Cell-penetrating peptides with enhanced solubility

Abstract

Provided are peptides having enhanced aqueous solubility due to the presence of a solubility enhancing region comprising at least two cell-penetrating sequences. Also provided are methods of preparing a peptide having enhanced aqueous solubility and of increasing solubility of an amino acid sequence of interest in an aqueous solvent.

Description

Sapience.024.WO1 PATENT CELL-PENETRATING PEPTIDES WITH ENHANCED SOLUBILITY BACKGROUND

[0001] A significant consideration for formulation scientists is the solubility of a drug substance in a pharmaceutically acceptable vehicle. Many drug substances are characterized by low aqueous solubility, which can complicate manufacturing and reduce the stability and bioavailability of a pharmaceutical product. For example, liquid drug products tend to degrade faster at lower concentrations. In addition, drugs that are poorly water soluble must often be administered at higher doses to achieve the desired pharmacological response.

[0002] Because therapeutic peptides are administered parenterally, aqueous solubility is particularly important for the development of drug products comprising a peptide as an active agent. Solubility of a peptide depends on its physical and chemical properties, including length and amino acid composition. For example, peptides having a higher percentage of polar amino acids are typically more water-soluble than those having more hydrophobic amino acids.

[0003] Strategies for increasing solubility of poorly soluble drugs include the use of solid dispersions, co-solvents, surfactants, pH adjustment, and reduction of drug particle size. However, improved methods are needed, particularly to enhance the solubility of therapeutic peptides. SUMMARY OF THE INVENTION

[0004] Some of the main aspects of the present invention are summarized below. Additional aspects are described in the Detailed Description of the Invention, Examples, Drawings, and Claims sections of this disclosure. The description in each section of this disclosure is intended to be read in conjunction with the other sections. Furthermore, the various embodiments described in each section of this disclosure can be combined in various different ways, and all such combinations are intended to fall within the scope of the present invention.

[0005] The invention provides peptides with enhanced aqueous solubility, due to the inclusion of at least two cell penetrating peptide (CPP) sequences, wherein solubility is compared with a control peptide. One embodiment is a peptide comprising a cargo region operably linked to a solubility enhancing region, wherein the cargo region comprises an amino acid sequence selected from the group consisting ofSapience.024.WO1 PATENT [FRWLLRQLARLAQLA]D(SEQ ID NO: 1), [MSFTLDADF]L(SEQ ID NO: 2), [MDTAFSFLD]L (SEQ ID NO: 3), and [DISGINASVVNIQKEIDRLNEVAKNLNESLIDLQEL]L(SEQ ID NO: 140); and wherein the solubility enhancing region comprises an amino acid sequence selected from the group consisting of [KLTPVKLTPV]D (SEQ ID NO: 4) and [VPTLKVPTLK]L (SEQ ID NO: 5); wherein D and L subscripts indicate chirality of the amino acids in the sequence; wherein the peptide displays greater solubility in an aqueous solvent compared with a control peptide. In some embodiments, the peptide comprises a sequence selected from the group consisting of: [FRWLLRQLARLAQLAKLTPVKLTPV]D (SEQ ID NO: 6), [MSFTLDADFVPTLKVPTLK]L(SEQ ID NO: 7), [MDTAFSFLDVPTLKVPTLK]L(SEQ ID NO: 8), and [DISGINASVVNIQKEIDRLNEVAKNLNESLIDLQELVPTLKVPTLK]L(SEQ ID NO: 141).

[0006] In one embodiment, the peptide comprises an N-terminal group selected from the group consisting of acetyl, naphthyl, octanoyl, phenyl, and isovaleryl. In a preferred embodiment, the N-terminal group is octanoyl.

[0007] In certain embodiments, the peptide comprises a C-terminal amide group.

[0008] A particular embodiment is a peptide comprising a cargo region comprising the D-amino acid sequence FRWLLRQLARLAQLA (SEQ ID NO: 1) operably linked to a solubility enhancing region comprising the D-amino acid sequence KLTPVKLTPV (SEQ ID NO: 4). In one embodiment, the peptide comprises an N-terminal octanoyl group. In one embodiment, the solubility of the peptide in an aqueous solvent is at least about 2-fold higher than the solubility of a control peptide in the aqueous solvent.

[0009] Also provided are compositions comprising a peptide of the invention having enhanced aqueous solubility and an aqueous carrier. In one embodiment, the aqueous carrier comprises a buffer. In certain embodiments, the composition is a pharmaceutical composition.

[0010] Further provided is a kit comprising a peptide of the invention or a composition of the invention.

[0011] Additional aspects include methods of enhancing the solubility of a peptide. One embodiment is a method of enhancing the solubility of a cargo peptide, wherein the cargo peptide comprises an amino acid sequence selected from the group consisting of [FRWLLRQLARLAQLA]D (SEQ ID NO: 1), [MSFTLDADF]L (SEQ ID NO: 2),Sapience.024.WO1 PATENT [MDTAFSFLD] (SEQ ID NO: 3) and [DISGINASVVNIQKEIDRLNEVAKNLNESLIDLQEL]L (SEQ ID NO: 140), the method comprising modifying the cargo peptide with a solubility enhancing region comprising an amino acid sequence selected from the group consisting of [KLTPVKLTPV]D (SEQ ID NO: 4) and [VPTLKVPTLK]L (SEQ ID NO: 5) to form a modified peptide; wherein the solubility enhancing region is operably linked to the cargo peptide; wherein D and L subscripts indicate the form of the amino acid sequence; and wherein the modified peptide displays greater solubility in an aqueous solvent compared with a control peptide.

[0012] In one embodiment, the modified peptide comprises a sequence selected from the group consisting of: [FRWLLRQLARLAQLAKLTPVKLTPV]D (SEQ ID NO: 6), [MSFTLDADFVPTLKVPTLK]L(SEQ ID NO: 7), [MDTAFSFLDVPTLKVPTLK]L(SEQ ID NO: 8), and [DISGINASVVNIQKEIDRLNEVAKNLNESLIDLQELVPTLKVPTLK]L(SEQ ID NO: 141). In a particular embodiment, the cargo region comprises the D-amino acid sequence FRWLLRQLARLAQLA (SEQ ID NO: 1), and wherein the solubility enhancing region comprises the D-amino acid sequence KLTPVKLTPV (SEQ ID NO: 4).

