID NO: 28), CPPR9-Tat (GRRRRRRRRRPPQ; SEQ ID NO: 29), MAP's CPP (KLALKLALKLALALKLA; SEQ ID NO: 30), SBP's CPP (MGLGLHLLVLAAALQGAWSQPKKKRKV; SEQ ID NO: 31), CPP of FBP (GALFLGWLGAAGSTMGAWSQPKKKRKV;SEQ ID NO: 32), CPP of MPG (ac-GALFLGFLGAAGSTMGAWSQPKKKRKV-cya; SEQ ID NO: 33), CPP of MPG(ENLS) (ac-GALFLGFLGAAGSTMGAWSQPKSKRKV-cya; SEQ ID NO: 34), CPP of Pep-1 (ac-KETWWETWWTEWSQPKKKRKV-cya; SEQ ID NO: 35), CPP of Pep-2 (ac-KETWFETWFTEWSQPKKKRKV-cya; SEQ ID NO: 36), polyarginine sequences with structural RN (where N is between 4 and 17), GRKKRRQRRR sequence (SEQ ID NO: 37), RRRRRRLR sequence (SEQ ID NO: 38), RRQRRTSKLMKR sequence (SEQ ID NO: 39); transport protein GWTLNSAGYLLGKINLKALAALAKKIL (SEQ ID NO: 39). 40); KALAWEAKLAKALAKALAKHLAKALAKALKCEA (SEQ ID NO: 41); RQIKIWFQNRRMKWKK (SEQ ID NO: 42), YGRKKRRQRRR sequence (SEQ ID NO: 43); RKKRRQRR sequence (SEQ ID NO: 44); YARAAARQARA sequence (SEQ ID NO: 45); THRPRRRRRR sequence (SEQ ID NO: 40); NO:46); GGRRARRRRRR sequence (SEQ ID NO:47). ;

[0085] In a preferred embodiment, the cell-penetrating peptide is not an endogenous peptide contained in SEQ ID NO: 1.

[0086] In a preferred embodiment, the cell-penetrating peptide sequence is selected from the group consisting of: SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:37 and SEQ ID NO:38.

[0087] In another preferred embodiment, the CPP is the CPP of the HIV-1 TAT protein, which consists of amino acids 49-57 (RKKRRQRRR, SEQ ID NO: 16). In another preferred embodiment, the CPP is a GRKKRRQRRR sequence (SEQ ID NO: 37) or RRRRRLR (SEQ ID NO: 38). In another preferred embodiment, the CPP is a GRKKRRQRRR sequence (SEQ ID NO: 37) or RRRRRRR (SEQ ID NO: 65).

[0088] In some implementations, the CPP is the CPP as described in WO2019 / 018898, the contents of which are incorporated herein by reference in their entirety.

[0089] In one embodiment, the cell-penetrating peptide sequence is fused to the N-terminus of the polypeptide of the present invention or a functionally equivalent variant of the polypeptide. In another embodiment, the cell-penetrating peptide is fused to the C-terminus of the polypeptide of the present invention or a functionally equivalent variant of the polypeptide.

[0090] In a preferred embodiment, the conjugate or fusion protein of the compound for use according to the invention comprises, in addition to the self-cell-penetrating peptide found in the polypeptide of SEQ ID NO: 1 or a functionally equivalent variant of the 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.

[0091] Suitable fusion proteins of the present invention include the peptides Omomyc*TAT and Omomyc*LZArg as defined below:

[0092] Therefore, in a preferred embodiment, the fusion protein is a polypeptide selected from SEQ ID NO: 11 and 12.

[0093] Suitable assays for determining whether a conjugate retains the cell membrane translocation ability of Omomyc include, but are not limited to, assays that measure the ability of the conjugate to transduce cells in a culture. Such assays are based on contacting the conjugate with cultured cells and detecting the presence of the conjugate at its intracellular location.

[0094] In another preferred embodiment, the conjugate of the compound used according to the invention further contains other nuclear localization signals.

[0095] As used herein, the term “nuclear localization signal” (NLS) refers to an amino acid sequence of approximately 4–20 amino acid residues in length that guides a protein to the cell nucleus. Typically, NLS sequences are rich in basic amino acids, and exemplary sequences are well known in the art (Gorlich D. (1998) EMBO 5.17:2721–7). In some embodiments, the NLS is selected from the group consisting of: SV40 large T antigen NLS (PKKKRKV, SEQ ID NO: 48); nucleoplasmic protein NLS (KRPAATKKAGQAKKKK, SEQ ID NO: 49); CBP80 NLS (RRRHSDENDGGQPHKRRK, SEQ ID NO: 50); HIV-I Rev protein NLS (RQARRNRRRWE, SEQ ID NO: 51); HTLV-I Rex (MPKTRRRPRRSQRKRPPT, SEQ ID NO: 52); hnRNP A NLS (NQSSNFGPMKGGNFGGRSSGPYGGGGQYFKPRNQGGY, SEQ ID NO: 53); rpL23a NLS (VHSHKKKKIRTSPTFTTPKTLRLRRQPKYPRKSAPRRNKLDHY, SEQ ID NO: 54). In one embodiment of the present invention, the nuclear localization signal comprises the motif K (K / R) X (K / R) (SEQ ID NO:55).

[0096] In an even more preferred embodiment, the nuclear positioning signal is selected from the group consisting of PKKKRKV (SEQ ID NO: 48), PAAKRVKLD (SEQ ID NO: 56) and KRPAATKKAGQAKKKK (SEQ ID NO: 49).

[0097] In another preferred embodiment, NLS may be the N-terminus or C-terminus of a conjugate or fusion protein comprising the polypeptide of SEQ ID NO: 1 or a functionally equivalent variant thereof.

[0098] Those skilled in the art will understand that it may be desirable for the conjugate according to the invention to further comprise one or more flexible peptides, the linker of which comprises the polypeptide of SEQ ID NO: 1 or a functionally equivalent variant thereof, a cell-penetrating peptide sequence, and / or an NLS. Thus, in a particular embodiment, the polypeptide comprising SEQ ID NO: 1 or a functionally equivalent variant thereof is directly linked to the cell-penetrating peptide sequence. In another particular embodiment, the polypeptide comprising SEQ ID NO: 1 or a functionally equivalent variant thereof is linked to the cell-penetrating peptide sequence via a flexible peptide. In one embodiment, the polypeptide comprising SEQ ID NO: 1 or a functionally equivalent variant thereof is directly linked to an NLS. In another embodiment, the polypeptide comprising SEQ ID NO: 1 or a functionally equivalent variant thereof is linked to an NLS via a flexible peptide.

[0099] In a particular embodiment, the polypeptide of the conjugate according to the present invention is directly linked to a cell-penetrating peptide sequence and an NLS.

[0100] In one embodiment, the NLS is one of the NLS that occurs endogenously in the Myc sequence, such as the M1 peptide (PAAKRVKLD, SEQ ID NO: 56) or the M2 peptide (RQRRNELKRSF, SEQ ID NO: 57).

[0101] In another embodiment, the additional NLS refers to an NLS that is different from the endogenous NLS found in the polypeptide containing SEQ ID NO: 1 or a functionally equivalent variant of SEQ ID NO: 1.

[0102] In a preferred embodiment, the conjugate or fusion protein used according to the invention contains, in addition to the endogenous NLS found in the polypeptide or its functionally equivalent variants of the invention, at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, or at least 10 NLS.

[0103] In another specific embodiment, the polypeptide of the conjugate according to the present invention is linked to a cell-penetrating peptide sequence via a first flexible peptide linker and to an NLS via a second flexible peptide linker.

[0104] As used herein, the terms "flexible peptide," "interval peptide," or "linker peptide" refer to a peptide that covalently binds to two proteins or portions of but not all of a polypeptide, allowing one polypeptide to move relative to the other without materially or detrimentally affecting the function of the protein or portion. Therefore, flexible linkers do not affect the activity of the polypeptide sequence, the cell-penetrating activity of cell-penetrating peptides, or the nuclear localization ability of NLS.

[0105] The flexible peptide comprises at least one amino acid, at least two amino acids, at least three amino acids, at least four amino acids, at least five amino acids, at least six amino acids, at least seven amino acids, at least eight amino acids, at least nine amino acids, at least 10 amino acids, at least 12 amino acids, at least 14 amino acids, at least 16 amino acids, at least 18 amino acids, at least 20 amino acids, at least 25 amino acids, at least 30 amino acids, at least 35 amino acids, at least 40 amino acids, at least 45 amino acids, at least 50 amino acids, at least 60 amino acids, at least 70 amino acids, at least 80 amino acids, at least 90 amino acids, or about 100 amino acids. In some embodiments, the flexible peptide will allow one protein to move relative to another protein to increase protein solubility and / or improve its activity. Suitable linker regions include polyglycine regions, i.e., the GPRRRR sequence of a combination of glycine, proline, and alanine residues (SEQ ID NO: 58).

[0106] In certain embodiments, the conjugates used according to the invention comprise a tag that binds to a C-terminal or N-terminal domain of the conjugate or the said polypeptide or fusion protein or a variant thereof. The tag is typically a peptide or amino acid sequence that can be used to isolate or purify the fusion protein. Therefore, the tag is capable of binding with high affinity to one or more ligands, such as one or more ligands of an affinity matrix (such as a chromatographic support or beads). Examples of such tags are histidine tags (His tags or HT), such as tags containing 6 histidine residues (His6 or H6), which can bind with high affinity to nickel (Ni... 2+ ) or cobalt (Co) 2+ His tags have the desirable characteristic of binding to their ligands under conditions that denature most proteins and disrupt most protein-protein interactions. Therefore, they can be used to remove H6-tagged bait proteins after the protein-protein interactions involved in the bait have been disrupted.

[0107] Additional exemplary, non-limiting examples of tags for isolating or purifying conjugates or peptides containing SEQ ID NO: 1, or variants thereof, or fusion proteins include Arg tags, FLAG tags (DYKDDDDK; SEQ ID NO: 59), Strep tags (WSHPQFEK, SEQ ID NO: 60), epitopes that can be recognized by antibodies such as c-myc tags (recognized by anti-c-myc antibodies), HA tags (YPYDVPDYA, SEQ ID NO: 61), V5 tags (GKPIPNPLLGLDST, SEQ ID NO: 62), SBP tags, S tags, calmodulin-binding peptides, cellulose-binding domains, chitin-binding domains, glutathione S-transferase tags, maltose-binding proteins, NusA, TrxA, DsbA, Avi tags, etc. (Terpe K., Appl. Microbiol. Biotechnol. 2003, 60:523-525), and amino acid sequences such as AHGHRP (SEQ ID NO: 1). 63) or PIHDHDHPHLVIHSGMTCXXC (SEQ ID NO: 64), β-galactosidase, etc.

[0108] If necessary, tags can be used to isolate or purify the fusion protein.

[0109] In another preferred embodiment, the compound used according to the invention is (c) a polynucleotide, hereinafter referred to as the polynucleotide of the invention, encoding the polypeptide or fusion protein disclosed above. In a preferred embodiment, the compound used according to the invention is a polynucleotide encoding a polypeptide comprising the sequence SEQ ID NO: 1 or a functionally equivalent variant thereof. In another embodiment, the compound used according to the invention is a polynucleotide encoding a conjugate containing a polypeptide comprising the sequence SEQ ID NO: 1 or a functionally equivalent variant thereof and a chemical moiety promoting cellular uptake of the polypeptide or a functionally equivalent variant thereof; more preferably, it is a polynucleotide encoding a fusion protein comprising a polypeptide comprising the sequence SEQ ID NO: 1 or a functionally equivalent variant thereof and a cell-penetrating peptide sequence.

[0110] The terms “polynucleotide,” “nucleic acid,” and “nucleic acid molecule” are used interchangeably to refer to a polymer of nucleotides of any length. Polynucleotides may contain deoxyribonucleotides, ribonucleotides, and / or their analogues. Nucleotides can have any three-dimensional structure and can perform any known or unknown function. The term “polynucleotide” includes, for example, single-stranded, double-stranded, and triple-stranded molecules, genes or gene fragments, exons, introns, mRNA, tRNA, rRNA, ribozymes, cDNA, recombinant polynucleotides, branched polynucleotides, plasmids, vectors, isolated DNA of any sequence, isolated RNA of any sequence, nucleic acid probes, and primers. In addition to natural nucleic acid molecules, the nucleic acid molecules of this invention may also comprise modified nucleic acid molecules. As used herein, mRNA refers to RNA that can be translated in cells.

