EP4735043A1 - Pro-polypeptides and methods of using and designing the same - Google Patents

Abstract

Provided are pro-polypeptides for delivery of a polypeptide of interest (POI) to a biological target. In some embodiments, the pro-polypeptides comprise the POI and a masking structure linked to the POI through one or more protease-cleavable sites. Such pro-polypeptides comprise a stably-folded tertiary structure that masks the POI from the biological target, and where cleavage of the one or more protease-cleavable sites decreases the conformational stability of the tertiary structure leading to release of the POI and availability of the POI to interact with the biological target. Aspects of the present disclosure further include computer-implemented methods of designing such pro-polypeptides. Also provided are nucleic acids, cells and methods that find use in producing the pro-polypeptides of the present disclosure, as well as methods of using the pro-polypeptides, e.g., to deliver a POI to a biological target in a subject in need thereof.

Description

PRO-POLYPEPTIDES ND METHODS OF USING AND DESIGNING THE SAME

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of U.S. Provisional Patent Application No. 63/524,593, filed June 30, 2023, which application is incorporated herein by reference in its entirety.

STATEMENT OF GOVERNMENT SUPPORT

This invention was made with government support under R35 GM122603 awarded by the National Institutes of Health. The government has certain rights in the invention.

SUMMARY

Provided are pro-polypeptides for delivery of a polypeptide of interest (POI) to a biological target. In some embodiments, the pro-polypeptides comprise the POI and a masking structure linked to the POI through one or more protease-cleavable sites. Such pro-polypeptides comprise a stably-folded tertiary structure that masks the POI from the biological target, and where cleavage of the one or more protease-cleavable sites decreases the conformational stability of the tertiary structure leading to release of the POI and availability of the POI to interact with the biological target. Aspects of the present disclosure further include computer-implemented and/or rational design methods of designing such pro-polypeptides. Also provided are nucleic acids, cells and methods that find use in producing the pro-polypeptides of the present disclosure, as well as methods of using the pro-polypeptides, e.g., to deliver a POI to a biological target in a subject in need thereof.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1A-1 D: Schematic illustrations of an approach for pro-polypeptide design according to some embodiments of the present disclosure.

FIG. 2A-2D: Schematic illustrations of an approach for pro-polypeptide design according to some embodiments of the present disclosure.

FIG. 3A-3D: Schematic illustrations of a single scaffold to mask a POI from several POI families.

FIG. 4A-4H: Schematic illustrations of various quaternary and tertiary scaffolds which may be designed/implemented to mask the same POI.

FIG. 5A-5E: Schematic illustrations and confocal microscopy images relating to a proof- of-concept pro-polypeptide designed according to the methods of the present disclosure.

FIG. 6A-6C: Schematic illustration of structural blocks of a useful design according to some embodiments.

FIG. 7A-7C: Schematic illustration of a variation of the design shown in FIG. 6 in which the pro-polypeptide does not include a cargo/payload. FIG. 8: Schematic illustration of a uPA-cleavable pro-polypeptide in which the POI is an antimicrobial peptide, and minimum inhibitory concentration (MIC) data demonstrating that the masking/unmasking mechanism works as intended.

FIG. 9: Schematic illustration of a neutrophil elastase-cleavable pro-polypeptlde In which the POI is an antimicrobial peptide, and minimum inhibitory concentration (MIC) data demonstrating that the masking/unmasking mechanism works as intended.

FIG. 10: Schematic illustration of a pro-polypeptide and masking/unmasking mechanism according to embodiments of the present disclosure.

DETAILED DESCRIPTION

Before the pro-polypeptides and methods of the present disclosure are described in greater detail, it is to be understood that the pro-polypeptides and methods are not limited to particular embodiments described, as such may, of course, vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting, since the scope of the pro-polypeptides and methods will be limited only by the appended claims.

Where a range of values is provided, it is understood that each intervening value, to the tenth of the unit of the lower limit unless the context clearly dictates otherwise, between the upper and lower limit of that range and any other stated or intervening value in that stated range, is encompassed within the pro-polypeptides and methods. The upper and lower limits of these smaller ranges may independently be included in the smaller ranges and are also encompassed within the pro-polypeptides and methods, subject to any specifically excluded limit in the stated range. Where the stated range includes one or both of the limits, ranges excluding either or both of those included limits are also included in the pro-polypeptides and methods.

Certain ranges are presented herein with numerical values being preceded by the term “about.” The term “about” is used herein to provide literal support for the exact number that it precedes, as well as a number that is near to or approximately the number that the term precedes. In determining whether a number is near to or approximately a specifically recited number, the near or approximating unrecited number may be a number which, in the context in which it is presented, provides the substantial equivalent of the specifically recited number.

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 the pro-polypeptides and methods belong. Although any pro-polypeptides and methods similar or equivalent to those described herein can also be used in the practice or testing of the pro-polypeptides and methods, representative illustrative pro-polypeptides and methods are now described.

All publications and patents cited in this specification are herein incorporated by reference as if each individual publication or patent were specifically and individually indicated to be incorporated by reference and are incorporated herein by reference to disclose and describe the materials and/or methods in connection with which the publications are cited. The citation of any publication is for its disclosure prior to the filing date and should not be construed as an admission that the present pro-polypeptides and methods are not entitled to antedate such publication, as the date of publication provided may be different from the actual publication date which may need to be independently confirmed.

It is noted that, as used herein and in the appended claims, the singular forms “a”, “an”, and “the” include plural referents unless the context clearly dictates otherwise. It is further noted that the claims may be drafted to exclude any optional element. As such, this statement is intended to serve as antecedent basis for use of such exclusive terminology as “solely,” “only” and the like in connection with the recitation of claim elements, or use of a “negative” limitation.

It is appreciated that certain features of the pro-polypeptides and methods, which are, for clarity, described in the context of separate embodiments, may also be provided in combination in a single embodiment. Conversely, various features of the pro-polypeptides and methods, which are, for brevity, described in the context of a single embodiment, may also be provided separately or in any suitable sub-combination. All combinations of the embodiments are specifically embraced by the present disclosure and are disclosed herein just as if each and every combination was individually and explicitly disclosed, to the extent that such combinations embrace operable processes and/or compositions. In addition, all sub-combinations listed in the embodiments describing such variables are also specifically embraced by the present pro- polypeptides and methods and are disclosed herein just as if each and every such subcombination was individually and explicitly disclosed herein.

As will be apparent to those of skill in the art upon reading this disclosure, each of the individual embodiments described and illustrated herein has discrete components and features which may be readily separated from or combined with the features of any of the other several embodiments without departing from the scope or spirit of the present methods. Any recited method can be carried out in the order of events recited or in any other order that is logically possible.

PRO-POLYPEPTIDES

Aspects of the present disclosure include pro-polypeptides. The pro-polypeptides find use for delivery (e.g., specific delivery) of a polypeptide of interest (POI) to a biological target. In some embodiments, the pro-polypeptides comprise the POI and a masking structure linked to the POI through one or more protease-cleavable sites (e.g., one or more flexible protease-cleavable linkers). Such pro-polypeptides comprise a stably-folded tertiary structure that masks the POI from the biological target, and where cleavage of the one or more protease-cleavable sites decreases the conformational stability of the tertiary structure leading to release of the POI and availability of the POI to interact with the biological target. The terms “polypeptide”, “peptide”, or “protein” are used interchangeably herein to designate a linear series of amino acid residues connected one to the other by peptide bonds between the alpha-amino and carboxy groups of adjacent residues. The amino acids may include the 20 “standard” genetically encodable amino acids, amino acid analogs, or a combination thereof.

The term “amino acid” includes, but is not limited to, naturally-occurring amino acids and their stereoisomers. “Stereoisomers” of amino acids refer to mirror image isomers of the amino acids, such as L-amino acids or D-amino acids. For example, a stereoisomer of a naturally- occurring amino acid refers to the mirror image isomer of the naturally-occurring amino acid (i.e., the D-amino acid). A pro-polypeptide of the present disclosure may comprise only of L-amino acids, may comprise only of D-amino acids, or may comprise a mixture of L-amino acids and D- amino acids. Pro-polypeptides comprised of D-amino acids (e.g., all-D amino acids) may be advantageous compared to their all-L counterparts in terms of PK, reduced immunogenicity, and/or the like. See Mandal et al. (2012) PNAS 109:14779-14784. Moreover, any of the pro- polypeptides of the present disclosure may comprise one or more beta amino acids, one or more gamma amino acids, and/or one or more of any other amino acid types that suitably replace alpha-amino acids. See, e.g., Sang et al. Acc. Chem. Res. 53, 10, 2425-2442. In certain embodiments, s pro-polypeptide of the present disclosure comprises one or more amino acid analogs available from Bachem.

The term “protease-cleavable site” refers to a site comprising a bond (e.g., a scissile bond) cleavable by a protease of interest. A cleavage site for a protease includes a specific amino acid sequence recognized by the protease during proteolytic cleavage and may include surrounding amino acids (e.g., from one to six amino acids) on either side of the scissile bond, which bind to the active site of the protease and are needed for recognition as a substrate. A protease- cleavable site is typically cleavable under physiological conditions. Any of the protease-cleavable sites described herein may be provided within a linker, e.g., a protease-cleavable site may be provided as a flexible protease-cleavable linker.

In some instances, a pro-polypeptide of the present disclosure comprises two or more POIs. According to some embodiments, the stably-folded tertiary structure is selected from the group consisting of: a three-helix bundle, a single-chain helix-loop-helix, a four-helix bundle, and an Alpha-Beta protein bundle. In certain embodiments, the pro-polypeptide comprises two or more of the stably-folded tertiary structures flexibly linked together.

According to some embodiments, a pro-polypeptide of the present disclosure is a multimeric assembly. By way of example, a pro-polypeptide may comprise a quaternary structure comprising a multimeric assembly. For example, the quaternary structure may comprise a dimeric assembly, a non-limiting example of which is a dimeric assembly comprising two helix-loop-helix motifs. Two examples of such designs can be found in the following (a schematic representation can also be found Figures 4C-4F):

Schematic illustrations of various quaternary and tertiary scaffolds which may be designed/implemented to mask the same POI are provided in Figures 4A-4H. Examples given are for primarily helical masks, though the designs are not limited to this, an example of this is, but is not limited to, what is exemplified in Figure 4E. In Figure 4A, the POI is masked with two helical peptides to form a three-helix bundle. A variation on this (not shown) is the masking of two POIs with a single masking helix. In Figure 4B, a POI is masked with a single masking helix to give a bundle dimer. In Figure 4C-4F, the bundle dimer concept is extended to a dimer of dimers which can be connected in a number of different ways. In Figure 4G, two POIs are physically linked to two masking sequences to form a four-helix bundle. The two POIs can be the same or different, and the cleavage sequences can be the same or different. A variation on this, not shown, is a single POI with a masking sequence of three helices. In Figure 4H, the masking sequence includes a beta strand.