[0013] In certain embodiments, the modified peptide comprises an N-terminal group selected from the group consisting of acetyl, naphthyl, octanoyl, phenyl, and isovaleryl and / or a C-terminal amide group. In a specific embodiment, the N-terminal group is octanoyl.

[0014] In one embodiment, the solubility of the peptide in an aqueous solvent is at least about 2-fold higher than the solubility of a control peptide in the aqueous solvent. DETAILED DESCRIPTION OF THE INVENTION

[0015] The practice of the present invention will employ, unless otherwise indicated, conventional techniques of pharmaceutics, formulation science, biochemistry, protein chemistry, and molecular biology, which are within the skill of the art.

[0016] In order that the present invention can be more readily understood, certain terms are first defined. Additional definitions are set forth throughout the disclosure. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention is related.Sapience.024.WO1 PATENT

[0017] Any headings provided herein are not limitations of the various aspects or embodiments of the invention, which can be had by reference to the specification as a whole. Accordingly, the terms defined immediately below are more fully defined by reference to the specification in its entirety.

[0018] All references cited in this disclosure are hereby incorporated by reference in their entireties. In addition, any manufacturers’ instructions or catalogues for any products cited or mentioned herein are incorporated by reference. Documents incorporated by reference into this text, or any teachings therein, can be used in the practice of the present invention. Documents incorporated by reference into this text are not admitted to be prior art. I. Definitions

[0019] The phraseology or terminology in this disclosure is for the purpose of description and not of limitation, such that the terminology or phraseology of the present specification is to be interpreted by the skilled artisan in light of the teachings and guidance.

[0020] As used in this specification and the appended claims, the singular forms “a,” “an,” and “the” include plural referents, unless the context clearly dictates otherwise. The terms “a” (or “an”) as well as the terms “one or more” and “at least one” can be used interchangeably.

[0021] Furthermore, “and / or” is to be taken as specific disclosure of each of the two specified features or components with or without the other. Thus, the term “and / or” as used in a phrase such as “A and / or B” is intended to include A and B, A or B, A (alone), and B (alone). Likewise, the term “and / or” as used in a phrase such as “A, B, and / or C” is intended to include A, B, and C; A, B, or C; A or B; A or C; B or C; A and B; A and C; B and C; A (alone); B (alone); and C (alone).

[0022] Wherever embodiments are described with the language “comprising,” otherwise analogous embodiments described in terms of “consisting of” and / or “consisting essentially of” are included.

[0023] Units, prefixes, and symbols are denoted in their Système International de Unites (SI) accepted form. Numeric ranges are inclusive of the numbers defining the range, and any individual value provided herein can serve as an endpoint for a range that includes other individual values provided herein. For example, a set of values such as 1,Sapience.024.WO1 PATENT 2, 3, 8, 9, and 10 is also a disclosure of a range of numbers from 1-10, from 1-8, from 3-9, and so forth. Likewise, a disclosed range is a disclosure of each individual value encompassed by the range. For example, a stated range of 5-10 is also a disclosure of 5, 6, 7, 8, 9, and 10.

[0024] As used herein, “operably linked” means that two or more components are arranged in a functional relationship, such that one component exerts its effect on one or more other components. In some embodiments, the components are nucleic acid sequences and / or amino acid sequences. For example, a promoter sequence and a gene sequence are operably linked if the two sequences are arranged such that the promoter is able to control expression of the gene.

[0025] A “polynucleotide,” as used herein can include one or more “nucleic acids,” “nucleic acid molecules,” or “nucleic acid sequences,” and refers to a polymer of nucleotides of any length, and includes DNA and RNA. The polynucleotides can be deoxyribonucleotides, ribonucleotides, modified nucleotides or bases, and / or their analogs, or any substrate that can be incorporated into a polymer by DNA or RNA polymerase. A polynucleotide can comprise modified nucleotides, such as methylated nucleotides and their analogs. The preceding description applies to all polynucleotides referred to herein, including RNA and DNA.

[0026] The terms “polypeptide,” “peptide,” and “protein” are used interchangeably herein to refer to polymers of amino acids of any length. The polymer can be linear or branched, can comprise modified amino acids, and can be interrupted by non-amino acids. Except where indicated otherwise, e.g., for the abbreviations for the uncommon or unnatural amino acids set forth herein, the three-letter and one-letter abbreviations, as used in the art, are used herein to represent amino acid residues. Except where indicated by a “D,” the amino acid is an L-amino acid. Groups or strings of amino acid abbreviations are used to represent peptides. Unless specifically indicated, peptides are indicated with the N-terminus of the left and the sequence is written from the N-terminus to the C-terminus.

[0027] Polypeptides, peptides, and proteins can encompass natural or synthetic modifications, for example, disulfide bonds, lactam bridges, glycosylation, lipidation, acetylation, acylation, amidation, phosphorylation, or other manipulation or modification, such as conjugation with a labeling component or addition of a protecting group. Also included are, for example, polypeptides containing one or more analogs of an amino acidSapience.024.WO1 PATENT (including, for example, amino-isobutyric acid (Aib), unnatural amino acids, such as naphthylalanine (Nal), etc.) and polypeptides comprising or consisting of D-amino acids, as well as other modifications known in the art.

[0028] Polypeptides can be in one or multiple salt forms. Preferred salt forms include acetate, chloride or trifluoroacetate. In certain embodiments, the polypeptides can occur as single chains, covalent dimers, or non-covalent associated chains.

[0029] Polypeptides can also be in cyclic form. Cyclic polypeptides can be prepared, for example, by bridging free amino and free carboxyl groups. Formation of the cyclic compounds can be achieved by treatment with a dehydrating agent, with suitable protection if needed. The open chain (linear form) to cyclic form reaction can involve intramolecular-cyclization. Cyclic polypeptides can also be prepared by other methods known in the art, for example, using one or more lactam bridges, hydrogen bond surrogates (Patgiri et al. 2008), hydrocarbon staples (Schafmeister et al.2000), triazole staples (Le Chevalier Isaad et al.2009), or disulfide bridges (Wang et al.2006). Bridges or staples can be spaced, for example, 3, 4, 7, or 8 amino acids apart.