[0111] In a preferred embodiment, the polynucleotide used according to the invention is mRNA. mRNA can be chemically synthesized, obtained by in vitro transcription, or synthesized in vivo in target cells. The nucleotide sequences forming the polynucleotide encoding the conjugate or fusion protein according to the invention are located in the same correct reading frame for their expression.

[0112] In a preferred embodiment, the compound used according to the invention is an mRNA encoding a polypeptide consisting of the sequence SEQ ID NO: 1, a polypeptide consisting of a functionally equivalent variant of SEQ ID NO: 1, or a polypeptide consisting of SEQ ID NO: 4.

[0113] In another embodiment, the compound used according to the invention is (d) a carrier containing the polynucleotide of the invention.

[0114] As used herein, the term "vector" refers to a nucleic acid sequence containing the necessary sequence such that, upon transcription and translation of said sequence in a cell, a polypeptide encoded by the polynucleotide of the present invention is produced. The sequence is operatively linked to additional fragments that provide autonomous replication within the target host cell. Preferably, the vector is an expression vector, defined as containing, in addition to a region that autonomously replicates within the host cell, a region operatively linked to the nucleic acid of the present invention and capable of enhancing the expression of the nucleic acid product according to the present invention. Vectors for use according to the present invention can be obtained using techniques widely known in the art.

[0115] Examples of vectors include, but are not limited to, viral vectors, naked DNA or RNA expression vectors, plasmids, granules or phage vectors, DNA or RNA expression vectors associated with cationic condensers, DNA or RNA expression vectors encapsulated in liposomes, and certain eukaryotic cells such as production cells. Suitable vectors containing the polynucleotides of the present invention are derived from the following: expression vectors in prokaryotes, such as pUC18, pUC19, pBluescript and its derivatives, mp18, mp19, pBR322, pMB9, ColE1, pCR1, RP4; bacteriophages and "shuttle" vectors, such as pSA3 and pAT28; expression vectors in yeast, such as 2-micron plasmid vectors, integrative plasmids, YEP vectors, centromere plasmids, etc.; expression vectors in insect cells, such as pAC and pVL series vectors; expression vectors in plants, such as pIBI, pEarleyGate, pAVA, pCAMBIA, pGSA, pGWB, pMDC, pMY, pORE, etc.; expression vectors in superior eukaryotic cells based on viral vectors (adenovirus, adenovirus-associated virus, etc., and retroviruses, and especially lentiviruses); and 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 polynucleotides used according to the invention are contained in a vector selected from the group consisting of pEGFP or pBabe retroviral vectors and pTRIPZ or pSLIK lentiviral vectors.

[0116] The vector according to the present invention can be used to transform, transfect, or infect cells that can be transformed, transfected, or infected by said vector. The cells can be prokaryotic or eukaryotic cells.

[0117] The vector preferably comprises a polynucleotide according to the present invention, which is operatively bound to a sequence regulating the expression of the polynucleotide. The regulatory sequence used in the present invention may be a nuclear promoter, or alternatively, an enhancer sequence 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 the promoter is compatible with the cell in which the polynucleotide is to be expressed. Therefore, promoters suitable for implementing the present invention include, but are not limited to, constitutive promoters, such as derivatives of eukaryotic viral genomes, such as polyomavirus, adenovirus, SV40, CMV, avian sarcoma virus, hepatitis B virus, metallothionein gene promoters, herpes simplex virus thymidine kinase gene promoters, LTR regions of retroviruses, immunoglobulin gene promoters, actin gene promoters, EF-1α gene promoters, and inducible promoters, wherein protein expression depends on the addition of molecular or exogenous signals, such as tetracycline systems, NFκB / UV light systems, Cre / Lox systems, heat shock gene promoters, regulated RNA polymerase II promoters and tissue-specific promoters described in WO / 2006 / 135436.

[0118] In another embodiment, the compound used according to the invention is (e) a cell capable of secreting the polypeptide or conjugate of the invention, preferably the polypeptide or fusion protein of the invention, into a culture medium.

[0119] Suitable cells capable of secreting the polypeptides of the present invention include, but are not limited to, cardiomyocytes, adipocytes, endothelial cells, epithelial cells, lymphocytes (B cells and T cells), mast cells, eosinophils, vascular endothelial cells, primary cultures of isolated cells from different organs, preferably cells isolated from Langerhans islets, hepatocytes, leukocytes (including monocytes), mesenchymal cells, umbilical cord or adult (skin, lung, kidney, and liver) cells, osteoclasts, chondrocytes, and other connective tissue cells. Cells from established cell lines, such as Jurkat T cells, NIH-3T3, CHO, Cos, VERO, BHK, HeLa, COS, MDCK, 293, 3T3 cells, C2C12 myoblasts, and W138 cells, are also suitable. Those skilled in the art will understand that cells capable of secreting the polypeptides of the present invention into a culture medium can form microparticles or microcapsules, thereby extending the cell lifespan in the patient. Materials suitable for forming the particulate objects of the present invention include any biocompatible polymeric material that allows for the continuous secretion of therapeutic products and acts as a carrier for cells. Therefore, the biocompatible polymeric material can be, for example, a thermoplastic polymer or a hydrogen polymer. Examples of thermoplastic polymers are acrylic acid, acrylamide, 2-aminoethyl methacrylate, poly(tetrafluoroethylene-co-hexafluoropropylene), methacrylate-(7-coumarinoxy)ethyl acetate, N-isopropylacrylamide, polyacrylic acid, polyacrylamide, polyamide amine, poly(amino)-p-xylene, poly(chloroethyl vinyl ether), polycaprolactone, poly(caprolactone-co-trimethylene carbonate), poly(carbonate urea) polyurethane, poly(carbonate) polyurethane, polyethylene, polyethylene and acrylamide copolymer, polyethylene glycol, polyethylene glycol methacrylate, poly(p-xylene)-co-xylene, and poly(chloroethyl vinyl ether). Polyethylene phthalate, poly(4-hydroxybutyl acrylate), poly(hydroxyethyl methacrylate), poly(N-2-hydroxypropyl methacrylate), poly(glycolic acid lactate), poly(L-lactic acid), poly(γ-methyl,L-glutamic acid), poly(methyl methacrylate), poly(propylene glycol fumarate), poly(propylene oxide), polypyrrole, polystyrene, polytetrafluoroethylene, polyurethane, polyvinyl alcohol, ultra-high molecular weight polyethylene, 6-(p-vinylbenzamide)-hexanoic acid N-p-vinylbenzyl-D-maltamide, and copolymers containing one or more of the above polymers. Examples of hydrogel polymers are natural materials such as alginate, agarose, collagen, starch, hyaluronic acid, bovine serum albumin, cellulose and its derivatives, pectin, chondroitin sulfate, fibroin, and silk fibroin, as well as synthetic hydrogels such as Sepharose® and Sephadex®.

[0120] The compounds according to the present invention are used for the prevention and / or treatment of autoimmune diseases in subjects.

[0121] Therefore, in one aspect, the present invention relates to a compound selected from the group consisting of: (a) A polypeptide containing the sequence SEQ ID NO: 1 or a functionally equivalent variant thereof, (b) A conjugate comprising a polypeptide comprising the sequence SEQ ID NO: 1 or a functionally equivalent variant thereof and a chemical moiety that promotes cellular uptake of said polypeptide or a functionally equivalent variant thereof. (c) Polynucleotides encoding a) polypeptides or b) conjugates. (d) A vector containing the polynucleotides according to (c), and (e) Cells capable of secreting the polypeptide according to (a) or the conjugate according to (b) into the culture medium. Used for the prevention of autoimmune diseases in subjects.

[0122] The term "prevention" is understood to mean administering a compound according to the invention at the initial or early stage of a disease, or simultaneously preventing its onset. In a particular embodiment, the term "prevention" means administering the compound before the onset of an autoimmune disease, i.e., before the appearance of symptoms.

[0123] In another respect, the present invention relates to a compound selected from the group consisting of: (a) A polypeptide containing the sequence SEQ ID NO: 1 or a functionally equivalent variant thereof, (b) A conjugate comprising a polypeptide comprising the sequence SEQ ID NO: 1 or a functionally equivalent variant thereof and a chemical moiety that promotes cellular uptake of said polypeptide or a functionally equivalent variant thereof. (c) Polynucleotides encoding a) polypeptides or b) conjugates. (d) A vector containing the polynucleotides according to (c), and (e) Cells capable of secreting the polypeptide according to (a) or the conjugate according to (b) into the culture medium. Used for the treatment of autoimmune diseases in subjects.

[0124] The term "treatment" is used to refer to the administration of a compound according to the invention for the purpose of controlling disease progression before or after the onset of clinical signs. Controlling disease progression is understood as a beneficial or desired clinical outcome, including but not limited to: symptom relief, reduction of disease duration, stabilization of pathological condition (partially avoiding additional damage), delay of disease progression, improvement of pathological condition, and remission (partial and complete remission). Controlling disease progression also involves an extension of survival compared to expected survival without treatment. In a preferred embodiment, control of disease progression is measured by a reduction in weight loss. In another preferred embodiment, control of disease progression is measured by a lower clinical score, preferably according to an example of the invention. In another preferred embodiment, control of disease progression is measured by a lower neurological dysfunction score, preferably according to an example of the invention. In another embodiment, control of disease progression is measured by a delay in the onset of disease.

[0125] The term "autoimmune disease" as used in this invention refers to a pathogenic condition in which a patient's immune system develops a disease due to its own antigens (autoimmunity). Autoimmune diseases are a group of diseases caused by a compromised immune system, which triggers a reaction of the immune system against the body's own tissues. Autoimmunity is present in everyone to some extent. It is generally harmless and may be a common phenomenon in vertebrate life. However, autoimmunity can be the cause of a wide variety of human diseases known as autoimmune diseases. Changes in cellular recognition mechanisms that typically enable the body to distinguish between "self" and "non-self"—that is, to differentiate between elements belonging to the body and elements foreign to the body—lead to antibody production and the differentiation of specific T cells and B cells. These cells can target a single organ (organ-specific disease) or trigger systemic illness, thereby impairing the function of the entire individual. Therefore, when benign autoimmunity progresses to pathogenic autoimmunity, it is defined as an autoimmune disease. This progression is determined by both genetic influences and environmental triggers. The autoimmune diseases that this invention can treat and / or prevent include systemic lupus erythematosus (SLE), lupus nephritis, central nervous system (CNS) lupus, type I diabetes, asthma, ulcerative colitis, Crohn's disease, Graves' disease, Addison's disease, celiac disease, alopecia areata, arthritis (including rheumatoid arthritis and osteoarthritis), pernicious anemia, latent autoimmune diabetes in adults (LADA), and multiple sclerosis.

[0126] The compounds according to the present invention can be used to treat a variety of autoimmune diseases, including autoimmune blood disorders such as pernicious anemia, autoimmune hemolytic anemia, aplastic anemia, idiopathic thrombocytopenic purpura, and ankylosing spondylitis; muscle tissue autoimmune diseases such as polymyositis and dermatomyositis; ear autoimmune diseases such as autoimmune hearing loss and Meniere's syndrome; and autoimmune eye diseases such as Mooren's disease, Reiter's syndrome, Vogt-Koyanagi-Harada disease, and acute anterior uveitis. (e.g., urethritis); autoimmune kidney diseases, including glomerulonephritis, IgA nephropathy, and lupus nephritis; type I diabetes; autoimmune skin diseases, including pemphigus (autoimmune bullous diseases), such as pemphigus vulgaris, pemphigus foliaceus, pemphigus erythematosus, bullous pemphigoid, vitiligo, epidermolysis bullosa acquisita, psoriasis, and alopecia areata;Cardiovascular autoimmune diseases, including autoimmune myocarditis, vasculitis including Churg-Strauss syndrome, giant cell arteritis, Kawasaki disease, polyarteritis nodosa, Takayasu's arteritis, and Wegener's granulomatosis; endocrine autoimmune diseases, including Addison's disease, autoimmune hypoparathyroidism, autoimmune hypophysitis, autoimmune oophoritis, autoimmune orchitis, Graves' disease, and Hashimoto's thyroiditis. Autoimmune diseases include: thyroiditis, polyglandular autoimmune syndrome type 1 (PAS-I), polyglandular autoimmune syndrome type 2 (PAS-2), and polyglandular autoimmune syndrome type 3 (PAS-3); autoimmune gastrointestinal diseases, including autoimmune hepatitis, primary biliary cirrhosis, inflammatory bowel disease, celiac disease, and Crohn's disease; and autoimmune neurological diseases, including multiple sclerosis, myasthenia gravis, Guillain-Barré syndrome, and chronic inflammatory demyelinating neuropathy.And systemic autoimmune diseases, including systemic lupus erythematosus, antiphospholipid syndrome, autoimmune lymphoproliferative disease, autoimmune polyendocrinopathy, Bechet's disease, Goodpasture's disease, arthritis (including rheumatoid arthritis, osteoarthritis, and septic arthritis), sarcoidosis, scleroderma, Sjogren's syndrome, and psoriasis.

