CELL-PENETRATING PEPTIDES

PRIORITY CLAIM

[0001] This application claims priority to U.S. Provisional Patent Application No. 62/803,123, filed February 8, 2019, the entire contents of which are incorporated herein by reference.

SEQUENCE LISTINGS

[0002] This disclosure includes a sequence listing, which was submitted in ASCII format via EFS-Web, and is hereby incorporated by reference in its entirety. The ASCII copy, created on February 7, 2020, is named 130268 Sequence Listing.txt and is 12 kilobytes in size.

TECHNICAL FIELD

[0003] The embodiments disclosed herein relate generally to HoxD12-PDE8A cell- penetrating peptides, compositions thereof and methods for treating and preventing disease, such as cancer through, administration of the HoxD12-PDE8A cell-penetrating peptides. These peptides facilitate entry into tissues, cells and the nucleus of cells thus allowing the PDE8A fusion protein to reach its site of action for therapeutic purposes.

BACKGROUND

[0004] In diseases, such as cancer, genes may be regulated through the direct delivery of biologically active molecules, such as nucleic acids, peptides and proteins, to their intracellular and intranuclear sites of action to influence gene expression either directly or indirectly through interference with transcription, translation or transcription factor production and action and also missing or defective protein products may be replaced to provide these types of molecules in individuals with germ-line or somatic mutations. The use of biologically important proteins is hampered by the inability of these proteins to reach intracellular sites and tissue sites where they normally function. Furthermore, patients having certain genotypes, including genetic mutations, are cannot be treated with conventional therapeutics and/or develop a resistance to conventional therapeutics over time. [0005] As such, there remains a need for developing novel effective treatments to treat diseases, such as cancer.

SUMMARY

[0006] In some aspects provided herein are conjugates comprising: a first region comprising: (i) a homeodomain structure and having a polypeptide sequence set forth in SEQ ID No. 1 or a variant thereof or (ii) at least one of a HOX D12 homeodomain; and a second region comprising a cAMP-degrading phosphodiesterase-8A (PDE8A) - C-Raf complex disruptor.

[0007] In some aspects provided herein are compositions comprising a conjugate according to any embodiment disclosed and described herein and a pharmaceutically acceptable carrier.

[0008] In some aspects provided herein are methods of treating cancer or an autoimmune disease in a subject comprising administering a formulation comprising a conjugate or composition according to any embodiment disclosed and described herein to the subject.

[0009] In some aspects provided herein are methods of treating cancer in a subject comprising administering a conjugate for a period of time sufficient to increase C-Raf- S259 phosphorylation and/or reduce ERK signaling.

[0010] In some embodiments, the PDE8A - C-Raf complex disruptor is PDE8A or a fragment thereof. In some embodiments, the PDE8A has a polypeptide sequence set forth in SEQ. ID No. 2 or a variant thereof.

[0011] In some embodiments, the second region is conjugated to the C-terminus of the first region. In other embodiments, the second region is conjugated to the N- terminus of the first region.

[0012] In some embodiments, the conjugate is in the form of a fusion protein.

[0013] In some embodiments, the conjugate has a polypeptide sequence set forth in SEQ. ID No. 3.

[0014] In some embodiments, the conjugate further comprises a linker sequence between the first and second regions. In some embodiments, the first region is linked to the second region with a peptidic bond or a non-peptidic bond. In other embodiments, the linker sequence is a thioester linker.

[0015] In some embodiments, the composition is in the form of an inhalable composition, an enema, a topical composition, or an injectable composition including injectable implants for sustained release.

[0016] In some embodiments, the composition further comprises a B-Raf inhibitor.

[0017] In some embodiments, the formulation is administered as an intravenous solution.

[0018] In some embodiments, the conjugate elicits slow cancer cell growth and/or stops cancer cell growth compared to a control. In some embodiments, the control is a peptide comprising the second region when not conjugated to the first region.

[0019] In some embodiments, the cancer is melanoma, malignant melanoma, colon cancer, colorectal cancer, lung cancer, urinary bladder carcinoma, and/or thyroid carcinoma. In some embodiments, the cancer is B-Raf resistant melanoma. In some embodiments, the cancer is metastatic cancer.

[0020] In some embodiments, the subject has previously been administered a B- Raf inhibitor. In some embodiments, the subject is resistant to a B-Raf inhibitor.

[0021] In some embodiments, the conjugate interacts with and/or prevents endogenous PDE8A binding with endogenous C-Raf. In some embodiments, the conjugate potentiates PKA-mediated inhibitory phosphorylation. In some embodiments, at least a portion of total C-Raf remains complexed with PDE8A.

[0022] In some embodiments, the subject is administered a B-Raf inhibitor, for example, the B-Raf inhibitor is administering prior to, simultaneously with, and/or following administration of a conjugate or composition disclosed and described herein.

[0023] In some embodiments, the subject has wildtype BRAF, NRAS gain-of- function mutations, KRAS gain-of-function mutations, and/or HRAS gain-of-function mutations.

[0024] In some embodiments, the autoimmune disease is multiple sclerosis or rheumatoid arthritis. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] FIG. 1 shows exemplary conjugates of HoxD12 (SEQ. ID Nos, 23, 25, 27, and 29) and PDE8A (residues 454-465) (SEQ. ID Nos 24, 26, 28, and 30) formed by thioester or disulfide linkages.

[0026] FIGs. 2A-2C show the effects of HoxD12-PDE8A cell-penetrating conjugates (also referred to herein as“PPL008”) on pERK levels and rate of cell proliferation. Normalized phospho-ERK (mean ± SEM) following treatment with DMSO (lane 1 ), PLX4032 B-Raf inhibitor (lane 2), PPL008 conjugates (lanes 1 1 -14) or PLX co-treatments with PDE8A - C-Raf peptide disrupters (original stearylated‘disrupter’ lane 3, or scrambled control , lane 4) or PPL-008 conjugates (lanes 7-10) in: (A) MM415 (NRAS Q61 L) and (B) A375 (BRAF V600E) human malignant melanoma cell lines. Respective pERK and GAPDH immunoblot examples shown below (N > 3, ^* P < 0.05, ^** P < 0.01 ). (C) Real-time cell analyses (xCELLigence platform) of MM415 cell proliferation following treatments described above. Treatments occurred at 21 hours and the slope of normalized cell index (mean ± STDEV) was measured between 21 - 39 hours, with (ii) representing DMSO vs. peptide disrupter treatments only (10mM) and (iii) representing PLX (1 mM) vs. co-treatments of peptide disrupters and PLX4032 (n = 3, ^*** P < 0.001 ). D, stearylated disrupter; S, stearylated scrambled; N, PPL-008N; C, PPL-008C; NSS, PPL-008NSS; CSS, PPL-008CSS.

[0027] FIGs. 3A-3B show that single-treatment of 10mM PPL-008C and PPL- 008CSS significantly attenuated A375 cell growth. However, pERK suppression is not seen in A375 cells following the same treatments with PPL-008C or PPL-008CSS. (mean ± STDEV, ^** P < 0.01 , ^*** P < 0.001 , n = 3).

[0028] FIGs. 4A-4D show PPL008-C/N dose response in MM415 (NRAS Q61 L) human malignant melanoma cell line. FIGs. 4A and 4B demonstrate normalized pERK expression (mean ± SEM) in MM415 cells following dose response co-treatment with PLX4032 (1 mM) and PPL-008N or PPL-008C (1 nM - 10mM). Respective pERK and GAPDH immunoblot examples shown below. (N = 3, ^* P < 0.05, ^** P < 0.01 , ^*** P < 0.001 ), DMSO vs. PPL008 only, PLX vs. PLX + PPL008). FIGs. 4C(i) and 4D(i) show real-time cell analyses (xCELLigence platform) of MM415 cell proliferation following dose response co-treatments with PPL-008N or PPL-008C (1 nM - 10mM) and PLX4032 (1 mM). Treatments occurred at 32 hours and the slope of normalized cell index (mean ± STDEV) was measured between 32 - 48 hours (n = 3, ^*** P < 0.001 ). FIGs. 4C(ii) and 4D(ii) show representative traces of normalized cell index of each treatment shown below. N, PPL-008N; C, PPL-008C.

[0029] FIGs. 5A-5C demonstrate in vivo suppression of phospho-ERK signaling in a MM415 murine xenograft model. In vivo suppression of phospho-ERK signaling in an MM415 murine xenograft model. FIG. 5A shows normalized total pERK1/2, FIG. 5B shows pERK1 (T202, 44kDa) and FIG. 5C shows pERK2 (Y204, 42kDa) levels (mean ± SEM) in MM415 (Q61 L) tumor xenografts from NSG immuno-deficient mice following PPL-008C treatment, at multiple time points, with either 25mg/kg or 100mg/kg doses (control N = 3, treated N = 4, ^* P < 0.05). Control mice were treated with a 5% dextrose in dH ₂ 0 solution and PPL-008C was administered via subcutaneous injection at the site of tumor. Representative pERK1/2 and GAPDH immunoblot examples are shown below FIG. 5A.

[0030] FIGs. 6A-6B are schematics showing the dual inhibition of B-Raf and C-Raf that inhibits melanoma tumor progression. FIG. 6A shows B-Raf inhibition leads to the Ras negative feedback mechanism switching to C-Raf driven tumorigenesis via potentiation of the Raf/MEK/ERK signalling axis. FIG. 6B shows PPL-008 cell- penetrating peptide (PDE8A - C-Raf disrupter peptide) binds to C-Raf, preventing PDE8A localization within the C-Raf cAMP microdomain and exposing serine 259 - C- Raf to inhibitory phosphorylation by PKA. Co-treatment with B-Raf inhibitor and PPL- 008 blocks onco-Ras driven tumor progression via inhibition of the Raf/MEK/ERK axis.

DETAILED DESCRIPTION

[0031] Melanoma is the most aggressive form of skin cancer, with a wide range of treatments currently available and many more at pre-clinical and clinical phases ⁸ · ³² · ³³ . First line B-Raf inhibitors are capable of managing the majority of melanoma patients that express the BRAF V600E mutation ³³ . However, in patients expressing wildtype BRAF and NRAS or KRAS gain-of-function mutations, B-Raf inhibitors become ineffective and tumors persist - warranting the development of novel effective treatments ^{10 18} . Cell-penetrating conjugates

[0032] Provided herein are novel cell-penetrating peptides and compositions thereof. In particular, provided herein are conjugates comprising: a first region comprising: (i) a homeodomain structure and having a polypeptide sequence set forth in SEQ ID No. 1 or a variant thereof or (ii) at least one of a HOX D12 homeodomain; and a second region comprising a cAMP-degrading phosphodiesterase-8A (PDE8A) - C-Raf complex disruptor.

[0033] The terms “peptide(s),” “protein(s),” and “polypeptide(s)” are used synonymously.

[0034] The term“homeodomain structure” refers to HOX-derived homeodomains, such as the human HOX D12 and HOX C12 sequences shown in Table 1 as SEQ ID No. 1 and SEQ ID No. 4, respectively, or variants or portions thereof.

[0035] Table 1 : Peptide Sequence Identities.

[0036] A person of ordinary skill in the molecular biology/biotechnology art would appreciate that numerous variations of the sequences shown in Table 1 would fall within the embodiments disclosed herein. As used herein, homology refers to identity or near identity of nucleotide or amino acid sequences. As is understood in the art, nucleotide mismatches can occur at the third or wobble base in the codon without causing amino acid substitutions in the translated polypeptide sequence. Also, minor nucleotide modifications (e.g., substitutions, insertions or deletions) in certain regions of the gene sequence can be tolerated whenever such modifications result in changes in amino acid sequence that do not alter functionality of the final gene product. Homologs of specific DNA sequences may be identified by those skilled in the art using the test of cross hybridization of nucleic acids under conditions of stringency as is well understood m the art (as described in Hames et al. , Nucleic Acid Hybridisation, (1985) IRL Press, Oxford, UK). Extent of homology is often measured in terms of percentage of identity between the sequences compared.

[0037] The term "variant" refers to a polypeptide or protein that differs from a reference polypeptide or protein, but retains essential properties. A typical variant of a polypeptide differs in amino acid sequence from another, reference polypeptide. Generally, differences are limited so that the sequences of the reference polypeptide and the variant are closely similar overall (homologous) and, in many regions, identical. A variant and reference polypeptide may differ in amino acid sequence by one or more modifications (e.g., substitutions, additions, and/or deletions). A substituted or inserted amino acid residue may or may not be one encoded by the genetic code. A variant of a polypeptide may be naturally occurring such as an allelic variant, or it may be a variant that is not known to occur naturally.

[0038] Modifications and changes can be made in the structure of the polypeptides and proteins of this disclosure and still result in a molecule having similar characteristics as the polypeptide (e.g., a conservative amino acid substitution). For example, certain amino acids can be substituted for other amino acids in a sequence without appreciable loss of activity. Because it is the interactive capacity and nature of a polypeptide that defines that polypeptide's or protein's biological functional activity, certain amino acid sequence substitutions can be made in a polypeptide or protein sequence and nevertheless obtain a polypeptide or protein with like properties.

[0039] Amino acid substitutions are generally based on the relative similarity of the amino acid side-chain substituents, for example, their hydrophobicity, hydrophilicity, charge, size, and the like. Exemplary substitutions that take one or more of the foregoing characteristics into consideration are well known to those of skill in the art and include, but are not limited to (original residue: exemplary substitution): (Ala: Gly, Ser), (Arg: Lys), (Asn: Gin, His), (Asp: Glu, Cys, Ser), (Gin: Asn), (Glu: Asp), (Gly: Ala), (His: Asn, Gin), (lie: Leu, Val), (Leu: lie, Val), (Lys: Arg), (Met: Leu, Tyr), (Ser: Thr), (Thr: Ser), (Tip: Tyr), (Tyr: Trp, Phe), and (Val: lie, Leu). Embodiments of this disclosure, therefore, consider functional or biological equivalents of a polypeptide or protein as set forth above. In some embodiments, and proteins can include variants having about 50%, 60%, 70%, 80%, 90%, and 95% sequence identity to the polypeptide and protein of interest. In some embodiments, D-amino acids can be used for the first region and/or the second region. It is contemplated that using D-amino acids for at least the second region will reduce intracellular degradation and/or increase the pharmacological half- life.

[0040] "Identity," as known in the art, is a relationship between two or more polypeptide or protein sequences, as determined by comparing the sequences. In the art, "identity" also refers to the degree of sequence relatedness between polypeptides or proteins, as determined by the match between strings of such sequences. "Identity" can be readily calculated by known bioinformational methods.

[0041] First Regiorr. The first region of the conjugate or fusion protein embodiments disclosed may comprise a natural or synthetic 60-amino acid peptide or variant or portion thereof derived from the HOX D12 or HOX C12 gene or variants or portions thereof.

[0042] In one embodiment, the HOX D12 (SEQ ID No. 1 ) amino acid sequence is:

ARKKRKPYTKQQIAELENEFLVNEFINRQKRKELSNRLNLSDQQVKIWFQNRR

MKKKRVV

[0043] In another embodiment, the HOX C12 (SEQ ID No. 4) amino acid sequence is:

SRKKRKPYSKLQLAELEGEFLVNEFITRQRRRELSDRLNLSDQQVKIWFQNR

RMKKKRLL

[0044] In addition, synthetic variants may be used provided that they retain the ability to translocate the membrane. Synthetic variants will generally differ from the naturally-occurring proteins by substitution, particularly conservative substitution. The phrase“conservative amino acid changes” herein means replacing an amino acid from one of the amino acid groups, namely hydrophobic, polar, acidic or basic, with an amino acid from within the same group. An example of such a change is the replacement of valine by methionine and vice versa. Other examples of conservative substitutions may be seen by reference to Table 2 below: [0045] Table 2: Conservative Amino Acid Substitutions.