[0030] A “retro inverso” polypeptide has a reversed amino acid sequence, relative to a native L-amino acid sequence, and is made up of D-amino acids (inverting the α-center chirality of the amino acid subunits) to help maintain side-chain topology similar to that of the original L-amino acid peptide.

[0031] The term “variant” refers to a polypeptide having one or more amino acid substitutions, deletions, and / or insertions compared to a reference sequence. Deletions and insertions can be internal and / or at one or more termini. Substitution can include the replacement of one or more amino acids with a similar or homologous amino acid(s) or a dissimilar amino acid(s). For example, some variants include alanine substitutions at one or more amino acid positions. Other substitutions include conservative substitutions that have little or no effect on the overall net charge, polarity, or hydrophobicity of the protein. Some variants include non-conservative substitutions that change the charge or polarity of the amino acid. Substitution can be with either the L- or the D-form of an amino acid.

[0032] The term “conservative substitution” as used herein denotes that one or more amino acids are replaced by another, biologically similar residue. Examples include substitution of amino acid residues with similar characteristics, e.g., small amino acids, acidic amino acids, polar amino acids, basic amino acids, hydrophobic amino acids, andSapience.024.WO1 PATENT aromatic amino acids. For further information concerning phenotypically silent substitutions in peptides and proteins, see, for example, Bowie et. al.1990. In the table below, conservative substitutions of amino acids are grouped by physicochemical properties; I: neutral and / or hydrophilic, II: acids and amides, III: basic, IV: hydrophobic, V: aromatic, bulky amino acids. Table I I II III IV V

[0033] In the tableamino acids are grouped by physicochemical properties; VI: neutral or hydrophobic, VII: acidic, VIII: basic, IX: polar, X: aromatic. Table II VI VII VIII IX X

[0034] Methods ofy g and amino acid substitutions which do not affect protein function are well-known in the art (see, e.g., Brummell et al. 1993; Kobayashi et al.1999 ; Burks et al.1997).

[0035] The terms “identical” or percent “identity” in the context of two or more nucleic acids or polypeptides, refers to two or more sequences or subsequences that are the same or have a specified percentage of nucleotides or amino acid residues that are the same, when compared and aligned (introducing gaps, if necessary) for maximum correspondence, not considering any conservative amino acid substitutions as part of theSapience.024.WO1 PATENT sequence identity. The percent identity can be measured using sequence comparison software or algorithms, or by visual inspection. Various algorithms and software are known in the art that can be used to obtain alignments of amino acid or nucleotide sequences.

[0036] An “isolated” molecule is one that is in a form not found in nature, including those which have been purified.

[0037] An “active agent” is an ingredient that is intended to furnish biological activity. The active agent can be in association with one or more other ingredients. An active agent that is a peptide can also be referred to as an “active peptide.”

[0038] An “effective amount” of an active agent is an amount sufficient to carry out a specifically stated purpose.

[0039] The term “pharmaceutical composition” refers to a preparation that is in such form as to permit the biological activity of the active ingredient to be effective and which contains no additional components that are unacceptably toxic to a subject to which the composition would be administered. Such composition can be sterile and can comprise a pharmaceutically acceptable carrier, such as physiological saline. Suitable pharmaceutical compositions can comprise one or more of a buffer (e.g. acetate, phosphate or citrate buffer), a surfactant (e.g. polysorbate), a stabilizing agent (e.g. polyol or amino acid), a preservative (e.g. sodium benzoate), and / or other conventional solubilizing or dispersing agents.

[0040] “Solubility” is used herein in accordance with its usual meaning in the art and refers, for example, to the ability of a first substance, the “solute,” to dissolve in second substance, the “solvent.” Commonly, the solute is a solid and the solvent is a liquid, although both the solute and solvent can be in other forms. Solubility can be expressed in a variety of conventions, for example, mass or volume of solute per mass or volume of solvent, e.g., mg / mL v / v, or w / w, the latter two of which can also be expressed as a percentage. Solubility can also be expressed, for example, as quantity of solute per quantity of solution, e.g., mol / L. Solubility depends on temperature, pressure, and on the physical properties of the solution components, such as pH and polarity. For instance, charged or ionic solutes are more soluble in polar solvents than in non-polar solvents.Sapience.024.WO1 PATENT II. Peptides and Compositions

[0041] The invention provides peptides with enhanced aqueous solubility, comprising a cargo region and a solubility enhancing regions, wherein the solubility enhancing region comprises at least two copies of a cell-penetrating peptide (CPP). Accordingly, peptides of the invention are cell-penetrating peptides.

[0042] Peptides of the invention comprising a cargo region and a solubility enhancing region display greater solubility in an aqueous solvent than a control peptide. As used herein, a “control peptide” comprises the same cargo region as a peptide of the invention, but lacks a solubility enhancing region. In one embodiment, the control peptide has one CPP. In one embodiment, the control peptide has no CPP.

[0043] In some embodiments, a peptide of the invention is at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% more soluble than a control peptide having no CPP or one CPP. In one embodiment, a peptide of the invention is at least 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, or 10-fold more soluble than a control peptide having no CPP or one CPP. In a particular embodiment, the peptide is at least 3-fold more soluble in water than a control peptide. In another particular embodiment, the peptide is at least 10-fold more soluble in water than a control peptide. When comparing solubility of a peptide of the invention with a control peptide, solubility is measured in the same aqueous solvent, under the same conditions.

[0044] Solubility can be measured by any method known in the art. Exemplary methods include spectrophotometry, high-performance liquid chromatography based on ultraviolet absorbance or fluorescence (HPLC-UV fluorescence), and light scattering (turbidity). Comparative solubility measurements should be performed at the same temperature and pH, and in the same solvent. In one embodiment, solubility is measured in an aqueous buffer, such as a trehalose buffer or phosphate buffered saline (PBS), at pH 6.5 and 20^C.