A pro-polypeptide of the present disclosure may include one or any combination of protein folds of any class, non-limiting examples of which include all-alpha, mixed alpha/beta, all-beta, and alpha-PPII (where PPII is a poly-proline II helix, e.g., rcsb I PPT.pdb), which can be placed before or after the POI.

In certain embodiments, the biological target is a cell membrane. In some instances, the biological target is a protein.

According to some embodiments, the POI is selected from a cell-penetrating peptide (CPP), an antimicrobial peptide (AMP), a cytotoxic peptide, an anticancer peptide, an endosomal escape peptide, a cellular localization peptide, a peptide hormone, a membrane-associating peptide, a lytic peptide, and a pore-forming peptide. In some instances, the POI comprises or is substituted with a ligand for radioimaging or therapy.

In some instances, the POI is an antimicrobial peptide (AMP). AMPs are multifaceted in nature and have been shown to have antiviral, antifungal and anticancer activity. Details regarding AMPs that may be implemented as POIs in the pro-polypeptides of the present disclosure may be found, e.g., in Gan BH, Gaynord J, Rowe SM, Deingruber T, Spring DR. The multifaceted nature of antimicrobial peptides: current synthetic chemistry approaches and future directions. Chem Soc Rev. 2021 Jul 5;50(13):7820-7880. doi: 10.1039/d0cs00729c. Erratum in: Chem Soc Rev. 2022 Jan 24;51 (2):792. doi: 10.1039/d1 cs90109e. PMID: 34042120; PMCID: PMC8689412; Vanzolini, T., Bruschi, M., Rinaldi, A. C., Magnani, M., & Fraternale, A. (2022). Multitalented Synthetic Antimicrobial Peptides and Their Antibacterial, Antifungal and Antiviral Mechanisms. International journal of molecular sciences, 23(1 ), 545; Buda De Cesare, G., Cristy, S. A., Garsin, D. A., & Lorenz, M. C. (2020). Antimicrobial Peptides: a New Frontier in Antifungal Therapy. mBio, 77(6), e02123-20; and Tornesello AL, Borrelli A, Buonaguro L, Buonaguro FM, Tornesello ML. Antimicrobial Peptides as Anticancer Agents: Functional Properties and Biological Activities. Molecules. 2020 Jun 19;25(12):2850. doi: 10.3390/molecules25122850. PMID: 32575664; PMCID: PMC7356147; the disclosures of which are incorporated herein by reference in their entireties for all purposes.

In certain embodiments, the protease-cleavable site is cleavable by a protease expressed on the surface of a cell. In some instances, the protease-cleavable site is cleavable by a protease secreted by a cell. According to some embodiments, the protease-cleavable site is cleavable by an intracellular protease. The cell may be any cell of interest, non-limiting examples of which include a cancer cell, an immune cell, and/or the like.

In some instances, the protease-cleavable site is cleavable by a protease present in a tumor microenvironment. According to some embodiments, the protease-cleavable site is cleavable by a cancer-associated protease. Non-limiting examples of cancer-associated proteases include urokinase-type plasminogen activator (uPA), cathepsin B, cathepsin D, cathepsin E, cathepsin G, cathepsin K, cathepsin L, cathepsin S, matrix metalloprotease 1 (MMP1 ), MMP2, MMP3, MMP9, granzyme B, a kallikrein, ADAM10, ADAM17, FAP, and a calpain.

According to some embodiments, the protease-cleavable site is cleavable by a protease of the coagulation cascade. A non-limiting example of such a protease is thrombin. In certain embodiments, the protease-cleavable site is cleavable by a protease present at the site of a thrombus.

In certain embodiments, the protease-cleavable site is cleavable by a protease present at the site of a bacterial infection. In some instances, the protease-cleavable site is cleavable by a bacterial protease. Non-limiting examples of such bacterial proteases include elastase, V8, gelatinase, proteinase K, aureolysin, staphopain A, staphopain B, hemagglutinin/protease (HapA), a bacterial serine protease, and a bacterial subtilisin protease.

In some instances, the protease-cleavable site is cleavable by a protease from an immune cell at the site of bacterial infection. For example, the protease may be neutrophil elastase, neutrophil-derived serine proteases (NSPs), proteinase-3, cathepsin G, cathepsin L, cathepsin D, tryptase, chymase, granzyme B, a metalloprotease (e.g., MMP012), a serine protease, or a cysteine protease.

Nucleic Acids, Cells, and Methods of Making Pro-Polypeptides

Aspects of the present disclosure further include nucleic acids. In certain embodiments, a nucleic acid of the present disclosure encodes any of the pro-polypeptides of the present disclosure. Because of the knowledge of the codons corresponding to the various amino acids, availability of an amino acid sequence of a polypeptide of interest provides a description of all the polynucleotides capable of encoding the polypeptide of interest. The degeneracy of the genetic code, where the same amino acids are encoded by alternative or synonymous codons allows an extremely large number of nucleic acids to be made, all of which encode the pro-polypeptides disclosed herein. Thus, having identified a particular amino acid sequence, those of ordinary skill in the art could make any number of different nucleic acids by simply modifying the sequence of one or more codons in a way which does not change the amino acid sequence of the polypeptide of interest. In this regard, the present disclosure specifically contemplates each and every possible variation of polynucleotides that could be made by selecting combinations based upon the possible codon choices, and all such variations are to be considered specifically disclosed for any pro-polypeptides disclosed herein.

The nucleotide sequences of the nucleic acids of the present disclosure may be codon- optimized. “Codon-optimized” refers to changes in the codons of the polynucleotide encoding a polypeptide to those preferentially used in a particular organism such that the encoded protein is efficiently expressed in the organism of interest. Although the genetic code is degenerate in that most amino acids are represented by several codons, called “synonyms” or “synonymous” codons, it is well known that codon usage by particular organisms is nonrandom and biased towards particular codon triplets. This codon usage bias may be higher in reference to a given gene, genes of common function or ancestral origin, highly expressed proteins versus low copy number proteins, and the aggregate protein coding regions of an organism's genome. In some embodiments, a nucleic acid of the present disclosure encoding a pro-polypeptide may be codon- optimized for optimal production from the host organism selected for expression, e.g., mammalian cells, such as rodent or human cells.

Also provided are expression vectors comprising any of the nucleic acids of the present disclosure. Expression of natural or synthetic nucleic acids encoding the pro-polypeptides of the present disclosure can be achieved by operably linking a nucleic acid encoding the pro- polypeptide to a promoter (which is either constitutive or inducible) and incorporating the construct into an expression vector to generate a recombinant expression vector. The vectors can be suitable for replication and integration in prokaryotes, eukaryotes, or both. Typical cloning vectors contain functionally appropriately oriented transcription and translation terminators, initiation sequences, and promoters useful for regulation of the expression of the nucleic acid encoding the pro-polypeptide. The vectors optionally contain generic expression cassettes containing at least one independent terminator sequence, sequences permitting replication of the cassette in both eukaryotes and prokaryotes, e.g., as found in shuttle vectors, and selection markers for both prokaryotic and eukaryotic systems.

Cells that comprise any of the nucleic acids and/or expression vectors of the present disclosure are also provided. Also provided are methods of making a pro-polypeptide of the present disclosure, the methods including culturing a cell of the present disclosure under conditions suitable for the cell to express the pro-polypeptide, where the pro-polypeptide is produced. The conditions for culturing the cell such that the pro-polypeptide is expressed may vary. Such conditions may include culturing the cell in a suitable container (e.g., a cell culture plate or well thereof), in suitable medium (e.g., cell culture medium, such as DMEM, RPMI, MEM, IMDM, DMEM/F-12, or the like) at a suitable temperature (e.g., 32°C - 42°C, such as 37°C) and pH (e.g., pH 7.0 - 7.7, such as pH 7.4) in an environment having a suitable percentage of CO2, e.g., 3% to 10%, such as 5%).

A pro-polypeptide of the present disclosure may be produced by any suitable method, including recombinant and non-recombinant methods (e.g., chemical synthesis). Where a pro- polypeptide is chemically synthesized, the synthesis may proceed via liquid-phase or solidphase. Solid-phase synthesis (SPPS) allows the incorporation of unnatural amino acids, peptide/protein backbone modification. Various forms of SPPS, such as Fmoc and Boc, are available for synthesizing pro-polypeptide of the present disclosure. Details of the chemical synthesis are known in the art (e.g., Ganesan A. 2006 Mini Rev. Med Chem. 6:3-10 and Camarero JA et al. 2005 Protein Pept Lett. 12:723-8). Briefly, small insoluble, porous beads are treated with functional units on which peptide chains are built. After repeated cycling of coupling/deprotection, the free N-terminal amine of a solid-phase attached peptide or amino acid is coupled to a single N-protected amino acid unit. This unit is then deprotected, revealing a new N-terminal amine to which a further amino acid may be attached. The pro-polypeptide remains immobilized on the solid-phase and undergoes a filtration process before being cleaved off.

In certain embodiments, a method of producing a pro-polypeptide of the present disclosure further comprises comprising conjugating an agent (sometimes referred to herein is a “cargo” or “payload”) to the POI. For example, a ligand for radioimaging or therapy may be conjugated to/ appended onto the POI. The terms “conjugation”, “conjugated”, and “appended” generally refer to a chemical linkage, usually covalent, that proximally associates one molecule of interest with a second molecule of interest. In some instances, the agent is a detectable label. A non-limiting example of a detectable label is one that finds use in in vivo imaging, such as near-infrared (NIR) optical imaging, singlephoton emission computed tomography (SPECT) ± CT imaging, positron emission tomography (PET) ± CT imaging, nuclear magnetic resonance (NMR) spectroscopy, or the like. Labeling agents that find use in such applications include, but are not limited to, fluorescent labels, radioisotopes, and the like. In certain aspects, the agent is a multi-modal in vivo imaging agent that permits in vivo imaging using two or more imaging approaches (e.g., see Thorp-Greenwood and Coogan (201 1 ) Dalton Trans. 40:6129-6143).

In certain embodiments, the agent is an in vivo imaging agent that finds use in nearinfrared (NIR) imaging applications. Such agents include, but are not limited to, a Kodak X- SIGHT dye, Pz 247, DyLight 750 and 800 Fluors, Cy 5.5 and 7 Fluors, Alexa Fluor 680 and 750 Dyes, IRDye 680 and 800CW Fluors. According to some embodiments, the agent is an in vivo imaging agent that finds use in SPECT imaging applications, non-limiting examples of which include ^99mTc, ¹¹¹1n, ¹²³l, ²⁰¹TI, and ¹³³Xe. In certain embodiments, the labeling agent is an in vivo imaging agent that finds use in PET imaging applications, e.g., ¹¹C, ¹³N, ¹⁵0, ¹⁸F, ⁶⁴Cu, ⁶²Cu, ¹²⁴l, ⁷⁶Br, ⁸²Rb, ⁶⁸Ga, or the like.