[0127] In a particular implementation, the autoimmune disease is selected from the group consisting of: celiac disease, type I diabetes, Graves' disease, inflammatory bowel disease, multiple sclerosis, alopecia areata, Addison's disease, pernicious anemia, psoriasis, rheumatoid arthritis, latent autoimmune diabetes mellitus in adults (LADA), and systemic lupus erythematosus.

[0128] In a more specific implementation, the autoimmune disease is selected from the group consisting of diabetes mellitus (type I), multiple sclerosis, and psoriasis.

[0129] In a preferred embodiment, the autoimmune disease is an autoimmune demyelinating disease of the central nervous system (CNS).

[0130] CNS autoimmune demyelinating diseases are those in which myelin-supporting cells (such as oligodendrocytes) and / or myelin lamellae in the CNS are destroyed. Demyelination leads to a disruption of neural signals between the brain and the rest of the body, ultimately resulting in a range of signs and symptoms, including physical, mental, and sometimes even psychiatric problems.

[0131] The CNS autoimmune demyelinating diseases treatable by this invention include, but are not limited to: multiple sclerosis (MS), clinically isolated syndrome (CIS), tumor-like MS, Marburg's acute MS, Balós's concentric sclerosis, acute disseminated encephalomyelitis (ADEM), acute hemorrhagic leukoencephalitis, Schilder's disease, solitary sclerosis, transverse myelitis, Susac's syndrome, leukoaraiosis, myalgic encephalomyelitis, Guillain-Barré syndrome, and progressive inflammatory neuropathy. Inflammatory neurophaathy, leukodystrophy, including adrenoleukodystrophy, adrenomyeloneuropathy, post-vaccinal encephalitis (PVE), post-infectious encephalomyelitis (PIE), neuromyelitis optica (NMO), optic neuritis, and myelin oligodendrocyte glycoprotein (MOG)-related disease (MOGAD). Preferably, the CNS autoimmune demyelinating disease is selected from the group consisting of: multiple sclerosis (MS), clinically isolated syndrome (CIS), tumor-like MS, Marburg acute MS, Barlow concentric sclerosis, acute disseminated encephalomyelitis (ADEM), post-vaccination encephalomyelitis (PVE), post-infectious encephalomyelitis (PIE), and neuromyelitis optica (NMO). In a more specific embodiment, the autoimmune disease is selected from multiple sclerosis (MS), acute disseminated encephalomyelitis (ADEM), and post-vaccination encephalomyelitis (PVE).In a still more preferred embodiment, the autoimmune disease is multiple sclerosis.

[0132] There is a lack of natural non-human counterparts for MS. Therefore, the EAE model is an acceptable animal model for MS and other CNS autoimmune demyelinating diseases characterized by demyelination and neurological dysfunction, as well as corresponding clinical symptoms (Baxter AG. The origin and application of experimental autoimmune encephalomyelitis. Nat Rev Immunol 2007, 7:904-912; Storch MK et al. Autoimmunity to myelin oligodendrocyte glycoprotein in rats mimics the spectrum of multiple sclerosis pathology. Brain Pathol. 1998, 8(4):681-94 and Hirano Y. Acute disseminated encephalomyelitis. Nippon Rinsho. 1997, 55(4):934-9.).

[0133] The experiments of this invention were conducted in an experimental autoimmune encephalomyelitis model, sometimes also referred to as an experimental allergic encephalomyelitis (EAE) model. EAE is an inflammatory demyelinating disease of the CNS that serves as an animal model of brain inflammation. It is primarily used in rodents and has been extensively studied as an animal model of human CNS demyelinating diseases, including multiple sclerosis (MS) and clinically isolated syndrome (CIS), particularly for the treatment of MS.

[0134] Multiple sclerosis presents in several forms, including episodic acute exacerbations (which are the most common initially), progressive neurological deterioration, or a combination of both. These different forms have been given standardized names: relapsing-remitting (RR), secondary progressive (SP), primary progressive (PP), and progressive relapsing (PR). RR-MS is defined as a well-defined relapse of the disease that results in either complete recovery, sequelae, or residual deficits after recovery; the period between relapses is characterized by the absence of disease progression. Approximately 85% of patients begin with RR-MS (Lublin FD et al., Defining the clinical course of multiple sclerosis: the 2013 revisions. Neurology. 2014 Jul 15;83(3):278-86).

[0135] As used herein, the term "subject" includes any animal that has an autoimmune disease or exhibits symptoms of an autoimmune disease, or any animal at risk of having an autoimmune disease or exhibiting symptoms of an autoimmune disease. Suitable subjects (patients) include laboratory animals (such as mice, rats, rabbits, or guinea pigs), farm animals, and livestock or pets (such as cats or dogs). Non-human primates are included, preferably human patients. Preferably, the subject is a mammal.

[0136] The term "mammal" includes, but is not limited to, domesticated and farm mammals, primates, and humans, such as human populations, non-human primates, cattle, horses, pigs, sheep, goats, dogs, cats, or rodents. In a preferred embodiment, the mammal is a human.

[0137] The compounds used according to the invention can be incorporated into pharmaceutical compositions for administration to a subject, wherein the pharmaceutical composition further comprises at least one pharmaceutically acceptable excipient.

[0138] The pharmaceutical composition comprises a therapeutically effective amount of the compound according to the purpose of the invention.

[0139] Therefore, in a particular embodiment of the first aspect of the invention, the compound for use according to the invention is included in a pharmaceutical composition, wherein the pharmaceutical composition further comprises at least one pharmaceutically acceptable excipient.

[0140] In another aspect, the present invention relates to a pharmaceutical composition comprising a compound according to the invention and a pharmaceutically acceptable excipient, for the prevention and / or treatment of an autoimmune disease in a subject.

[0141] The term "pharmaceutically acceptable excipient" is understood to refer to a therapeutically inactive substance used to incorporate an active ingredient, and which is pharmacologically / toxicologically acceptable to the patient and, from a physical / chemical perspective, acceptable to the medicinal chemist who manufactures the excipient for the composition, formulation, stability, patient acceptability, and bioavailability. An excipient can be a carrier. As used herein, "carrier" means any substance used to improve the delivery and efficacy of the active ingredient within a pharmaceutical composition. In preferred embodiments, the carrier does not allow direct delivery of the compound into the cytoplasm of the cell; that is, the carrier does not fuse with the plasma membrane of the target cell. Examples of pharmaceutically acceptable carriers include one or more of the following: water, saline, phosphate-buffered saline, glucose (dextrose), glycerol, ethanol, and combinations thereof. In many cases, the composition preferably includes an isotonic agent, such as sugars, polyols such as mannitol, sorbitol, or sodium chloride. Pharmaceutically acceptable carriers may also include small amounts of adjuvants, such as wetting agents or emulsifiers, preservatives, or buffers, which can extend the shelf life or effectiveness of components forming compositions used in the present invention. Examples of suitable carriers are well known in the literature (see, for example, Remington's Pharmaceutical Sciences, 19th ed., Mack Publishing Company, Easton, PA, 1995). Examples of carriers include, but are not limited to, a range of sugars such as lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, and maltitol; a range of starches such as corn starch, wheat starch, rice starch, and potato starch; a range of celluloses such as cellulose, methylcellulose, sodium carboxymethylcellulose, and hydroxypropyl methylcellulose; and a range of fillers such as gelatin and polyvinylpyrrolidone. In some cases, disintegrants such as croscarmellose, agar, alginate, or sodium alginate may be added.

[0142] The quantity and properties of pharmaceutically acceptable excipients depend on the desired dosage form. Pharmaceutically acceptable excipients are known to those skilled in the art (Faulí y Trillo C. (1993) “Tratado de Farmacia Galénica”, Luzán 5, SA Ediciones, Madrid). The composition can be prepared by conventional methods known in the prior art (“Remington: The Science and Practice of Pharmacy”, 20th edition (2003) Genaro AR, ed., Lippincott Williams & Wilkins, Philadelphia, US).

[0143] For pharmaceutical compositions containing an agent, the agent is a nucleic acid molecule that can be present in any of the various delivery systems known to those skilled in the art, including nucleic acid and bacterial, viral, and mammalian expression systems, such as, for example, recombinant expression constructs provided herein. Techniques for integrating DNA into such expression systems are well known to those skilled in the art. The DNA may also be “naked,” for example, as described by Ulmer et al. in Science 259:1745-49, 1993, and commented on by Cohen in Science 259:1691-1692, 1993. The uptake of naked DNA can be increased by coating the DNA onto biodegradable beads that are efficiently transported into cells.

[0144] Nucleic acid molecules can be delivered into cells according to any of the 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 Patent Application Publication No. WO 94 / 02595); Selbo et al., Int. J. Cancer 87:853-59 (2000); Selbo et al., Tumour Biol. 23:103-12 (2002); U.S. Patent Application Publication 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, via iontophoresis, or by 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 Application Publications WO 03 / 47518 and WO 03 / 46185); poly(lactic-co-hydroxyacetic acid) (PLGA) and PLCA microspheres (also used for delivering peptides and polypeptides and other substances) (see, for example, U.S. Patent No. 6,447,796; U.S. Patent Application Publication No. 2002 / 130430); biodegradable nanocapsules; and bioadhesive microspheres, or via protein carriers (International Application Publication No. WO 00 / 53722). In another embodiment, the nucleic acid molecule may also be formulated or compounded with polyethyleneimine and its derivatives, such as polyethyleneimine-polyethylene glycol-N-acetylgalactosamine (PEI-PEG-GAL) or polyethyleneimine-polyethylene glycol-tri-N-acetylgalactosamine (PEI-PEG-triGAL) derivatives (see, for example, U.S. Patent Application Publication No. 2003 / 0077829).

[0145] In certain embodiments, when the compound used according to the invention is a nucleic acid, the pharmaceutical composition may be formulated as a composition for gene therapy; for example, and not limited to, the pharmaceutical composition may contain a viral or non-viral vector comprising a suitable polynucleotide or gene construct. For example, and not limited to, the vector may be a viral vector, such as those based on retroviruses, adenoviruses, etc., or a non-viral vector, such as ADN-liposomes, ADN-polymers, ADN-polymer-liposome complexes, etc. [see “Nonviral Vectors for Gene Therapy”, edited by Huang, Hung and Wagner, Academic Press (1999)]. The vector containing the corresponding polynucleotide or gene construct may be administered directly to a subject by conventional methods. Alternatively, the vector may be used to transform, transfect, or infect cells, such as mammalian cells, including human cells, ex vivo cells, and subsequently implanted into a human or animal to obtain the desired therapeutic effect. For administration to a human or animal, the cells will be formulated in a suitable culture medium that will not adversely affect cell viability.

[0146] A pharmaceutical composition comprising a compound for use according to the present invention comprises a therapeutically effective amount of the compound.

[0147] As used herein, the term "therapeutically effective amount" is understood to mean the amount that provides a therapeutic effect and can be determined by those skilled in the art using conventional methods. The amount of Omomyc peptides, their functionally equivalent variants, conjugates, fusion proteins, polynucleotides, carriers, or cells varies depending on the subject and the specific route of administration. Those skilled in the art will understand that the dosage can also be determined under the guidance of Goodman and Goldman's *The Pharmacological Basis of Therapeutics*, Ninth Edition (1996), Appendix II, pp. 1707-1711, and Goodman and Goldman's *The Pharmacological Basis of Therapeutics*, Tenth Edition (2001), Appendix II, pp. 475-493.

[0148] The appropriate dosage of one or more active elements in the pharmaceutical composition will depend on the type of autoimmune disease to be treated, the severity and course of the disease, whether the composition is administered for preventive or therapeutic purposes, prior treatment, the patient's clinical history and response to the peptide or polypeptide, and the judgment of the attending physician.

[0149] The dosage of the compound for use according to the present invention can be expressed in mg per kg body weight or mg per square meter of body surface area. (See the article by Reagan-Shaw S. et al.) Dose translation from animal to human studies revisited” FASEB J 2008, 22(3):659-661) provides a conversion of mg / kg to mg / m³. 2 The standard conversion factor.

[0150]

[0151] The article also explains that this conversion is the basis for converting the dose of one animal species to the dose of another (allometric dose conversion). Therefore, the animal dose (AD) in mg / kg can be converted to the human equivalent dose (HED) in mg / kg using the following formula:

[0152] K for each species m The results are shown in Table 1 (data extracted from Reagan-Shaw S. et al.). Dose translation from animal to human studies revisited”. FASEB J 2008, 22(3):659-661).