[0045] Synthesis of shorter peptides is generally more efficient than that of longer peptides. Yield is unavoidably lost at each step of synthesis, so an increasing number of amino acids typically results in decreased synthetic yield. The present inventors surprisingly discovered that the yield of peptides comprising a solubility enhancing region was improved, compared to control peptides, in spite of the increased length. Therefore, in addition to improving peptide solubility, the solubility enhancing region also improves the efficiency of peptide synthesis.Sapience.024.WO1 PATENT

[0046] In some embodiments, the synthetic yield of a peptide of the invention is at least 10%, 15%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100% higher than that of a control peptide having no CPP or one CPP. In one embodiment, the synthetic yield of a peptide of the invention is at least 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, or 10-fold higher than that of a control peptide having no CPP or one CPP. In a particular embodiment, the synthetic yield of the peptide is at least 10% higher than that of a control peptide. In another particular embodiment, the synthetic yield of the peptide is at least 15% higher than that of a control peptide. In one preferred embodiment, the synthetic yield of the peptide is at least 2-fold higher than that of a control peptide. In another preferred embodiment, the synthetic yield of the peptide is at least 3-fold higher than that of a control peptide. When comparing synthetic yield of a peptide of the invention with a control peptide, synthesis is carried out in the same manner and under the same conditions. Percent yield is the actual yield divided by the theoretical yield and multiplied by 100.

[0047] Peptides of the invention can have a modified N-terminus and / or a modified C-terminus. For example, peptides can optionally include an N-terminal acetyl group and / or a C-terminal amide group. Other examples of optional N-terminal and / or C- terminal groups include hydrophobic groups, such as a linear or cyclic C2-C18aliphatic or aromatic hydrocarbon, a naphthyl group, a phenyl group, an octanoyl group, and a valeryl group, including an isovaleryl group. In some embodiments, the peptide comprises a linker or spacer between the peptide and the hydrophobic group. Such linkers or spacers include, for example, aminohexanoic acid, beta-alanine, substituted alkyls, substituted cycloalkyls, and polyethylene glycol.

[0048] Peptides of the invention are preferably 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50 amino acids in length, including ranges having any of those lengths as endpoints, for example, 22-47 amino acids.

[0049] In certain aspects, the invention provides a composition comprising a peptide of the invention and an aqueous carrier. In one embodiment, the composition has a pH of 4.0 to 7.5, preferably 6.0 to 7.0. In a particular embodiment, the pH of the composition is 6.5.

[0050] The aqueous carrier can be any suitable solvent, including, for example, water or saline. In one embodiment, the aqueous carrier comprises 0.9% sodium. In someSapience.024.WO1 PATENT embodiments, the aqueous carrier is a buffer, such as an acetate, phosphate, citrate, or trehalose buffer. In a particular embodiment, the aqueous carrier is PBS.

[0051] In some embodiments, the composition is a pharmaceutical composition.

[0052] Also within the scope of the invention are kits comprising the peptides and compositions as provided herein and, optionally, instructions for use. Kits typically include a label indicating the intended use of the contents of the kit. In this context, the term “label” includes any writing or recorded material supplied on or with the kit, or that otherwise accompanies the kit. Cell-Penetrating Peptides

[0053] CPPs are short (typically about 6-40 amino acids) peptides that can cross cell membranes and transport covalently or non-covalently linked molecular cargo, such as polypeptides, polynucleotides, and nanoparticles, across cell membranes and the blood brain barrier. The transport can be endocytotic or energy-independent (i.e., non- endocytotic) via translocation.

[0054] Numerous CPPs are described and characterized in the literature (see, e.g., Handbook of Cell-Penetrating Peptides (2d ed. Ulo Langel ed., 2007); Hervé et al.2008; Heitz et al.2009; Munyendo et al.2012; Zou et al.2013; Krautwald et al. 2016). A curated database of CPPs is maintained at crdd.osdd.net / raghava / cppsite (Gautam et al. 2012).

[0055] Peptides referred to as nuclear localization sequences (NLSs) are a subset of CPPs. The classical NLS contains one (monopartite) or two (bipartite) regions of basic amino acids. Consensus sequences of classical monopartite and bipartite NLSs are, respectively, K(K / R)X(K / R) and (K / R)(K / R)X10-12(K / R)3 / 5, where 3 / 5 indicates that at least 3 of 5 consecutive amino acids are lysine or arginine (Kosugi et al.2009). An NLS sequence from SV40 large T antigen, PKKKRKV (SEQ ID NO: 9), is an example of a classical monopartite NLS, while an NLS sequence from nucleoplasmin, KRPAATKKAGQAKKK (SEQ ID NO: 10) is an example of a classical bipartite NLS (Lange et al.2007; Kosugi et al.2009). There are also numerous non-classical NLSs, such as those from ribonucleoproteins (RNPs) hnRNP A1, hnRNP K, and U snRNP (Mattaj et al.1998).

[0056] In one embodiment, the CPP is a sequence from a Bax-inhibiting peptide, such as VPTLK (SEQ ID NO: 11) or KLPVM (SEQ ID NO: 12), which target the cytoplasm. Because the function of CPPs depends on their physical characteristics rather thanSapience.024.WO1 PATENT sequence-specific interactions, they can have the reverse sequence (“retro”) and / or the reverse chirality (“inverso”) as those known in the art. For example, retro inverso forms of the CPPs (reverse sequence and reverse chirality) are suitable for use in the invention. Examples of retro inverso CPPs include those having the D-amino acid sequence KLTPV (SEQ ID NO: 13), MVPLK (SEQ ID NO: 14), or OLTPV (SEQ ID NO: 15).

[0057] Variants of the CPP sequences having one or more amino acid additions, deletions, and / or substitutions that retain the ability to cross cell membranes and / or the BBB are also suitable for use in the invention. Conservative substitutions are particularly preferred. Proline residues should not be substituted, except with proline analogs. Examples of proline analogs include α-methyl-L-proline, α-benzyl-L-proline, trans-4- hydroxy-L-proline, cis-4-hydroxy-L-proline, trans-3-hydroxy-L-proline, cis-3-hydroxy-L- proline, trans-4-amino-L-proline, 3,4-dehydro-DL-proline, and inverso forms of any of the foregoing. Solubility Enhancing Region

[0058] The solubility enhancing region comprises at least two CPPs, and can comprise, for example, two, three, or four CPPs. The at least two CPPs can each be the same CPP sequence or can be different CPP sequences. The CPPs in the solubility enhancing region can have the same or opposite chirality relative to one another, or can be of mixed chirality.