[0046] Such variants may be synthesized directly or prepared using standard recombinant DNA techniques such as site-directed mutagenesis. Where insertions are to be made, synthetic DNA encoding the insertion together with 5' and 3' flanking regions corresponding to the naturally-occurring sequence either side of the insertion site. The flanking regions will contain convenient restriction sites corresponding to sites in the naturally-occurring sequence so that the sequence may be cut with the appropriate enzyme(s) and the synthetic DNA ligated into the cut. The DNA is then expressed to make the encoded protein. These methods are only illustrative of the numerous standard techniques known in the art for manipulation of DNA sequences and other known techniques may also be used. Variants that retain at least 50% sequence identity with the claimed 60-amino acid sequences or variants or portions thereof derived from HOX-D12 will likely maintain their cell permeability characteristics and retain their human characteristics resulting in low immunogenicity potential. The ability of a naturally occurring or synthetic HOX sequence to translocate the membrane may be tested by routine methods known in the art.

[0047] Second Region Peptides or Proteins. The second region of the conjugate or fusion protein embodiments disclosed may comprise a cAMP-degrading phosphodiesterase-8A (PDE8A) - C-Raf complex disruptor. The second region also may or may not be from the same species as the first region, but the first and second regions will be present in the conjugate or fusion protein embodiments in a manner different from the natural situation.

[0048] The second region of the fusion protein or conjugate embodiments may be a cAMP-degrading phosphodiesterase-8A (PDE8A) - C-Raf complex disruptor peptide or protein of any length as long as it is biologically active on its target when included in the fusion protein or conjugate. [0049] In some embodiments, the PDE8A - C-Raf complex disruptor is PDE8A or a fragment thereof, for example, in certain embodiments, the PDE8A has a polypeptide sequence set forth in SEQ. ID No. 2 or a variant thereof. In other embodiments, the conjugate comprises polypeptide sequence set forth in SEQ. ID No. 3.

[0050] The term “conjugate” or “conjugates” herein comprises a category of structures, including fusion proteins, in which the first region, a homeodomain sequence or variant or portion thereof, is conjugated to the PDE8A - C-Raf complex disruptor directly via a peptide bond or other type of bond including both covalent and non- covalent bonds. Conjugates may include a linker region that connects the homeodomain sequence to the PDE8A - C-Raf complex disruptor that is not naturally associated with the first region.

[0051] The phrase“not naturally associated with” means that entire sequence of the conjugate or fusion protein is not found in nature, and that the entire sequence is not encoded for by a single gene found in nature.

[0052] Any of a wide variety of linkers (short, connecting sequences) known in the art may be utilized to form the conjugate provided that function of the conjugate is not compromised by its addition. Thus, translocation of the second region is enabled through a cellular or nuclear membrane. For example, see a wide variety of linkers known in the art in Chen et al. “Fusion protein linkers: property, design and functionality.” Advanced Drug Delivery Reviews. http://dx.doi.Org/10.1016/J.addr.2012.09.039. In alternative embodiments the term “fusion protein” is used to refer to a particular subcategory of conjugate that exists when no such linkers are used to form the conjugate and the domains are linked entirely by peptide bonds.

[0053] The first region and the second regions may be linked by a cleavable linker region this may be any region suitable for this purpose provided the function of the conjugate is not compromised by its addition. In some embodiments, the second region is conjugated to the C-terminus of the first region. In other embodiments, the second region is conjugated to the N-terminus of the first region.

[0054] The cleavable linker region is a protease cleavable linker, although other linkers, cleavable for example by small molecules, may be used. These include Met-X sites, cleavable by cyanogen bromide, Asn-Gly, cleavable by hydroxylamine, Asp-Pro, cleavable by weak acid and Trp-X cleavable by, inter alia, NBS-skatole. Protease cleavage sites require milder cleavage conditions and are found in, for example, factor Xa, thrombin and collagenase. Any of these may be used. The precise sequences are available in the art and the skilled person will have no difficulty in selecting a suitable cleavage site. The cleavable linker region may be one that is targeted by endocellular proteases. Linkers may not be required for function but linkers may be included between first and second regions to allow targeted release of the second region without compromising function or to enhance biological activity of the second region with linker cleavage.

[0055] Table 3 - showing partial listing of linkers known in the art, adapted from Chen et al., 2012.

[0056] The conjugates of the present disclosure may comprise a linker sequence between the first and second regions. In some embodiments, the first region is linked to the second region with a peptidic bond or a non-peptidic bond. In some embodiments, the linker sequence is a thioester linker.

Compositions

[0057] Composition embodiments disclosed herein may comprise one or more of the cell-penetrating conjugates disclosed and described herein and a pharmaceutically acceptable carrier, diluent or excipient. The term "pharmaceutically acceptable carrier diluent or excipient" refers to any substance, not itself a therapeutic agent, used as a carrier or vehicle for delivery of a therapeutic agent to a subject or added to a pharmaceutical composition to improve its handling or storage properties or to permit or facilitate formation of a unit dose of the composition, and that does not produce unacceptable toxicity or interaction with other components in the composition.

[0058] The choice of pharmaceutically acceptable carrier, excipient or diluent may be selected based on the formulation and the intended route of administration, as well as standard pharmaceutical practice. Such compositions may comprise any agents that may aid, regulate, release or increase entry into the body compartment, tissue, intracellular or intranuclear target site, such as binder(s), lubricant(s), suspending agent(s), coating agent(s), solubilizing agent(s), or other agents. An injectable implant for the sustained release of the protein may also be used to obtain prolonged exposure and action. The term "sustained release" refers to formulations from which the conjugate is released at a slow rate allowing for a longer period of exposure at active concentrations.

[0059] The compositions comprising one or more cell-penetrating conjugates disclosed herein can be administered, depending on condition to be treated or other considerations, in any number of ways, for example without limitation, by any one or more of the following: (1 ) inhalation; (2) in the form of a suppository or pessary; (3) in the form of a topical lotion, solution, cream, ointment or dusting powder; (4) by use of a skin patch; (5) orally in the form of tablets containing excipients such as starch or lactose, or in capsules or ovules either alone or in admixture with excipients, or in the form of elixirs, solutions or suspensions containing flavoring or coloring agents; (6) injected parenterally, for example intracavernosally, intravenously, intramuscularly or subcutaneously; (7) for ophthalmic diseases, they may be formulated as eye drops or for intraocular injection; (8) for parenteral administration, they may be in the form of a sterile aqueous solution or injectable implant which may contain other substances, for example, with adequate salt or monosaccharide content to make the solution isotonic with blood or substances that allow slow release; and (9) for buccal or sublingual administration the compositions may be administered in the form of tablets or lozenges which can be formulated in a conventional manner. In some embodiments, the composition is in the form of an inhalable composition, an enema, a topical composition, or an injectable composition including injectable implants for sustained release. [0060] In some embodiments, the cell-penetrating conjugates further comprise a B- Raf inhibitor. Non-limiting examples of suitable B-Raf inhibitors include vemurafenib (aslso known as PLX4032 and Zelboraf, Daiichi-Sankyo), encorafenib, XL281 (also known as BMS-908662, Bristol-Myers Squibb), LGX818 (Novartis), PLX3603 (Hofmann-LaRoche), RAF265 (Novartis), R05185426 (Hofmann-LaRoche), and GSK21 18436 (also known as dabrafenib and Tafinlar, GlaxoSmithKline).

[0061] In one embodiment, the active concentration of cell-penetrating conjugates in cell culture is less than about 1 15 mM, less than about 100 pM, less than about 90 pM, less than about 80 pM, less than about 70 pM, less than about 65 pM, less than about 60 pM, less than about 55 pM, less than about 50 pM, less than about 45 pM, less than about 40 pM, less than about 35 pM, less than about 30 pM, less than about 25 pM, less than about 20 pM, less than about 15 pM, less than about 10 pM, less than about 5 pM, or less than about 1 pM for example, less than about 1 pM to about 3 pM, less than about 1 pM to about 6 pM, less than about 1 pM to about 8 pM, less than about 1 pM to about 15 pM, less than about 1 pM to about 25 pM, less than about 1 pM to about 50 pM, less than about 1 pM to about 70 pM, less than about 1 pM to about 85 pM, less than about 1 pM to about 1 10 pM, less than about 10 pM to about 1 10 pM, less than about 15 pM to about 70 pM, less than about 15 pM to about 60 pM, less than about 20 pM to about 55 pM, or less than about 25 pM to about 45 pM.

[0062] In other embodiments, the dosage delivered (daily or as required) in mouse models (per 20 g mouse) through (1 ) an intravenous (i.v.) or intraperitoneal (i.p.) injection, (2) a topical formulation, or (3) an inhaled formulation is at least 500 pg, at least 450 pg, at least 400 pg, at least 350 pg, at least 300 pg, at least 250 pg, at least 200 pg, at least 150 pg, at least 100 pg, at least 80 pg, at least 70 pg, at least 60 pg, at least 50 pg, at least 40 pg, at least 30 pg, at least 20 pg, at least 10 pg, or at least 1 pg, for example, about 1 pg to about 500 pg, about 10 pg to about 450 pg, about 20 pg to about 400 pg about 30 pg to about 350 pg, about 30 pg to about 200 pg, about 30 pg to about 100 pg, about 40 pg to about 300 pg, about 40 pg to about 200 pg, about 50 pg to about 100 pg, about 50 pg to about 90 pg, about 55 pg to about 85 pg, about 60 pg to about 80 pg, about 60 pg to about 100 pg; about 1 pg to about 200 pg; about 1 pg to about 100 pg, about 1 pg to about 90 pg, about 1 pg to about 80 pg, about 1 pg to about 70 pg, about 1 to about 60 pg, about 1 to about 50 pg, about 1 to about 40 pg, about 1 to about 30 pg, about 1 to about 20 pg, about 1 to about 15 pg, about 1 to about 12 pg, about 1 to about 10 pg, about 1 to about 8 pg about 1 to about 6 pg, about 1 to about 4 pg, or about 1 to about 3 pg.

[0063] In other embodiments, the dosage delivered (daily or as required) through an intravenous (i.v.) or intraperitoneal (i.p.) injection in a mouse model is at least 100 mg/kg, less than about 80 mg/kg, less than about 45 mg/kg, less than about 40 mg/kg, less than about 30 mg/kg, less than about 25 mg/kg, less than about 20 mg/kg, less than about 15 mg/kg, less than about 12 mg/kg, less than about 10 mg/kg, less than about 8 mg/kg, less than about 4 mg/kg, less than about 2 mg/kg, or less than about 1 mg/kg, for example, less than about 1 mg/kg to about 50 mg/kg, about 5 mg/kg to about 40 mg/kg, about 8 mg/kg to about 30 mg/kg, about 10 mg/kg to about 20 mg/kg, or about 12 mg/kg to about 15 mg/kg, about 8 mg/kg to about 12 mg/kg, about 5 mg/kg to about 9 mg/kg, about 3 mg/kg to about 6 mg/kg, about 2 mg/kg to about 5 mg/kg, about 2 mg/kg to about 4 mg/kg, about 1 mg/kg to about 3 mg/kg, about 1 mg/kg to about 2.0 mg/kg.

[0064] In other embodiments, the dosage delivered (daily or as required) through an inhaled formulation in humans (70 kg weight) is at least about 600 mg, at least about 500 mg, at least about 450 mg, at least about 400 mg, at least about 350 mg, at least about 300 mg, at least about 250 mg, at least about 200 mg, at least about 150 mg, at least about 125 mg, at least about 100 mg, at least about 75 mg, at least about 50 mg, at least about 25 mg, at least about 20 mg, at least about 15 mg, at least about 10 mg, at least about 5 mg, at least about 1 mg, at least about 500 pg, at least about 450 pg, at least about 400 pg, at least about 350 pg, at least about 300 pg, at least about 250 pg, at least about 200 pg, at least about 150 pg, at least about 100 pg, at least about 80 pg, at least about 70 pg, at least about 60 pg, at least about 50 pg, at least about 40 pg, at least about 30 pg, at least about 20 pg, at least about 10 pg, or at least about 1 pg, for example, between about 1 pg to about 750 pg; about 1 pg to about 500 pg, about 10 pg to about 450 pg, about 20 pg to about 400 pg about 30 pg to about 350 pg, about 30 pg to about 200 pg, about 30 pg to about 100 pg, about 40 pg to about 300 pg, about 40 pg to about 200 pg, about 50 pg to about 100 pg, about 50 pg to about 90 pg, about 55 pg to about 85 pg, about 60 pg to about 80 pg, about 60 pg to about 100 pg; about 1 pg to about 200 pg; about 1 pg to about 100 pg, about 1 pg to about 90 pg, about 1 pg to about 80 pg, about 1 pg to about 70 pg, about 1 to about 60 pg, about 1 to about 50 pg, about 1 to about 40 pg, about 1 to about 30 pg, about 1 to about 20 pg, about 1 to about 15 pg, about 1 to about 12 pg, about 1 to about 10 pg, about 1 to about 8 pg about 1 to about 6 pg, about 1 to about 4 pg, about 1 pg to about 3 pg, about 1 pg to about 1 mg, about 1 pg to about 2 mg, about 1 pg to about 5 mg; about 1 pg to about 10 mg; about 1 mg to about 10 mg, about 1 mg to about 15 mg; about 2 mg to about 20 mg, about 3 mg to about 30 mg, about 4 mg to about 40 mg, about 5 mg to about 50 mg, about 5 mg to about 80 mg, about 5 mg to about 1 10 mg, about 10 mg to about 150 mg, about 10 mg to about 80 mg, about 20 mg to about 70 mg, about 20 mg to about 60 mg, about 30 mg to about 60 mg, about 120 mg to about 190 mg, about 130 mg to about 180 mg, about 130 mg to about 200 mg, about 140 mg to about 250 mg, about 180 mg to about 300 mg, about 190 mg to about 350 mg, about 220 mg to about 400 mg, about 250 mg to about 425 mg, about 280 mg to about 460 mg, about 300 mg to about 480 mg, about 350 mg to about 490 mg, about 380 mg to about 550 mg, about 400 mg to about 580 mg, about 480 mg to about 590 mg, or about 520 mg to about 600 mg.

[0065] In other embodiments, the dosage delivered (daily or as required) through topical formulation in humans and in mouse models is less than about 5% wt/vol, less than about 4.5% wt/vol, less than about 3.5% wt/vol, less than about 2.5% wt/vol, less than about 1 .5% wt/vol, less than about 0.5% wt/vol, less than about 0.4% wt/vol, less than about 0.3% wt/vol, less than about 0.2%, less than about 0.1 % wt/vol, less than about 0.09% wt/vol, less than about 0.08% wt/vol, less than about 0.07% wt/vol, less than about 0.06% wt/vol, less than about 0.05% wt/vol, less than about 0.04% wt/vol, less than about 0.03% wt/vol, less than about 0.02% wt/vol, less than about 0.01 % wt/vol, less than about 0.008% wt/vol, less than about 0.006% wt/vol, less than about 0.004% wt/vol, or less than about 0.002% wt/vol, for example between about 0.002% wt/vol and about 5% wt/vol, about 0.01 % wt/vol and about 4% wt/vol, about 0.05% wt/vol and about 3% wt/vol, about 0.02% wt/vol and about 2.5% wt/vol, about 0.03% wt/vol and about 2% wt/vol, about 0.05% wt/vol and about 1 % wt/vol, about 0.06% wt/vol and about 0.9% wt/vol, about 0.07% wt/vol and about 0.6% wt/vol, about 0.08% wt/vol and about 0.4% wt/vol, about 0.09% wt/vol and about 0.2% wt/vol or about 0.09 wt/vol and about 0.1 % wt/vol.

[0066] In other embodiments the dosage delivered (daily or as required) through a topical formulation in humans (70 kg weight) is less than about 70 pg, less than about 50 pg, less than about 45 pg, less than about 40 pg, less than about 30 pg, less than about 25 pg, less than about 20 pg, less than about 15 pg, less than about 12 pg, less than about 10 pg, less than about 8 pg, less than about 4 pg, less than about 2 pg, or less than about 1 pg, for example, about 1 pg to about 50 pg, about 5 pg to about 40 pg, about 8 pg to about 30 pg, about 10 pg to about 20 pg, or about 12 pg to about 15 pg, about 8 pg to about 12 pg, about 5 pg to about 9, about 3 pg to about 6 pg, about 2 pg to about 5 pg, or less than about 1 pg to about 3 pg.