[0153]

[0154] Table 1. K values ​​for converting AD to HED m coefficient The amount of the polypeptide containing the sequence SEQ ID NO: 1, its functionally equivalent variants, fusion proteins, conjugates, polynucleotides, carriers, or cells is suitable for administration to a patient in a single dose or in a series of treatments. Depending on the type and severity of the disease, an appropriate dose level is typically from about 0.01 to 500 mg per kg of patient body weight per day, which can be administered as a single or multiple doses. Preferably, the dose level is from about 0.1 to about 250 mg / kg per day; more preferably, from about 0.5 to about 100 mg / kg per day. Alternatively, the dose 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 dose may be from 0.05 to 0.5, 0.5 to 5, or 5 to 50 mg / kg per day. For oral administration, the composition is preferably provided in tablet form, the tablets containing 1.0 to 1000 mg of the active ingredient, particularly 1.0, 5.0, 10.0, 15.0, 20.0, 25.0, 50.0, 75.0, 100.0, 150.0, 200.0, 250.0, 300.0, 400.0, 500.0, 600.0, 750.0, 800.0, 900.0 and 1000.0 mg of the active ingredient, for symptomatic adjustment of the dosage for the patient being treated.

[0155] In certain embodiments, the compound or pharmaceutical composition comprising the compound according to the invention is administered in a regimen comprising multiple administrations (i.e., at least two administrations). In preferred embodiments, the regimen comprises at least two administrations, at least three administrations, at least four administrations, at least five administrations, at least six administrations, at least seven administrations, at least eight administrations, at least nine administrations, or at least ten administrations. In more specific embodiments, the regimen comprises at least three administrations. In still more preferred embodiments, the regimen comprises four administrations.

[0156] In one embodiment, the compound is administered daily. In another embodiment, the compound is administered every other day.

[0157] In one embodiment, the compound can be administered once a week, twice a week, three times a week, four times a week, five times a week, six times a week, or seven times a week. In another embodiment, the compound can be administered once a week. In yet another embodiment, the compound can be administered twice a week. In yet another embodiment, the compound can be administered three times a week.

[0158] The duration of treatment can be at least 3 days, at least 4 days, at least 5 days, at least 6 days, at least one week, at least two weeks, at least three weeks, at least four weeks, at least five weeks, at least six weeks, at least seven weeks, at least eight weeks, at least nine weeks, at least ten weeks, or longer. Preferably, the duration of treatment is at least 7 days.

[0159] In a particular embodiment, the compound used according to the invention or a pharmaceutical composition containing the compound used according to the invention is administered in an early stage of an autoimmune response.

[0160] In a particular embodiment, the compound used according to the invention is administered at a dose level between 0.1 and 100 mg / kg per day, preferably between 1 and 85 mg / kg per day, and more preferably between 5 and 75 mg / kg per day.

[0161] In a particular embodiment, the amount comprising the compound of sequence SEQ ID NO: 1, its functionally equivalent variant, fusion protein, conjugate, polynucleotide, carrier, or cell, preferably the amount of peptide or its functionally equivalent variant, is about 50 mg / kg of the subject's body weight per day, preferably administered daily, preferably intraperitoneally, and more preferably intravenously. In a preferred embodiment, the amount comprising the compound of sequence SEQ ID NO: 1, its functionally equivalent variant, fusion protein, conjugate, polynucleotide, carrier, or cell, preferably the amount of peptide or its functionally equivalent variant, is about 75 to 200 mg / kg of the subject's body weight per day. 2 Preferred concentration: 100 to 175 mg / m³ 2 More preferably 150 mg / m 2 It is preferred to administer the medication daily, preferably intraperitoneally, and more preferably intravenously.

[0162] In another specific embodiment, the amount comprising the compound of sequence SEQ ID NO: 1, its functionally equivalent variant, fusion protein, conjugate, polynucleotide, carrier, or cell, preferably the amount of peptide or its functionally equivalent variant, is about 75 mg / kg of the subject's body weight per day, preferably administered 3 days a week, preferably intravenously. In a preferred embodiment, the amount comprising the compound of sequence SEQ ID NO: 1, its functionally equivalent variant, fusion protein, conjugate, polynucleotide, carrier, or cell, preferably the amount of peptide or its functionally equivalent variant, is about 75 to 275 mg / kg of the subject's body weight per day. 2 Preferred concentration: 100 to 250 mg / m 2 More preferably 225 mg / m 2 It is preferred to administer the medication 3 days a week, preferably intravenously.

[0163] In another specific embodiment, the amount comprising the compound of sequence SEQ ID NO: 1, its functionally equivalent variant, fusion protein, conjugate, polynucleotide, carrier, or cell, preferably the amount of peptide or its functionally equivalent variant, is about 75 mg / kg of the subject's body weight per day, preferably administered every other day, preferably four times, preferably intravenously. In a preferred embodiment, the amount comprising the compound of sequence SEQ ID NO: 1, its functionally equivalent variant, fusion protein, conjugate, polynucleotide, carrier, or cell, preferably the amount of peptide or its functionally equivalent variant, is about 75 to 275 mg / kg of the subject's body weight per day, preferably administered every other day, preferably four times, preferably intravenously. 2 Preferred concentration: 100 to 250 mg / m 2 More preferably 225 mg / m 2 It is preferred to administer the medication every other day, for a total of 4 doses, preferably intravenously.

[0164] In some embodiments, when a compound comprising the sequence SEQ ID NO: 1, its functionally equivalent variants, fusion proteins, conjugates, polynucleotides, vectors, or cells, preferably peptides or their functionally equivalent variants, is used for the prevention and / or treatment of human patients, the dosage range is 0.01 mg / kg / day to 8 mg / kg / day, preferably 0.5 mg / kg / day to 7.5 mg / kg / day, more preferably 1 mg / kg / day to 7 mg / kg / day, more preferably 1.5 mg / kg / day to 6.75 mg / kg / day, more preferably 2 mg / kg / day to 6.5 mg / kg / day, more preferably 3 mg / kg / day to 6.25 mg / kg / day, and more preferably 4.05 mg / kg / day to 6 mg / kg / day. In preferred embodiments, the drug is administered daily, preferably intraperitoneally, and even more preferably intravenously.

[0165] In some embodiments, when a compound comprising the sequence SEQ ID NO: 1, a functionally equivalent variant thereof, a fusion protein, a conjugate, a polynucleotide, a vector or cell, preferably a polypeptide or a functionally equivalent variant thereof, is used for the prevention and / or treatment of human patients, the dosage range is 50 to 500 mg / m². 2 Preferred concentration: 60 to 450 mg / m 2 More preferably 70 to 400 mg / m 2 More preferably 80 to 350 mg / m 2 More preferably 90 to 300 mg / m 2 More preferably 95 to 250 mg / m 2 More preferably 100 to 200 mg / m 2 More preferably 105 to 190 mg / m 2 More preferably 110 to 185 mg / m 2More preferably 115 to 180 mg / m 2 More preferably 120 to 170 mg / m 2 More preferably 125 to 160 mg / m 2 In a preferred embodiment, the medication is administered daily, preferably intraperitoneally, and more preferably intravenously.

[0166] In another specific embodiment, when a compound comprising the sequence SEQ ID NO: 1, its functionally equivalent variants, fusion proteins, conjugates, polynucleotides, carriers, or cells, preferably peptides or their functionally equivalent variants, is used for the prevention and / or treatment of human patients, the dosage range is 0.1 mg / kg / day to 15 mg / kg / day, preferably 1 mg / kg / day to 14 mg / kg / day, more preferably 2 mg / kg / day to 13 mg / kg / day, more preferably 3 mg / kg / day to 12 mg / kg / day, more preferably 4 mg / kg / day to 11 mg / kg / day, more preferably 5 mg / kg / day to 10 mg / kg / day, more preferably 6 mg / kg / day to 9.5 mg / kg / day, more preferably 6.08 mg / kg / day to 9 mg / kg / day. In a preferred embodiment, the drug is administered three times a week or four doses every other day, more preferably intravenously.

[0167] In another specific embodiment, when a compound comprising the sequence SEQ ID NO: 1, a functionally equivalent variant thereof, a fusion protein, a conjugate, a polynucleotide, a carrier or cell, preferably a polypeptide or a functionally equivalent variant thereof, is used for the prevention and / or treatment of human patients, the dosage range is 50 to 400 mg / m². 2 Preferred concentration: 100 to 380 mg / m 2 More preferably 110 to 370 mg / m 2 More preferably 120 to 360 mg / m 2 More preferably 130 to 350 mg / m 2 More preferably 140 to 340 mg / m 2 More preferably 150 to 330 mg / m 2 More preferably 160 to 320 mg / m 2 More preferably 170 to 310 mg / m 2 More preferably 180 to 300 mg / m 2 More preferably 190 to 290 mg / m 2 More preferably 200 to 275 mg / m 2 More preferably 220 to 250 mg / m 2 More preferably 210 to 230 mg / m 2In a preferred embodiment, the drug is administered three times a week or four times every other day, more preferably intravenously.

[0168] The compound of the purpose according to the invention, or a pharmaceutical composition comprising the compound of the purpose, may be administered by any suitable route of administration, such as by oral route, local route, inhalation or parenteral route, in order to include pharmaceutically acceptable excipients necessary for the formulation of the desired dosage form. Other routes of administration may be rectal, intracerebral, or vaginal.

[0169] In an embodiment, the compound used according to the invention, or a pharmaceutical composition containing the compound used for that purpose, is administered systemically.

[0170] "Systemic route" is understood as administration via oral, intravenous, intraperitoneal, and intramuscular routes. The amount of compound required for therapeutic or preventative effects will naturally vary depending on the compound chosen, the nature and severity of the disease being treated, and the patient.

[0171] "Oral route" is understood to mean that the compound or pharmaceutical composition enters the body after being swallowed. In certain embodiments, the compound or pharmaceutical composition of the present invention may be a dosage form suitable for administration via the oral route, whether it is solid or liquid. Dosage forms suitable for administration via the oral route may be tablets, capsules, syrups, or solutions, and may contain any conventional excipients known in the art, such as binders, e.g., syrups, gum arabic, gelatin, sorbitol, or polyvinylpyrrolidone; fillers, e.g., lactose, sugar, corn starch, calcium phosphate, sorbitol, or glycine; tableting lubricants, e.g., magnesium stearate; disintegrants, e.g., starch, polyvinylpyrrolidone, sodium starch glycolate, or microcrystalline cellulose; or pharmaceutically acceptable wetting agents, e.g., sodium lauryl sulfate. Solid oral compositions may be prepared by conventional processes of mixing, filling, or compression. Repeated mixing operations may be used to fully distribute the active agent in those compositions using large amounts of fillers. Such operations are conventional in the art. Tablets can be prepared, for example, by means of wet or dry granulation, and optionally coated according to processes known in common pharmaceutical practice, particularly by enteric coating.

[0172] As used herein, the term "parenteral" includes administration via intravenous, intraperitoneal, intramuscular, or subcutaneous routes. In a preferred embodiment, the compound for use according to the invention or a pharmaceutical composition comprising the compound is administered via an intravenous, intraperitoneal, intramuscular, or subcutaneous route, preferably via an intraperitoneal or intravenous route; more preferably via an intravenous route. In an embodiment, administration is performed via subcutaneous infusion.

[0173] In one embodiment, a compound or pharmaceutical composition comprising the compound according to the invention may be suitable for parenteral administration, such as a sterile solution, suspension, or lyophilized product in appropriate dose units. Compounds or pharmaceutical compositions suitable for injection include sterile aqueous solutions (when they are soluble in water) or dispersions and sterile powders for the provisional preparation of sterile injectable solutions or dispersions. For intravenous administration, some suitable carriers include phosphate-buffered saline (PBS). In all cases, the compound or composition must be sterile and must be a fluid to achieve injectability. It must be stable under preparation and storage conditions and must be free from contamination by microorganisms such as bacteria and fungi. The carrier may be a solvent or dispersion medium containing, for example, water, ethanol, pharmaceutically acceptable polyols (such as glycerol, propylene glycol, liquid polyethylene glycol), and suitable mixtures thereof. Appropriate flowability may be maintained, for example, by using coatings (such as lecithin), by maintaining the desired particle size of the dispersion, and by using surfactants. Microbial activity can be prevented by various antimicrobial and antifungal agents, such as parabens, chlorobutanol, phenol, ascorbic acid, and thimerosal. In most cases, it is preferable to include isotonic agents in the composition, such as sugars; polyols, such as mannitol and sorbitol; or sodium chloride. Prolonged absorption of injectable compositions may be due to the inclusion of absorption-retarding agents, such as aluminum monostearate and gelatin monostearate.