[0059] Solubility enhancing regions include any combination of CPP sequences. Non-limiting examples of solubility enhancing regions include the sequences: VPTLKVPTLK (SEQ ID NO: 5), VPTLKKLPVM (SEQ ID NO: 16), KLPVMVPTLK (SEQ ID NO: 17), KLPVMKLPVM (SEQ ID NO: 18), VPTLKVPTLKVPTLK (SEQ ID NO: 19), VPTLKVPTLKKLPVM (SEQ ID NO: 20), VPTLKKLPVMVPTLK (SEQ ID NO: 21), VPTLKKLPVMKLPVM (SEQ ID NO: 22), KLPVMVPTLKVPTLK (SEQ ID NO: 23), KLPVMVPTLKKLPVM (SEQ ID NO: 24), KLPVMKLPVMVPTLK (SEQ ID NO: 25), KLPVMKLPVMKLPVM (SEQ ID NO: 26), and so forth.

[0060] Additional non-limiting examples of examples of solubility enhancing regions include the D-amino acid sequences: KLTPVKLTPV (SEQ ID NO: 4), KLTPVMVPLK (SEQ ID NO: 27), KLTPVOLTPV (SEQ ID NO: 28), MVPLKKLTPV (SEQ ID NO: 29), MVPLKMVPLK (SEQ ID NO: 30), MVPLKOLTPV (SEQ ID NO: 31), OLTPVKLTPV (SEQ ID NO: 32), OLTPVMVPLK (SEQ ID NO: 33), OLTPVOLTPV (SEQ ID NO: 34), KLTPVKLTPVKLTPV (SEQ ID NO: 35), KLTPVKLTPVMVPLKSapience.024.WO1 PATENT (SEQ ID NO: 36), KLTPVKLTPVOLTPV (SEQ ID NO: 37), KLTPVMVPLKKLTPV (SEQ ID NO: 38), KLTPVMVPLKMVPLK (SEQ ID NO: 39), KLTPVMVPLKOLTPV (SEQ ID NO: 40), KLTPVOLTPVKLTPV (SEQ ID NO: 41), KLTPVOLTPVMVPLK (SEQ ID NO: 42), KLTPVOLTPVOLTPV (SEQ ID NO: 43), MVPLKKLTPVKLTPV (SEQ ID NO: 44), MVPLKKLTPVMVPLK (SEQ ID NO: 45), MVPLKKLTPVOLTPV (SEQ ID NO: 46), MVPLKMVPLKKLTPV (SEQ ID NO: 47), MVPLKMVPLKMVPLK (SEQ ID NO: 48), MVPLKMVPLKOLTPV (SEQ ID NO: 49), MVPLKOLTPVKLTPV (SEQ ID NO: 50), MVPLKOLTPVMVPLK (SEQ ID NO: 51), MVPLKOLTPVOLTPV (SEQ ID NO: 52), OLTPVKLTPVKLTPV (SEQ ID NO: 53), OLTPVKLTPVMVPLK (SEQ ID NO: 54), OLTPVKLTPVOLTPV (SEQ ID NO: 55), OLTPVMVPLKKLTPV (SEQ ID NO: 56), OLTPVMVPLKMVPLK (SEQ ID NO: 57), OLTPVMVPLKOLTPV (SEQ ID NO: 58), OLTPVOLTPVKLTPV (SEQ ID NO: 59), OLTPVOLTPVMVPLK (SEQ ID NO: 60), OLTPVOLTPVOLTPV (SEQ ID NO: 61), and so forth.

[0061] Further non-limiting examples of examples of solubility enhancing regions include CPP sequences having mixed chirality, wherein D and L subscripts denote chirality of the amino acids: [VPTLK]L[KLTPV]D(SEQ ID NO: 62), [VPTLK]L[MVPLK]D(SEQ ID NO: 63), [VPTLK]L[OLTPV]D(SEQ ID NO: 64), [KLPVM]L[KLTPV]D (SEQ ID NO: 65), [KLPVM]L[MVPLK]D (SEQ ID NO: 66), [KLPVM]L[OLTPV]D (SEQ ID NO: 67), [KLTPV]D[VPTLK]L (SEQ ID NO: 68), [MVPLK]D[VPTLK]L(SEQ ID NO: 69), [OLTPV]D[VPTLK]L(SEQ ID NO: 70), [KLTPV]D[KLPVM]L (SEQ ID NO: 71), [MVPLK]D[KLPVM]L (SEQ ID NO: 72), [OLTPV]D[KLPVM]L(SEQ ID NO: 73), [VPTLK]L[KLTPV]D[VPTLK]L(SEQ ID NO: 74), [VPTLK]L[MVPLK]D[VPTLK]L (SEQ ID NO: 75), [VPTLK]L[OLTPV]D[VPTLK]L (SEQ ID NO: 76), [KLPVM]L[KLTPV]D[VPTLK]L (SEQ ID NO: 77), [KLPVM]L[MVPLK]D[VPTLK]L (SEQ ID NO: 78), [KLPVM]L[OLTPV]D[VPTLK]L (SEQ ID NO: 79), [KLTPV]D[VPTLK]L[KLTPV]D(SEQ ID NO: 80), [MVPLK]D[VPTLK]L[KLTPV]D (SEQ ID NO: 81), [OLTPV]D[VPTLK]L[KLTPV]D (SEQ ID NO: 82), [KLTPV]D[KLPVM]L[KLTPV]D (SEQ ID NO: 83), [MVPLK]D[KLPVM]L[KLTPV]D (SEQ ID NO: 84), [OLTPV]D[KLPVM]L[KLTPV]D (SEQ ID NO: 85), and so forth.

[0062] In the solubility enhancing region, the at least two copies of the CPP can be covalently linked to one another, for example, via a peptide bond, a disulfide bond, aSapience.024.WO1 PATENT thioether bond, or a linker known in the art. Exemplary linkers include, but are not limited to, a substituted alkyl, a substituted cycloalkyl, polyethylene glycol, and derivatives thereof. Linkers can be cleavable after the peptide is delivered into a cell.