[0067] In another embodiment, the systemic dosage delivered (daily or as required) through an intravenous, subcutaneous or intramuscular injection or an injectable implant for sustained release formulations in humans is less than about 100 mg/kg, less than about 80 mg/kg, less than about 45 mg/kg, less than about 40 mg/kg, less than about 30 mg/kg, less than about 25 mg/kg, less than about 20 mg/kg, less than about 15 mg/kg, less than about 12 mg/kg, less than about 10 mg/kg, less than about 8 mg/kg, less than about 4 mg/kg, less than about 2 mg/kg, less than about 1 mg/kg, less than about 0.1 mg/kg, or less than about 0.01 mg/kg, for example, less than about 0.01 mg/kg to about 50 mg/kg, less than about 5 mg/kg to about 40 mg/kg, less than about 8 mg/kg to about 30 mg/kg, less than about 10 mg/kg to about 20 mg/kg, or less than about 12 mg/kg to about 15 mg/kg, less than about 8 mg/kg to about 12 mg/kg, less than about 5 mg/kg to about 9 mg/kg, less than about 3 mg/kg to about 6 mg/kg, less than about 2 mg/kg to about 5 mg/kg, less than about 2 mg/kg to about 4 mg/kg, less than about 1 mg/kg to about 3 mg/kg, less than about 0.2 mg/kg to about 2.0 mg/kg, less than about 0.1 mg/kg to about 1.5 mg/kg, or less than about 0.01 mg/kg to about 2.00 mg/kg.

[0068] In other embodiments, the dosage delivered (daily or as required) to humans (based on 70 kg weight) through any formulation other than an intravenous, subcutaneous, or intramuscular injection or injectable implant for the sustained release, inhaled or topical formulation is at least about 600 mg, at least about 500 mg, at least about 450 mg, at least about 400 mg, at least about 350 mg, at least about 300 mg, at least about 250 mg, at least about 200 mg, at least about 150 mg, at least about 125 mg, at least about 100 mg, at least about 75 mg, at least about 50 mg, at least about 25 mg, at least about 20 mg, at least about 15 mg, at least about 10 mg, at least about 5 mg, at least about 1 mg, at least about 500 pg, at least about 450 pg, at least about 400 pg, at least about 350 pg, at least about 300 pg, at least about 250 pg, at least about 200 pg, at least about 150 pg, at least about 100 pg, at least about 80 pg, at least about 70 pg, at least about 60 pg, at least about 50 pg, at least about 40 pg, at least about 30 pg, at least about 20 pg, at least about 10 pg, or at least about 1 pg, for example, between about 1 pg to about 750 pg; about 1 pg to about 500 pg, about 10 pg to about 450 pg, about 20 pg to about 400 pg about 30 pg to about 350 pg, about 30 pg to about 200 pg, about 30 pg to about 100 pg, about 40 pg to about 300 pg, about 40 pg to about 200 pg, about 50 pg to about 100 pg, about 50 pg to about 90 pg, about 55 pg to about 85 pg, about 60 pg to about 80 pg, about 60 pg to about 100 pg; about 1 pg to about 200 pg; about 1 pg to about 100 pg, about 1 pg to about 90 pg, about 1 pg to about 80 pg, about 1 pg to about 70 pg, about 1 to about 60 pg, about 1 to about 50 pg, about 1 to about 40 pg, about 1 to about 30 pg, about 1 to about 20 pg, about 1 to about 15 pg, about 1 to about 12 pg, about 1 to about 10 pg, about 1 to about 8 pg about 1 to about 6 pg, about 1 to about 4 pg, about 1 pg to about 3 pg, about 1 pg to about 1 mg, about 1 pg to about 2 mg, about 1 pg to about 5 mg; about 1 pg to about 10 mg; about 1 mg to about 10 mg, about 1 mg to about 15 mg; about 2 mg to about 20 mg, about 3 mg to about 30 mg, about 4 mg to about 40 mg, about 5 mg to about 50 mg, about 5 mg to about 80 mg, about 5 mg to about 1 10 mg, about 10 mg to about 150 mg, about 10 mg to about 80 mg, about 20 mg to about 70 mg, about 20 mg to about 60 mg, about 30 mg to about 60 mg, about 120 mg to about 190 mg, about 130 mg to about 180 mg, about 130 mg to about 200 mg, about 140 mg to about 250 mg, about 180 mg to about 300 mg, about 190 mg to about 350 mg, about 220 mg to about 400 mg, about 250 mg to about 425 mg, about 280 mg to about 460 mg, about 300 mg to about 480 mg, about 350 mg to about 490 mg, about 380 mg to about 550 mg, about 400 mg to about 580 mg, about 480 mg to about 590 mg, or about 520 mg to about 600 mg.

Methods of Treatment

[0069] Provided herein are novel cell-penetrating peptides and compositions thereof for use in treating and/or preventing diseases and disorders.

[0070] Provided herein are methods of treating cancer or autoimmune diseases in a subject comprising administering a formulation comprising the conjugates or compositions disclosed herein.

[0071] Provided herein are methods of treating cancer in a subject comprising administering a conjugate for a period of time sufficient to increase C-Raf-S259 phosphorylation and/or reduce ERK signaling. [0072] The cell-penetrating conjugates, compositions, formulations, and methods can be used to treat any type of cancer. Non-limiting examples of cancer include skin cancer (e.g., melanoma, malignant melanoma), lung cancer (e.g., non-small lung cell carcinoma), urinary bladder carcinoma, thyroid cancer, prostate cancer, endometrial cancer, lung cancer, breast cancer, colon cancer, and colorectal cancer. In some embodiments, the cancer is melanoma. In some embodiments, the cancer is a gain-of- function onco-ras mutation cancer. In some embodiments, the cancer is a B-Raf resistant cancer, for example, B-Raf resistant melanoma. In some embodiments, the cancer is metastatic cancer.

[0073] The cell-penetrating conjugates, compositions, formulations, and methods can be used to treat any type of autoimmune disease. Non-limiting examples of autoimmune diseases include multiple sclerosis and rheumatoid arthritis.

[0074] Any appropriate method can be used to administer the cell-penetrating peptides. For example, cell-penetrating peptides can be administered orally or via injection (e.g., subcutaneous injection, intramuscular injection, intravenous injection, or intrathecal injection). In some cases, cell-penetrating peptides can be administered by different routes. For example, one cell-penetrating peptide can be administered orally and a second cell-penetrating peptide can be administered via injection. In some cases, cell-penetrating peptides can be administered following resection of a tumor. Cell- penetrating peptides can be administered to a mammal in any amount, at any frequency, and for any duration effective to achieve a desired outcome (e.g., to increase progression-free survival or to increase the time to progression).

[0075] In some cases, cell-penetrating peptides can be administered to a mammal having skin cancer to reduce the progression rate of melanoma by 5, 10, 25, 50, 75, 100, or more percent. For example, the progression rate can be reduced such that no additional cancer progression is detected. Any method can be used to determine whether or not the progression rate of skin cancer is reduced. For example, the progression rate of skin cancer can be assessed by imaging tissue at different time points and determining the amount of cancer cells present. The amounts of cancer cells determined within tissue at different times can be compared to determine the progression rate. After treatment as described herein, the progression rate can be determined again over another time interval. In some cases, the stage of skin cancer after treatment can be determined and compared to the stage before treatment to determine whether or not the progression rate was reduced.

[0076] In some embodiments, the subject has previously been administered a B- Raf inhibitor. Subjects previously administered a B-Raf inhibit may develop a resistance. In some embodiments, the subject has or is developing resistance to a B- Raf inhibitor.

[0077] In one embodiment, cell-penetrating peptides, composition, or formulation is administered systemically or locally (e.g. intraocular injection, enema formulation, inhalation, intratumor injection) at intervals of 6 hours, 12 hours, daily or every other day or on a weekly or monthly basis to elicit the desired benefit or otherwise provide a therapeutic effect. In another embodiment, the cell-penetrating peptides, composition, or formulation is administered as required to elicit the desired benefit or otherwise provide a therapeutic effect.

[0078] In one embodiment, upon treatment of one or more human or animal subjects with any of the cell-penetrating peptides, compositions, or formulations thereof the subject(s) will exhibit one or more of the following outcomes:

(a) an increase in progression-free survival;

(b) an increase in time to progression;

(c) an increase in time to progression without producing significant toxicity to the subject;

(d) a reduction in progression rate of cancer;

(e) stoppage of cancer cell growth;

(f) a reduction in tumor growth;

(g) a decrease in interaction between C-Raf and PDE8A (i.e., endogenous PDE8A);

(h) an increase in PKA-mediated inhibitory phosphorylation of C-Raf (e.g., serine 259 phosphorylation); and/or

(i) a reduction in ERK1/2 phosphorylation/signaling. [0079] In another embodiment, the subject is treated over a period, for example, of about 1 day through the lifetime of the subject, over a period of about 1 day to about 200 weeks, about 1 day to about 100 weeks, about 1 day to about 80 weeks, about 1 day to about 50 weeks, about 1 day to about 40 weeks, about 1 day to about 20 weeks, about 1 day to about 15 weeks, about 1 day to about 12 weeks, about 1 day to about 10 weeks, about 1 day to about 5 weeks, about 1 week to about 4 weeks, about 2 weeks to about 3 weeks, about 1 day to about 2 weeks, about 1 week, about 1 to 5 days, about 1 to 3 days, or about 1 to 2 days.

[0080] In another embodiment comprising a cell-penetrating peptide formulation utilized in any of the proposed studies in the examples provided, in other research and treatment, including animal research for human and animal applications, and veterinary treatment, the treatment group members, or the treatment group(s) will exhibit one or more of the following outcomes, each compared to baseline or control, unless otherwise indicated:

(a) an increase in progression-free survival;

(b) an increase in time to progression;

(c) an increase in time to progression without producing significant toxicity to the subject;

(d) a reduction in progression rate of cancer;

(e) stoppage of cancer cell growth;

(f) a reduction in tumor growth;

(g) a decreased interaction between C-Raf and PDE8A (i.e., endogenous

PDE8A);

(h) an increase in PKA-mediated inhibitory phosphorylation of C-Raf (e.g., serine

259 phosphorylation); and/or

(i) a reduction in ERK1/2 phosphorylation/signaling.

[0081] In another embodiment, upon treatment with a formulation comprising a cell- penetrating peptide disclosed herein, the (1 ) subject(s) or (2) treatment group(s) as disclosed in the studies in the examples, including experimental animals such as mice in animal models, exhibit one or more of the following outcomes compared to controls: (a) an increase in progression-free survival of at least about 1 week, about 2 weeks, about 3 weeks, about 4 weeks, about 2 months, 6 about months, about 1 year, about 2 years, about 3 years, about 4 years, about 5 years, about 6 years, about 7 years, about 8 years, about 9 years, about 10 years, or more;

(b) an increase in time to progression of at least about 1 week, about 2 weeks, about 3 weeks, about 4 weeks, about 2 months, 6 about months, about 1 year, about 2 years, about 3 years, about 4 years, about 5 years, about 6 years, about 7 years, about 8 years, about 9 years, about 10 years, or more;

(c) an increase in time to progression without producing significant toxicity to the subject of at least about 1 week, about 2 weeks, about 3 weeks, about 4 weeks, about 2 months, 6 about months, about 1 year, about 2 years, about 3 years, about 4 years, about 5 years, about 6 years, about 7 years, about 8 years, about 9 years, about 10 years, or more;

(d) a reduction in progression rate of cancer of at least about 99%, at least about 95%, at least about 90%, at least about 80%, at least about 70%, at least about 60%, at least about 50%, at least about 40%, at least about 35%, at least about 30%, at least about 20%, at least about 15%, at least about 10%, or at least about 5%, for example, about 30% to about 99%, about 80% to about 90%, about 70% to about 90%, about 60% to about 90%, about 50% to about 90%, about 40% to about 90%, about 35% to about 90%, about 30% to about 90%, about 25% to about 90%, about 5% to about 85%, or about 10% to about 80% (actual % change or median % change compared to baseline or control);

(e) a stoppage of cancer cell growth;

(f) a reduction in tumor growth of at least about 99%, at least about 95%, at least about 90%, at least about 80%, at least about 70%, at least about 60%, at least about 50%, at least about 40%, at least about 35%, at least about 30%, at least about 20%, at least about 15%, at least about 10%, or at least about 5%, for example, about 30% to about 99%, about 80% to about 90%, about 70% to about 90%, about 60% to about 90%, about 50% to about 90%, about 40% to about 90%, about 35% to about 90%, about 30% to about 90%, about 25% to about 90%, about 5% to about 85%, or about 10% to about 80% (actual % change or median % change compared to baseline or control); (g) decreased interaction between C-Raf and PDE8A (i.e., endogenous PDE8A) of at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, or at least about 99%, for example, about 30% to about 99%, about 80% to about 90%, about 70% to about 90%, about 60% to about 90%, about 50% to about 90%, about 40% to about 90%, about 35% to about 90%, about 30% to about 90%, about 25% to about 90%, about 5% to about 85%, or about 10% to about 80% (actual % change or median % change compared to baseline or control);

(h) an increase in PKA-mediated inhibitory phosphorylation of C-Raf (e.g., serine 259 phosphorylation) of at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, or at least about 99%, for example, about 30% to about 99%, about 80% to about 90%, about 70% to about 90%, about 60% to about 90%, about 50% to about 90%, about 40% to about 90%, about 35% to about 90%, about 30% to about 90%, about 25% to about 90%, about 5% to about 85%, or about 10% to about 80% (actual % change or median % change compared to baseline or control); and/or

(i) a reduction in ERK1/2 phosphorylation/signaling of at least about 99%, at least about 95%, at least about 90%, at least about 80%, at least about 70%, at least about 60%, at least about 50%, at least about 40%, at least about 35%, at least about 30%, at least about 20%, at least about 15%, at least about 10%, or at least about 5%, for example, about 30% to about 99%, about 80% to about 90%, about 70% to about 90%, about 60% to about 90%, about 50% to about 90%, about 40% to about 90%, about 35% to about 90%, about 30% to about 90%, about 25% to about 90%, about 5% to about 85%, or about 10% to about 80% (actual % change or median % change compared to baseline or control). [0082] In some embodiments, at least a portion of total C-Raf remains complexed with PDE8A. In some embodiments at least about 99%, at least about 95%, at least about 90%, at least about 80%, at least about 70%, at least about 60%, at least about 50%, at least about 40%, at least about 35%, at least about 30%, at least about 20%, at least about 15%, at least about 10%, or at least about 5%, for example, about 30% to about 99%, about 80% to about 90%, about 70% to about 90%, about 60% to about 90%, about 50% to about 90%, about 40% to about 90%, about 35% to about 90%, about 30% to about 90%, about 25% to about 90%, about 5% to about 85%, or about 10% to about 80% of total C-Raf remains complexed with PDE8A. In some embodiments less than about 90%, less than about 80%, less than about 70%, less than about 60%, less than about 50%, less than about 40%, less than about 30%, less than about 20%, less than about 10%, or less than about 5% of total C-Raf remains complexed with PDE8A.

[0083] In some embodiments, the subject is administered a B-Raf inhibitor. The B- Raf inhibitor may be administered prior to, simultaneously with, and/or following administration of the cell-penetrating conjugates, compositions, or formulations disclosed herein.

[0084] Various features and embodiments and select modifications will now be described by way of non-limiting examples. In all examples where an initiating methionine is included in a complete sequence to allow recombinant synthesis, the initiating methionine may be removed during purification to derive the active product with the indicated human homeodomain sequence. References to sequence numbers for complete conjugates in paragraphs describing their use are referring to the conjugate after removal of the initiating methionine.

EXAMPLES

Example 1

[0085] Malignant melanoma, an aggressive and lethal form of skin cancer, leaves metastatic patients with a 15 - 20% chance of surviving 5 years with the disease ¹ . Over 50% of melanoma patients carry a mutation in their BRAF gene, with the V600E (valine to glutamic acid) missense mutation being responsible for 80-90% of BRAF mutations ^{2 4} . B-Raf is a serine/threonine protein kinase that is part of the RAS - RAF - MEK - ERK signalling axis, involved in regulating many cellular processes including: differentiation, proliferation, survival and apoptosis ⁵ . This signalling pathway is believed to be crucial to melanoma progression, with the V600E mutation resulting in B-Raf protein conformational changes that constitutively activate B-Raf and downstream MEK - ERK signalling ⁵ .