[0174] Injectable sterile solutions can be prepared by incorporating the desired amount of the active compound with one or a combination of the above-mentioned components (as needed) into a suitable solvent, followed by sterilization through sterile membrane filtration. Typically, dispersions are prepared by incorporating the active compound into a sterile loading medium containing a basic dispersion medium and the remaining desired components listed above. In the case of sterile powders used to prepare injectable sterile solutions, a preferred preparation process is vacuum drying and lyophilization, which produces a powder containing the active ingredient as well as any desired additional components from its previously filtered sterile solution.

[0175] The compounds or pharmaceutical compositions of the present invention can be appropriately administered by means of pulse infusion, for example, to reduce the dosage of the composition.

[0176] In certain embodiments, the compound or pharmaceutical composition comprising the compound according to the present invention is administered intradermally, subcutaneously, intravenously, intraperitoneally, intramuscularly, intranasally, or orally, or topically.

[0177] In more specific embodiments, the compound or pharmaceutical composition comprising the compound according to the invention is administered intravenously or intraperitoneally; intravenous administration is preferred.

[0178] Intravenous delivery methods include, but are not limited to, bolus injection and infusion. Examples of intravenous dosage forms include, but are not limited to, those contained in water, preferably sterile water for injection (USP); aqueous carriers include, but are not limited to, sodium chloride injection, Ringer's injection, dextrose injection, dextrose sodium chloride injection, and lactated Ringer's injection; water-miscible vehicles include, but are not limited to, ethanol, polyethylene glycol, and polypropylene glycol; and non-aqueous carriers include, but are not limited to, corn oil, cottonseed oil, peanut oil, sesame oil, ethyl oleate, isopropyl myristate, and benzyl benzoate.

[0179] Intraperitoneal delivery methods include, but are not limited to, injection.

[0180] On the other hand, "topical route" is understood as administration via a non-systemic route, including external application of the compounds or pharmaceutical compositions of the present invention to the epidermis, oral cavity, and instillation of said composition into the ear, eye, and nose, wherein it does not significantly enter the bloodstream. Dosage forms of the compounds of the present invention for topical or transdermal administration include ointments, pastes, creams, lotions, gels, powders, solutions, sprays, inhalants, or patches.

[0181] Ophthalmic preparations, ear drops, and eye drops are also considered to fall within the scope of this invention. Furthermore, the invention contemplates the use of transdermal patches, which offer the additional advantage of providing controlled delivery of the compound into the body. Such dosage forms can be prepared by dissolving or dispersing the compound in a suitable medium. Absorption enhancers can also be used to increase the flux of the compound across the skin. The rate can be controlled by providing a rate-controlled membrane or by dispersing the compound in a polymer matrix or gel.

[0182] In another embodiment, the compound or pharmaceutical composition is administered intranasally. In a preferred embodiment, intranasal administration is performed by instillation or nasal inhalation.

[0183] "Inhalation" is understood to mean administration via the intranasal route and oral inhalation. Dosage forms suitable for this administration can be prepared using conventional techniques, such as aerosols or meter-dose inhalers. In this embodiment, the route of administration is intranasal.

[0184] The dosage form of compositions intended for intranasal and intrapulmonary administration is preferably a liquid, suspension, or solid. A suspension is a liquid formulation containing solid particles dispersed in a liquid load. The dosage form is preferably metered. For example, metered drops / sprays mean that a dispenser comprising drops / sprays delivers drops / sprays containing a metered (pre-defined) amount of the compound or composition intended for use according to the invention.

[0185] In the context of intranasal administration, a preferred dosage form includes nasal drops. Drops deposit primarily in the posterior nasal cavity, thus rapidly reaching the nasopharynx. A common challenge with drops is precisely controlling the drug dosage, which is particularly important for the administration of compounds or compositions.

[0186] Other intranasal dosage forms that can be used to administer compounds or pharmaceutical compositions for the purposes of this invention are nasal sprays. Nasal sprays typically contain compounds dissolved or suspended in a solution or mixture of excipients (e.g., preservatives, viscosity modifiers, emulsifiers, buffers) located in a non-pressurized dispenser. Nasal sprays offer several advantages, including a compact and convenient delivery device, ease of use, and accurate delivery of doses from 25 to 200 pL. They deposit in the anterior nasal cavity and are slowly cleared into the nasopharynx by mucociliary clearance. Nasal sprays, as used herein, can be liquids or suspensions.

[0187] Other intranasal dosage forms are nasal aerosols. Nasal aerosols differ from nasal sprays in the method of compound distribution: in aerosols, the compound is distributed due to excessive pressure and released through a valve. In sprays, the compound is distributed due to the forced propulsion of a miniature pump, with the pressure in the vial similar to atmospheric pressure. Aerosols offer similar advantages to sprays.

[0188] The compounds or compositions used according to the invention may alternatively be administered via nasal emulsions, ointments, gels, pastes, or creams. These are high-viscosity solutions or suspensions applied to the nasal mucosa.

[0189] Because of the limited volume of compositions that can be effectively delivered to the nasal mucosa, liquid intranasal dosage forms typically have higher concentrations than their corresponding intravenous dosage forms. When a substance exhibits poor solubility or instability in liquid form, powders can be used to administer the compounds or compositions used in this invention. A further advantage of powders is that they do not require preservatives and generally exhibit greater stability compared to liquid formulations. The main limitation of intranasal powder application relates to its irritant effect on the nasal mucosa.

[0190] In the context of intrapulmonary administration, one dosage form is an inhaled aerosol. Inhaled aerosols are typically packaged under pressure and contain a compound or composition according to the invention, which is released into the respiratory tract, particularly the lungs, upon activation of a valve system. The released aerosol is a colloidal mixture of fine solid particles (suspension) or droplets (solution) in air or other gases. Thus, the aerosol can be a solution or a suspension aerosol. The droplets or solid particles preferably have a diameter of less than 100 pm, more preferably less than 10 pm, and most preferably less than 1 pm.

[0191] Other dosage forms in the context of intrapulmonary administration are inhaled sprays. Inhaled sprays are typically water-based and do not contain any propellants. They deliver the conjugate to the lungs via oral inhalation.

[0192] Nebulized inhalation solutions and suspensions can also be used to deliver compounds or compositions via the intrapulmonary route. Nebulized inhalation solutions and suspensions are typically water-based formulations containing compounds or compositions intended for use according to the invention. Nebulized inhalation solutions and suspensions deliver compounds or compositions to the lungs via oral inhalation to produce a systemic effect and are used in conjunction with a nebulizer.

[0193] Dry powder inhalation is an alternative to aerosol inhalation. The composition is typically contained in capsules or inhalers for manual loading. The dry powder is usually delivered to the lungs via oral inhalation through an inhaler. Dry powders as used herein can be formulated purely. Pure formulations contain only or nearly only the drug, such as spray-dried powders. Dry powders as used herein can also be formulated with a carrier, such as lactose.

[0194] The preferred intrapulmonary dosage form is a metered form, i.e., delivered to the lungs in a predetermined amount.

[0195] In the context of this invention, intranasal delivery devices include spray pump systems, drop delivery pipettes, metered-dose spray pumps, nasal pressurized metered-dose inhalers, powder spray systems, respiratory-driven powder inhalers, and nasal powder blowers. Intranasal delivery devices may be filled with single-dose or multi-dose intranasal formulations.

[0196] Using the intrapulmonary route, compounds or compositions can be administered via a metered-dose inhaler (MDI). A metered-dose inhaler (MDI) delivers a fine mist of the compound, typically with an aerodynamic particle size of less than 5 pm.

[0197] Dry powder inhalers can be used alternatively for intrapulmonary delivery of compounds or compositions. Dry powder inhalers deliver powder in the form of single-dose or multi-dose powder.

[0198] Other intrapulmonary delivery devices are nebulizers, including ultrasonic nebulizers and air jet nebulizers. In an ultrasonic nebulizer, ultrasound is generated by ceramic piezoelectric crystals vibrating when electrically excited within the nebulizer chamber. This creates an aerosol cloud on the solution surface. In an air jet nebulizer, aerosols are generated when compressed air is forced through an orifice. Liquid can be drawn from a vertical nozzle (Bernoulli effect) to mix with the air jet, which uses baffles to atomize and promote aerosol cloud formation.

[0199] In one embodiment, a compound or pharmaceutical composition according to the invention is prepared together with a delivery vehicle that protects the ingredient from rapid elimination from the body, such as controlled-release formulations, including implants and microencapsulated delivery systems. Biodegradable, biocompatible polymers such as ethylenevinylacetate, polyanhydride, polyglycolic acid, collagen, polyorthoesters, and polylactic acid can be used. The processes for preparing the formulations are clear to those skilled in the art. These materials are also available from Alza and Nova Pharmaceuticals, Inc.

[0200] Sustained-release compositions also include crystal formulations suspended in suitable formulations that maintain crystal suspension. These formulations produce a sustained-release effect when injected subcutaneously or intraperitoneally. Other compositions include components encapsulated in liposomes. Liposomes containing such components are prepared by known methods, such as Epstein et al., Proc. Natl. Acad. Sci. USA, (1985) 82:3688-3692; Hwang et al., Proc. Natl. Acad. Sci. USA, (1980) 77:4030-4034; EP 52,322; EP 36,676; EP 88,046; EP 143,949. In a preferred embodiment, the compounds of the present invention are contained in liposomes.

[0201] Although it is true that Omomyc, its functionally equivalent variants, conjugates, and fusion proteins of the present invention can translocate across biological membranes, Omomyc, any functionally equivalent variants, conjugates, polynucleotides, carriers, or cells can be formulated into nanoparticles. Nanoparticles can help maintain the integrity of components in biological fluids until they reach the target organ. Furthermore, nanoparticles can be modified to include portions that allow them to target the target organ. In this way, the compounds of the present invention will be delivered near the target organ, facilitating the entry of the compounds into the cells where their biological activity is required.

[0202] Therefore, in another embodiment, the compound of the present invention is provided in the form of a portion of nanoparticles.

[0203] As used herein, the term "nanoparticle" refers to any material with a size in the range of 1-1,000 nm. In some embodiments, the nanoparticles have a size in the range of 2-200 nm, preferably in the range of 2-150 nm, and even more preferably in the range of 2-100 nm. Nanoparticles that can be used in this invention include nanoscale materials such as lipid-based nanoparticles, superparamagnetic nanoparticles, nanoshells, semiconductor nanocrystals, quantum dots, polymer-based nanoparticles, silicon-based nanoparticles, silica-based nanoparticles, metal-based nanoparticles, fullerenes, and nanotubes. Molecules may be embedded in the nanoparticle matrix or adsorbed on its surface, preferably embedded in the nanoparticles.

[0204] In a preferred embodiment, the nanoparticles are liposomes.

[0205] Targeted delivery can be achieved by adding ligands without compromising the nanoparticles' ability to deliver their contents. This is expected to enable delivery to specific cells, tissues, and organs. The targeting specificity of ligand-based delivery systems is based on the distribution of ligand receptors across different cell types. Targeting ligands can be non-covalently or covalently associated with nanoparticles and can be conjugated to nanoparticles using a variety of methods discussed in this paper.

[0206] Examples of proteins or peptides that can be used to target nanoparticles include transferrin, lactoferrin, TGF-β, nerve growth factor, albumin, HIV Tat peptide, RGD peptide, and insulin.

[0207] It will be understood that the formulations in the nanoparticles of the present invention are not intended, or are not only intended, to promote the entry of components into cells, but rather to protect components from degradation and / or to promote the targeting of nanoparticles to target organs.

[0208] In one example, the nanoparticles can be made from biodegradable polymers such as poly(butyl cyanoacrylate) (PBCA). Examples of elemental nanoparticles include carbon nanoparticles and iron oxide nanoparticles, which can then be coated with oleic acid (OA)-Pluronic®. In this method, a drug (e.g., a hydrophobic or water-insoluble drug) is loaded into the nanoparticles. Other nanoparticles are made of silica.

[0209] Nanoparticles can be formed from any useful polymer. Examples of polymers include biodegradable polymers such as poly(butyl cyanoacrylate), poly(lactide), poly(glycolic acid), poly-S-caprolactone, poly(butylene succinate), poly(ethylene glycol succinate), and poly(p-dioxanone); poly(ethylene glycol); poly(HEMA) 2-hydroxyethyl methacrylate; copolymers such as poly(lactide-co-glycolic acid), poly(lactide)-poly(ethylene glycol), poly(ethylene glycol) cyanoacrylate-co-hexadecyl cyanoacrylate, and poly[HEMA-co-methacrylic acid]; proteins such as fibrinogen, collagen, gelatin, and elastin; and polysaccharides such as amylopectin, amylose, and chitosan.