[0063] Alternatively, each CPP of the solubility enhancing region can be independently linked to the cargo region at the N-terminus, C-terminus, and / or via a side chain. For example, one CPP of the solubility enhancing region can be linked to the N- terminus of the cargo region while the other CPP is linked to the C-terminus of the cargo region.

[0064] The solubility enhancing region is operably linked to the cargo region. In some embodiments, the regions are covalently linked, for example, via a peptide bond, a disulfide bond, a thioether bond, or a linker known in the art. Exemplary linkers include, but are not limited to, a substituted alkyl, a substituted cycloalkyl, polyethylene glycol, and derivatives thereof. Linkers can be cleavable after the peptide is delivered into a cell. The solubility enhancing region can be linked to the N-terminus or the C-terminus of the cargo region, or via a residue side chain. The solubility enhancing region and the cargo region can have the same or opposite chirality. Each region can independently comprise amino acids of mixed chirality. Cargo Region

[0065] As used herein, a “cargo region” is an amino acid sequence of interest, for example an amino acid sequence that is an active agent, such as a therapeutic peptide. Exemplary cargo regions for use in the invention can include BCL9 mimetic sequences (see, e.g., WO 2021 / 007158) and ribonucleotide reductase inhibitor sequences (see, e.g., Huff et al.2022).

[0066] BCL9 is a 149 kDa eukaryotic protein involved in signal transduction through the Wnt pathway. BCL9 binds to and promotes the transcriptional activity of β-catenin. The β-catenin binding region or “HD2 domain” of BCL9 is 24-residue α-helix at amino acids 351-374 of BCL9: LSQEQLEHRERSLQTLRDIQRMLF (SEQ ID NO: 86). The full amino acid sequence of wild-type human BCL9 is set forth in NCBI Accession No. NP_004317.2.

[0067] In some embodiments, the cargo region comprises at least positions 16-23 of the wild-type HD2 domain, with at least one addition, deletion, or substitution relative to the wild-type sequence. The cargo region can comprise, for example, an amino acid sequence shown in Table 1.Sapience.024.WO1 PATENT Table 1 L-Amino Acid Sequence LSQEQLEHRERSLQTLRDIQRMLF (SEQ ID NO: 86)

[0068] ve, for example, a D-amino acid sequence X1LX2X3QLX4X5LX6X7LA (SEQ ID NO: 91), wherein each amino acid at positions 1-13 is independently selected from those shown in Table 2. Table 2 1 2 3 4 5 6 7 8 9 10 11 12 13 [0064 or 8 can be alanine, i.e., if position 4 is A, position 8 is not A, and vice versa. The cargo region can optionally comprise at position -1 a D-amino acid or an L-amino acid selected from the group consisting of F, 1-Nal, 2-Nal, R, and W. The cargo region can further optionally comprise a D-amino acid or an L-amino acid selected from the group consisting of F, 1-Nal, 2-Nal, and W at position -2. In one embodiment, if position -1 is R, position 1 is F or W and / or position -2 is F, 1-Nal, 2-Nal, or W.

[0070] Specific examples of retro inverso cargo regions are shown in Table 3. The retro inverso sequence of a portion of the wild-type BCL9 HD2 region is listed first as a point of reference. Table 3 D-Amino Acid SequenceD-Amino Acid Sequence7)Sapience.024.WO1 PATENT D-Amino Acid SequenceD-Amino Acid SequenceWWLARQLARLAQLA (SEQ ID NO: 97) WWLERQLARLAQLA (SEQ ID NO: 112) 19)

[0071] The cargo region can comprise amino acids of mixed chirality, such that one or more amino acids in the peptide are in the L form and one or more amino acids are in the D form. For example, an L-peptide can comprise one or more D-amino acids. Likewise, a retro inverso D-peptide can comprise one or more L-amino acids. In certain exemplary embodiments, the cargo region has a sequence selected from the group consisting of: (i) FDRL[WLARQLARLAQLA]D(SEQ ID NO: 121); (ii) FDRL[WLVRQLARLAQLA]D (SEQ ID NO: 122); (iii) FDWL[WLVRQLARLAQLA]D (SEQ ID NO: 123); (iv) FDWL[WLARQLARLAALA]D(SEQ ID NO: 124); (v) FDWL[WLARQLAALAQLA]D(SEQ ID NO: 125); (vi) WL-[WLARQLARLAQLA]D (SEQ ID NO: 126); (vii) WL-[WLARQLARLRQLA]D (SEQ ID NO: 127); (viii) WL-[WLARQLERLRRLA]D(SEQ ID NO: 128); (ix) WL-[WLARQLERLARLA]D (SEQ ID NO: 129); (x) FL-[WLARQLARLAQLA]D(SEQ ID NO: 130); (xi) R L-[WLARQLARLAQLA]D (SEQ ID NO: 131); (xii) FD-WL-[WLARQLARLAQLA]D (SEQ ID NO: 132); and (xiii) WL-[WLVRQLARLAQLA]D (SEQ ID NO: 133); wherein D and L subscripts denote chirality of the amino acids.Sapience.024.WO1 PATENT

[0072] Peptides that inhibit the interaction between subunits of the ribonucleotide reductase (RR) enzyme complex represent another class of exemplary cargo regions. Particular examples include the sequences MSFTLDADF (SEQ ID NO: 2) and MDTAFSFLD (SEQ ID NO: 3).

[0073] Variant peptides comprising a modified portion of the heptad repeat 2 (HR2) domain of a coronavirus spike (S) protein that can inhibit viral fusion with a target cell represent still another class of exemplary cargo regions. One example includes the sequence DISGINASVVNIQKEIDRLNEVAKNLNESLIDLQEL (SEQ ID NO: 140).

[0074] Variants of the foregoing cargo region sequences are also included in the scope of the invention.

[0075] Exemplary cargo regions comprising BCL9 mimetic sequences or RR inhibitors can be used, for example, to inhibit proliferation of and / or to promote cytotoxicity in a neoplastic cell. Proliferation and cytotoxicity can be measured by known assays, including the cell kill assays known in the art. III. Methods of Preparation

[0076] Peptides of the invention can be chemically synthesized, for example, using solid-phase peptide synthesis or solution-method peptide synthesis, or can be expressed using recombinant methods. Synthesis or expression may occur as fragments of the peptide which are subsequently combined either chemically or enzymatically.