[0086] As a result, B-Raf-specific small molecule inhibitors (and eventually MEK inhibitors) were developed and found to dramatically improve patient prognosis, survival rate and lead to tumor regression through suppression of downstream ERK signalling ^{6- 9} . Unexpectedly, B-Raf inhibitor resistance was developed in many patients through paradoxical activation of ERK; allowing the cancer to persist ^10-14 . Pathway reactivation is believed to occur as a result of oncogenic mutations in a number of genes, including NRAS (20% of cases; Q61 K/R/L most frequent) and KRAS (2% of cases) gain-of- function mutations ^15-18 . As B-Raf preferentially heterodimerises to C-Raf (vs. other A, B or C-Raf homo/heterodimers), B-Raf inhibition results in a negative feedback mechanism that switches from B-Raf to C-Raf activation by Ras and subsequent tumor invasion and metastasis ^{19 20} . In light of this, C-Raf has become a key therapeutic target for the development of new treatments able to suppress RAS-mediated tumor progression in B-Raf inhibitor resistant melanoma.

[0087] Previously, it was demonstrated that cAMP degrading enzyme, PDE8A, played an important role in protecting C-Raf from PKA-mediated inhibition ²¹ (reviewed ²² ). PDE8A, believed to be responsible for regulating basal cAMP fluctuations, was found to directly interact with C-Raf. The association of C-Raf with a PDE markedly inhibited the ability of local PKA pools to phosphorylate and inhibit the kinase, increasing the likelihood of C-Raf activation. Peptide mapping of the PDE8A-C-Raf interface allowed for the rational development of a cell penetrating peptide disrupter based on the C-Raf binding site on PDE8A ³² ’ ³³ . This disrupter was found to inhibit the PDE8A - C-Raf protein-protein interaction (PPI) and significantly increase C-Raf-S259 phosphorylation while concomitantly supressing phospho-ERK signalling. This concept was verified at an organismal level in both PDE8A knock out mice and a drosophila model, where basal ERK activation was attenuated compared to wild type ²¹ .

[0088] Further verification of the PDE8A - C-Raf PPI inhibitor concept has been supplied by a recent study, which demonstrated that the disrupter was able to attenuate T-effector cell adhesion and migration in an auto-immune multiple sclerosis mouse model. The inhibition of T-effector cell function was a direct result of increased levels of inhibitory C-Raf-S259 phosphorylation and subsequent suppression of ERK activation ²³ . The disrupter produced a more potent effect than highly-selective PDE8 enzyme inhibitors and highlighted a novel approach to targeting T-effector cells in inflammatory disorders.

Generation of HoxD12-PDE8 A cell-penetrating peptides

[0089] To determine whether cell-penetrating conjugates provided superior activity to delivery of PDE8A alone, various HoxD12-PDE8A cell-penetrating peptides (also referred to herein as“PPL-008 conjugates”) were generated. Briefly, HoxD12 (also referred to herein as“Cell Porter®”, Portage Pharmaceuticals) was conjugated onto the C or N-terminal of the disrupter (PDE8A) via either thioester (C or N giving PPL-008C (conjugate of SEQ. ID Nos. 25 and 26) or PPL-008N (conjugate of SEQ. ID Nos 29 and 30)) or disulfide bonds (CSS or NSS giving PPL-008CSS (conjugate of SEQ. ID Nos 23 and 24) or PPL-008NSS (conjugate of SEQ. ID Nos 27 and 28)). Figure 1. Linkers were intended to provide more molecular flexibility for the cargo (i.e. , PDE8A) to bind to its target without interference by the larger HoxD12 structure. PPL-008-CSS and PPL- 008-NSS were designed to include a disulfide bond between the disruptor peptide and the HoxD12 cell penetrating peptide sequence. Intracellular release of the cargo by cleavage of the disulfide bond occurs because of the reducing environment within the cell and not any enzymatic cleavage process. It was contemplated that CCS and NSS cell-penetrating conjugates would allow the PDE8A peptide to function inside the cell without being attached to the larger HoxD12 peptide.

Animals

[0090] Generation of a MM415 melanoma murine xenograft model and in vivo treatment was carried out by Ml Bioresearch (Michigan, USA). All protocols involving animals used were approved by the Institutional Animal Care and Use Committee of the University of Washington in accordance with the National Institutes of Health. In vivo 5- 6 week old female NSG - immunodeficient mice (Jackson Laboratory) were subcutaneously injected with 3.3e+8 MM415 malignant melanoma cells, at the SC - axilla (high) and tumors were allowed to grow for 30 days (-200 - 400mm ³ ). Mice were subcutaneously injected at the site of tumor with PPL-008 peptide drug dissolved in a 5% dextrose - water solution at either 25mg/kg or 100mg/kg. Mice were euthanized and the tumors were harvested at varying time points post-treatment: 30 min, 1 h, 2 h, 4 h, 8 h, 12 h. Tumors were frozen down at -80°C prior to preparation into lysates for follow-up western blot analyses.

MM415 Cell Culture and Drug Treatments

[0091] A375 (V600E) and MM415 (BRAF wt, KRAS wt, NR AS Q61 L) human malignant melanoma epithelial skin cell lines were cultured with RPMI 1640 medium, supplemented with 10% fetal bovine serum (FBS, v/v), 1 % L-glutamine (v/v), 1 % penicillin-streptomycin (v/v) (all Sigma-Aldrich) and incubated at 37°C, 5% C02 and 95% humidity. Cells were split at -80% confluency, using 0.05% trypsin-EDTA, 1 :5.

[0092] The original PDE8A - C-Raf disrupter, and its scrambled isoform, were synthesized with a C-terminal stearic acid group [CH ₃ (CH2)i6COOH] (GenScript) ²¹ . PPL-008 (i.e. PDE8A - C-Raf disrupter, without stearic acid) was synthesized with Cell Porter® conjugated to the C or N-terminus via either thioester or disulphide bonds. All peptides were dissolved to the appropriate concentration in DMSO for in vitro experimentation. PLX4032 (Vemurafenib) was dissolved in DMSO to a final concentration of 1 mM (Sellekchem). Peptides were added to cells for 2 h, and PLX for 1 h, before cells were harvested. In cases where co-treatments were administered, cells were first treated with peptides for 2 h, followed by 1 hr PLX.

Western Blotting

[0093] MM415 and A375 cells harvested from In vitro experiments were lysed in

3T3 lysis buffer, whilst MM415 melanoma murine xenograft tissue was homogenized and lysed in 1X RIPA buffer (both supplemented with protease cocktail inhibitor tablets (Roche)). Soluble fraction of lysate was resolved via SDS/PAGE using 4-12% Bis-Tris gels (NuPAGE). Proteins were transferred at 30V for 1 h onto 0.45pm nitrocellulose membrane (Protran) and blocked for 1 h in 5% non-fat dry milk solution (Marvel, w/v) in 1x TBS-T (20mM Tris-CI pH 7.6, 150mM NaCI, 0.1 % Tween-20). Blocked membranes were incubated in primary antibody (diluted in 1x TBS-T, 1 % marvel) overnight at 4°C. Membranes were washed three times in 1x TBS-T before membranes were incubated in secondary antibody (diluted in 1x TBS-T, 1 % marvel) for 1 h at room temperature. Membranes were washed a final three times in 1x TBS-T and fluorescent intensity of Li-Cor secondary antibody was measured using a Li-Cor Odyssey scanner. Cell Proliferation Measurements

[0094] Real-Time cellular growth analyses of MM415 cells, using the xCELLigence platform (Roche Applied Science), allowed for the label-free measurement of cell proliferation. 96 well E-plates, containing gold microelectrode sensors on the bottom of the plate, were used to measure cellular impedance inside each well as per manufacturer’s instructions. Cellular impedance measurements were translated into ‘cell index’, an arbitrary measurement that increases as MM415 cells adhere and spread-out/grow (and vice versa), giving quantitative information on cell proliferation and viability that were analyzed using RTCA software (Roche). All protocols carried out using the xCELLigence platform were based on prior publications ²¹ ’ ^{27 31} . Following MM415 cell adhesion, cells were treated with one of the peptide disrupters for 2 h, followed by PLX (1 mM). The slope (i.e. rate of cell proliferation/growth) was measured based on the normalized cell index from the point in which treatments were administered, until the response had plateaued appropriately.

Statistical Analyses

[0095] Results from western blot analyses are represented as mean ± SEM (n > 3). Results from xCELLigence cell proliferation assay are represented as mean ± STDEV (n > 3). P < 0.05 indicates data are significant, with significance determined via unpaired t-test using GraphPad Prism software.

[0096] All antibodies and chemical treatments used herein are listed in Table 4 below:

Attenuation of paradoxical activation of pERK signalling and MM415 cell proliferation with HoxD12-PDE8A cell-penetrating peptides

[0097] The cell-penetrating peptide conjugates shown in Figure 1 were used to treat the B-Raf inhibitor resistant MM415 (human malignant melanoma, BRAF wt, KRAS wt,

NRAS Q61 L) cell line. To determine if PPL-008 conjugates (10mM) could suppress phospho-ERK signalling in MM415 cells, pERK levels were determined via western blot

(pERK expression normalized to GAPDH, mean ± SEM, n = 4, Figure 2). MM415 B-Raf inhibitor treatment (PLX4032, 1 mM) clearly induced a paradoxical activation of ERK and this was significantly reduced following treatment with all the analogues (PPL-008N,

PPL-008C, PPL-008NSS and PPL-008CSS) ( ^** P < 0.01 or ^* P < 0.05; Figure 2A, lanes

7-10 inclusive). In addition, pERK was significantly reduced in the human A375 malignant melanoma cell line (BRAF V600E) following PLX treatment (1 mM) (Figure 2B, lanes 2,3,4) with PPL-008 analogues providing no ERK inhibition as a mono-treatment

(Figure 2B, lanes 1 1-14 inclusive) or further ERK inhibition as a co-treatment with PLX

(Figure 2B, lanes 7-10 inclusive). As B-Raf inhibition sufficiently suppressed pERK expression in A375 cells, and as MM415 cells were resistant to PLX (resembling the clinical phenotype of interest), MM415 cells were used for the remainder of the study.

[0098] To assess the ability of PPL-008 conjugates to inhibit cell growth, real-time measurements of MM415 cellular impedance was recorded on the xCELLigence platform as an indicator of cellular proliferation (slope (1/hr), mean ± STD, n = 3). All PPL-008-conjugates (10pM) significantly slowed cell proliferation in PLX treated MM415 cells ( ^* P < 0.05, Figure 2C, lanes 7-14 inclusive). This data suggests treatment (10pM) with each of the PPL-008 analogues suppressed both pERK and cell growth compared with DMSO treated control (Figures 2A and 2C), indicating PPL-008 has potential as an effective therapy in this context. Surprisingly, PPL-008C and PPL- 008CSS mono-treatments significantly attenuated A375 growth (Figure 3A-B) without affecting the phospho-ERK profile (Figure 2B, lanes 1 1-14 inclusive,). B-Raf inhibitors (including PLX4032) are known to potently reduce A375 cell growth. It is noteworthy that, the original stearylated PDE8A - C-Raf disrupter caused no significant reduction in pERK signalling (O’, Figure 2A, lane 5) or cell proliferation (O’, Figure 2C, lane 5) in MM415, similar to its scrambled control (‘S’, Figure 2A, lane 6 and Figure 2C, lane 6 ), suggesting the stearic acid group was insufficient in facilitating cell-penetration in MM415 cells. As all PPL-008 conjugates attenuated both pERK expression and cell proliferation, this indicates that Cell Porter® greatly improves intracellular delivery of PPL-008 conjugates compared with the original disrupter’s stearate group.

[0099] Both PPL-008CSS/NSS significantly inhibited pERK expression and cell proliferation in MM415 cells at 10uM as shown in Figure 2. However, in follow up dose- response experiments (using the same assays, data not shown), both PPL- 008CSS/NSS (PPL-008 conjugated to CellPorter® via disulphide linkage) failed to consistently inhibit pERK expression at all concentrations but remained to attenuate growth - with PPL-008CSS 10uM causing quite a dramatic negative slope result. It was contemplated that the inconsistent results for the CSS / NSS PPL-008 conjugates may be due to cytotoxicity to the MM415 melanoma cell line.

Inhibition of pERK expression and MM415 cell proliferation over multiple doses with HoxD12-PDE8A cell-penetrating peptides

[00100] To determine whether the HoxD12-PDE8A cell-penetrating peptides could inhibit pERK expression and cell proliferation, MM415 cells were co-treated with PLX (1 mM), following pre-treatment with a dose range of PPL-008C or PPL-008N (1 nM - 10mM). The levels of pERK and cell proliferation rates determined as previously described (Figure 4). The levels of pERK triggered by PLX (Figure 4A and 4B, lane 1 vs lane 2) were reduced at all concentrations following PPL-008N treatment, with the higher [10mM] dose causing the most significant reduction ( ^*** P < 0.001 , Figure 4A). This effect was recapitulated in the xCELLigence cell proliferation assay, where PPL- 008N reduced the rate of cell proliferation at all concentrations; most significantly at 10mM ( ^*** P < 0.001 , Figure 4C (i and ii)). In addition, PPL-008C-conjugate reduced MM415 pERK levels and rate of cell proliferation at all concentrations, with [10mM] producing the most significant inhibition ( ^*** P < 0.001 , Figure 4B and 4D (i and ii)). In vivo PPL-008C suppression of pERK in an MM415 melanoma murine xenograft model

[00101] Preliminary in vivo investigation of the effects of PPL-008C were carried out in an immuno-deficient NSG - MM415 melanoma murine xenograft model. PPL-008C was chosen as the lead peptide disrupter due to its consistency in attenuating pERK signalling and cell proliferation as both a single treatment and co-treatment with PLX. Briefly, PPL-008C was administered subcutaneously at the site of the tumor as a single treatment. Tumors were removed at varying time points post-treatment and pERK expression was assessed via western blot (N > 3, Figure 5).

[00102] PPL-008C significantly suppressed pERK1 , pERK2 and total pERK levels over all time-points in the time course and at both doses (25mg/kg and 100mg/kg), excluding the 25mg/kg - 12Hr treatment ( ^* P < 0.05, Figure 5). This shows that a single PPL-008C treatment can attenuate Raf - MEK- ERK signalling relatively quickly (within 30 minutes) and can maintain this inhibition for at least 12 hours at higher concentrations (100mg/kg, Figure 5). Maximal pERK inhibition occurred 2 hours post treatment with 100mg/kg PPL-008C.

[00103] Together this data demonstrates the PDE8A - C-Raf complex as a point of cross-talk between the MAP kinase signalling and cAMP signalling systems, that can be manipulated by a disrupter peptide to promote the inhibition of C-Raf via increased S259 phosphorylation ^{21 23} (Figure 4). This action can counteract C-Raf driven paradoxical activation of ERK in B-Raf inhibitor resistant melanoma cell lines resulting in a retardation of cell proliferation (Figures 2 and 4). Inhibition of C-Raf by targeting its binding to anchoring proteins rather than kinase activity is a novel approach for this kinase and an aim is to displace only a small percentage of total C-Raf that is in complex with PDE8A. Protein-protein interactions (PPIs) are increasingly being regarded as tractable molecular targets for the development of therapeutics, and peptides that mimic docking sites with protein complexes are often the ideal scaffold starting point for such agents ³⁴ .

[00104] The effectiveness of peptide delivery systems is context specific and the data shown herein demonstrates that CellPorter® has directed intracellular delivery of a novel C-Raf - PDE8A peptide disrupter leading to significant suppression of paradoxical ERK activation in a clinically relevant B-Raf inhibitor resistant human melanoma cell line and an apt xenograft model of the disease. It is further contemplated that PPL-008 conjugates represent potential agents for development of co-therapies for resistant melanoma to be administered with an appropriate B-Raf inhibitor in order to overcome B-Raf inhibitor resistance and attenuate ERK activation in melanocytes.

[00105] From the foregoing, it will be appreciated that specific embodiments have been described herein for purposes of illustration, but that various modifications may be made without deviating from the spirit and scope of the disclosure. Accordingly, the disclosure is not limited except as by the appended claims.