According to some embodiments, the agent is a therapeutic agent. As used herein, a “therapeutic agent” is a physiologically or pharmacologically active substance that can produce a desired biological effect in a targeted site in an animal, such as a mammal or in a human. The therapeutic agent may be any inorganic or organic compound. A therapeutic agent may decrease, suppress, attenuate, diminish, arrest, or stabilize the development or progression of disease, disorder, or cell growth in an animal such as a mammal or human.

According to some embodiments, the therapeutic agent is a radioisotope, e.g., a radioisotope chelated through a metal binding moiety. Non-limiting examples of radioisotopes include Actinium-225, Astatine-211 , Bismuth-212, Bismuth-213, Bromine-76, Bromine-77, Calcium-47, Carbon-1 1 , Carbon-14, Chromium-51 , Cobalt-57, Cobalt-58, Copper-64, Erbium- 169, Fluorine-18, Gallium-67, Gallium-68, Hydrogen-3, lndium-1 1 1 , lodine-123, lodine-125, lodine-131 , Iron-59, Krypton-81 m, Lead-212, Lutetium-177, Nitrogen-13, Oxygen-15, Phosphorus-32, Radium-223, Radium-224, Samarium-153, Selenium-75, Sodium-22, Sodium- 24, Strontium-89, Technetium-99m, Thallium-201 , Thorium-226, Thorium-227, Xenon-133, or Yttrium-9.

COMPOSITIONS

Aspects of the present disclosure further include compositions. According to some embodiments, a composition of the present disclosure includes a pro-polypeptide of the present disclosure. For example, the pro-polypeptide may be any of the pro-polypeptides described in the pro-polypeptides section hereinabove and/or in the Experimental section below, which descriptions are incorporated but not reiterated herein for purposes of brevity. In certain aspects, a composition of the present disclosure includes the pro-polypeptide present in a liquid medium. The liquid medium may be an aqueous liquid medium, such as water, a buffered solution, or the like. One or more additives such as a salt (e.g., NaCI, MgCI₂, KCI, MgSO4), a buffering agent (a Tris buffer, N-(2-Hydroxyethyl)piperazine-N'-(2-ethanesulfonic acid) (HEPES), 2-(N-Morpholino)ethanesulfonic acid (MES), 2-(N-Morpholino)ethanesulfonic acid sodium salt (MES), 3-(N-Morpholino)propanesulfonic acid (MOPS), N-tris[Hydroxymethyl]methyl- 3-aminopropanesulfonic acid (TAPS), etc.), a solubilizing agent, a detergent (e.g., a non-ionic detergent such as Tween-20, etc.), a nuclease inhibitor, a protease inhibitor, glycerol, a chelating agent, and the like may be present in such compositions.

A composition of the present disclosure may be formulated for administration to a subject (e.g., a mammalian subject such as a human subject) in need thereof. Aspects of the present disclosure further include pharmaceutical compositions. In some embodiments, a pharmaceutical composition of the present disclosure includes a pro-polypeptide of the present disclosure, and a pharmaceutically acceptable carrier.

The pro-polypeptide can be incorporated into a variety of formulations for therapeutic administration. More particularly, the pro-polypeptides can be formulated into pharmaceutical compositions by combination with appropriate, pharmaceutically acceptable excipients or diluents, and may be formulated into preparations in solid, semi-solid, liquid or gaseous forms, such as tablets, capsules, powders, granules, ointments, solutions, injections, inhalants and aerosols.

Formulations of the pro-polypeptides for administration to a subject (e.g., suitable for human administration) are generally sterile and may further be free of detectable pyrogens or other contaminants contraindicated for administration to a patient according to a selected route of administration.

In pharmaceutical dosage forms, the pro-polypeptides can be administered in the form of their pharmaceutically acceptable salts, or they may also be used alone or in appropriate association, as well as in combination, with other pharmaceutically active compounds. The following methods and carriers/excipients are merely examples and are in no way limiting.

For oral preparations, the pro-polypeptides can be used alone or in combination with appropriate additives to make tablets, powders, granules or capsules, for example, with conventional additives, such as lactose, mannitol, corn starch or potato starch; with binders, such as crystalline cellulose, cellulose derivatives, acacia, corn starch or gelatins; with disintegrators, such as corn starch, potato starch or sodium carboxymethylcellulose; with lubricants, such as talc or magnesium stearate; and if desired, with diluents, buffering agents, moistening agents, preservatives and flavoring agents.

The pro-polypeptides can be formulated for parenteral (e.g., intravenous, intra-arterial, intraosseous, intramuscular, intracerebral, intracerebroventricular, intrathecal, subcutaneous, etc.) administration. In certain embodiments, the pro-polypeptides are formulated for injection by dissolving, suspending or emulsifying the pro-polypeptides in an aqueous or non-aqueous solvent, such as vegetable or other similar oils, synthetic aliphatic acid glycerides, esters of higher aliphatic acids or propylene glycol; and if desired, with conventional additives such as solubilizers, isotonic agents, suspending agents, emulsifying agents, stabilizers and preservatives.

Pharmaceutical compositions that include the pro-polypeptides may be prepared by mixing the Pro-polypeptides having the desired degree of purity with optional physiologically acceptable carriers, excipients, stabilizers, surfactants, buffers and/or tonicity agents. Acceptable carriers, excipients and/or stabilizers are nontoxic to recipients at the dosages and concentrations employed, and include buffers such as phosphate, citrate, and other organic acids; antioxidants including ascorbic acid, glutathione, cysteine, methionine and citric acid; preservatives (such as ethanol, benzyl alcohol, phenol, m-cresol, p-chlor-m-cresol, methyl or propyl parabens, benzalkonium chloride, or combinations thereof); amino acids such as arginine, glycine, ornithine, lysine, histidine, glutamic acid, aspartic acid, isoleucine, leucine, alanine, phenylalanine, tyrosine, tryptophan, methionine, serine, proline and combinations thereof; monosaccharides, disaccharides and other carbohydrates; low molecular weight (less than about 10 residues) polypeptides; proteins, such as gelatin or serum albumin; chelating agents such as EDTA; sugars such as trehalose, sucrose, lactose, glucose, mannose, maltose, galactose, fructose, sorbose, raffinose, glucosamine, N-methylglucosamine, galactosamine, and neuraminic acid; and/or nonionic surfactants such as Tween, Brij Pluronics, Triton-X, or polyethylene glycol (PEG).

The pharmaceutical composition may be in a liquid form, a lyophilized form or a liquid form reconstituted from a lyophilized form, wherein the lyophilized preparation is to be reconstituted with a sterile solution prior to administration. The standard procedure for reconstituting a lyophilized composition is to add back a volume of pure water (typically equivalent to the volume removed during lyophilization); however solutions comprising antibacterial agents may be used for the production of pharmaceutical compositions for parenteral administration.

An aqueous formulation of the pro-polypeptides may be prepared in a pH-buffered solution, e.g., at pH ranging from about 4.0 to about 7.0, or from about 5.0 to about 6.0, or alternatively about 5.5. Examples of buffers that are suitable for a pH within this range include phosphate-, histidine-, citrate-, succinate-, acetate-buffers and other organic acid buffers. The buffer concentration can be from about 1 mM to about 100 mM, or from about 5 mM to about 50 mM, depending, e.g., on the buffer and the desired tonicity of the formulation.

A tonicity agent may be included to modulate the tonicity of the formulation. Example tonicity agents include sodium chloride, potassium chloride, glycerin and any component from the group of amino acids, sugars as well as combinations thereof. In some embodiments, the aqueous formulation is isotonic, although hypertonic or hypotonic solutions may be suitable. The term "isotonic" denotes a solution having the same tonicity as some other solution with which it is compared, such as physiological salt solution or serum. Tonicity agents may be used in an amount of about 5 mM to about 350 mM, e.g., in an amount of 100 mM to 350 mM.

A surfactant may also be added to the formulation to reduce aggregation and/or minimize the formation of particulates in the formulation and/or reduce adsorption. Example surfactants include polyoxyethylensorbitan fatty acid esters (Tween), polyoxyethylene alkyl ethers (Brij), alkylphenylpolyoxyethylene ethers (Triton-X), polyoxyethylene-polyoxypropylene copolymer (Poloxamer, Pluronic), and sodium dodecyl sulfate (SDS). Examples of suitable polyoxyethylenesorbitan-fatty acid esters are polysorbate 20, (sold under the trademark Tween 20™) and polysorbate 80 (sold under the trademark Tween 80™). Examples of suitable polyethylene-polypropylene copolymers are those sold under the names Pluronic® F68 or Poloxamer 188™. Examples of suitable Polyoxyethylene alkyl ethers are those sold under the trademark Brij™. Example concentrations of surfactant may range from about 0.001% to about 1% w/v.

A lyoprotectant may also be added in order to protect the pro-polypeptides against destabilizing conditions during a lyophilization process. For example, known lyoprotectants include sugars (including glucose and sucrose); polyols (including mannitol, sorbitol and glycerol); and amino acids (including alanine, glycine and glutamic acid). Lyoprotectants can be included, e.g., in an amount of about 10 mM to 500 nM.

In some embodiments, a pharmaceutical composition includes the pro-polypeptide, and one or more of the above-identified components (e.g., a surfactant, a buffer, a stabilizer, a tonicity agent) and is essentially free of one or more preservatives, such as ethanol, benzyl alcohol, phenol, m-cresol, p-chlor-m-cresol, methyl or propyl parabens, benzalkonium chloride, and combinations thereof. In other embodiments, a preservative is included in the formulation, e.g., at concentrations ranging from about 0.001 to about 2% weight/volume (w/v).

METHODS OF USE

Aspects of the present disclosure further include methods of using the pro-polypeptides of the present disclosure. For example, in certain embodiments, provided are methods of delivering a POI to a biological target in a subject in need thereof. Such methods comprise administering a composition of the present disclosure to the subject, wherein a protease that cleaves the protease-cleavable site is present on the biological target, secreted by the biological target, within the biological target, or in the microenvironment of the biological target. In some embodiments, the biological target is a cell (e.g., a cancer cell, a bacterial cell, or the like), a site of infection, a site of a thrombus, or the like.

In some instances, the subject has cancer, and the protease-cleavable site is cleavable by a cancer-associated protease. The subject methods may be employed for the delivery of the POI to cells of a large variety of cancers, e.g., to treat and/or image the cancer. “Tumor”, as used herein, refers to all neoplastic cell growth and proliferation, whether malignant or benign, and all pre-cancerous and cancerous cells and tissues. The terms “cancer” and “cancerous” refer to or describe the physiological condition in mammals that is typically characterized by unregulated cell growth/proliferation. Examples of cancers that may be treated using the subject methods include, but are not limited to, carcinoma, lymphoma, blastoma, and sarcoma.