[0077] The invention provides a method of preparing a peptide having enhanced aqueous solubility, the method comprising operably linking a solubility enhancing region to cargo region to produce a peptide, wherein the peptide produced displays greater solubility in an aqueous solvent compared with a peptide comprising the cargo region and lacking a solubility enhancing region.

[0078] Also provided is a method of increasing the solubility of a cargo region in an aqueous solvent, the method comprising operably linking a solubility enhancing region to the cargo region to produce a cell-penetrating peptide, wherein the cell-penetrating peptide displays greater solubility in the aqueous solvent compared with a peptide comprising the cargo region and lacking a solubility enhancing region. EXAMPLES

[0079] Embodiments of the present disclosure can be further defined by reference to the following non-limiting examples. It will be apparent to those skilled in the art thatSapience.024.WO1 PATENT many modifications, both to materials and methods, can be practiced without departing from the scope of the present disclosure. Example 1. Peptides Comprising Multiple CPPs Display Increased Aqueous Solubility

[0080] To determine the maximum solubility, super-saturated solutions of the peptides were prepared in water or PBS. At least 100 μL of water or PBS was added to lyophilized peptide powder to attempt to achieve a concentration of approximately 200 mg / mL. In the case of particularly insoluble peptides, more liquid was added until the peptide was mostly dissolved. The saturated solutions were vortexed for 30 seconds and then centrifuged for 2 minutes at 14,000xg.

[0081] The concentration of the supernatant was determined using UV absorbance measured with a Nanodrop. Absorbance measurements were made at 276, 280, and 288 nm for tryptophan-containing peptides and at 252, 258, and 262 nm for peptides which contained only phenylalanine. The concentration was calculated at each wavelength using the extinction coefficient of tryptophan or phenylalanine (Tryptophan: 5431 at 276 nm, 5690 at 280 nm, and 4815 at 288 nm; Phenylalanine: 153.9 at 252 nm, 193.4 at 258 nm, and 128.1 and 262 nm). The concentrations determined at each wavelength were then averaged to determine the final concentration. This was determined to be the maximum concentration in water or PBS when dissolving powder directly.

[0082] Results are shown in Table 4; solubilities are given in mg / mL. CPPs are shown in bold, italicized type. Solubility enhancing regions are underlined. Percent yield is also shown for a representative synthesis of each peptide.1 T ONW.E Tdldl42 A0.PecneipaS 4elba Td V i P V V c PIV V Q d 1 P T:DID P Pi IN N c K K A T L T T L L ES T L KAO Q Q LNE E A A o n K i K K o S S V A A A(A K A A L L Lni )V 2:DI ()3:(E E K K N Nm L A- Q L Q Q Q L Q m A A A AA- O Q L Q E T O L T L L R R D L L L AL A L LNS R R R R R D(PNP L V D VDIDEIEA A A R AIK KIK L L L A A Q L L Q L L T T Q L K K L E E T)R Q Q Q Q Q R S(P P V V S PQ(IQI1 p R R R V N N4 u 1oRrL V VF F FV L L:L D D gV LL L L V VD D D L LLlOL S SF F yA A AWW W W WN)oS S A AW R D D DR R R R 0F FnDaL L L F F FF N N4F WI IIA AtT T T 1- - -c) ) )G GT T Q:F F Ft t t oc c cS SES S S D DI IOlaO O O S( ( ( (M M M M M D N Dnimr1 2 8 0 0e7e e0 0 0 2 1t-7-d d 61 1 1 1 1- - - - -1 5 6 2 7 2i itPt2NP P P P PP P P P P S S =Ap pF D D D D DA A A A Ae eF)CC C C C C P PtcB B B B B BO(Sapience.024.WO1 PATENT

[0083] Peptides comprising a solubility enhancing region had substantially improved solubility, compared to analogous peptides comprising 0 or 1 CPP. This improvement was especially pronounced in PBS. In addition, the solubility enhancing region unexpectedly increased the yield of synthesis, despite the increased peptide length. REFERENCES Bowie JU, et. al., Deciphering the Message in Protein Sequences: Tolerance to Amino Acid Substitutions. Science 247:1306-1310 (1990). Brummell DA et al., Probing the combining site of an anti-carbohydrate antibody by saturation- mutagenesis: role of the heavy-chain CDR3 residues. Biochem.32:1180-1187 (1993). Burks EA et al., In vitro scanning saturation mutagenesis of an antibody binding pocket. Proc. Natl. Acad. Sci. U.S.A.94:412-417 (1997). Gautam A, et al. CPPsite: a curated database of cell penetrating peptides. Database doi:10.1093 / database / bas015 (2012). Heitz F, et al. Twenty years of cell-penetrating peptides: from molecular mechanisms to therapeutics. Brit. J. Pharmacol.157:195-206 (2009). Hervé F, et al. CNS delivery via adsorptive transcytosis. AAPS J.10:455-472 (2008). Huff SE, et al. Inhibitors of the Cancer Target Ribonucleotide Reductase, Past and Present. Kobayashi H, et al., Tryptophan H33 plays an important role in pyrimidine (6-4) pyrimidone photoproduct binding by a high-affinity antibody. Protein Eng.12(10):879-884 (1999). Kosugi S, et al. Six Classes of Nuclear Localization Signals Specific to Different Binding Grooves of Importin α. J. Biol. Chem.284:478-485 (2009). Krautwald S, et al. Inhibition of regulated cell death by cell-penetrating peptides. Cell. Mol. Life Sci.73:2269-2284 (2016). Lange A, et al. Classical Nuclear Localization Signals: Definition, Function, and Interaction with Importin α. J. Biol. Chem.282:5101-5105 (2007). Le Chevalier Isaad A, et al. Side chain-to-side chain cyclization by click reaction. J. Pept. Sci. 15:451-454 (2009). Mattaj IW, et al. Nucleocytoplasmic Transport: The Soluble Phase. Ann. Rev. Biochem.67:265- 306 (1998). Munyendo WLL, et al. Cell penetrating peptides in the delivery of biopharmaceuticals. Biomolecules 2:187-202 (2012). Patgiri A, et al. A hydrogen bond surrogate approach for stabilization of short peptide sequences in alpha helical conformation. Acc. Chem. Res.41:1289-1300 (2008). Schafmeister CE, et al. An All-Hydrocarbon Cross-Linking System for Enhancing the Helicity and Metabolic Stability of Peptides. J. Am. Chem. Soc.122:5891-5892 (2000).Sapience.024.WO1 PATENT Wang X-Y, et al. Synthesis of small cyclic peptides containing the disulfide bond. ARKIVOC xi:148-154 (2006). Zou LL, et al. Cell-penetrating peptide-mediated therapeutic molecule delivery into the central nervous system. Curr. Neuropharmacol.11:197-208 (2013). *** The present invention is further described by the following claims.