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STRUCTURE, MANUFACTURING AND USES OF HOXD12-PDE8A

CELL-PENETRATING PEPTIDES

PRIORITY CLAIM

[0001] This application claims priority to U.S. Provisional Patent Application No. 62/803,123, filed February 8, 2019, the entire contents of which are incorporated herein by reference.

SEQUENCE LISTINGS

[0002] This disclosure includes a sequence listing, which was submitted in ASCII format via EFS-Web, and is hereby incorporated by reference in its entirety. The ASCII copy, created on February 7, 2020, is named 130268 Sequence Listing.txt and is 12 kilobytes in size.

TECHNICAL FIELD

[0003] The embodiments disclosed herein relate generally to HoxD12-PDE8A cell- penetrating peptides, compositions thereof and methods for treating and preventing disease, such as cancer through, administration of the HoxD12-PDE8A cell-penetrating peptides. These peptides facilitate entry into tissues, cells and the nucleus of cells thus allowing the PDE8A fusion protein to reach its site of action for therapeutic purposes.

BACKGROUND

[0004] In diseases, such as cancer, genes may be regulated through the direct delivery of biologically active molecules, such as nucleic acids, peptides and proteins, to their intracellular and intranuclear sites of action to influence gene expression either directly or indirectly through interference with transcription, translation or transcription factor production and action and also missing or defective protein products may be replaced to provide these types of molecules in individuals with germ-line or somatic mutations. The use of biologically important proteins is hampered by the inability of these proteins to reach intracellular sites and tissue sites where they normally function. Furthermore, patients having certain genotypes, including genetic mutations, are cannot be treated with conventional therapeutics and/or develop a resistance to conventional therapeutics over time. [0005] As such, there remains a need for developing novel effective treatments to treat diseases, such as cancer.

SUMMARY

[0006] In some aspects provided herein are conjugates comprising: a first region comprising: (i) a homeodomain structure and having a polypeptide sequence set forth in SEQ ID No. 1 or a variant thereof or (ii) at least one of a HOX D12 homeodomain; and a second region comprising a cAMP-degrading phosphodiesterase-8A (PDE8A) - C-Raf complex disruptor.

[0007] In some aspects provided herein are compositions comprising a conjugate according to any embodiment disclosed and described herein and a pharmaceutically acceptable carrier.

[0008] In some aspects provided herein are methods of treating cancer or an autoimmune disease in a subject comprising administering a formulation comprising a conjugate or composition according to any embodiment disclosed and described herein to the subject.

[0009] In some aspects provided herein are methods of treating cancer in a subject comprising administering a conjugate for a period of time sufficient to increase C-Raf- S259 phosphorylation and/or reduce ERK signaling.

[0010] In some embodiments, the PDE8A - C-Raf complex disruptor is PDE8A or a fragment thereof. In some embodiments, the PDE8A has a polypeptide sequence set forth in SEQ. ID No. 2 or a variant thereof.

[0011] In some embodiments, the second region is conjugated to the C-terminus of the first region. In other embodiments, the second region is conjugated to the N- terminus of the first region.

[0012] In some embodiments, the conjugate is in the form of a fusion protein.

[0013] In some embodiments, the conjugate has a polypeptide sequence set forth in SEQ. ID No. 3.

[0014] In some embodiments, the conjugate further comprises a linker sequence between the first and second regions. In some embodiments, the first region is linked to the second region with a peptidic bond or a non-peptidic bond. In other embodiments, the linker sequence is a thioester linker.

[0015] In some embodiments, the composition is in the form of an inhalable composition, an enema, a topical composition, or an injectable composition including injectable implants for sustained release.

[0016] In some embodiments, the composition further comprises a B-Raf inhibitor.

[0017] In some embodiments, the formulation is administered as an intravenous solution.

[0018] In some embodiments, the conjugate elicits slow cancer cell growth and/or stops cancer cell growth compared to a control. In some embodiments, the control is a peptide comprising the second region when not conjugated to the first region.

[0019] In some embodiments, the cancer is melanoma, malignant melanoma, colon cancer, colorectal cancer, lung cancer, urinary bladder carcinoma, and/or thyroid carcinoma. In some embodiments, the cancer is B-Raf resistant melanoma. In some embodiments, the cancer is metastatic cancer.

[0020] In some embodiments, the subject has previously been administered a B- Raf inhibitor. In some embodiments, the subject is resistant to a B-Raf inhibitor.

[0021] In some embodiments, the conjugate interacts with and/or prevents endogenous PDE8A binding with endogenous C-Raf. In some embodiments, the conjugate potentiates PKA-mediated inhibitory phosphorylation. In some embodiments, at least a portion of total C-Raf remains complexed with PDE8A.

[0022] In some embodiments, the subject is administered a B-Raf inhibitor, for example, the B-Raf inhibitor is administering prior to, simultaneously with, and/or following administration of a conjugate or composition disclosed and described herein.

[0023] In some embodiments, the subject has wildtype BRAF, NRAS gain-of- function mutations, KRAS gain-of-function mutations, and/or HRAS gain-of-function mutations.

[0024] In some embodiments, the autoimmune disease is multiple sclerosis or rheumatoid arthritis. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] FIG. 1 shows exemplary conjugates of HoxD12 (SEQ. ID Nos, 23, 25, 27, and 29) and PDE8A (residues 454-465) (SEQ. ID Nos 24, 26, 28, and 30) formed by thioester or disulfide linkages.

[0026] FIGs. 2A-2C show the effects of HoxD12-PDE8A cell-penetrating conjugates (also referred to herein as“PPL008”) on pERK levels and rate of cell proliferation. Normalized phospho-ERK (mean ± SEM) following treatment with DMSO (lane 1 ), PLX4032 B-Raf inhibitor (lane 2), PPL008 conjugates (lanes 1 1 -14) or PLX co-treatments with PDE8A - C-Raf peptide disrupters (original stearylated‘disrupter’ lane 3, or scrambled control , lane 4) or PPL-008 conjugates (lanes 7-10) in: (A) MM415 (NRAS Q61 L) and (B) A375 (BRAF V600E) human malignant melanoma cell lines. Respective pERK and GAPDH immunoblot examples shown below (N > 3, ^* P < 0.05, ^** P < 0.01 ). (C) Real-time cell analyses (xCELLigence platform) of MM415 cell proliferation following treatments described above. Treatments occurred at 21 hours and the slope of normalized cell index (mean ± STDEV) was measured between 21 - 39 hours, with (ii) representing DMSO vs. peptide disrupter treatments only (10mM) and (iii) representing PLX (1 mM) vs. co-treatments of peptide disrupters and PLX4032 (n = 3, ^*** P < 0.001 ). D, stearylated disrupter; S, stearylated scrambled; N, PPL-008N; C, PPL-008C; NSS, PPL-008NSS; CSS, PPL-008CSS.

[0027] FIGs. 3A-3B show that single-treatment of 10mM PPL-008C and PPL- 008CSS significantly attenuated A375 cell growth. However, pERK suppression is not seen in A375 cells following the same treatments with PPL-008C or PPL-008CSS. (mean ± STDEV, ^** P < 0.01 , ^*** P < 0.001 , n = 3).

[0028] FIGs. 4A-4D show PPL008-C/N dose response in MM415 (NRAS Q61 L) human malignant melanoma cell line. FIGs. 4A and 4B demonstrate normalized pERK expression (mean ± SEM) in MM415 cells following dose response co-treatment with PLX4032 (1 mM) and PPL-008N or PPL-008C (1 nM - 10mM). Respective pERK and GAPDH immunoblot examples shown below. (N = 3, ^* P < 0.05, ^** P < 0.01 , ^*** P < 0.001 ), DMSO vs. PPL008 only, PLX vs. PLX + PPL008). FIGs. 4C(i) and 4D(i) show real-time cell analyses (xCELLigence platform) of MM415 cell proliferation following dose response co-treatments with PPL-008N or PPL-008C (1 nM - 10mM) and PLX4032 (1 mM). Treatments occurred at 32 hours and the slope of normalized cell index (mean ± STDEV) was measured between 32 - 48 hours (n = 3, ^*** P < 0.001 ). FIGs. 4C(ii) and 4D(ii) show representative traces of normalized cell index of each treatment shown below. N, PPL-008N; C, PPL-008C.

[0029] FIGs. 5A-5C demonstrate in vivo suppression of phospho-ERK signaling in a MM415 murine xenograft model. In vivo suppression of phospho-ERK signaling in an MM415 murine xenograft model. FIG. 5A shows normalized total pERK1/2, FIG. 5B shows pERK1 (T202, 44kDa) and FIG. 5C shows pERK2 (Y204, 42kDa) levels (mean ± SEM) in MM415 (Q61 L) tumor xenografts from NSG immuno-deficient mice following PPL-008C treatment, at multiple time points, with either 25mg/kg or 100mg/kg doses (control N = 3, treated N = 4, ^* P < 0.05). Control mice were treated with a 5% dextrose in dH ₂ 0 solution and PPL-008C was administered via subcutaneous injection at the site of tumor. Representative pERK1/2 and GAPDH immunoblot examples are shown below FIG. 5A.

[0030] FIGs. 6A-6B are schematics showing the dual inhibition of B-Raf and C-Raf that inhibits melanoma tumor progression. FIG. 6A shows B-Raf inhibition leads to the Ras negative feedback mechanism switching to C-Raf driven tumorigenesis via potentiation of the Raf/MEK/ERK signalling axis. FIG. 6B shows PPL-008 cell- penetrating peptide (PDE8A - C-Raf disrupter peptide) binds to C-Raf, preventing PDE8A localization within the C-Raf cAMP microdomain and exposing serine 259 - C- Raf to inhibitory phosphorylation by PKA. Co-treatment with B-Raf inhibitor and PPL- 008 blocks onco-Ras driven tumor progression via inhibition of the Raf/MEK/ERK axis.

DETAILED DESCRIPTION

[0031] Melanoma is the most aggressive form of skin cancer, with a wide range of treatments currently available and many more at pre-clinical and clinical phases ⁸ · ³² · ³³ . First line B-Raf inhibitors are capable of managing the majority of melanoma patients that express the BRAF V600E mutation ³³ . However, in patients expressing wildtype BRAF and NRAS or KRAS gain-of-function mutations, B-Raf inhibitors become ineffective and tumors persist - warranting the development of novel effective treatments ^{10 18} . Cell-penetrating conjugates

[0032] Provided herein are novel cell-penetrating peptides and compositions thereof. In particular, provided herein are conjugates comprising: a first region comprising: (i) a homeodomain structure and having a polypeptide sequence set forth in SEQ ID No. 1 or a variant thereof or (ii) at least one of a HOX D12 homeodomain; and a second region comprising a cAMP-degrading phosphodiesterase-8A (PDE8A) - C-Raf complex disruptor.

[0033] The terms “peptide(s),” “protein(s),” and “polypeptide(s)” are used synonymously.

[0034] The term“homeodomain structure” refers to HOX-derived homeodomains, such as the human HOX D12 and HOX C12 sequences shown in Table 1 as SEQ ID No. 1 and SEQ ID No. 4, respectively, or variants or portions thereof.

[0035] Table 1 : Peptide Sequence Identities.

[0036] A person of ordinary skill in the molecular biology/biotechnology art would appreciate that numerous variations of the sequences shown in Table 1 would fall within the embodiments disclosed herein. As used herein, homology refers to identity or near identity of nucleotide or amino acid sequences. As is understood in the art, nucleotide mismatches can occur at the third or wobble base in the codon without causing amino acid substitutions in the translated polypeptide sequence. Also, minor nucleotide modifications (e.g., substitutions, insertions or deletions) in certain regions of the gene sequence can be tolerated whenever such modifications result in changes in amino acid sequence that do not alter functionality of the final gene product. Homologs of specific DNA sequences may be identified by those skilled in the art using the test of cross hybridization of nucleic acids under conditions of stringency as is well understood m the art (as described in Hames et al. , Nucleic Acid Hybridisation, (1985) IRL Press, Oxford, UK). Extent of homology is often measured in terms of percentage of identity between the sequences compared.

[0037] The term "variant" refers to a polypeptide or protein that differs from a reference polypeptide or protein, but retains essential properties. A typical variant of a polypeptide differs in amino acid sequence from another, reference polypeptide. Generally, differences are limited so that the sequences of the reference polypeptide and the variant are closely similar overall (homologous) and, in many regions, identical. A variant and reference polypeptide may differ in amino acid sequence by one or more modifications (e.g., substitutions, additions, and/or deletions). A substituted or inserted amino acid residue may or may not be one encoded by the genetic code. A variant of a polypeptide may be naturally occurring such as an allelic variant, or it may be a variant that is not known to occur naturally.

[0038] Modifications and changes can be made in the structure of the polypeptides and proteins of this disclosure and still result in a molecule having similar characteristics as the polypeptide (e.g., a conservative amino acid substitution). For example, certain amino acids can be substituted for other amino acids in a sequence without appreciable loss of activity. Because it is the interactive capacity and nature of a polypeptide that defines that polypeptide's or protein's biological functional activity, certain amino acid sequence substitutions can be made in a polypeptide or protein sequence and nevertheless obtain a polypeptide or protein with like properties.

[0039] Amino acid substitutions are generally based on the relative similarity of the amino acid side-chain substituents, for example, their hydrophobicity, hydrophilicity, charge, size, and the like. Exemplary substitutions that take one or more of the foregoing characteristics into consideration are well known to those of skill in the art and include, but are not limited to (original residue: exemplary substitution): (Ala: Gly, Ser), (Arg: Lys), (Asn: Gin, His), (Asp: Glu, Cys, Ser), (Gin: Asn), (Glu: Asp), (Gly: Ala), (His: Asn, Gin), (lie: Leu, Val), (Leu: lie, Val), (Lys: Arg), (Met: Leu, Tyr), (Ser: Thr), (Thr: Ser), (Tip: Tyr), (Tyr: Trp, Phe), and (Val: lie, Leu). Embodiments of this disclosure, therefore, consider functional or biological equivalents of a polypeptide or protein as set forth above. In some embodiments, and proteins can include variants having about 50%, 60%, 70%, 80%, 90%, and 95% sequence identity to the polypeptide and protein of interest. In some embodiments, D-amino acids can be used for the first region and/or the second region. It is contemplated that using D-amino acids for at least the second region will reduce intracellular degradation and/or increase the pharmacological half- life.

[0040] "Identity," as known in the art, is a relationship between two or more polypeptide or protein sequences, as determined by comparing the sequences. In the art, "identity" also refers to the degree of sequence relatedness between polypeptides or proteins, as determined by the match between strings of such sequences. "Identity" can be readily calculated by known bioinformational methods.

[0041] First Regiorr. The first region of the conjugate or fusion protein embodiments disclosed may comprise a natural or synthetic 60-amino acid peptide or variant or portion thereof derived from the HOX D12 or HOX C12 gene or variants or portions thereof.

[0042] In one embodiment, the HOX D12 (SEQ ID No. 1 ) amino acid sequence is:

ARKKRKPYTKQQIAELENEFLVNEFINRQKRKELSNRLNLSDQQVKIWFQNRR

MKKKRVV

[0043] In another embodiment, the HOX C12 (SEQ ID No. 4) amino acid sequence is:

SRKKRKPYSKLQLAELEGEFLVNEFITRQRRRELSDRLNLSDQQVKIWFQNR

RMKKKRLL

[0044] In addition, synthetic variants may be used provided that they retain the ability to translocate the membrane. Synthetic variants will generally differ from the naturally-occurring proteins by substitution, particularly conservative substitution. The phrase“conservative amino acid changes” herein means replacing an amino acid from one of the amino acid groups, namely hydrophobic, polar, acidic or basic, with an amino acid from within the same group. An example of such a change is the replacement of valine by methionine and vice versa. Other examples of conservative substitutions may be seen by reference to Table 2 below: [0045] Table 2: Conservative Amino Acid Substitutions.