[0210] Other nanoparticles include solid lipid nanoparticles (SLNs). Examples of lipid molecules in solid lipid nanoparticles include stearic acid and modified stearic acid, such as stearic acid-PEG 2000; soybean lecithin; and emulsified waxes. Solid lipid nanoparticles may optionally include other components, including surfactants such as Epicuron® 200, Pluronic® F68, Brij 72, Brij 78, polysorbate 80 (Tween 80); and salts such as sodium taurocholate. Agents can be introduced into solid lipid nanoparticles by various methods discussed for liposomes, wherein these methods may further include high-pressure homogenization and microemulsion dispersion.

[0211] Nanoparticles may also comprise nanoscale micelles. Micelles can be formed from any polymer described herein. Exemplary polymers for forming micelles include block copolymers such as poly(ethylene glycol) and poly(ε-caprolactone). (For example, a PEO-b-PCL block copolymer comprising ε-caprolactone and α-methoxy-ω-hydroxy-poly(ethylene glycol)).

[0212] In some embodiments, the properties of the nanoparticles are modified by coating them with a surfactant. Any biocompatible surfactant can be used, such as polysorbate surfactants, such as polysorbate 20, 40, 60 and 80 (Tween 80); Epicuron® 200; poloxamer surfactants, such as 188 (Pluronic® F68), poloxamer 908 and 1508; and Brij surfactants, such as Brij 72 and Brij 78.

[0213] Nanoparticles may optionally be modified to include hydrophilic polymer groups (e.g., poly(ethylene glycol) or poly(propylene glycol)), for example, by covalently attaching the hydrophilic polymer groups to the surface or by using polymers containing such hydrophilic polymer groups (e.g., poly[methoxy poly(ethylene glycol) cyanoacrylate-co-hexadecyl cyanoacrylate]). Nanoparticles may optionally be crosslinked, which is particularly useful for protein-based nanoparticles.

[0214] In another embodiment, the pharmaceutical composition of the present invention is a nanoemulsion. As used herein, "nanoemulsion" means a colloidal dispersion of droplets (or particles) wherein at least some droplets have diameters in the nanometer range. Nanoemulsions comprise an oil rich in ω-3, ω-6, or ω-9 fatty acids in an aqueous phase and are thermodynamically stabilized by an amphiphilic surfactant forming an interfacial film, produced using a high-shear microfluidization process, with droplet diameters typically in the range of about 80-220 nm.

[0215] In a particular embodiment, the compound used according to the invention is administered in combination with at least other therapeutic agents for treating autoimmune diseases, wherein the at least other therapeutic agents are administered simultaneously, sequentially, or separately.

[0216] "Simultaneous administration" encompasses the co-administration of two therapeutic agents, regardless of their relative dosing frequencies or timings. Therefore, simultaneous administration includes the co-administration of two therapeutic agents at the same time and with the same dosing frequency. Furthermore, simultaneous administration refers to the co-administration of two therapeutic agents in which one agent is administered more frequently than one or more other agents. Additionally, simultaneous administration refers to the co-administration of two therapeutic agents in which one agent is administered only once during the administration of one or more other agents.

[0217] "Sequential" dosing means stopping the administration of the first component before starting the administration of the second component.

[0218] If administered separately, the therapeutic agents can be administered at intervals of time, for example, within 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, or 24 hours. In some embodiments, they can be administered within 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, or 24 days.

[0219] Combinations and pharmaceutical compositions of the present invention In another aspect, the present invention relates to a combination comprising: (i) the compounds according to the invention and (ii) Agents used to treat autoimmune diseases.

[0220] In another respect, the present invention relates to pharmaceutical compositions comprising pharmaceutically effective amounts of the combination according to the invention and pharmaceutically acceptable excipients.

[0221] In another respect, the present invention relates to combinations or pharmaceutical compositions according to the present invention for medical use.

[0222] In another respect, the present invention relates to the use of combinations or pharmaceutical compositions according to the present invention for the prevention and / or treatment of autoimmune diseases.

[0223] The embodiments defined with respect to the first aspect of the invention also apply to these additional aspects.

[0224] Furthermore, the embodiments defined in the context of the combinations and pharmaceutical compositions of the present invention also apply to the first aspect of the present invention.

[0225] According to the present invention, the term "combination" refers to various combinations of compounds (i) and (ii), such as compositions formulated as single formulations, mixtures of individual formulations of each component (e.g., "tank-mixes" that can be combined as a combined preparation), and combinations of single active ingredients applied sequentially, i.e., administered one after another or simultaneously over a considerably short period of time (e.g., hours or days). In the present invention, compound (i) refers to a therapeutically effective amount of a polypeptide comprising the sequence SEQ ID NO: 1 or a functionally equivalent variant thereof, or a conjugate containing a polypeptide comprising the sequence SEQ ID NO: 1 or a functionally equivalent variant thereof and a chemical moiety that promotes cellular uptake of said polypeptide or functionally equivalent variant thereof, or a polynucleotide encoding said polypeptide or said conjugate, or a carrier containing said polynucleotide, or cells capable of secreting said polypeptide or said conjugate into a culture medium. In the present invention, compound (ii) refers to a therapeutically effective amount of a compound suitable for treating autoimmune diseases. Preferably, the order of administration of compounds (i) and (ii) is not essential for carrying out the present invention.

[0226] The combination can be a kit-of-parts in which each component is individually formulated and packaged.

[0227] The combination of (i) and (ii) can be formulated for simultaneous, separate or sequential administration, as explained above.

[0228] Specifically, if not administered simultaneously, the compounds are administered at approximately the same time. Furthermore, the dosage forms of the compounds can be the same or different, and the routes of administration can also be the same or different; for example, one compound can be administered orally, and another compound can be administered intravenously. Preferably, compound (i) is administered intravenously, compound (ii) is administered systemically, more preferably parenterally, and even more preferably orally.

[0229] The combination of the two compounds (i) and (ii) can be administered in the following manner: - As part of the same drug formulation, the two compounds are then always administered simultaneously.

[0230] - As a combination of two units, each unit has a substance, which can be administered simultaneously, sequentially or separately.

[0231] In a particular embodiment, compound (i) and compound (ii) in the combination of the present invention are administered independently, i.e., administered as two units but simultaneously.

[0232] In another specific embodiment, compound (i) of the combination of the present invention is administered first, followed by compound (ii), i.e., compound (ii) is administered separately or sequentially.

[0233] Pharmaceutical compositions according to the invention, containing compounds derived from the invention and compounds suitable for treating autoimmune diseases, can be presented as a single formulation (e.g., as tablets or capsules containing a fixed amount of each component), or on the other hand, as separate formulations for later combination for combined, sequential, or separate administration. The compositions of the invention also include formulations as kits, wherein the components are formulated separately but packaged in the same container. Those skilled in the art will understand that formulations of different components in the pharmaceutical compositions according to the invention can be similar, in other words, similarly formulated (in tablets or pills), thereby allowing them to be administered via the same route. In the case where the different components of the invention are formulated separately, the two components can be presented in blister packs. Each blister pack contains the amount of medicine that must be taken throughout the day. If the medicine must be administered several times a day, the medicine corresponding to each administration can be placed in different portions of the blister pack, preferably with the time of administration recorded in each portion of the blister pack. Alternatively, the components of the compositions of the invention can be formulated in different ways so that different components are administered in different ways. Thus, a first group can be dispensed as tablets or capsules for oral administration, while a second group is dispensed for intravenous administration, and vice versa. Those skilled in the art can adjust the ratio between components that are part of a combination or pharmaceutical composition according to the invention, depending on the agent used in each specific situation and the desired indication. Therefore, the invention contemplates compositions in which the ratio between the amounts of component (i) and component (ii) can range from 50:1 to 1:50, particularly 20:1 to 1:20, 1:10 to 10:1, or 5:1 to 1:5. In a more specific embodiment, the ratio between the amounts ranges from 1:1 to 1:5, preferably 1:1 to 1:3. In a more preferred embodiment, the ratio ranges from 1:1 to 1:1.5, preferably 1:1.3 to 1:1.4, more preferably 1:1.34. In another preferred embodiment, the ratio ranges from 1:1 to 1:2.8, preferably 1:2.6 to 1:2.7, more preferably 1:2.67. In yet another specific embodiment, the ratio between the amounts ranges from 30:1 to 5:1, preferably 30:1 to 8:1, more preferably 25:1 to 15:1, more preferably 20:1 to 10:1. In one embodiment, the ratio is 20:1. In another embodiment, the ratio is 10:1. Preferably, these ratios are w / w ratios.

[0234] As used in this article, the expression "therapeutic agent for the treatment of autoimmune diseases" refers to an agent suitable for the treatment of one of the aforementioned diseases.

[0235] In a more specific embodiment, at least the other agents used to treat autoimmune diseases are selected from the group consisting of nonsteroidal anti-inflammatory drugs (NSAIDs), steroids, and immunosuppressants.

[0236] In a more specific embodiment, at least one other therapeutic agent for treating autoimmune diseases is selected from the group consisting of: corticosteroids, ofatumumab, fingolimod, siponimod, ozanimod, ponsimod, laquinimod, teriflunomide, fumaric acid ester (dimethyl fumarate, diroximel fumarate, monomethyl fumarate), cladribine, and mycophenolate. The following drugs are listed: mofetil, atorvastatin, interferon-β, glatiramer acetate, mitoxantrone, cyclophosphamide, natalizumab, daclizumab, rituximab, ustekinumab, ocrelizumab, alemtuzumab, valacyclovir, CTLA4Ig, and atacicept. In a preferred embodiment, at least the other therapeutic agents for treating multiple sclerosis are selected from the group consisting of: corticosteroids, olfatumumab, fingolimod, sinimod, ozamod, posamod, laquimod, teriflunomide, fumarates (dimethyl fumarate, diloximel fumarate, monomethyl fumarate), clavibine, mycophenolate mofetil, atorvastatin, interferon-β, glatiramer acetate, mitoxantrone, cyclophosphamide, natelizumab, dalizumab, rituximab, ustekinumab, ozenafil, alenzumab, valacyclovir, CTLA4Ig, copaxone, tocilizumab, and atacicept. In a more preferred embodiment, at least the other therapeutic agents for treating multiple sclerosis are selected from the group consisting of: interferon, copaxone, teriflunomide, fumarate, natelizumab, fingolimod, alenzumab, rituximab, ozoglucon, clavibine, posamod, sinimod, ozagimide, tocilizumab, and olfatumumab.

[0237] In a preferred embodiment, the therapeutic agent for treating systemic lupus erythematosus, preferably lupus nephritis, is selected from the group consisting of: cyclophosphamide, mycophenolate mofetil, calcineurin inhibitor, rituximab, ozoglucillin, belimumab, atasip, abatacept, alenzusab, sirukumab, tocilizumab, etanercept, eculizumab, epratuzumab, abetimus / LJP-394, BG9588 / IDEC 131, intravenous immunoglobulin, hydroxychloroquine, tacrolimus, and corticoids.

[0238] In another preferred embodiment, the therapeutic agent for treating rheumatoid arthritis is selected from the group consisting of infliximab, adalimumab, certolizumab, golimumab, and etanercept.

[0239] In another preferred embodiment, the therapeutic agent for treating inflammatory bowel disease is selected from the group consisting of cyclosporine A, tacrolimus, methotrexate, thiopurine, and anti-TNF agents.

[0240] For the treatment of autoimmune diseases, particularly diabetes (type 1), agents may be selected from the following group: insulin, teplizumab, otelixizumab, alexicept, abatacept, rituximab, infliximab, adalimumab, etanercept, golimumab, tocilizumab, verapamil, dapagliflozin, empagliflozin, sotagliflozin, exenatide, liraglutide, dulaglutide, and semaglutide.

[0241] Treatment agents for autoimmune diseases, particularly Graves' disease, can be selected from the following group: antithyroid drugs (such as propylthiouracil and methimazole), beta blockers (such as propranolol, atenolol, metoprolol, and nadolol), corticosteroids, and teprotumumab.

[0242] For the treatment of autoimmune diseases, especially alopecia areata, agents can be selected from the following groups: topical corticosteroids, intralesional corticosteroids, anthralin, minoxidil, topical tacrolimus, topical retinoids, prostaglandin analogs, azathioprine, cyclosporine, methotrexate, and sulfasalazine.