Claims

Sapience.024.WO1 PATENT CLAIMS 1. A peptide comprising a cargo region and a solubility enhancing region, wherein a) the cargo region comprises an amino acid sequence selected from the group consisting of [FRWLLRQLARLAQLA]D (SEQ ID NO: 1), [MSFTLDADF]L (SEQ ID NO: 2), [MDTAFSFLD]L(SEQ ID NO: 3) and [DISGINASVVNIQKEIDRLNEVAKNLNESLIDLQEL]L(SEQ ID NO: 140); and b) the solubility enhancing region comprises an amino acid sequence selected from the group consisting of [KLTPVKLTPV]D (SEQ ID NO: 4) and [VPTLKVPTLK]L (SEQ ID NO: 5); wherein the solubility enhancing region is operably linked to the cargo region; wherein D and L subscripts indicate chirality of the amino acids in the sequence; and wherein the peptide displays greater solubility in an aqueous solvent compared with a control peptide.

2. The peptide according to claim 1, comprising a sequence selected from the group consisting of: [FRWLLRQLARLAQLAKLTPVKLTPV]D(SEQ ID NO: 6), [MSFTLDADFVPTLKVPTLK]L(SEQ ID NO: 7), [MDTAFSFLDVPTLKVPTLK]L(SEQ ID NO: 8), and [DISGINASVVNIQKEIDRLNEVAKNLNESLIDLQELVPTLKVPTLK]L (SEQ ID NO: 141).

3. The peptide according to claim 1 or claim 2, wherein the peptide comprises an N- terminal group selected from the group consisting of acetyl, naphthyl, octanoyl, phenyl, and isovaleryl.

4. The peptide according to claim 3, wherein the N-terminal group is octanoyl.

5. The peptide according to any preceding claim, wherein the peptide comprises a C- terminal amide group.

6. A peptide comprising: a) a cargo region comprising the D-amino acid sequence FRWLLRQLARLAQLA (SEQ ID NO: 1) and b) a solubility enhancing region comprising the D-amino acid sequence KLTPVKLTPV (SEQ ID NO: 4);Sapience.024.WO1 PATENT wherein the solubility enhancing region is operably linked to the cargo region; wherein the peptide displays greater solubility in an aqueous solvent compared with a control peptide.

7. The peptide according to claim 6, further comprising an N-terminal octanoyl group.

8. The peptide according to claim 7, wherein the solubility of the peptide in an aqueous solvent is at least about 2-fold higher than the solubility of a control peptide in the aqueous solvent.

9. A composition comprising the peptide according to any preceding claim and an aqueous carrier.

10. The composition according to claim 9, wherein the aqueous carrier comprises a buffer.

11. The composition according to claim 9 or claim 10, which is a pharmaceutical composition.

12. A kit comprising the peptide according to any one of claims 1 to 8 or the composition according to any one of claims 9 to 11.

13. A method of enhancing the solubility of a cargo peptide, wherein the cargo peptide comprises an amino acid sequence selected from the group consisting of [FRWLLRQLARLAQLA]D(SEQ ID NO: 1), [MSFTLDADF]L(SEQ ID NO: 2), [MDTAFSFLD] (SEQ ID NO: 3), and [DISGINASVVNIQKEIDRLNEVAKNLNESLIDLQEL]L (SEQ ID NO: 140), the method comprising modifying the cargo peptide with a solubility enhancing region comprising an amino acid sequence selected from the group consisting of [KLTPVKLTPV]D(SEQ ID NO: 4) and [VPTLKVPTLK]L (SEQ ID NO: 5) to form a modified peptide; wherein the solubility enhancing region is operably linked to the cargo peptide; wherein D and L subscripts indicate chirality of the amino acids in the sequence; and wherein the modified peptide displays greater solubility in an aqueous solvent compared with a control peptide.Sapience.024.WO1 PATENT 14. The method according to claim 13, wherein the modified peptide comprises a sequence selected from the group consisting of: [FRWLLRQLARLAQLAKLTPVKLTPV]D(SEQ ID NO: 6), [MSFTLDADFVPTLKVPTLK]L(SEQ ID NO: 7), [MDTAFSFLDVPTLKVPTLK]L (SEQ ID NO: 8) and [DISGINASVVNIQKEIDRLNEVAKNLNESLIDLQELVPTLKVPTLK]L(SEQ ID NO: 141).

15. The method according to claim 13 or claim 14, wherein the modified peptide comprises an N-terminal group selected from the group consisting of acetyl, naphthyl, octanoyl, phenyl, and isovaleryl.

16. The method according to claim 15, wherein the N-terminal group is octanoyl.

17. The peptide according to any one of claims 13 to 16, wherein the modified peptide comprises a C-terminal amide group.

18. The method according to any one of claims 13 to 17, wherein the cargo region comprises the D-amino acid sequence FRWLLRQLARLAQLA (SEQ ID NO: 1), and wherein the solubility enhancing region comprises the D-amino acid sequence KLTPVKLTPV (SEQ ID NO: 4).

19. The method according to any one of claims 13 to 18, wherein the solubility of the peptide in an aqueous solvent is at least about 2-fold higher than the solubility of a control peptide in the aqueous solvent.

20. The method according to any one of claims 13 to 19, wherein synthetic yield of the peptide is higher than the synthetic yield of a control peptide.

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