More particular examples of cancers to which a POI may be delivered according to the methods of the present disclosure (e.g., to treat and/or image the cancer) include renal cancer; kidney cancer; glioblastoma multiforme; metastatic breast cancer; breast carcinoma; breast sarcoma; neurofibroma; neurofibromatosis; pediatric tumors; neuroblastoma; malignant melanoma; carcinomas of the epidermis; leukemias such as but not limited to, acute leukemia, acute lymphocytic leukemia, acute myelocytic leukemias such as myeloblastic, promyelocytic, myelomonocytic, monocytic, erythroleukemia leukemias and myelodysplastic syndrome, chronic leukemias such as but not limited to, chronic myelocytic (granulocytic) leukemia, chronic lymphocytic leukemia, hairy cell leukemia; polycythemia vera; lymphomas such as but not limited to Hodgkin's disease, non-Hodgkin's disease; multiple myelomas such as but not limited to smoldering multiple myeloma, nonsecretory myeloma, osteosclerotic myeloma, plasma cell leukemia, solitary plasmacytoma and extramedullary plasmacytoma; Waldenstrom's macroglobulinemia; monoclonal gammopathy of undetermined significance; benign monoclonal gammopathy; heavy chain disease; bone cancer and connective tissue sarcomas such as but not limited to bone sarcoma, myeloma bone disease, multiple myeloma, cholesteatoma-induced bone osteosarcoma, Paget's disease of bone, osteosarcoma, chondrosarcoma, Ewing's sarcoma, malignant giant cell tumor, fibrosarcoma of bone, chordoma, periosteal sarcoma, soft- tissue sarcomas, angiosarcoma (hemangiosarcoma), fibrosarcoma, Kaposi's sarcoma, leiomyosarcoma, liposarcoma, lymphangio sarcoma, neurilemmoma, rhabdomyosarcoma, and synovial sarcoma; brain tumors such as but not limited to, glioma, astrocytoma, brain stem glioma, ependymoma, oligodendroglioma, nonglial tumor, acoustic neurinoma, craniopharyngioma, medulloblastoma, meningioma, pineocytoma, pineoblastoma, and primary brain lymphoma; breast cancer including but not limited to adenocarcinoma, lobular (small cell) carcinoma, intraductal carcinoma, medullary breast cancer, mucinous breast cancer, tubular breast cancer, papillary breast cancer, Paget's disease (including juvenile Paget's disease) and inflammatory breast cancer; adrenal cancer such as but not limited to pheochromocytom and adrenocortical carcinoma; thyroid cancer such as but not limited to papillary or follicular thyroid cancer, medullary thyroid cancer and anaplastic thyroid cancer; pancreatic cancer such as but not limited to, insulinoma, gastrinoma, glucagonoma, vipoma, somatostatin-secreting tumor, and carcinoid or islet cell tumor; pituitary cancers such as but limited to Cushing's disease, prolactinsecreting tumor, acromegaly, and diabetes insipius; eye cancers such as but not limited to ocular melanoma such as iris melanoma, choroidal melanoma, and ciliary body melanoma, and retinoblastoma; vaginal cancers such as squamous cell carcinoma, adenocarcinoma, and melanoma; vulvar cancer such as squamous cell carcinoma, melanoma, adenocarcinoma, basal cell carcinoma, sarcoma, and Paget's disease; cervical cancers such as but not limited to, squamous cell carcinoma, and adenocarcinoma; uterine cancers such as but not limited to endometrial carcinoma and uterine sarcoma; ovarian cancers such as but not limited to, ovarian epithelial carcinoma, borderline tumor, germ cell tumor, and stromal tumor; cervical carcinoma; esophageal cancers such as but not limited to, squamous cancer, adenocarcinoma, adenoid cyctic carcinoma, mucoepidermoid carcinoma, adenosquamous carcinoma, sarcoma, melanoma, plasmacytoma, verrucous carcinoma, and oat cell (small cell) carcinoma; stomach cancers such as but not limited to, adenocarcinoma, fungating (polypoid), ulcerating, superficial spreading, diffusely spreading, malignant lymphoma, liposarcoma, fibrosarcoma, and carcinosarcoma; colon cancers; colorectal cancer, KRAS mutated colorectal cancer; colon carcinoma; rectal cancers; liver cancers such as but not limited to hepatocellular carcinoma and hepatoblastoma, gallbladder cancers such as adenocarcinoma; cholangiocarcinomas such as but not limited to papillary, nodular, and diffuse; lung cancers such as KRAS-mutated non-small cell lung cancer, non-small cell lung cancer, squamous cell carcinoma (epidermoid carcinoma), adenocarcinoma, large-cell carcinoma and small-cell lung cancer; lung carcinoma; testicular cancers such as but not limited to germinal tumor, seminoma, anaplastic, classic (typical), spermatocytic, nonseminoma, embryonal carcinoma, teratoma carcinoma, choriocarcinoma (yolk-sac tumor), prostate cancers such as but not limited to, androgen-independent prostate cancer, androgendependent prostate cancer, adenocarcinoma, leiomyosarcoma, and rhabdomyosarcoma; penal cancers; oral cancers such as but not limited to squamous cell carcinoma; basal cancers; salivary gland cancers such as but not limited to adenocarcinoma, mucoepidermoid carcinoma, and adenoidcystic carcinoma; pharynx cancers such as but not limited to squamous cell cancer, and verrucous; skin cancers such as but not limited to, basal cell carcinoma, squamous cell carcinoma and melanoma, superficial spreading melanoma, nodular melanoma, lentigo malignant melanoma, acrallentiginous melanoma; kidney cancers such as but not limited to renal cell cancer, adenocarcinoma, hypernephroma, fibrosarcoma, transitional cell cancer (renal pelvis and/or uterer); renal carcinoma; Wilms' tumor; and bladder cancers such as but not limited to transitional cell carcinoma, squamous cell cancer, adenocarcinoma, carcinosarcoma. In some embodiments, the cancer is myxosarcoma, osteogenic sarcoma, endotheliosarcoma, lymphangioendotheliosarcoma, mesothelioma, synovioma, hemangioblastoma, epithelial carcinoma, cystadenocarcinoma, bronchogenic carcinoma, sweat gland carcinoma, sebaceous gland carcinoma, papillary carcinoma, or papillary adenocarcinomas.

The pro-polypeptides of the present disclosure may be administered via a route of administration selected from oral (e.g., in tablet form, capsule form, liquid form, or the like), parenteral (e.g., by intravenous, intra-arterial, subcutaneous, intramuscular, epidural injection), topical, intra-nasal, intra-tumoral administration, or intraperitoneal (IP) administration. The pro-polypeptides of the present disclosure may be administered (e.g., in a pharmaceutical composition) in a therapeutically effective amount. By “therapeutically effective amount” is meant a dosage sufficient to produce a desired result, e.g., an amount sufficient to effect beneficial or desired therapeutic (including preventative) results, such as a reduction in a symptom of a cancer, a reduction in or slowing of an infection, and/or the like, as compared to a control. With respect to cancer, in some embodiments, the therapeutically effective amount is sufficient to slow the growth of a tumor, reduce the size of a tumor, and/or the like. An effective amount can be administered in one or more administrations.

Provided are methods of treating a condition (e.g., cancer, an infection, etc.) where by treatment is meant at least an amelioration of one or more symptoms associated with the condition of the subject, where amelioration is used in a broad sense to refer to at least a reduction in the magnitude of a parameter, e.g., symptom, associated with the condition. As such, treatment also includes situations where the condition, or at least one or more symptoms associated therewith, are completely inhibited, e.g., prevented from happening, or stopped, e.g., terminated, such that the subject no longer suffers from the condition, or at least the symptoms that characterize the condition.

A pro-polypeptide of the present disclosure may be administered to the individual alone or in combination with a second agent. Second agents of interest include, but are not limited to, agents approved by the United States Food and Drug Administration and/or the European Medicines Agency (EMA) for use in treating cancer, for use in treating an infection, or the like. In some embodiments, the second agent is a chemotherapeutic agent. Non-limiting examples of such chemotherapeutic agents include Capecitabine, Carboplatin, Fluorouracil, Busulfan, Cyclophosphamide, Daunorubicin, Gemcitabine, Altretamine, Fludarabine, Cytarabine, Docetaxel, Etoposide, Mercaptopurine, Methotrexate, Alkylating antineoplastic agent, Azacitidine, Carmustine, Chlorambucil, Dacarbazine, Floxuridine, Melphalan, Paclitaxel, Pentostatin, Temozolomide, or any combination thereof. In some embodiments, the second agent is an immune checkpoint inhibitor. Immune checkpoint inhibitors of interest include, but are not limited to, a cytotoxic T-lymphocyte-associated antigen 4 (CTLA-4) inhibitor, a programmed cell death-1 (PD-1) inhibitor, a programmed cell death ligand-1 (PD-L1 ) inhibitor, a lymphocyte activation gene-3 (LAG-3) inhibitor, a T-cell immunoglobulin domain and mucin domain 3 (TIM- 3) inhibitor, an indoleamine (2,3)-dioxygenase (IDO) inhibitor, a T cell immunoreceptor with Ig and ITIM domains (TIGIT) inhibitor, a V-domain Ig suppressor of T cell activation (VISTA) inhibitor, a B7-H3 inhibitor, and any combination thereof.

When a pro-polypeptide of the present disclosure is administered with a second agent, the pro-polypeptide and the second agent may be administered to the subject according to any suitable administration regimen. According to certain embodiments, the pro-polypeptide and the second agent are administered according to a dosing regimen approved for individual use. In some embodiments, the administration of the pro-polypeptide permits the second agent to be administered according to a dosing regimen that involves one or more lower and/or less frequent doses, and/or a reduced number of cycles as compared with that utilized when the second agent is administered without administration of the pro-polypeptide. In certain aspects, the administration of the second agent permits the pro-polypeptide to be administered according to a dosing regimen that involves one or more lower and/or less frequent doses, and/or a reduced number of cycles as compared with that utilized when the pro-polypeptide is administered without administration of the second agent.

In some embodiments, one or more doses of the pro-polypeptide and the second agent are administered concurrently to the individual. By “concurrently” is meant the pro-polypeptide and the second agent are either present in the same pharmaceutical composition, or the pro- polypeptide and the second agent are administered as separate pharmaceutical compositions within 1 hour or less, 30 minutes or less, or 15 minutes or less.

In some embodiments, one or more doses of the pro-polypeptide and the second agent are administered sequentially to the individual.