[0046] Such variants may be synthesized directly or prepared using standard recombinant DNA techniques such as site-directed mutagenesis. Where insertions are to be made, synthetic DNA encoding the insertion together with 5' and 3' flanking regions corresponding to the naturally-occurring sequence either side of the insertion site. The flanking regions will contain convenient restriction sites corresponding to sites in the naturally-occurring sequence so that the sequence may be cut with the appropriate enzyme(s) and the synthetic DNA ligated into the cut. The DNA is then expressed to make the encoded protein. These methods are only illustrative of the numerous standard techniques known in the art for manipulation of DNA sequences and other known techniques may also be used. Variants that retain at least 50% sequence identity with the claimed 60-amino acid sequences or variants or portions thereof derived from HOX-D12 will likely maintain their cell permeability characteristics and retain their human characteristics resulting in low immunogenicity potential. The ability of a naturally occurring or synthetic HOX sequence to translocate the membrane may be tested by routine methods known in the art.

[0047] Second Region Peptides or Proteins. The second region of the conjugate or fusion protein embodiments disclosed may comprise a cAMP-degrading phosphodiesterase-8A (PDE8A) - C-Raf complex disruptor. The second region also may or may not be from the same species as the first region, but the first and second regions will be present in the conjugate or fusion protein embodiments in a manner different from the natural situation.

[0048] The second region of the fusion protein or conjugate embodiments may be a cAMP-degrading phosphodiesterase-8A (PDE8A) - C-Raf complex disruptor peptide or protein of any length as long as it is biologically active on its target when included in the fusion protein or conjugate. [0049] In some embodiments, the PDE8A - C-Raf complex disruptor is PDE8A or a fragment thereof, for example, in certain embodiments, the PDE8A has a polypeptide sequence set forth in SEQ. ID No. 2 or a variant thereof. In other embodiments, the conjugate comprises polypeptide sequence set forth in SEQ. ID No. 3.

[0050] The term “conjugate” or “conjugates” herein comprises a category of structures, including fusion proteins, in which the first region, a homeodomain sequence or variant or portion thereof, is conjugated to the PDE8A - C-Raf complex disruptor directly via a peptide bond or other type of bond including both covalent and non- covalent bonds. Conjugates may include a linker region that connects the homeodomain sequence to the PDE8A - C-Raf complex disruptor that is not naturally associated with the first region.

[0051] The phrase“not naturally associated with” means that entire sequence of the conjugate or fusion protein is not found in nature, and that the entire sequence is not encoded for by a single gene found in nature.

[0052] Any of a wide variety of linkers (short, connecting sequences) known in the art may be utilized to form the conjugate provided that function of the conjugate is not compromised by its addition. Thus, translocation of the second region is enabled through a cellular or nuclear membrane. For example, see a wide variety of linkers known in the art in Chen et al. “Fusion protein linkers: property, design and functionality.” Advanced Drug Delivery Reviews. http://dx.doi.Org/10.1016/J.addr.2012.09.039. In alternative embodiments the term “fusion protein” is used to refer to a particular subcategory of conjugate that exists when no such linkers are used to form the conjugate and the domains are linked entirely by peptide bonds.

[0053] The first region and the second regions may be linked by a cleavable linker region this may be any region suitable for this purpose provided the function of the conjugate is not compromised by its addition. In some embodiments, the second region is conjugated to the C-terminus of the first region. In other embodiments, the second region is conjugated to the N-terminus of the first region.

[0054] The cleavable linker region is a protease cleavable linker, although other linkers, cleavable for example by small molecules, may be used. These include Met-X sites, cleavable by cyanogen bromide, Asn-Gly, cleavable by hydroxylamine, Asp-Pro, cleavable by weak acid and Trp-X cleavable by, inter alia, NBS-skatole. Protease cleavage sites require milder cleavage conditions and are found in, for example, factor Xa, thrombin and collagenase. Any of these may be used. The precise sequences are available in the art and the skilled person will have no difficulty in selecting a suitable cleavage site. The cleavable linker region may be one that is targeted by endocellular proteases. Linkers may not be required for function but linkers may be included between first and second regions to allow targeted release of the second region without compromising function or to enhance biological activity of the second region with linker cleavage.

[0055] Table 3 - showing partial listing of linkers known in the art, adapted from Chen et al., 2012.

[0056] The conjugates of the present disclosure may comprise a linker sequence between the first and second regions. In some embodiments, the first region is linked to the second region with a peptidic bond or a non-peptidic bond. In some embodiments, the linker sequence is a thioester linker.

Compositions

[0057] Composition embodiments disclosed herein may comprise one or more of the cell-penetrating conjugates disclosed and described herein and a pharmaceutically acceptable carrier, diluent or excipient. The term "pharmaceutically acceptable carrier diluent or excipient" refers to any substance, not itself a therapeutic agent, used as a carrier or vehicle for delivery of a therapeutic agent to a subject or added to a pharmaceutical composition to improve its handling or storage properties or to permit or facilitate formation of a unit dose of the composition, and that does not produce unacceptable toxicity or interaction with other components in the composition.

[0058] The choice of pharmaceutically acceptable carrier, excipient or diluent may be selected based on the formulation and the intended route of administration, as well as standard pharmaceutical practice. Such compositions may comprise any agents that may aid, regulate, release or increase entry into the body compartment, tissue, intracellular or intranuclear target site, such as binder(s), lubricant(s), suspending agent(s), coating agent(s), solubilizing agent(s), or other agents. An injectable implant for the sustained release of the protein may also be used to obtain prolonged exposure and action. The term "sustained release" refers to formulations from which the conjugate is released at a slow rate allowing for a longer period of exposure at active concentrations.

[0059] The compositions comprising one or more cell-penetrating conjugates disclosed herein can be administered, depending on condition to be treated or other considerations, in any number of ways, for example without limitation, by any one or more of the following: (1 ) inhalation; (2) in the form of a suppository or pessary; (3) in the form of a topical lotion, solution, cream, ointment or dusting powder; (4) by use of a skin patch; (5) orally in the form of tablets containing excipients such as starch or lactose, or in capsules or ovules either alone or in admixture with excipients, or in the form of elixirs, solutions or suspensions containing flavoring or coloring agents; (6) injected parenterally, for example intracavernosally, intravenously, intramuscularly or subcutaneously; (7) for ophthalmic diseases, they may be formulated as eye drops or for intraocular injection; (8) for parenteral administration, they may be in the form of a sterile aqueous solution or injectable implant which may contain other substances, for example, with adequate salt or monosaccharide content to make the solution isotonic with blood or substances that allow slow release; and (9) for buccal or sublingual administration the compositions may be administered in the form of tablets or lozenges which can be formulated in a conventional manner. In some embodiments, the composition is in the form of an inhalable composition, an enema, a topical composition, or an injectable composition including injectable implants for sustained release. [0060] In some embodiments, the cell-penetrating conjugates further comprise a B- Raf inhibitor. Non-limiting examples of suitable B-Raf inhibitors include vemurafenib (aslso known as PLX4032 and Zelboraf, Daiichi-Sankyo), encorafenib, XL281 (also known as BMS-908662, Bristol-Myers Squibb), LGX818 (Novartis), PLX3603 (Hofmann-LaRoche), RAF265 (Novartis), R05185426 (Hofmann-LaRoche), and GSK21 18436 (also known as dabrafenib and Tafinlar, GlaxoSmithKline).

[0061] In one embodiment, the active concentration of cell-penetrating conjugates in cell culture is less than about 1 15 mM, less than about 100 pM, less than about 90 pM, less than about 80 pM, less than about 70 pM, less than about 65 pM, less than about 60 pM, less than about 55 pM, less than about 50 pM, less than about 45 pM, less than about 40 pM, less than about 35 pM, less than about 30 pM, less than about 25 pM, less than about 20 pM, less than about 15 pM, less than about 10 pM, less than about 5 pM, or less than about 1 pM for example, less than about 1 pM to about 3 pM, less than about 1 pM to about 6 pM, less than about 1 pM to about 8 pM, less than about 1 pM to about 15 pM, less than about 1 pM to about 25 pM, less than about 1 pM to about 50 pM, less than about 1 pM to about 70 pM, less than about 1 pM to about 85 pM, less than about 1 pM to about 1 10 pM, less than about 10 pM to about 1 10 pM, less than about 15 pM to about 70 pM, less than about 15 pM to about 60 pM, less than about 20 pM to about 55 pM, or less than about 25 pM to about 45 pM.

[0062] In other embodiments, the dosage delivered (daily or as required) in mouse models (per 20 g mouse) through (1 ) an intravenous (i.v.) or intraperitoneal (i.p.) injection, (2) a topical formulation, or (3) an inhaled formulation is at least 500 pg, at least 450 pg, at least 400 pg, at least 350 pg, at least 300 pg, at least 250 pg, at least 200 pg, at least 150 pg, at least 100 pg, at least 80 pg, at least 70 pg, at least 60 pg, at least 50 pg, at least 40 pg, at least 30 pg, at least 20 pg, at least 10 pg, or at least 1 pg, for example, about 1 pg to about 500 pg, about 10 pg to about 450 pg, about 20 pg to about 400 pg about 30 pg to about 350 pg, about 30 pg to about 200 pg, about 30 pg to about 100 pg, about 40 pg to about 300 pg, about 40 pg to about 200 pg, about 50 pg to about 100 pg, about 50 pg to about 90 pg, about 55 pg to about 85 pg, about 60 pg to about 80 pg, about 60 pg to about 100 pg; about 1 pg to about 200 pg; about 1 pg to about 100 pg, about 1 pg to about 90 pg, about 1 pg to about 80 pg, about 1 pg to about 70 pg, about 1 to about 60 pg, about 1 to about 50 pg, about 1 to about 40 pg, about 1 to about 30 pg, about 1 to about 20 pg, about 1 to about 15 pg, about 1 to about 12 pg, about 1 to about 10 pg, about 1 to about 8 pg about 1 to about 6 pg, about 1 to about 4 pg, or about 1 to about 3 pg.

[0063] In other embodiments, the dosage delivered (daily or as required) through an intravenous (i.v.) or intraperitoneal (i.p.) injection in a mouse model is at least 100 mg/kg, less than about 80 mg/kg, less than about 45 mg/kg, less than about 40 mg/kg, less than about 30 mg/kg, less than about 25 mg/kg, less than about 20 mg/kg, less than about 15 mg/kg, less than about 12 mg/kg, less than about 10 mg/kg, less than about 8 mg/kg, less than about 4 mg/kg, less than about 2 mg/kg, or less than about 1 mg/kg, for example, less than about 1 mg/kg to about 50 mg/kg, about 5 mg/kg to about 40 mg/kg, about 8 mg/kg to about 30 mg/kg, about 10 mg/kg to about 20 mg/kg, or about 12 mg/kg to about 15 mg/kg, about 8 mg/kg to about 12 mg/kg, about 5 mg/kg to about 9 mg/kg, about 3 mg/kg to about 6 mg/kg, about 2 mg/kg to about 5 mg/kg, about 2 mg/kg to about 4 mg/kg, about 1 mg/kg to about 3 mg/kg, about 1 mg/kg to about 2.0 mg/kg.

[0064] In other embodiments, the dosage delivered (daily or as required) through an inhaled formulation in humans (70 kg weight) is at least about 600 mg, at least about 500 mg, at least about 450 mg, at least about 400 mg, at least about 350 mg, at least about 300 mg, at least about 250 mg, at least about 200 mg, at least about 150 mg, at least about 125 mg, at least about 100 mg, at least about 75 mg, at least about 50 mg, at least about 25 mg, at least about 20 mg, at least about 15 mg, at least about 10 mg, at least about 5 mg, at least about 1 mg, at least about 500 pg, at least about 450 pg, at least about 400 pg, at least about 350 pg, at least about 300 pg, at least about 250 pg, at least about 200 pg, at least about 150 pg, at least about 100 pg, at least about 80 pg, at least about 70 pg, at least about 60 pg, at least about 50 pg, at least about 40 pg, at least about 30 pg, at least about 20 pg, at least about 10 pg, or at least about 1 pg, for example, between about 1 pg to about 750 pg; about 1 pg to about 500 pg, about 10 pg to about 450 pg, about 20 pg to about 400 pg about 30 pg to about 350 pg, about 30 pg to about 200 pg, about 30 pg to about 100 pg, about 40 pg to about 300 pg, about 40 pg to about 200 pg, about 50 pg to about 100 pg, about 50 pg to about 90 pg, about 55 pg to about 85 pg, about 60 pg to about 80 pg, about 60 pg to about 100 pg; about 1 pg to about 200 pg; about 1 pg to about 100 pg, about 1 pg to about 90 pg, about 1 pg to about 80 pg, about 1 pg to about 70 pg, about 1 to about 60 pg, about 1 to about 50 pg, about 1 to about 40 pg, about 1 to about 30 pg, about 1 to about 20 pg, about 1 to about 15 pg, about 1 to about 12 pg, about 1 to about 10 pg, about 1 to about 8 pg about 1 to about 6 pg, about 1 to about 4 pg, about 1 pg to about 3 pg, about 1 pg to about 1 mg, about 1 pg to about 2 mg, about 1 pg to about 5 mg; about 1 pg to about 10 mg; about 1 mg to about 10 mg, about 1 mg to about 15 mg; about 2 mg to about 20 mg, about 3 mg to about 30 mg, about 4 mg to about 40 mg, about 5 mg to about 50 mg, about 5 mg to about 80 mg, about 5 mg to about 1 10 mg, about 10 mg to about 150 mg, about 10 mg to about 80 mg, about 20 mg to about 70 mg, about 20 mg to about 60 mg, about 30 mg to about 60 mg, about 120 mg to about 190 mg, about 130 mg to about 180 mg, about 130 mg to about 200 mg, about 140 mg to about 250 mg, about 180 mg to about 300 mg, about 190 mg to about 350 mg, about 220 mg to about 400 mg, about 250 mg to about 425 mg, about 280 mg to about 460 mg, about 300 mg to about 480 mg, about 350 mg to about 490 mg, about 380 mg to about 550 mg, about 400 mg to about 580 mg, about 480 mg to about 590 mg, or about 520 mg to about 600 mg.

[0065] In other embodiments, the dosage delivered (daily or as required) through topical formulation in humans and in mouse models is less than about 5% wt/vol, less than about 4.5% wt/vol, less than about 3.5% wt/vol, less than about 2.5% wt/vol, less than about 1 .5% wt/vol, less than about 0.5% wt/vol, less than about 0.4% wt/vol, less than about 0.3% wt/vol, less than about 0.2%, less than about 0.1 % wt/vol, less than about 0.09% wt/vol, less than about 0.08% wt/vol, less than about 0.07% wt/vol, less than about 0.06% wt/vol, less than about 0.05% wt/vol, less than about 0.04% wt/vol, less than about 0.03% wt/vol, less than about 0.02% wt/vol, less than about 0.01 % wt/vol, less than about 0.008% wt/vol, less than about 0.006% wt/vol, less than about 0.004% wt/vol, or less than about 0.002% wt/vol, for example between about 0.002% wt/vol and about 5% wt/vol, about 0.01 % wt/vol and about 4% wt/vol, about 0.05% wt/vol and about 3% wt/vol, about 0.02% wt/vol and about 2.5% wt/vol, about 0.03% wt/vol and about 2% wt/vol, about 0.05% wt/vol and about 1 % wt/vol, about 0.06% wt/vol and about 0.9% wt/vol, about 0.07% wt/vol and about 0.6% wt/vol, about 0.08% wt/vol and about 0.4% wt/vol, about 0.09% wt/vol and about 0.2% wt/vol or about 0.09 wt/vol and about 0.1 % wt/vol.

[0066] In other embodiments the dosage delivered (daily or as required) through a topical formulation in humans (70 kg weight) is less than about 70 pg, less than about 50 pg, less than about 45 pg, less than about 40 pg, less than about 30 pg, less than about 25 pg, less than about 20 pg, less than about 15 pg, less than about 12 pg, less than about 10 pg, less than about 8 pg, less than about 4 pg, less than about 2 pg, or less than about 1 pg, for example, about 1 pg to about 50 pg, about 5 pg to about 40 pg, about 8 pg to about 30 pg, about 10 pg to about 20 pg, or about 12 pg to about 15 pg, about 8 pg to about 12 pg, about 5 pg to about 9, about 3 pg to about 6 pg, about 2 pg to about 5 pg, or less than about 1 pg to about 3 pg.