[0243] For the treatment of autoimmune diseases, especially Addison's disease, the following groups of agents may be selected: hydrocortisone, prednisone, methylprednisolone, and fludrocortisone acetate.

[0244] For the treatment of autoimmune diseases, particularly psoriasis, agents may be selected from the following groups: corticosteroids, vitamin D analogs (such as calcipotriene and calcitriol), retinoids (such as tazarotene), calcineurin inhibitors (such as tacrolimus and pimecrolimus), salicylic acid, anthralin, steroids, retinoids, apremilast (Otezla®), etanercept (Enbrel®), infliximab (Remicade®), adalimumab (Humira®), and ulcerative colitis. Stelara®, secukinumab (Cosentyx®), ixekizumab (Taltz®), guselkumab (Tremfya®), tildrakizumab (Ilumya®), certolizumab (Cimzia®), methotrexate, cyclosporine, thioguanine (Tabloid®), and hydroxyurea (Droxia®, Hydrea®).

[0245] For the treatment of autoimmune diseases, especially autoimmune nephropathy, and even more so lupus nephritis, agents can be selected from the group consisting of: immunosuppressants, steroids, vitamin D, VIP (vasoactive intestinal peptide), hydroxychloroquine, chloroquine, and T-cell vaccination.

[0246] In this implementation, the immunosuppressant is selected from the group consisting of cyclophosphamide, mycophenolate mofetil, azathioprine, and anticalcineurin.

[0247] In this embodiment, the steroid is selected from the group consisting of prednisolone and methylprednisolone.

[0248] Therapeutic agents for the treatment of autoimmune diseases, particularly autoimmune nephropathy, and especially lupus nephritis, may be compounds targeting targets selected from the group consisting of: T cells, B cells, co-stimulatory inhibitors, inflammatory cytokines, cell adhesion molecules, and complement components.

[0249] In this embodiment, the T-cell target may be selected from the group consisting of: CD3, CD52, IL-2, and LFA-1. Preferably, the compound targeting T cells is selected from the group consisting of: OKT3, alenzusmab, basilximab, efalizumab, laquinimod, and rapamycin.

[0250] In another embodiment, the B-cell target may be selected from the group consisting of: CD20, CD22, BAFF, BAFF and APRIL, FcγRIIb and CD74. Preferably, the compound targeting B cells is selected from the group consisting of: rituximab, ozoglucomancil, oflamb, vetozumab, ozoglucomancil, epazolizumab, belimumab, blisibimod, attacip, soluble FcγRIIb, milatuzumab and tabalumab.

[0251] In this embodiment, the co-stimulatory inhibitor can be selected from the group consisting of CD40 and CD80 / 86. Preferably, the compound targeting the co-stimulatory inhibitor is selected from the group consisting of ASKP1240, abatacept, and belacept.

[0252] In this implementation, the inflammatory cytokine targets may be selected from the group consisting of: IL-1β, IL-6, IL-6R, TNF, TWEAK, IFNα, IL-17A, IL-12 / IL-23, IFNγ, MIF, and IL-5. Preferably, the compound targeting inflammatory cytokines is selected from the group consisting of: canakinumab, anakinra, cilukumab, tocilizumab, etanercept, infliximab, adalimumab, golimumab, sertozumab, BIIB023, cifamumab, rontalizumab, sekunumab, ustekunumab, AMG 811, imalumab, mepolizumab, brodalumab, briakinumab, sarilumab, rilonacept, and anifrolumab.

[0253] In this embodiment, the cell adhesion molecule target is VLA-1. Preferably, the compound targeting the cell adhesion molecule is natalizumab.

[0254] In this embodiment, the complement component target is selected from the group consisting of C5 and C5aR. Preferably, the compound targeting the complement component is selected from the group consisting of eculizumab, mubodina, LFG316, and CCX168.

[0255] Table 2 shows the different compounds and their targets mentioned above.

[0256]

[0257] Table 2. Compounds used to treat autoimmune diseases. Adapted from Holdsworth SR et al. 2016. Nat. Rev. 12:217-231.

[0258] In a preferred embodiment, the therapeutic agent for treating autoimmune diseases is selected from the group consisting of: cyclosporine A, tacrolimus, methotrexate, thiopurine, anti-TNF agents, infliximab, adalimumab, sertozumab, golimumab, etanercept, rituximab, epazolizumab, belimumab, rapamycin, anti-interferon antibody, tocilizumab, laquinimod, taberucizumab, oflamimumab, ixekizumab, brolimumab, brezhnezumab, thalidomab, linazolizumab, avolimumab, cyclophosphamide, mycophenolate mofetil, azathioprine, anti-calcineurin, prednisolone, methylprednisolone, vitamin D, vasoactive intestinal peptide, hydroxychloroquine, chloroquine, ozograzumab, atazolizumab, abatacept, alenzumab, cilukumab, eculizumab, and T-cell vaccines.

[0259] In a further aspect, the present invention relates to: 1. Select compounds from the following groups: (a) A polypeptide containing the sequence SEQ ID NO: 1 or a functionally equivalent variant thereof, (b) A conjugate comprising a polypeptide comprising the sequence SEQ ID NO: 1 or a functionally equivalent variant thereof, and a chemical moiety that promotes cellular uptake of said polypeptide or a functionally equivalent variant thereof. (c) A polynucleotide encoding the polypeptide of (a) or the conjugate of (b). (d) A vector comprising the polynucleotide according to (c), and (e) Cells capable of secreting the polypeptide according to (a) or the conjugate according to (b) into a culture medium. Used for the prevention and / or treatment of autoimmune diseases in the subject.

[0260] 2. The compound for use according to aspect 1, wherein the functionally equivalent variant of SEQ ID NO: 1 is selected from the group consisting of: SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9 and SEQ ID NO: 10.

[0261] 3. A compound for use according to any one of aspects 1 or 2, wherein the chemical portion that promotes cellular uptake of the polypeptide or a functionally equivalent variant thereof is a cell-penetrating peptide sequence, and wherein the cell-penetrating peptide sequence and the polypeptide or a functionally equivalent variant thereof form a fusion protein.

[0262] 4. The compound for use according to aspect 3, wherein the cell-penetrating peptide sequence is selected from the group consisting of: SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 37 and SEQ ID NO: 38.

[0263] 5. The compound for use according to any one of aspects 1 to 4, wherein the conjugate additionally contains other nuclear localization signals.

[0264] 6. The compound for use according to any one of aspects 1 to 5, wherein the compound is a polypeptide comprising the sequence SEQ ID NO: 1.

[0265] 7. The compound for use according to any one of aspects 1 to 6, wherein the subject is a mammal.

[0266] 8. The compound used according to aspect 7, wherein the subject is a human.

[0267] 9. The compound for use according to any one of aspects 1 to 8, wherein the autoimmune disease is selected from the group consisting of: celiac disease, type I diabetes, latent autoimmune diabetes mellitus (LADA) in adults, Graves' disease, inflammatory bowel disease, multiple sclerosis, alopecia areata, Addison's disease, pernicious anemia, psoriasis, rheumatoid arthritis, and systemic lupus erythematosus.

[0268] 10. The compound for use according to aspect 9, wherein the autoimmune disease is selected from the group consisting of diabetes mellitus (type I), multiple sclerosis, and psoriasis.

[0269] 11. The compound used according to aspect 10, wherein the autoimmune disease is multiple sclerosis.

[0270] 12. The compound for use according to any one of aspects 1 to 8, wherein the autoimmune disease is an autoimmune demyelinating disease of the central nervous system (CNS).

[0271] 13. The compound for use according to aspect 12, wherein the CNS autoimmune demyelinating disease is selected from the group consisting of: multiple sclerosis (MS), clinically isolated syndrome (CIS), tumor-like MS, Marburg acute MS, Barlow concentric sclerosis, acute disseminated encephalomyelitis (ADEM), acute hemorrhagic leukoencephalitis, Heard's disease, isolated sclerosis, transverse myelitis, Sussac syndrome, leukoaraiosis, myalgic encephalomyelitis, Guillain-Barré syndrome, progressive inflammatory neuropathy, leukodystrophy, including adrenoleukodystrophy, adrenal neuropathy, post-vaccination encephalitis (PVE), post-infectious encephalomyelitis (PIE), neuromyelitis optica (NMO), optic neuritis, and myelin oligodendrocyte glycoprotein (MOG)-related disease (MOGAD).

[0272] 14. The compound for use according to any one of aspects 1 to 13, wherein the compound is administered intradermally, subcutaneously, intravenously, intraperitoneally, intramuscularly, intranasally, or orally, or topically.

[0273] 15. The compound for use according to aspect 14, wherein the compound is administered intravenously or intraperitoneally.

[0274] 16. The compound for use according to any one of aspects 1 to 15, wherein the compound is administered at a dose level between 0.1 and 100 mg / kg per day, preferably between 1 and 85 mg / kg per day, more preferably between 5 and 75 mg / kg per day.

[0275] 17. A compound for use according to any one of aspects 1 to 16, wherein the compound is administered in combination with at least one other therapeutic agent for treating an autoimmune disease, wherein the at least one other therapeutic agent is administered simultaneously, sequentially, or separately.

[0276] 18. The compound for use according to aspect 17, wherein the at least other therapeutic agent for treating autoimmune diseases is selected from the group consisting of: corticosteroids, olfatumumab, fingolimod, sinimod, ozamod, posamod, latoimod, teriflunomide, fumarates (dimethyl fumarate, diloxicame fumarate, monomethyl fumarate), clavibine, mycophenolate mofetil, atorvastatin, interferon-β, glatiramer acetate, mitoxantrone, cyclophosphamide, natelizumab, dalizumab, rituximab, ustekinumab, ozenafil, alenzumab, valacyclovir, CTLA4Ig, copaxone, tocilizumab, and atacicept.

[0277] 19. The compound for use according to aspect 17, wherein at least one other therapeutic agent for treating autoimmune diseases is selected from the group consisting of: interferon, copaxone, teriflunomide, fumarate, natelizumab, fingolimod, alenzumab, rituximab, ozoglucon, clavibine, posamod, sinimod, ozagimide, tocilizumab, and olfatumumab.

[0278] 20. A compound for use according to any one of aspects 1 to 19, wherein the compound is contained in a pharmaceutical composition, wherein the pharmaceutical composition further comprises at least one pharmaceutically acceptable excipient.

[0279] 21. A combination comprising: (i) Choose compounds from the following groups: (a) A polypeptide containing the sequence SEQ ID NO: 1 or a functionally equivalent variant thereof, (b) A conjugate comprising a polypeptide comprising the sequence SEQ ID NO: 1 or a functionally equivalent variant thereof, and a chemical moiety that promotes cellular uptake of said polypeptide or a functionally equivalent variant thereof. (c) A polynucleotide encoding the polypeptide of (a) or the conjugate of (b). (d) A vector comprising the polynucleotide according to (c), and (e) Cells capable of secreting the polypeptide according to (a) or the conjugate according to (b) into a culture medium; and (ii) Agents used to treat autoimmune diseases.

[0280] 22. The combination according to claim 21, wherein the agent for treating autoimmune diseases is selected from the group consisting of: interferon, copaxone, teriflunomide, fumarate, natelizumab, fingolimod, alenzumab, rituximab, ozenafil, clavibine, posamod, sinimod, ozamod, tocilizumab, and olfatumumab; more preferably selected from the group consisting of: copaxone, teriflunomide, fumarate, natelizumab, fingolimod, ozenafil, posamod, sinimod, ozamod, and tocilizumab.

[0281] 23. A pharmaceutical composition comprising a pharmaceutically effective amount of the combination according to aspect 21 or 22 and a pharmaceutically acceptable excipient.

[0282] 24. The combination according to aspect 21 or 22 or the pharmaceutical composition according to aspect 23, for medical use.

[0283] 25. Use of the combination according to aspect 21 or 22 or the pharmaceutical composition according to aspect 23 for the prevention and / or treatment of autoimmune diseases.

[0284] The present invention is described through the following embodiments, which are for illustrative purposes only and do not limit the scope of the invention.