In some embodiments, the pro-polypeptide and the second agent are administered to the individual in different compositions and/or at different times. For example, the pro-polypeptide may be administered prior to administration of the second agent, e.g., in a particular cycle. Alternatively, the second agent may be administered prior to administration of the pro- polypeptide, e.g., in a particular cycle. The second agent to be administered may be administered a period of time that starts at least 1 hour, 3 hours, 6 hours, 12 hours, 24 hours, 48 hours, 72 hours, or up to 5 days or more after the administration of the first agent to be administered.

In one example, the second agent is administered to the individual for a desirable period of time prior to administration of the pro-polypeptide. In certain aspects, when the individual has cancer, such a regimen “primes” the cancer cells to potentiate the anti-cancer effect of the pro- polypeptide. Such a period of time separating a step of administering the second agent from a step of administering the pro-polypeptide is of sufficient length to permit priming of the cancer cells, desirably so that the anti-cancer effect of the pro-polypeptide is increased.

In some embodiments, administration of one agent is specifically timed relative to administration of the other agent. For example, in some embodiments, the pro-polypeptide is administered so that a particular effect is observed (or expected to be observed, for example based on population studies showing a correlation between a given dosing regimen and the particular effect of interest).

In certain aspects, desired relative dosing regimens for agents administered in combination may be assessed or determined empirically, for example using ex vivo, in vivo and/or in vitro models; in some embodiments, such assessment or empirical determination is made in vivo, in a patient population (e.g., so that a correlation is established), or alternatively in a particular individual of interest. In some embodiments, the pro-polypeptide and the second agent are administered according to an intermittent dosing regimen including at least two cycles. Where two or more agents are administered in combination, and each by such an intermittent, cycling, regimen, individual doses of different agents may be interdigitated with one another. In certain aspects, one or more doses of a second agent is administered a period of time after a dose of the first agent. In some embodiments, each dose of the second agent is administered a period of time after a dose of the first agent. In certain aspects, each dose of the first agent is followed after a period of time by a dose of the second agent. In some embodiments, two or more doses of the first agent are administered between at least one pair of doses of the second agent; in certain aspects, two or more doses of the second agent are administered between at least one pair of doses of the first agent. In some embodiments, different doses of the same agent are separated by a common interval of time; in some embodiments, the interval of time between different doses of the same agent varies. In certain aspects, different doses of the pro-polypeptide and the second agent are separated from one another by a common interval of time; in some embodiments, different doses of the different agents are separated from one another by different intervals of time.

One exemplary protocol for interdigitating two intermittent, cycled dosing regimens may include: (a) a first dosing period during which a therapeutically effective amount the pro- polypeptide is administered to the individual; (b) a first resting period; (c) a second dosing period during which a therapeutically effective amount of the second agent is administered to the individual; and (d) a second resting period. A second exemplary protocol for interdigitating two intermittent, cycled dosing regimens may include: (a) a first dosing period during which a therapeutically effective amount the second agent is administered to the individual; (b) a first resting period; (c) a second dosing period during which a therapeutically effective amount of the pro-polypeptide is administered to the individual; and (d) a second resting period.

In some embodiments, the first resting period and second resting period may correspond to an identical number of hours or days. Alternatively, in some embodiments, the first resting period and second resting period are different, with either the first resting period being longer than the second one or, vice versa. In some embodiments, each of the resting periods corresponds to 120 hours, 96 hours, 72 hours, 48 hours, 24 hours, 12 hours, 6 hours, 30 hours, 1 hour, or less. In some embodiments, if the second resting period is longer than the first resting period, it can be defined as a number of days or weeks rather than hours (for instance 1 day, 3 days, 5 days, 1 week, 2, weeks, 4 weeks or more).

If the first resting period’s length is determined by existence or development of a particular biological or therapeutic event, then the second resting period’s length may be determined on the basis of different factors, separately or in combination. Exemplary such factors may include type and/or stage of a cancer against which the therapy is administered; properties (e.g., pharmacokinetic properties) of the pro-polypeptide, and/or one or more features of the patient’s response to therapy with the pro-polypeptide. In some embodiments, length of one or both resting periods may be adjusted in light of pharmacokinetic properties (e.g., as assessed via plasma concentration levels) of one or the other of the administered agents. For example, a relevant resting period might be deemed to be completed when plasma concentration of the relevant agent is below a pre-determined level, optionally upon evaluation or other consideration of one or more features of the individual’s response.

In certain aspects, the number of cycles for which a particular agent is administered may be determined empirically. Also, in some embodiments, the precise regimen followed (e.g., number of doses, spacing of doses (e.g., relative to each other or to another event such as administration of another therapy), amount of doses, etc.) may be different for one or more cycles as compared with one or more other cycles.

The pro-polypeptide and the second agent may be administered together or independently via any suitable route of administration. The pro-polypeptide and the second agent may be administered via a route of administration independently selected from oral, parenteral (e.g., by intravenous, intra-arterial, subcutaneous, intramuscular, or epidural injection), topical, or intra-nasal administration. According to certain embodiments, the pro-polypeptide and the second agent are both administered orally (e.g., in tablet form, capsule form, liquid form, or the like) either concurrently (in the same pharmaceutical composition or separate pharmaceutical compositions) or sequentially.

KITS

Aspects of the present disclosure further include kits. In certain embodiments, the kits find use in practicing the methods of the present disclosure, e.g., methods of treating a condition in a subject in need thereof.

Accordingly, in certain embodiments, a kit of the present disclosure comprises any of the pro-polypeptides of the present disclosure (e.g., present in a pharmaceutical composition), and instructions for administering the pro-polypeptide to an individual in need thereof. As will be appreciated, the kits of the present disclosure may include any of the pro-polypeptides having any of the features described above in the section relating to the pro-polypeptides of the present disclosure, which are not reiterated herein for purposes of brevity.

The kits of the present disclosure may include a quantity of the pro-polypeptide, present in unit dosages, e.g., ampoules, or a multi-dosage format. As such, in certain embodiments, the kits may include one or more (e.g., two or more) unit dosages (e.g., ampoules) of an pro- polypeptide of the present disclosure. The term “unit dosage”, as used herein, refers to physically discrete units suitable as unitary dosages for human and animal subjects, each unit containing a predetermined quantity of the pro-polypeptide calculated in an amount sufficient to produce the desired effect. The amount of the unit dosage depends on various factors, such as the pro- polypeptide employed, the effect to be achieved, and the pharmacodynamics associated with the pro-polypeptide, in the subject. In yet other embodiments, the kits may include a single multi dosage amount of the pro-polypeptide.

The instructions (e.g., instructions for use (I FU)) included in the kits may be recorded on a suitable recording medium. For example, the instructions may be printed on a substrate, such as paper or plastic, etc. As such, the instructions may be present in the kits as a package insert, in the labeling of the container of the kit or components thereof (i.e., associated with the packaging or sub-packaging) etc. In other embodiments, the instructions are present as an electronic storage data file present on a suitable computer readable storage medium, e.g., portable flash drive, etc. In yet other embodiments, the actual instructions are not present in the kit, but means for obtaining the instructions from a remote source (e.g., via the internet) are provided. An example of this embodiment is a kit that includes a web address where the instructions can be viewed and/or from which the instructions can be downloaded. As with the instructions, the means for obtaining the instructions is recorded on a suitable substrate.

METHODS OF DESIGNING PRO-POLYPEPTIDES

Aspects of the present disclosure further include methods of designing pro-polypeptides. For example, provided are methods of designing pro-polypeptides comprising any combination or each of the features of any of the pro-polypeptides of the present disclosure.

The methods of designing the pro-polypeptides may be computer-implemented. By “computer-implemented” is meant at least one step of the method is implemented using one or more processors and one or more non-transitory computer-readable media. For example, in certain embodiments, provided are computer-implemented methods of designing a pro- polypeptide, the methods being implemented using one or more processors and one or more non-transitory computer-readable media comprising instructions stored thereon, which when executed by the one or more processors, cause the one or more processors to perform one or more steps of the methods described in detail below, e.g., any or each of the defining, identifying, linking or addition of linkers, converting and/or modifying steps of the methods of designing pro- polypeptides. Computer-implemented methods of the present disclosure may further comprise one or more steps that are not computer-implemented, e.g., steps in connection with producing a designed pro-polypeptide, using a designed pro-polypeptide (e.g., to deliver a POI to a biological target in a subject in need thereof), and the like.

Alternatively, or additionally, the pro-polypeptides can also be designed by a rational set of criteria, non-limiting examples of which include those described in Woolfson D. N. (2021 ) A Brief History of De Novo Protein Design: Minimal, Rational, and Computational. Journal of molecular biology, 433(20), 167160 (doi .org/10.1016/j.jmb.2O21 .167160) ; and Kamtekar, S., Schiffer, J. M., Xiong, H., Babik, J. M., & Hecht, M. H. (1993) Protein design by binary patterning of polar and nonpolar amino acids. Science (New York, N. Y.), 262(5140), 1680-1685 (doi.org/10.1126/science.8259512); the disclosures of which are incorporated herein by reference in their entireties for all purposes.

In some aspects, a computer-implemented method of designing a pro-polypeptide comprises identifying a polypeptide scaffold comprising a stably-folded tertiary structure for masking the POI with the masking structure, substituting a portion of the polypeptide scaffold with the POI, including one or more protease-cleavable sites through which the POI is linked to the masking structure, and modifying the sequence of the masking structure, the POI, the one or more protease-cleavable sites, or any combination thereof, thereby designing the pro- polypeptide. In certain embodiments, the modifying is performed to stabilize the folded tertiary structure.

In some instances, the one or more protease-cleavable sites are linked to, or part of, the POI, such that the substituting and including steps occur at the same time. In certain embodiments, the substituting step is based on substitution scores for the amino acids of the POI and corresponding amino acids of the substituted portion of the polypeptide scaffold.

According to some embodiments, the modifying comprises modifying the polypeptide scaffold after the substituting step, after the including step, or both. In certain embodiments, the modifying comprises modifying the POI after the substituting step, after the including step, or both. In some instances, the polypeptide scaffold comprises a naturally occurring polypeptide or fragment thereof, a de novo designed polypeptide, or a combination thereof.