[0067] In another embodiment, the systemic dosage delivered (daily or as required) through an intravenous, subcutaneous or intramuscular injection or an injectable implant for sustained release formulations in humans is less than about 100 mg/kg, less than about 80 mg/kg, less than about 45 mg/kg, less than about 40 mg/kg, less than about 30 mg/kg, less than about 25 mg/kg, less than about 20 mg/kg, less than about 15 mg/kg, less than about 12 mg/kg, less than about 10 mg/kg, less than about 8 mg/kg, less than about 4 mg/kg, less than about 2 mg/kg, less than about 1 mg/kg, less than about 0.1 mg/kg, or less than about 0.01 mg/kg, for example, less than about 0.01 mg/kg to about 50 mg/kg, less than about 5 mg/kg to about 40 mg/kg, less than about 8 mg/kg to about 30 mg/kg, less than about 10 mg/kg to about 20 mg/kg, or less than about 12 mg/kg to about 15 mg/kg, less than about 8 mg/kg to about 12 mg/kg, less than about 5 mg/kg to about 9 mg/kg, less than about 3 mg/kg to about 6 mg/kg, less than about 2 mg/kg to about 5 mg/kg, less than about 2 mg/kg to about 4 mg/kg, less than about 1 mg/kg to about 3 mg/kg, less than about 0.2 mg/kg to about 2.0 mg/kg, less than about 0.1 mg/kg to about 1.5 mg/kg, or less than about 0.01 mg/kg to about 2.00 mg/kg.

[0068] In other embodiments, the dosage delivered (daily or as required) to humans (based on 70 kg weight) through any formulation other than an intravenous, subcutaneous, or intramuscular injection or injectable implant for the sustained release, inhaled or topical formulation is at least about 600 mg, at least about 500 mg, at least about 450 mg, at least about 400 mg, at least about 350 mg, at least about 300 mg, at least about 250 mg, at least about 200 mg, at least about 150 mg, at least about 125 mg, at least about 100 mg, at least about 75 mg, at least about 50 mg, at least about 25 mg, at least about 20 mg, at least about 15 mg, at least about 10 mg, at least about 5 mg, at least about 1 mg, at least about 500 pg, at least about 450 pg, at least about 400 pg, at least about 350 pg, at least about 300 pg, at least about 250 pg, at least about 200 pg, at least about 150 pg, at least about 100 pg, at least about 80 pg, at least about 70 pg, at least about 60 pg, at least about 50 pg, at least about 40 pg, at least about 30 pg, at least about 20 pg, at least about 10 pg, or at least about 1 pg, for example, between about 1 pg to about 750 pg; about 1 pg to about 500 pg, about 10 pg to about 450 pg, about 20 pg to about 400 pg about 30 pg to about 350 pg, about 30 pg to about 200 pg, about 30 pg to about 100 pg, about 40 pg to about 300 pg, about 40 pg to about 200 pg, about 50 pg to about 100 pg, about 50 pg to about 90 pg, about 55 pg to about 85 pg, about 60 pg to about 80 pg, about 60 pg to about 100 pg; about 1 pg to about 200 pg; about 1 pg to about 100 pg, about 1 pg to about 90 pg, about 1 pg to about 80 pg, about 1 pg to about 70 pg, about 1 to about 60 pg, about 1 to about 50 pg, about 1 to about 40 pg, about 1 to about 30 pg, about 1 to about 20 pg, about 1 to about 15 pg, about 1 to about 12 pg, about 1 to about 10 pg, about 1 to about 8 pg about 1 to about 6 pg, about 1 to about 4 pg, about 1 pg to about 3 pg, about 1 pg to about 1 mg, about 1 pg to about 2 mg, about 1 pg to about 5 mg; about 1 pg to about 10 mg; about 1 mg to about 10 mg, about 1 mg to about 15 mg; about 2 mg to about 20 mg, about 3 mg to about 30 mg, about 4 mg to about 40 mg, about 5 mg to about 50 mg, about 5 mg to about 80 mg, about 5 mg to about 1 10 mg, about 10 mg to about 150 mg, about 10 mg to about 80 mg, about 20 mg to about 70 mg, about 20 mg to about 60 mg, about 30 mg to about 60 mg, about 120 mg to about 190 mg, about 130 mg to about 180 mg, about 130 mg to about 200 mg, about 140 mg to about 250 mg, about 180 mg to about 300 mg, about 190 mg to about 350 mg, about 220 mg to about 400 mg, about 250 mg to about 425 mg, about 280 mg to about 460 mg, about 300 mg to about 480 mg, about 350 mg to about 490 mg, about 380 mg to about 550 mg, about 400 mg to about 580 mg, about 480 mg to about 590 mg, or about 520 mg to about 600 mg.

Methods of Treatment

[0069] Provided herein are novel cell-penetrating peptides and compositions thereof for use in treating and/or preventing diseases and disorders.

[0070] Provided herein are methods of treating cancer or autoimmune diseases in a subject comprising administering a formulation comprising the conjugates or compositions disclosed herein.

[0071] Provided herein are methods of treating cancer in a subject comprising administering a conjugate for a period of time sufficient to increase C-Raf-S259 phosphorylation and/or reduce ERK signaling. [0072] The cell-penetrating conjugates, compositions, formulations, and methods can be used to treat any type of cancer. Non-limiting examples of cancer include skin cancer (e.g., melanoma, malignant melanoma), lung cancer (e.g., non-small lung cell carcinoma), urinary bladder carcinoma, thyroid cancer, prostate cancer, endometrial cancer, lung cancer, breast cancer, colon cancer, and colorectal cancer. In some embodiments, the cancer is melanoma. In some embodiments, the cancer is a gain-of- function onco-ras mutation cancer. In some embodiments, the cancer is a B-Raf resistant cancer, for example, B-Raf resistant melanoma. In some embodiments, the cancer is metastatic cancer.

[0073] The cell-penetrating conjugates, compositions, formulations, and methods can be used to treat any type of autoimmune disease. Non-limiting examples of autoimmune diseases include multiple sclerosis and rheumatoid arthritis.

[0074] Any appropriate method can be used to administer the cell-penetrating peptides. For example, cell-penetrating peptides can be administered orally or via injection (e.g., subcutaneous injection, intramuscular injection, intravenous injection, or intrathecal injection). In some cases, cell-penetrating peptides can be administered by different routes. For example, one cell-penetrating peptide can be administered orally and a second cell-penetrating peptide can be administered via injection. In some cases, cell-penetrating peptides can be administered following resection of a tumor. Cell- penetrating peptides can be administered to a mammal in any amount, at any frequency, and for any duration effective to achieve a desired outcome (e.g., to increase progression-free survival or to increase the time to progression).

[0075] In some cases, cell-penetrating peptides can be administered to a mammal having skin cancer to reduce the progression rate of melanoma by 5, 10, 25, 50, 75, 100, or more percent. For example, the progression rate can be reduced such that no additional cancer progression is detected. Any method can be used to determine whether or not the progression rate of skin cancer is reduced. For example, the progression rate of skin cancer can be assessed by imaging tissue at different time points and determining the amount of cancer cells present. The amounts of cancer cells determined within tissue at different times can be compared to determine the progression rate. After treatment as described herein, the progression rate can be determined again over another time interval. In some cases, the stage of skin cancer after treatment can be determined and compared to the stage before treatment to determine whether or not the progression rate was reduced.

[0076] In some embodiments, the subject has previously been administered a B- Raf inhibitor. Subjects previously administered a B-Raf inhibit may develop a resistance. In some embodiments, the subject has or is developing resistance to a B- Raf inhibitor.

[0077] In one embodiment, cell-penetrating peptides, composition, or formulation is administered systemically or locally (e.g. intraocular injection, enema formulation, inhalation, intratumor injection) at intervals of 6 hours, 12 hours, daily or every other day or on a weekly or monthly basis to elicit the desired benefit or otherwise provide a therapeutic effect. In another embodiment, the cell-penetrating peptides, composition, or formulation is administered as required to elicit the desired benefit or otherwise provide a therapeutic effect.

[0078] In one embodiment, upon treatment of one or more human or animal subjects with any of the cell-penetrating peptides, compositions, or formulations thereof the subject(s) will exhibit one or more of the following outcomes:

(a) an increase in progression-free survival;

(b) an increase in time to progression;

(c) an increase in time to progression without producing significant toxicity to the subject;

(d) a reduction in progression rate of cancer;

(e) stoppage of cancer cell growth;

(f) a reduction in tumor growth;

(g) a decrease in interaction between C-Raf and PDE8A (i.e., endogenous PDE8A);

(h) an increase in PKA-mediated inhibitory phosphorylation of C-Raf (e.g., serine 259 phosphorylation); and/or

(i) a reduction in ERK1/2 phosphorylation/signaling. [0079] In another embodiment, the subject is treated over a period, for example, of about 1 day through the lifetime of the subject, over a period of about 1 day to about 200 weeks, about 1 day to about 100 weeks, about 1 day to about 80 weeks, about 1 day to about 50 weeks, about 1 day to about 40 weeks, about 1 day to about 20 weeks, about 1 day to about 15 weeks, about 1 day to about 12 weeks, about 1 day to about 10 weeks, about 1 day to about 5 weeks, about 1 week to about 4 weeks, about 2 weeks to about 3 weeks, about 1 day to about 2 weeks, about 1 week, about 1 to 5 days, about 1 to 3 days, or about 1 to 2 days.

[0080] In another embodiment comprising a cell-penetrating peptide formulation utilized in any of the proposed studies in the examples provided, in other research and treatment, including animal research for human and animal applications, and veterinary treatment, the treatment group members, or the treatment group(s) will exhibit one or more of the following outcomes, each compared to baseline or control, unless otherwise indicated:

(a) an increase in progression-free survival;

(b) an increase in time to progression;

(c) an increase in time to progression without producing significant toxicity to the subject;

(d) a reduction in progression rate of cancer;

(e) stoppage of cancer cell growth;

(f) a reduction in tumor growth;

(g) a decreased interaction between C-Raf and PDE8A (i.e., endogenous

PDE8A);

(h) an increase in PKA-mediated inhibitory phosphorylation of C-Raf (e.g., serine

259 phosphorylation); and/or

(i) a reduction in ERK1/2 phosphorylation/signaling.

[0081] In another embodiment, upon treatment with a formulation comprising a cell- penetrating peptide disclosed herein, the (1 ) subject(s) or (2) treatment group(s) as disclosed in the studies in the examples, including experimental animals such as mice in animal models, exhibit one or more of the following outcomes compared to controls: (a) an increase in progression-free survival of at least about 1 week, about 2 weeks, about 3 weeks, about 4 weeks, about 2 months, 6 about months, about 1 year, about 2 years, about 3 years, about 4 years, about 5 years, about 6 years, about 7 years, about 8 years, about 9 years, about 10 years, or more;

(b) an increase in time to progression of at least about 1 week, about 2 weeks, about 3 weeks, about 4 weeks, about 2 months, 6 about months, about 1 year, about 2 years, about 3 years, about 4 years, about 5 years, about 6 years, about 7 years, about 8 years, about 9 years, about 10 years, or more;

(c) an increase in time to progression without producing significant toxicity to the subject of at least about 1 week, about 2 weeks, about 3 weeks, about 4 weeks, about 2 months, 6 about months, about 1 year, about 2 years, about 3 years, about 4 years, about 5 years, about 6 years, about 7 years, about 8 years, about 9 years, about 10 years, or more;

(d) a reduction in progression rate of cancer of at least about 99%, at least about 95%, at least about 90%, at least about 80%, at least about 70%, at least about 60%, at least about 50%, at least about 40%, at least about 35%, at least about 30%, at least about 20%, at least about 15%, at least about 10%, or at least about 5%, for example, about 30% to about 99%, about 80% to about 90%, about 70% to about 90%, about 60% to about 90%, about 50% to about 90%, about 40% to about 90%, about 35% to about 90%, about 30% to about 90%, about 25% to about 90%, about 5% to about 85%, or about 10% to about 80% (actual % change or median % change compared to baseline or control);

(e) a stoppage of cancer cell growth;

(f) a reduction in tumor growth of at least about 99%, at least about 95%, at least about 90%, at least about 80%, at least about 70%, at least about 60%, at least about 50%, at least about 40%, at least about 35%, at least about 30%, at least about 20%, at least about 15%, at least about 10%, or at least about 5%, for example, about 30% to about 99%, about 80% to about 90%, about 70% to about 90%, about 60% to about 90%, about 50% to about 90%, about 40% to about 90%, about 35% to about 90%, about 30% to about 90%, about 25% to about 90%, about 5% to about 85%, or about 10% to about 80% (actual % change or median % change compared to baseline or control); (g) decreased interaction between C-Raf and PDE8A (i.e., endogenous PDE8A) of at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, or at least about 99%, for example, about 30% to about 99%, about 80% to about 90%, about 70% to about 90%, about 60% to about 90%, about 50% to about 90%, about 40% to about 90%, about 35% to about 90%, about 30% to about 90%, about 25% to about 90%, about 5% to about 85%, or about 10% to about 80% (actual % change or median % change compared to baseline or control);

(h) an increase in PKA-mediated inhibitory phosphorylation of C-Raf (e.g., serine 259 phosphorylation) of at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, or at least about 99%, for example, about 30% to about 99%, about 80% to about 90%, about 70% to about 90%, about 60% to about 90%, about 50% to about 90%, about 40% to about 90%, about 35% to about 90%, about 30% to about 90%, about 25% to about 90%, about 5% to about 85%, or about 10% to about 80% (actual % change or median % change compared to baseline or control); and/or

(i) a reduction in ERK1/2 phosphorylation/signaling of at least about 99%, at least about 95%, at least about 90%, at least about 80%, at least about 70%, at least about 60%, at least about 50%, at least about 40%, at least about 35%, at least about 30%, at least about 20%, at least about 15%, at least about 10%, or at least about 5%, for example, about 30% to about 99%, about 80% to about 90%, about 70% to about 90%, about 60% to about 90%, about 50% to about 90%, about 40% to about 90%, about 35% to about 90%, about 30% to about 90%, about 25% to about 90%, about 5% to about 85%, or about 10% to about 80% (actual % change or median % change compared to baseline or control). [0082] In some embodiments, at least a portion of total C-Raf remains complexed with PDE8A. In some embodiments at least about 99%, at least about 95%, at least about 90%, at least about 80%, at least about 70%, at least about 60%, at least about 50%, at least about 40%, at least about 35%, at least about 30%, at least about 20%, at least about 15%, at least about 10%, or at least about 5%, for example, about 30% to about 99%, about 80% to about 90%, about 70% to about 90%, about 60% to about 90%, about 50% to about 90%, about 40% to about 90%, about 35% to about 90%, about 30% to about 90%, about 25% to about 90%, about 5% to about 85%, or about 10% to about 80% of total C-Raf remains complexed with PDE8A. In some embodiments less than about 90%, less than about 80%, less than about 70%, less than about 60%, less than about 50%, less than about 40%, less than about 30%, less than about 20%, less than about 10%, or less than about 5% of total C-Raf remains complexed with PDE8A.

[0083] In some embodiments, the subject is administered a B-Raf inhibitor. The B- Raf inhibitor may be administered prior to, simultaneously with, and/or following administration of the cell-penetrating conjugates, compositions, or formulations disclosed herein.

[0084] Various features and embodiments and select modifications will now be described by way of non-limiting examples. In all examples where an initiating methionine is included in a complete sequence to allow recombinant synthesis, the initiating methionine may be removed during purification to derive the active product with the indicated human homeodomain sequence. References to sequence numbers for complete conjugates in paragraphs describing their use are referring to the conjugate after removal of the initiating methionine.

EXAMPLES

Example 1

[0085] Malignant melanoma, an aggressive and lethal form of skin cancer, leaves metastatic patients with a 15 - 20% chance of surviving 5 years with the disease ¹ . Over 50% of melanoma patients carry a mutation in their BRAF gene, with the V600E (valine to glutamic acid) missense mutation being responsible for 80-90% of BRAF mutations ^{2 4} . B-Raf is a serine/threonine protein kinase that is part of the RAS - RAF - MEK - ERK signalling axis, involved in regulating many cellular processes including: differentiation, proliferation, survival and apoptosis ⁵ . This signalling pathway is believed to be crucial to melanoma progression, with the V600E mutation resulting in B-Raf protein conformational changes that constitutively activate B-Raf and downstream MEK - ERK signalling ⁵ .