[0285] Example Materials and methods Experimental autoimmune encephalomyelitis (EAE) induction and follow-up Eight-week-old female C57BL6 / JOlaHsd mice were purchased from Envigo (Netherlands). Anesthetized mice were immunized subcutaneously by injection of 100 µl phosphate-buffered saline (PBS) containing 100 µg of peptide 35-55 (MOG) derived from myelin oligodendrocyte glycoprotein. 35-55 This peptide, in 100 µl of complete Freund's adjuvant (containing 4 mg / ml Mycobacterium tuberculosis), Mycobacterium tuberculosisEmulsified in H37RA in an incomplete Freund's adjuvant. Mice were intravenously injected with 250 ng of pertussis toxin on days 0 and 2 post-immunization. Mice were weighed daily and examined for neurological signs in a blind manner, with the following criteria: 0, no clinical signs; 0.5, partial mild tail paralysis; 1, complete tail paralysis; 2, mild paraplegia of one or two hind limbs; 2.5, severe paraplegia or paralysis of the hind limbs; 3, mild quadriplegia (mild to moderate hind limb paralysis); 4, mild quadriplegia (severe hind limb paralysis); 4.5, severe quadriplegia; 5, quadriplegia; 6, death (Gutierrez-Franco A., et al. 2017. Semaphorin 7A as a Potential Therapeutic Target for Multiple Sclerosis. Mol Neurobiol 54(6): 4820-4831). Mice were subcutaneously injected with 0.5 ml of 10% glucose if they lost more than 15% of their body weight. A score of 5 and a weight loss exceeding 30% were defined as endpoints to minimize suffering and ensure animal welfare. All presented data conformed to the guidelines recommended by the EAE (Baker D. and Amor S. 2012. Publication guidelines for refereeing and reporting on animal use in experimental autoimmune encephalomyelitis. J Neuroimmunol 242(1-2): 78-83). In in vitro experiments, mice were followed up for 20 days, after which they were euthanized to obtain their spleens. In in vivo experiments, mice were followed up for 28 days.

[0286] EAE clinical parameters In in vivo experiments, the area under the curve (AUC) of the mean EAE clinical score was calculated as a measure of disease severity. Weight loss is a characteristic of EAE and is closely related to disease severity; therefore, the AUC of mean weight loss was also calculated and compared between groups. Other clinical parameters, such as the highest score achieved during clinical follow-up and the number of days since onset, were also compared.

[0287] Statistical analysis Statistical analyses were performed using GraphPad Prism 8 or SPSS Statistics v26 software packages. One-way ANOVA was used for in vitro experiments, with Dunnett's test as a post-hoc method. For in vivo experiments, t-tests were used to compare clinical parameters between the vehicle group and the Omomyc treatment group.

[0288] In EAE-affected mice, both PHA and MOG stimulate spleen cells. Omomyc can reduce [the body's] weight in a dose-dependent manner. Oligosplenic cells proliferate and induce cell death ( Figure 1 Spleens were collected from EAE-affected mice 20 days post-immunization (when mice presented with clinical symptoms). Spleen cells were isolated and seeded in 96-well plates. In the presence of increased concentrations of Omomyc (SEQ ID NO: 4) (1, 10, 100, and 200 µg / ml), 5 μg / ml MOG was used. 35-55 Spleen cell cultures were stimulated with either antigen-specific stimulation or 5 μg / ml PHA-L (polyclonal stimulation). For proliferation assays, MOG was applied to each experimental condition. 35-55 Five replicates were performed on the samples (PHA-L and control). The cultures were maintained for 72 h, with 1 μCi added to each well during the last 18 h. 3 H-thymidine. Radioactivity incorporation is measured in a β-scintillation counter. The stimulation index (SI) is determined by measuring the MOG... 35-55 Or, calculate by dividing the average count per minute (cpm) under the PHA-L condition by the average cpm under the control condition. Stimulation was performed at 1, 10, 100, and 200 µg / ml Omomyc (5 μg / ml MOG). 35-55 After 48 h, cell death was assessed using a staining agent that can fix dead and live cells, and the percentage of dead cells was determined by flow cytometry. (or 5 μg / ml PHA-L)

[0289] Omomyc treatment can delay disease onset and improve the clinical course of the disease. ( Figure 2 To determine the potential therapeutic effect of Omomyc on EAE clinical outcomes, mice were treated with 50 mg / kg Omomyc daily via intraperitoneal administration, starting on day 1 post-immunization. Clinical signs and weight loss in mice were monitored daily, and clinical progress was compared with a cohort of mice treated with the loading medium (20 mM sodium acetate, pH 5.5, 200 mM sorbitol, 0.02% w / v Tween® 20).

[0290] Omomyc treatment significantly improved the clinical course of the disease, and the animals exhibited milder clinical signs. ( Figure 3To test routes of administration compatible with those used in humans, mice were administered 75 mg / kg of Omomyc intravenously three times weekly, starting on day 1 post-immunization. The dose of Omomyc was increased to offset the reduction in dosing frequency. Clinical signs and weight loss in mice were monitored daily, and clinical progress was compared with a group of mice that had been administered the load.

[0291] The beneficial effects of Omomyc are long-lasting. ( Figure 4 Starting on day 1 post-immunization, mice were administered 75 mg / kg of Omomyc intraperitoneally every other day, and treatment was discontinued after the fourth dose. Mice were followed up for 28 days, with clinical signs and weight loss monitored daily, and clinical progress compared to a cohort of mice that had received the loaded formulation.

[0292] result All data were obtained using experimental models of multiple sclerosis and experimental autoimmune encephalomyelitis (EAE).

[0293] In the first set of experiments, the inventors tested the ability of Omomyc to regulate the proliferation of spleen cells from EAE-affected mice. They observed that increasing the concentration of Omomyc eliminated the proliferative capacity of spleen cells from EAE-affected mice stimulated with either their own antigens (35-55 myelin oligodendrocyte glycoprotein or MOG peptide) or polyclonal stimulants (phytohemagglutinin or PHA). Furthermore, the mortality rate of MOG- and PHA-stimulated EAE spleen cells increased with increased Omomyc concentrations, and both effects were dose-dependent. Figure 1 ).

[0294] The inventors then aimed to test the efficacy of Omomyc in vivo. Omomyc was administered daily to EAE mice via intraperitoneal injection at a dose of 50 mg / kg. Daily treatment with Omomyc delayed disease onset and improved clinical outcomes, as the mice exhibited milder neurological dysfunction, reaching its maximum therapeutic effect 17 days after the first administration. Weight loss was also a characteristic of EAE-affected mice, increasing with disease severity, becoming particularly pronounced when neurological dysfunction began and the disease reached its peak. In this regard, they also observed that the Omomyc-treated animals did not exhibit weight loss compared to their corresponding animals treated with their load, confirming the milder disease in the Omomyc-treated mice. Figure 2 ).

[0295] The inventors then attempted to use a human-approved route of administration. To this end, they administered Omomyc intravenously to EAE mice. Because the EAE model is highly sensitive to stress, the dosing frequency was reduced to three times per week to avoid stress from daily manipulation of the mice. To overcome the reduced dosing frequency, they used a dose of 75 mg / kg per administration. Treatment with Omomyc significantly reduced EAE outcomes, with less severe clinical course (AUC of the score) and lower levels of neurological dysfunction at the peak of the disease (reaching the maximum impairment score). In fact, the Omomyc-treated animals showed only slight weight loss and rapid recovery compared to their counterparts treated with their load, while the load-treated animals showed significant and sustained weight loss after the onset of neurological dysfunction. Figure 3 ).

[0296] The inventors also aimed to demonstrate whether the beneficial effects of Omomyc were durable. To this end, they administered four doses of Omomyc intraperitoneally before the onset of EAE but after disease induction. When EAE mice received only four doses of Omomyc, they observed clinical benefit. Mice treated with Omomyc experienced delayed disease onset and a milder clinical course (manifested as a decrease in daily AUC scores). The days with the greatest clinical benefit were 14 days after the start of treatment and seven days after the last dose, implying that the effects of Omomyc are durable. Furthermore, mice treated with Omomyc showed almost no weight loss compared to the group treated with a loading apparatus. Figure 4 ).

Claims

1. Use of a compound selected from the group consisting of, for the prevention and / or treatment of an autoimmune disease in a subject: (a) A polypeptide containing the sequence SEQ ID NO: 1 or a functionally equivalent variant thereof, (b) A conjugate comprising a polypeptide comprising the sequence SEQ ID NO: 1 or a functionally equivalent variant thereof, and a chemical moiety that promotes cellular uptake of said polypeptide or a functionally equivalent variant thereof. (c) A polynucleotide encoding the polypeptide of (a) or the conjugate of (b). (d) A vector comprising the polynucleotide according to (c), and (e) Cells capable of secreting the polypeptide according to (a) or the conjugate according to (b) into a culture medium.

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

10.

3. The compound for the use according to any one of claims 1 or 2, wherein, The chemical component that promotes cellular uptake of the polypeptide or a functionally equivalent variant thereof is a cell-penetrating peptide sequence, and wherein the cell-penetrating peptide sequence and the polypeptide or a functionally equivalent variant thereof form a fusion protein.

4. The compound for the use according to claim 3, wherein, The cell-penetrating peptide sequence is selected from the group consisting of: SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 37 and SEQ ID NO:

38.

5. The compound for use according to any one of claims 1 to 4, wherein, The subjects were humans.

6. The compound for use according to any one of claims 1 to 5, wherein, The compound is a polypeptide containing the sequence SEQ ID NO:

1.

7. The compound for use according to any one of claims 1 to 6, wherein, The autoimmune diseases are selected from the group consisting of: celiac disease, type I diabetes, latent autoimmune diabetes mellitus (LADA) in adults, Graves' disease, inflammatory bowel disease, multiple sclerosis, alopecia areata, Addison's disease, pernicious anemia, psoriasis, rheumatoid arthritis, and systemic lupus erythematosus.

8. The compound for use according to claim 7, wherein, The autoimmune disease mentioned is multiple sclerosis.

9. The compound for use according to any one of claims 1 to 6, wherein, The autoimmune disease is an autoimmune demyelinating disease of the central nervous system (CNS), preferably selected from the group consisting of: multiple sclerosis (MS), clinically isolated syndrome (CIS), tumor-like MS, Marburg acute MS, Barlow concentric sclerosis, acute disseminated encephalomyelitis (ADEM), acute hemorrhagic leukoencephalitis, Heard's disease, isolated sclerosis, transverse myelitis, Sussac syndrome, leukoaraiosis, myalgic encephalomyelitis, Guillain-Barré syndrome, progressive inflammatory neuropathy, leukodystrophy, including adrenoleukodystrophy, adrenal neuropathy, post-vaccination encephalitis (PVE), post-infectious encephalomyelitis (PIE), neuromyelitis optica (NMO), optic neuritis, and myelin oligodendrocyte glycoprotein (MOG)-related disease (MOGAD).

10. The compound for use according to any one of claims 1 to 9, wherein, The compound is administered intradermally, subcutaneously, intravenously, intraperitoneally, intramuscularly, intranasally, or orally, preferably intravenously or intraperitoneally.

11. The compound for use according to any one of claims 1 to 10, wherein, The compound is administered in combination with at least one other therapeutic agent for the treatment of autoimmune diseases, wherein the at least one other therapeutic agent is administered simultaneously, sequentially or separately, preferably selected from the group consisting of: corticosteroids, olfatumumab, fingolimod, sinimod, ozamod, posamod, latoimod, teriflunomide, fumarates (dimethyl fumarate, diloxicame fumarate, monomethyl fumarate), clavibine, mycophenolate mofetil, atorvastatin, interferon-β, glatiramer acetate, mitoxantrone, cyclophosphamide, natelizumab, dalizumab, rituximab, ustekinumab, ozenafil, alenzumab, valacyclovir, CTLA4Ig, copaxone, tocilizumab, and atacicept.

12. A combination comprising: (i) Choose compounds from the following groups: (a) A polypeptide containing the sequence SEQ ID NO: 1 or a functionally equivalent variant thereof, (b) A conjugate comprising a polypeptide comprising the sequence SEQ ID NO: 1 or a functionally equivalent variant thereof, and a chemical moiety that promotes cellular uptake of said polypeptide or a functionally equivalent variant thereof. (c) A polynucleotide encoding the polypeptide of (a) or the conjugate of (b). (d) A vector comprising the polynucleotide according to (c), and (e) Cells capable of secreting the polypeptide according to (a) or the conjugate according to (b) into a culture medium; and (ii) Agents for the treatment of autoimmune diseases, selected from the group consisting of: copaxone, teriflunomide, fumarate, natelizumab, fingolimod, ozagilumab, posamod, sinimod, ozagilmod, and tocilizumab.

13. A pharmaceutical composition comprising a pharmaceutically effective amount of the combination according to claim 12 and a pharmaceutically acceptable excipient.

14. Use in medicine of the combination according to claim 12 or the pharmaceutical composition according to claim 13.

15. Use of the combination according to claim 12 or the pharmaceutical composition according to claim 13 for the prevention and / or treatment of autoimmune diseases.

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