A non-limiting embodiment of the above-described aspect is schematically illustrated in FIG. 1 A-1 D. FIG. 1 A: The design begins with a known protein backbone, which may be a natural or de novo designed protein. FIG. 1 B: The sequence of the POI is substituted onto a section of a target protein fold. The alignment of the POI sequence onto the target 3D structure is chosen so that the residues in the POI will ultimately lead to a stable overall fold. For example, if the POI has a high propensity to form an alpha-helix, it will be threaded onto the helix of the target (rather than in a non-helical region). Also, apolar residues will frequently be positioned towards the interior of the tertiary structure (a 3-helix bundle in this example). The length of the secondary structural elements can be expanded or contracted to achieve a stable structure and accommodate the POI within the overall scaffold. FIG. 1 C: The design of the masking sequence is carried out using a set of rules as implemented manually or by programs that include, but are not limited to, Rosetta, ESM-IF1 and/or MPNN, keeping the sequence of the POI fixed. The possible sequences are selected using a scoring function which may include but is not limited to AMBER, CHARMM, Rosetta energy function and scores, etc. FIG. 1 D: The original sequence connecting the POI and cage is re-designed to include a known protease recognition site and flanking flexible linkers. At this stage, the secondary structural elements of both the POI and the mask can be extended as needed to maintain a stably-folded structure. In some embodiments, well-scoring sequences are then submitted to structure prediction methods for generating predicted 3D structure from sequence alone. Such programs include but are not limited to Alphafold3 and ESMFold3, and the resulting structures may again be scored. High-scoring sequences are now ready for experimental characterization. The connecting loop can alternatively be introduced prior to sequence selection, occurs in step c) in this flow diagram.

In a second aspect, a computer-implemented method of designing a pro-polypeptide comprises defining a backbone conformation of the POI, identifying a polypeptide scaffold comprising a stably-folded tertiary structure for masking the POI in its defined backbone conformation, linking the masking structure to the POI via one or more linkages, converting the one or more linkages to one or more protease-cleavable sites, and modifying the sequence of the masking structure, the POI, the one or more protease-cleavable sites, or any combination thereof, thereby designing the pro-polypeptide. In certain embodiments, the modifying is performed to stabilize the folded tertiary structure. According to some embodiments, the modifying comprises modifying the masking structure after the linking step, after the converting step, or both. In certain embodiments, the modifying comprises modifying the POI after the linking step, after the converting step, or both.

In certain embodiments, defining the backbone conformation of the POI comprises selecting a set of phi/psi angles that define the backbone conformation. According to some embodiments, the defining step is performed using a protein structure prediction algorithm. Nonlimiting examples of such algorithms that may be employed include a Chou Fasman method, Alphafold3, or ESMFold3. In some instances, a generative model is used to design the stably- folded tertiary structure to mask the POI. A diffusion-based algorithm may be used to design the stably-folded tertiary structure to mask the POI.

According to some embodiments, the modifying comprises substituting one or more amino acids of the masking structure, the POI, the protease-cleavable sites, or any combination thereof. In certain embodiments, the modifying comprises substituting one or more amino acids of the masking structure. In some instances, the modifying comprises substituting one or more amino acids of the one or more protease-cleavable sites. According to some embodiments, the modifying comprises increasing the length of one or more secondary structures of the masking structure, one or more of the one or more protease-cleavable sites, or both. In certain embodiments, the sequence of the POI is fixed (i.e., it’s amino acid sequence and length unchanged) during the modifying.

A non-limiting embodiment of the second aspect is schematically illustrated in FIG. 2A- 2D. FIG. 2A: The design begins with a known POI sequence. Structure prediction methods such as Alphafold3 and ESMFold3 (or experimental methods such as NMR or X-ray crystallography) can be used to predict the target 3D structure of the POI. With this starting point, a tertiary structure is created (step b) that incorporates the POI’s target structure. Alternatively, this step, involving the prediction of the 3D structure of the POI, can be bypassed, and only its sequence will be used as input into B. FIG. 2B: Generative models such as RFdiffusion and/or Chroma are used to build a protein cage around the POI. Restraints can be chosen to place the POI at the protein surface or other locations. In this example, 2 additional helices were diffused in to generate the scaffold sequence, creating an overall 3-helix bundle. FIG. 2C: The design of the masking sequence is carried out using a set of rules as implemented manually or by programs that include, but are not limited to, Rosetta, ESM-IF1 and/or MPNN, keeping the sequence of the POI fixed. The possible sequences are selected using a scoring function that includes but is not limited to AMBER, CHARMM, Rosetta energy function and scores, etc. FIG. 2D: The original sequence connecting the POI and cage is re-designed to include a known protease recognition site and flanking flexible linkers. At this stage the secondary structural elements of both the POI and the mask can be extended as needed to maintain a stably-folded structure. In some embodiments, well-scoring sequences are then submitted to structure prediction methods for generating predicted 3D structure from sequence alone. Such programs include Alphafold3 and ESMFold3, and the resulting structures are again scored. High-scoring sequences are now ready for experimental characterization. The connecting loop can alternatively be introduced prior to sequence selection, occurs in step c) in this flow diagram.

A single scaffold may be used to mask a POI from several POI families. FIG. 3 illustrates the use of a single scaffold or mask, here a 3-helix bundle (FIG. 3A) to cage 3 different peptides of interest. These peptides of interest include, but are not limited to, any peptide that can interact with a membrane or target protein, a cell-penetrating peptide (CPP), an antimicrobial peptide, a cytotoxic peptide, an anticancer peptide, an endosomal escape peptide, a cellular localization peptide, a peptide hormone, a membrane-associating peptide, a lytic peptide and a pore-forming peptide. A ligand for radioimaging or therapy may be appended onto the POI. The POI may also be substituted with ligands for radioimaging or therapy. For example here, the POIs are a cell penetrating peptide (FIG. 3B), an antimicrobial peptide (FIG. 3C) or a peptide hormone (FIG. 3D). Each begins with the same scaffold, they differ by the POI threaded onto the scaffold and the masking sequences that are designed to support the respective POI.

FIG. 6 illustrates structural blocks of one useful design. A “his tag” is included at or near the N-terminus of the design. This is a standard element of protein design, and facilitates selective separation of a peptide that includes such a his tag. As detailed in the text, here the tag is six sequential histidine residues. The his tag is linked to the remaining structure, possibly directly and possibly with a linker sequence. These linkers are generally as short as needed, but can be longer, as known to one skilled in the art. A common linkage structure is a sequence of G and S (glycine, serine) with relatively few design constraints, which are understood in the art. The Cargo block can be any of a variety of peptide sequences known in the art and yet to be developed. This can include semi-synthetic modifications that modify a peptide sequence to include a small molecule such as a drug or a chelating structure as in Evans et al. Here the cargo is mCherry, a peptide sequence with a MW of about 30 KDa that fluoresces red with suitable excitation. In Figure 5, there is no cargo block, for simplicity of illustration and also relevant when the POI itself has biological activity of interest without needing a cargo. The “Link (optional cleavage site)” is a peptide sequence designed to connect the cargo (if any) to the POI. An optional cleavage site such as a sequence susceptible to proteolysis can be included to permit easy post translational modification to remove the cargo when desired. A common cleavage site is sensitive to TEV. One suitable sequence here is LVPR/(G or S). The peptide of interest (POI) is described generally throughout this disclosure, and can take several forms. This is linked directly or through a linkage sequence to an “activation loop” or cleavable loop that includes a peptide sequence known or expected to be sensitive to proteolysis by a protease of interest. The activation loop is connected directly or through a linkage sequence to “Cage Peptide 1”, a component of the masking sequence. In one embodiment illustrated in Figure 3, this is the primary component of the masking sequence. In a three helix bundle embodiment, Cage Peptide 1 is connected directly or through a loop or linkage sequence to “Cage Peptide 2”. The cage peptides are illustrated as cylinders representing alpha helical structures in one preferred embodiment. This is constructed as N-terminal to the left moving to C terminal on the right, noting that Cage 1 is antiparallel in this illustration and the N terminus of Cage 1 is what is connected indirectly to the POI, right to left for Cage 1 as shown. Generally, the pro-polypeptide is composed in a modular manner of components that include a purification tag, one or more protease cleavable linkers, a masking sequence, linkers and a POI. In addition, there can be one or more POIs. These components can be arranged in different ways. For example, in Fig. 6A the POI occurs near the N-terminus in this example. Because protein sequences can be circularly permuted (see review: Das, D. and Ainavarapu, S.R.K. (2024), Protein engineering using circular permutation - structure, function, stability, and applications. FEBS J. https://doi.org/10.1 1 11/febs.17146), the POI can also be placed C-terminally to the caging peptides (Fig. 6B), or even between the caging peptides (Fig. 6C).

FIG. 7A is a schematic illustration of a variation of the design shown in FIG. 6 in which the pro-polypeptide does not include a cargo/payload. The optional cleavage site of FIG. 6 is here selected to be a TEV cleavage site. One common TEV cleavage sequence is ENLYFQ. This is connected in one embodiment by SGS to the POI. As shown in FIG. 7B, cleavage with TEV leaves the components illustrated, the “caged POI” with the masking sequence still connected. As shown in FIG. 7C, the activation loop has been cleaved by the protease of interest, releasing the POI. In this illustration, residues that connected the C-terminus of the POI to the protease cleavage sequence remain connected to the POI. The linkage residues can be designed to keep this relatively short, or otherwise design as desired.

Systems and Computer-Readable Media

Also provided are systems and computer-readable media for designing pro-polypeptides of the present disclosure. According to some embodiments, such systems comprising one or more processors and one or more computer-readable media. The one or more computer- readable media comprise instructions stored thereon, which when executed by the one or more processors, cause the one or more processors to execute one or more steps of the methods of designing pro-polypeptides described elsewhere herein, e.g., one or more of the identifying, substituting, including, defining, linking, converting, and/or modifying steps of the methods of designing the pro-polypeptides.

A variety of processor-based systems may be employed to implement the embodiments of the present disclosure. Such systems may include system architecture wherein the components of the system are in electrical communication with each other using a bus. System architecture can include a processing unit (CPU or processor), as well as a cache, that are variously coupled to the system bus. The bus couples various system components including system memory, (e.g., read only memory (ROM) and random access memory (RAM), to the processor.

System architecture can include a cache of high-speed memory connected directly with, in close proximity to, or integrated as part of the processor. System architecture can copy data from the memory and/or the storage device to the cache for quick access by the processor. In this way, the cache can provide a performance boost that avoids processor delays while waiting for data. These and other modules can control or be configured to control the processor to perform various actions. Other system memory may be available for use as well. Memory can include multiple different types of memory with different performance characteristics. Processor can include any general purpose processor and a hardware module or software module, such as first, second and third modules stored in the storage device, configured to control the processor as well as a special-purpose processor where software instructions are incorporated into the actual processor design. The processor may essentially be a completely self-contained computing system, containing multiple cores or processors, a bus, memory controller, cache, etc. A multi-core processor may be symmetric or asymmetric.

To enable user interaction with the computing system architecture, an input device can represent any number of input mechanisms, such as a microphone for speech, a touch-sensitive screen for gesture or graphical input, keyboard, mouse, motion input, speech and so forth. An output device can also be one or more of a number of output mechanisms. In some instances, multimodal systems can enable a user to provide multiple types of input to communicate with the computing system architecture. A communications interface can generally govern and manage the user input and system output. There is no restriction on operating on any particular hardware arrangement and therefore the basic features here may easily be substituted for improved hardware or firmware arrangements as they are developed.