[0086] As a result, B-Raf-specific small molecule inhibitors (and eventually MEK inhibitors) were developed and found to dramatically improve patient prognosis, survival rate and lead to tumor regression through suppression of downstream ERK signalling ^{6- 9} . Unexpectedly, B-Raf inhibitor resistance was developed in many patients through paradoxical activation of ERK; allowing the cancer to persist ^10-14 . Pathway reactivation is believed to occur as a result of oncogenic mutations in a number of genes, including NRAS (20% of cases; Q61 K/R/L most frequent) and KRAS (2% of cases) gain-of- function mutations ^15-18 . As B-Raf preferentially heterodimerises to C-Raf (vs. other A, B or C-Raf homo/heterodimers), B-Raf inhibition results in a negative feedback mechanism that switches from B-Raf to C-Raf activation by Ras and subsequent tumor invasion and metastasis ^{19 20} . In light of this, C-Raf has become a key therapeutic target for the development of new treatments able to suppress RAS-mediated tumor progression in B-Raf inhibitor resistant melanoma.

[0087] Previously, it was demonstrated that cAMP degrading enzyme, PDE8A, played an important role in protecting C-Raf from PKA-mediated inhibition ²¹ (reviewed ²² ). PDE8A, believed to be responsible for regulating basal cAMP fluctuations, was found to directly interact with C-Raf. The association of C-Raf with a PDE markedly inhibited the ability of local PKA pools to phosphorylate and inhibit the kinase, increasing the likelihood of C-Raf activation. Peptide mapping of the PDE8A-C-Raf interface allowed for the rational development of a cell penetrating peptide disrupter based on the C-Raf binding site on PDE8A ³² ’ ³³ . This disrupter was found to inhibit the PDE8A - C-Raf protein-protein interaction (PPI) and significantly increase C-Raf-S259 phosphorylation while concomitantly supressing phospho-ERK signalling. This concept was verified at an organismal level in both PDE8A knock out mice and a drosophila model, where basal ERK activation was attenuated compared to wild type ²¹ .

[0088] Further verification of the PDE8A - C-Raf PPI inhibitor concept has been supplied by a recent study, which demonstrated that the disrupter was able to attenuate T-effector cell adhesion and migration in an auto-immune multiple sclerosis mouse model. The inhibition of T-effector cell function was a direct result of increased levels of inhibitory C-Raf-S259 phosphorylation and subsequent suppression of ERK activation ²³ . The disrupter produced a more potent effect than highly-selective PDE8 enzyme inhibitors and highlighted a novel approach to targeting T-effector cells in inflammatory disorders.

Generation of HoxD12-PDE8 A cell-penetrating peptides

[0089] To determine whether cell-penetrating conjugates provided superior activity to delivery of PDE8A alone, various HoxD12-PDE8A cell-penetrating peptides (also referred to herein as“PPL-008 conjugates”) were generated. Briefly, HoxD12 (also referred to herein as“Cell Porter®”, Portage Pharmaceuticals) was conjugated onto the C or N-terminal of the disrupter (PDE8A) via either thioester (C or N giving PPL-008C (conjugate of SEQ. ID Nos. 25 and 26) or PPL-008N (conjugate of SEQ. ID Nos 29 and 30)) or disulfide bonds (CSS or NSS giving PPL-008CSS (conjugate of SEQ. ID Nos 23 and 24) or PPL-008NSS (conjugate of SEQ. ID Nos 27 and 28)). Figure 1. Linkers were intended to provide more molecular flexibility for the cargo (i.e. , PDE8A) to bind to its target without interference by the larger HoxD12 structure. PPL-008-CSS and PPL- 008-NSS were designed to include a disulfide bond between the disruptor peptide and the HoxD12 cell penetrating peptide sequence. Intracellular release of the cargo by cleavage of the disulfide bond occurs because of the reducing environment within the cell and not any enzymatic cleavage process. It was contemplated that CCS and NSS cell-penetrating conjugates would allow the PDE8A peptide to function inside the cell without being attached to the larger HoxD12 peptide.

Animals

[0090] Generation of a MM415 melanoma murine xenograft model and in vivo treatment was carried out by Ml Bioresearch (Michigan, USA). All protocols involving animals used were approved by the Institutional Animal Care and Use Committee of the University of Washington in accordance with the National Institutes of Health. In vivo 5- 6 week old female NSG - immunodeficient mice (Jackson Laboratory) were subcutaneously injected with 3.3e+8 MM415 malignant melanoma cells, at the SC - axilla (high) and tumors were allowed to grow for 30 days (-200 - 400mm ³ ). Mice were subcutaneously injected at the site of tumor with PPL-008 peptide drug dissolved in a 5% dextrose - water solution at either 25mg/kg or 100mg/kg. Mice were euthanized and the tumors were harvested at varying time points post-treatment: 30 min, 1 h, 2 h, 4 h, 8 h, 12 h. Tumors were frozen down at -80°C prior to preparation into lysates for follow-up western blot analyses.

MM415 Cell Culture and Drug Treatments

[0091] A375 (V600E) and MM415 (BRAF wt, KRAS wt, NR AS Q61 L) human malignant melanoma epithelial skin cell lines were cultured with RPMI 1640 medium, supplemented with 10% fetal bovine serum (FBS, v/v), 1 % L-glutamine (v/v), 1 % penicillin-streptomycin (v/v) (all Sigma-Aldrich) and incubated at 37°C, 5% C02 and 95% humidity. Cells were split at -80% confluency, using 0.05% trypsin-EDTA, 1 :5.

[0092] The original PDE8A - C-Raf disrupter, and its scrambled isoform, were synthesized with a C-terminal stearic acid group [CH ₃ (CH2)i6COOH] (GenScript) ²¹ . PPL-008 (i.e. PDE8A - C-Raf disrupter, without stearic acid) was synthesized with Cell Porter® conjugated to the C or N-terminus via either thioester or disulphide bonds. All peptides were dissolved to the appropriate concentration in DMSO for in vitro experimentation. PLX4032 (Vemurafenib) was dissolved in DMSO to a final concentration of 1 mM (Sellekchem). Peptides were added to cells for 2 h, and PLX for 1 h, before cells were harvested. In cases where co-treatments were administered, cells were first treated with peptides for 2 h, followed by 1 hr PLX.

Western Blotting

[0093] MM415 and A375 cells harvested from In vitro experiments were lysed in

3T3 lysis buffer, whilst MM415 melanoma murine xenograft tissue was homogenized and lysed in 1X RIPA buffer (both supplemented with protease cocktail inhibitor tablets (Roche)). Soluble fraction of lysate was resolved via SDS/PAGE using 4-12% Bis-Tris gels (NuPAGE). Proteins were transferred at 30V for 1 h onto 0.45pm nitrocellulose membrane (Protran) and blocked for 1 h in 5% non-fat dry milk solution (Marvel, w/v) in 1x TBS-T (20mM Tris-CI pH 7.6, 150mM NaCI, 0.1 % Tween-20). Blocked membranes were incubated in primary antibody (diluted in 1x TBS-T, 1 % marvel) overnight at 4°C. Membranes were washed three times in 1x TBS-T before membranes were incubated in secondary antibody (diluted in 1x TBS-T, 1 % marvel) for 1 h at room temperature. Membranes were washed a final three times in 1x TBS-T and fluorescent intensity of Li-Cor secondary antibody was measured using a Li-Cor Odyssey scanner. Cell Proliferation Measurements

[0094] Real-Time cellular growth analyses of MM415 cells, using the xCELLigence platform (Roche Applied Science), allowed for the label-free measurement of cell proliferation. 96 well E-plates, containing gold microelectrode sensors on the bottom of the plate, were used to measure cellular impedance inside each well as per manufacturer’s instructions. Cellular impedance measurements were translated into ‘cell index’, an arbitrary measurement that increases as MM415 cells adhere and spread-out/grow (and vice versa), giving quantitative information on cell proliferation and viability that were analyzed using RTCA software (Roche). All protocols carried out using the xCELLigence platform were based on prior publications ²¹ ’ ^{27 31} . Following MM415 cell adhesion, cells were treated with one of the peptide disrupters for 2 h, followed by PLX (1 mM). The slope (i.e. rate of cell proliferation/growth) was measured based on the normalized cell index from the point in which treatments were administered, until the response had plateaued appropriately.

Statistical Analyses

[0095] Results from western blot analyses are represented as mean ± SEM (n > 3). Results from xCELLigence cell proliferation assay are represented as mean ± STDEV (n > 3). P < 0.05 indicates data are significant, with significance determined via unpaired t-test using GraphPad Prism software.

[0096] All antibodies and chemical treatments used herein are listed in Table 4 below:

Attenuation of paradoxical activation of pERK signalling and MM415 cell proliferation with HoxD12-PDE8A cell-penetrating peptides

[0097] The cell-penetrating peptide conjugates shown in Figure 1 were used to treat the B-Raf inhibitor resistant MM415 (human malignant melanoma, BRAF wt, KRAS wt,

NRAS Q61 L) cell line. To determine if PPL-008 conjugates (10mM) could suppress phospho-ERK signalling in MM415 cells, pERK levels were determined via western blot

(pERK expression normalized to GAPDH, mean ± SEM, n = 4, Figure 2). MM415 B-Raf inhibitor treatment (PLX4032, 1 mM) clearly induced a paradoxical activation of ERK and this was significantly reduced following treatment with all the analogues (PPL-008N,

PPL-008C, PPL-008NSS and PPL-008CSS) ( ^** P < 0.01 or ^* P < 0.05; Figure 2A, lanes

7-10 inclusive). In addition, pERK was significantly reduced in the human A375 malignant melanoma cell line (BRAF V600E) following PLX treatment (1 mM) (Figure 2B, lanes 2,3,4) with PPL-008 analogues providing no ERK inhibition as a mono-treatment

(Figure 2B, lanes 1 1-14 inclusive) or further ERK inhibition as a co-treatment with PLX

(Figure 2B, lanes 7-10 inclusive). As B-Raf inhibition sufficiently suppressed pERK expression in A375 cells, and as MM415 cells were resistant to PLX (resembling the clinical phenotype of interest), MM415 cells were used for the remainder of the study.

[0098] To assess the ability of PPL-008 conjugates to inhibit cell growth, real-time measurements of MM415 cellular impedance was recorded on the xCELLigence platform as an indicator of cellular proliferation (slope (1/hr), mean ± STD, n = 3). All PPL-008-conjugates (10pM) significantly slowed cell proliferation in PLX treated MM415 cells ( ^* P < 0.05, Figure 2C, lanes 7-14 inclusive). This data suggests treatment (10pM) with each of the PPL-008 analogues suppressed both pERK and cell growth compared with DMSO treated control (Figures 2A and 2C), indicating PPL-008 has potential as an effective therapy in this context. Surprisingly, PPL-008C and PPL- 008CSS mono-treatments significantly attenuated A375 growth (Figure 3A-B) without affecting the phospho-ERK profile (Figure 2B, lanes 1 1-14 inclusive,). B-Raf inhibitors (including PLX4032) are known to potently reduce A375 cell growth. It is noteworthy that, the original stearylated PDE8A - C-Raf disrupter caused no significant reduction in pERK signalling (O’, Figure 2A, lane 5) or cell proliferation (O’, Figure 2C, lane 5) in MM415, similar to its scrambled control (‘S’, Figure 2A, lane 6 and Figure 2C, lane 6 ), suggesting the stearic acid group was insufficient in facilitating cell-penetration in MM415 cells. As all PPL-008 conjugates attenuated both pERK expression and cell proliferation, this indicates that Cell Porter® greatly improves intracellular delivery of PPL-008 conjugates compared with the original disrupter’s stearate group.

[0099] Both PPL-008CSS/NSS significantly inhibited pERK expression and cell proliferation in MM415 cells at 10uM as shown in Figure 2. However, in follow up dose- response experiments (using the same assays, data not shown), both PPL- 008CSS/NSS (PPL-008 conjugated to CellPorter® via disulphide linkage) failed to consistently inhibit pERK expression at all concentrations but remained to attenuate growth - with PPL-008CSS 10uM causing quite a dramatic negative slope result. It was contemplated that the inconsistent results for the CSS / NSS PPL-008 conjugates may be due to cytotoxicity to the MM415 melanoma cell line.

Inhibition of pERK expression and MM415 cell proliferation over multiple doses with HoxD12-PDE8A cell-penetrating peptides

[00100] To determine whether the HoxD12-PDE8A cell-penetrating peptides could inhibit pERK expression and cell proliferation, MM415 cells were co-treated with PLX (1 mM), following pre-treatment with a dose range of PPL-008C or PPL-008N (1 nM - 10mM). The levels of pERK and cell proliferation rates determined as previously described (Figure 4). The levels of pERK triggered by PLX (Figure 4A and 4B, lane 1 vs lane 2) were reduced at all concentrations following PPL-008N treatment, with the higher [10mM] dose causing the most significant reduction ( ^*** P < 0.001 , Figure 4A). This effect was recapitulated in the xCELLigence cell proliferation assay, where PPL- 008N reduced the rate of cell proliferation at all concentrations; most significantly at 10mM ( ^*** P < 0.001 , Figure 4C (i and ii)). In addition, PPL-008C-conjugate reduced MM415 pERK levels and rate of cell proliferation at all concentrations, with [10mM] producing the most significant inhibition ( ^*** P < 0.001 , Figure 4B and 4D (i and ii)). In vivo PPL-008C suppression of pERK in an MM415 melanoma murine xenograft model

[00101] Preliminary in vivo investigation of the effects of PPL-008C were carried out in an immuno-deficient NSG - MM415 melanoma murine xenograft model. PPL-008C was chosen as the lead peptide disrupter due to its consistency in attenuating pERK signalling and cell proliferation as both a single treatment and co-treatment with PLX. Briefly, PPL-008C was administered subcutaneously at the site of the tumor as a single treatment. Tumors were removed at varying time points post-treatment and pERK expression was assessed via western blot (N > 3, Figure 5).

[00102] PPL-008C significantly suppressed pERK1 , pERK2 and total pERK levels over all time-points in the time course and at both doses (25mg/kg and 100mg/kg), excluding the 25mg/kg - 12Hr treatment ( ^* P < 0.05, Figure 5). This shows that a single PPL-008C treatment can attenuate Raf - MEK- ERK signalling relatively quickly (within 30 minutes) and can maintain this inhibition for at least 12 hours at higher concentrations (100mg/kg, Figure 5). Maximal pERK inhibition occurred 2 hours post treatment with 100mg/kg PPL-008C.

[00103] Together this data demonstrates the PDE8A - C-Raf complex as a point of cross-talk between the MAP kinase signalling and cAMP signalling systems, that can be manipulated by a disrupter peptide to promote the inhibition of C-Raf via increased S259 phosphorylation ^{21 23} (Figure 4). This action can counteract C-Raf driven paradoxical activation of ERK in B-Raf inhibitor resistant melanoma cell lines resulting in a retardation of cell proliferation (Figures 2 and 4). Inhibition of C-Raf by targeting its binding to anchoring proteins rather than kinase activity is a novel approach for this kinase and an aim is to displace only a small percentage of total C-Raf that is in complex with PDE8A. Protein-protein interactions (PPIs) are increasingly being regarded as tractable molecular targets for the development of therapeutics, and peptides that mimic docking sites with protein complexes are often the ideal scaffold starting point for such agents ³⁴ .

[00104] The effectiveness of peptide delivery systems is context specific and the data shown herein demonstrates that CellPorter® has directed intracellular delivery of a novel C-Raf - PDE8A peptide disrupter leading to significant suppression of paradoxical ERK activation in a clinically relevant B-Raf inhibitor resistant human melanoma cell line and an apt xenograft model of the disease. It is further contemplated that PPL-008 conjugates represent potential agents for development of co-therapies for resistant melanoma to be administered with an appropriate B-Raf inhibitor in order to overcome B-Raf inhibitor resistance and attenuate ERK activation in melanocytes.

[00105] From the foregoing, it will be appreciated that specific embodiments have been described herein for purposes of illustration, but that various modifications may be made without deviating from the spirit and scope of the disclosure. Accordingly, the disclosure is not limited except as by the appended claims.

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