The storage device is typically a non-volatile memory and can be a hard disk or other types of computer-readable media which can store data that are accessible by a computer, such as magnetic cassettes, flash memory cards, solid state memory devices, digital versatile disks, cartridges, random access memories (RAMs), read only memory (ROM), and hybrids thereof. The storage device can include software modules for controlling the processor. Other hardware or software modules are contemplated. The storage device can be connected to the system bus. In one aspect, a hardware module that performs a particular function can include the software component stored in a computer-readable medium in connection with the necessary hardware components, such as the processor, bus, output device, and so forth, to carry out various functions of the disclosed technology.

Embodiments within the scope of the present disclosure may also include tangible and/or non-transitory computer-readable storage media or devices for carrying or having computerexecutable instructions or data structures stored thereon. Such tangible computer-readable storage devices can be any available device that can be accessed by a general purpose or special purpose computer, including the functional design of any special purpose processor as described above. By way of example, and not limitation, such tangible computer-readable devices can include RAM, ROM, EEPROM, CD-ROM or other optical disk storage, magnetic disk storage or other magnetic storage devices, or any other device which can be used to carry or store desired program code in the form of computer-executable instructions, data structures, or processor chip design. When information or instructions are provided via a network or another communications connection (either hardwired, wireless, or combination thereof) to a computer, the computer properly views the connection as a computer-readable medium. Thus, any such connection is properly termed a computer-readable medium. Combinations of the above should also be included within the scope of the computer-readable storage devices.

Computer-executable instructions include, for example, instructions and data which cause a general purpose computer, special purpose computer, or special purpose processing device to perform a certain function or group of functions. Computer-executable instructions also include program modules that are executed by computers in stand-alone or network environments. Generally, program modules include routines, programs, components, data structures, objects, and the functions inherent in the design of special-purpose processors, etc. that perform tasks or implement abstract data types. Computer-executable instructions, associated data structures, and program modules represent examples of the program code means for executing steps of the methods disclosed herein. The particular sequence of such executable instructions or associated data structures represents examples of corresponding acts for implementing the functions described in such steps.

Other embodiments of the disclosure may be practiced in network computing environments with many types of computer system configurations, including personal computers, hand-held devices, multi-processor systems, microprocessor-based or programmable consumer electronics, network PCs, minicomputers, mainframe computers, and the like. Embodiments may also be practiced in distributed computing environments where tasks are performed by local and remote processing devices that are linked (either by hardwired links, wireless links, or by a combination thereof) through a communications network. In a distributed computing environment, program modules may be located in both local and remote memory storage devices.

For purposes of completeness, non-limiting aspects and embodiments of the present disclosure are further disclosed in the following numbered clauses.

1 . A pro-polypeptide for delivery of a polypeptide of interest (POI) to a biological target, wherein the pro-polypeptide comprises: the POI; and a masking structure linked to the POI through one or more protease-cleavable sites, wherein the pro-polypeptide comprises a stably-folded tertiary structure that masks the POI from the biological target, and wherein cleavage of the one or more protease-cleavable sites decreases the conformational stability of the tertiary structure leading to release of the POI and availability of the POI to interact with the biological target.

2. The pro-polypeptide of clause 1 , wherein the masking structure is linked to the POI through one or more flexible protease-cleavable sites.

3. The pro-polypeptide of clause 1 or clause 2, wherein the pro-polypeptide comprises two or more POIs.

4. The pro-polypeptide of any one of clauses 1 to 3, wherein the stably-folded tertiary structure is selected from the group consisting of: a three-helix bundle, a single-chain helixloop-helix, a four-helix bundle, and an Alpha-Beta protein bundle.

5. The pro-polypeptide of clause 4, wherein the pro-polypeptide comprises two or more of the stably-folded tertiary structures flexibly linked together.

6. The pro-polypeptide of any one of clauses 1 to 5, wherein the pro-polypeptide is a multimeric assembly.

7. The pro-polypeptide of clause 6, wherein the pro-polypeptide comprises a quaternary structure comprising a multimeric assembly.

8. The pro-polypeptide of clause 7, wherein the quaternary structure comprises a dimeric assembly.

9. The pro-polypeptide of clause 8, wherein the dimeric assembly comprises two helixloop-helix motifs.

10. The pro-polypeptide of any one of clauses 1 to 9, wherein the biological target is a cell membrane.

11 . The pro-polypeptide of any one of clauses 1 to 10, wherein the biological target is a protein. 12. The pro-polypeptide of any one of clauses 1 to 11 , wherein the POI is selected from a cell-penetrating peptide (CPP), an antimicrobial peptide, a cytotoxic peptide, an anticancer peptide, an endosomal escape peptide, a cellular localization peptide, a peptide hormone, a membrane-associating peptide, a lytic peptide, and a pore-forming peptide.

13. The pro-polypeptide of any one of clauses 1 to 12, wherein a ligand for radioimaging or therapy is appended onto the POI.

14. The pro-polypeptide of any one of clauses 1 to 12, wherein the POI comprises or is substituted with a ligand for radioimaging or therapy.

15. The pro-polypeptide of any one of clauses 1 to 14, wherein the protease-cleavable site is cleavable by a protease expressed on the surface of a cell.

16. The pro-polypeptide of any one of clauses 1 to 15, wherein the protease-cleavable site is cleavable by a protease secreted by a cell.

17. The pro-polypeptide of any one of clauses 1 to 14, wherein the protease-cleavable site is cleavable by an intracellular protease.

18. The pro-polypeptide of any one of clauses 15 to 17, wherein the cell is a cancer cell.

19. The pro-polypeptide of any one of clauses 15 to 18, wherein the cell is an immune cell.

20. The pro-polypeptide of any one of clauses 1 to 19, wherein the protease-cleavable site is cleavable by a protease present in a tumor microenvironment.

21 . The pro-polypeptide of any one of clauses 1 to 20, wherein the protease-cleavable site is cleavable by a cancer-associated protease.

22. The pro-polypeptide of clause 21 , wherein the cancer-associated protease is selected from the group consisting of: urokinase-type plasminogen activator (uPA), cathepsin B, cathepsin D, cathepsin E, cathepsin G, cathepsin K, cathepsin L, cathepsin S, matrix metalloprotease 1 (MMP1 ), MMP2, MMP3, MMP9, granzyme B, a kallikrein, ADAM10, ADAM17, FAP, and a caipain.

23. The pro-polypeptide of any one of clauses 1 to 19, wherein the protease-cleavable site is cleavable by a protease of the coagulation cascade.

24. The pro-polypeptide of clause 23, wherein the protease-cleavable site is cleavable by thrombin.

25. The pro-polypeptide of clause 23 or 24, wherein the protease-cleavable site is cleavable by a protease present at the site of a thrombus.

26. The pro-polypeptide of any one of clauses 1 to 14, wherein the protease-cleavable site is cleavable by a protease present at the site of a bacterial infection.

27. The pro-polypeptide of any one of clauses 1 to 14 or 26, wherein the protease-cleavable site is cleavable by a bacterial protease. 28. The pro-polypeptide of clause 27, wherein the bacterial protease is elastase, V8, gelatinase, proteinase K, aureolysin, staphopain A, staphopain B, or Vibrio cholerae hemagglutinin/protease (HapA).

29. The pro-polypeptide of clause 26, wherein the protease-cleavable site is cleavable by a protease from an immune cell.

30. The pro-polypeptide of clause 29, wherein the protease is neutrophil elastase, neutrophil-derived serine proteases (NSPs), a metalloprotease, a serine protease, or a cysteine protease.

31 . A computer-implemented method of designing the pro-polypeptide of any one of clauses 1 to 30, the method comprising:

(a) identifying a polypeptide scaffold comprising a folded tertiary structure for masking the POI;

(b) substituting a portion of the polypeptide scaffold with the POI;

(c) including one or more protease-cleavable sites through which the POI is linked to the masking structure; and

(d) modifying the sequence of the masking structure, the POI, the one or more protease-cleavable sites, or any combination thereof, thereby designing the pro- polypeptide.

32. The method of clause 31 , wherein the substituting at step (b) is based on substitution scores for the amino acids of the POI and corresponding amino acids of the substituted portion of the polypeptide scaffold.

33. The method of clause 31 or 32, wherein the one or more protease-cleavable sites are linked to, or part of, the POI, such that steps (b) and (c) occur at the same time.

34. The computer-implemented method of any one of clauses 31 to 33, wherein the modifying comprises modifying the sequence of the masking structure after step (b), after step (c), or both.

35. The computer-implemented method of any one of clauses 31 to 34, wherein the modifying comprises modifying the sequence of the POI after step (b), after step (c), or both.

36. The computer-implemented method of any one of clauses 31 to 35, wherein the modifying is performed to stabilize the folded tertiary structure.

37. The computer-implemented method according to any one of clauses 31 to 36, wherein the polypeptide scaffold comprises a naturally occurring polypeptide or fragment thereof, a de novo designed polypeptide, or a combination thereof.

38. A computer-implemented method of designing the pro-polypeptide of any one of clauses 1 to 30, the method comprising:

(a) defining a backbone conformation of the POI; (b) identifying a polypeptide scaffold comprising a folded tertiary structure for masking the POI in its defined backbone conformation;

(c) linking the masking structure to the POI via one or more linkages;

(d) converting the one or more linkages to one or more protease-cleavable sites; and

(e) modifying the sequence of the masking structure, the POI, the one or more flexible protease-cleavable sites, or any combination thereof, thereby designing the propolypeptide.

39. The computer-implemented method of clause 38, wherein the modifying comprises modifying the sequence of the masking structure after step (c), after step (d), or both.

40. The computer-implemented method of clause 39, wherein the modifying comprises modifying the sequence of the POI after step (c), after step (d), or both.

41 . The computer-implemented method according to any one of clauses 38 to 40, wherein defining the backbone conformation of the POI comprises selecting a set of phi/psi angles that define the backbone conformation.

42. The computer-implemented method according to clause 38 or 41 , wherein step (a) is performed using a protein structure prediction algorithm.

43. The computer-implemented method according to clause 42, wherein the protein structure prediction algorithm is a Chou Fasman method, Alphafold3, or ESMFold3.

44. The computer-implemented method according to any one of clauses 38 to 43, wherein a generative model is used to design the stably-folded tertiary structure to mask the POI.

45. The computer-implemented method according to any one of clauses 38 to 43, wherein a diffusion-based algorithm is used to design the stably-folded tertiary structure to mask the POI.

46. The computer-implemented method according to any one of clauses 38 to 45, wherein the modifying is performed to stabilize the folded tertiary structure.

47. The computer-implemented method according to any one of clauses 31 to 46, wherein the modifying comprises substituting one or more amino acids of the masking structure, the POI, the flexible protease-cleavable sites, or any combination thereof.

48. The computer-implemented method according to any one of clauses 31 to 47, wherein the modifying comprises substituting one or more amino acids of the masking structure.

49. The computer-implemented method according to any one of clauses 31 to 48, wherein the modifying comprises substituting one or more amino acids of the one or more protease- cleavable sites.

50. The computer-implemented method according to any one of clauses 31 to 49, wherein the modifying comprises increasing the length of one or more secondary structures of the masking structure. 51 . The computer-implemented method according to any one of clauses 31 to 50, wherein the modifying comprises increasing the length of one or more of the protease-cleavable sites.

52. The computer-implemented method according to any one of clauses 31 to 51 , wherein the modifying comprises decreasing the length of one or more secondary structures of the masking structure.

53. The computer-implemented method according to any one of clauses 31 to 52, wherein the modifying comprises decreasing the length of one or more of the one or more protease- cleavable sites.

54. The computer-implemented method according to any one of clauses 31 to 53, wherein the sequence of the POI is fixed during the modifying.

55. The computer-implemented method according to any one of clauses 31 to 54, further comprising producing the designed pro-polypeptide.

56. The computer-implemented method according to clause 55, further comprising administering the produced pro-polypeptide to a subject in need thereof, thereby delivering the POI to the biological target in the subject.

57. A nucleic acid encoding the pro-polypeptide of any one of clauses 1 to 30.

58. The nucleic acid of clause 57 operably linked to a promoter.

59. An expression vector comprising the nucleic acid of clause 58.

60. A cell comprising the expression vector of clause 59.

61 . A method of producing a pro-polypeptide, the method comprising culturing the cell of clause 60 under conditions suitable for the cell to express the pro-polypeptide, wherein the pro- polypeptide is produced.

62. A method of producing the pro-polypeptide of any one of clauses 1 to 30, the method comprising synthesizing the pro-polypeptide by solid-phase synthesis.

63. The method according to clause 61 or clause 62, further comprising conjugating an agent to the POI.

64. The method according to clause 63, wherein the agent is a ligand for radioimaging or therapy.

65. The method according to clause 63, wherein the agent is a detectable label.

66. The method according to clause 65, wherein the detectable label is an in vivo imaging agent.

67. The method according to clause 63, wherein the agent is a therapeutic agent.

68. The method according to any one of clauses 63 to 67, wherein the agent is a radioisotope. 69. A pharmaceutical composition comprising the pro-polypeptide of any one of clauses 1 to 30, or a pro-polypeptide produced according to the method of any one of clauses 61 to 68.

70. A method of delivering a POI to a biological target in a subject in need thereof, the method comprising administering the pharmaceutical composition of clause 69 to the subject, wherein a protease that cleaves the protease-cleavable site is present on the biological target, secreted by the biological target, within the biological target, or in the microenvironment of the biological target.

71 . The method according to clause 70, wherein the biological target is a cell.

72. The method according to clause 71 , wherein the cell is a cancer cell.

73. The method according to any one of clauses 70 to 72, wherein the subject has cancer, and wherein the protease-cleavable site is cleavable by a cancer-associated protease.

74. The method according to clause 71 , wherein the cell is a bacterial cell.

75. The method according to any one of clauses 70 to 72, wherein the subject has a bacterial infection, and wherein the protease-cleavable site is cleavable by a bacterial protease.

76. The method according to any one of clauses 70 to 72, wherein the subject has a bacterial infection, and wherein the protease-cleavable site is cleavable by a host protease.

The following examples are offered by way of illustration and not by way of limitation.

EXPERIMENTAL

Design and Use of Pro- Polypeptides for Delivery of POIs to Biological Targets

Described herein is a modular method to deliver cargos (e.g., proteins) to membranes and (optionally) through membranes. Cell-penetrating peptides (CPP) have been shown to be effective at delivering genetically fused protein partners into cells. Additionally, anticancer peptides (ACP) and antimicrobial peptides (AMP) have been shown as promising alternatives to treat cancer and antimicrobial infection, respectively. However, these constructs lack tissue- and cell-specificity, which has precluded their use in vivo as drugs despite initial interest. Described herein is de novo protein design to design a protein domain that is fused to the POI and protects the POI from binding non-specifically until the construct reaches the biological target (e.g., cell, tissue) of interest.

Cell-Penetrating Peptide

In a first non-limiting example, and solely as proof of concept, the cargo molecule is the fluorescent mCherry protein sequence (including an N-terminus tag and linker) which is connected at the N-terminus of the POI via a cargo linker sequence. At the C-terminus of the POI is a protease cleavable loop/linker, here a thrombin cleavable loop, connecting it to a masking sequence of two helices to form a three-helix bundle (Figure 5A-B). Upon treatment with the thrombin protease, the thrombin cleavable loop is cleaved to release the mCherry-POl construct (including residual residues from the cleavable loop) from the masking sequence (Figure 5C).

Shown in Figures 5D-5E is data demonstrating that the pro-polypeptide works as designed. HeLa cells were treated with samples containing either the untreated pro-polypeptide (Figure 5D) or the pro-polypeptide treated with thrombin for 1 hour at room temperature (Figure 5E). Images obtained via confocal microscopy with staining only done for the nucleus, all other fluorescent signal results from the mCherry. The data demonstrates successful delivery of the mCherry-POl to the cells.

The amino acid sequences of the pro-polypeptide and domains thereof of this example are provided in the table below. Antimicrobial Peptide

In a second non-limiting example, and also solely as proof of concept, the pro-polypeptide molecule is a three-helical bundle protein, where the POI is the N-terminal first helix, which is attached to a TEV recognition sequence, connected to a N-terminus “His tag” via a short linker sequence. At the C-terminus of the POI is a protease cleavable loop/linker, here a (a) urokinasetype plasminogen activator (uPA), and (b) neutrophil elastase, connecting it to a masking sequence of two helices to form a three-helix bundle (Figure 3C). Upon treatment with the protease of interest, the loop is cleaved to release the POI construct (including residual residues from the cleavable loop) from the masking sequence.

Shown in FIGs. 8 and 9 are data demonstrating that the pro-polypeptides work as designed. E coli (K12), and P aeruginosa (35151 ) cells were treated with samples containing either the untreated pro-polypeptide, or the pro-polypeptide treated with protease of interest (uPA and neutrophile elastase as schematically illustrated in FIG. 8 and FIG. 9, respectively) at least for 3 hour at room temperature. Minimum Inhibitory concentration (MIC) analyses were performed monitoring bacterial growth for 16 hours. The data demonstrates peptide toxicity masking ranging from 10-to-50-fold by the designed protein scaffold (FIGs. 8 and 9).

The amino acid sequences of the pro-polypeptide and domains thereof of this example are provided in the table below.

Accordingly, the preceding merely illustrates the principles of the present disclosure. It will be appreciated that those skilled in the art will be able to devise various arrangements which, although not explicitly described or shown herein, embody the principles of the invention and are included within its spirit and scope. Furthermore, all examples and conditional language recited herein are principally intended to aid the reader in understanding the principles of the invention and the concepts contributed by the inventors to furthering the art, and are to be construed as being without limitation to such specifically recited examples and conditions. Moreover, all statements herein reciting principles, aspects, and embodiments of the invention as well as specific examples thereof, are intended to encompass both structural and functional equivalents thereof. Additionally, it is intended that such equivalents include both currently known equivalents and equivalents developed in the future, i.e., any elements developed that perform the same function, regardless of structure. The scope of the present invention, therefore, is not intended to be limited to the exemplary embodiments shown and described herein.

Claims

WHAT IS CLAIMED IS:

1 . A pro-polypeptide for delivery of a polypeptide of interest (POI) to a biological target, wherein the pro-polypeptide comprises: the POI; and a masking structure linked to the POI through one or more protease-cleavable sites, wherein the pro-polypeptide comprises a stably-folded tertiary structure that masks the POI from the biological target, and wherein cleavage of the one or more protease-cleavable sites decreases the conformational stability of the tertiary structure leading to release of the POI and availability of the POI to interact with the biological target.

2. The pro-polypeptide of claim 1 , wherein the masking structure is linked to the POI through one or more flexible protease-cleavable sites.

3. The pro-polypeptide of claim 1 or claim 2, wherein the pro-polypeptide comprises two or more POIs.

4. The pro-polypeptide of any one of claims 1 to 3, wherein the stably-folded tertiary structure is selected from the group consisting of: a three-helix bundle, a single-chain helixloop-helix, a four-helix bundle, and an Alpha-Beta protein bundle.

5. The pro-polypeptide of claim 4, wherein the pro-polypeptide comprises two or more of the stably-folded tertiary structures flexibly linked together.

6. The pro-polypeptide of any one of claims 1 to 5, wherein the pro-polypeptide is a multimeric assembly.

7. The pro-polypeptide of claim 6, wherein the pro-polypeptide comprises a quaternary structure comprising a multimeric assembly.

8. The pro-polypeptide of claim 7, wherein the quaternary structure comprises a dimeric assembly.

9. The pro-polypeptide of claim 8, wherein the dimeric assembly comprises two helix-loop- helix motifs.

10. The pro-polypeptide of any one of claims 1 to 9, wherein the biological target is a cell membrane.

11 . The pro-polypeptide of any one of claims 1 to 10, wherein the biological target is a protein.

12. The pro-polypeptide of any one of claims 1 to 11 , wherein the POI is selected from a cell-penetrating peptide (CPP), an antimicrobial peptide, a cytotoxic peptide, an anticancer peptide, an endosomal escape peptide, a cellular localization peptide, a peptide hormone, a membrane-associating peptide, a lytic peptide, and a pore-forming peptide.

13. The pro-polypeptide of any one of claims 1 to 12, wherein a ligand for radioimaging or therapy is appended onto the POI.

14. The pro-polypeptide of any one of claims 1 to 12, wherein the POI comprises or is substituted with a ligand for radioimaging or therapy.

15. The pro-polypeptide of any one of claims 1 to 14, wherein the protease-cleavable site is cleavable by a protease expressed on the surface of a cell.

16. The pro-polypeptide of any one of claims 1 to 15, wherein the protease-cleavable site is cleavable by a protease secreted by a cell.

17. The pro-polypeptide of any one of claims 1 to 14, wherein the protease-cleavable site is cleavable by an intracellular protease.

18. The pro-polypeptide of any one of claims 15 to 17, wherein the cell is a cancer cell.

19. The pro-polypeptide of any one of claims 15 to 18, wherein the cell is an immune cell.

20. The pro-polypeptide of any one of claims 1 to 19, wherein the protease-cleavable site is cleavable by a protease present in a tumor microenvironment.

21 . The pro-polypeptide of any one of claims 1 to 20, wherein the protease-cleavable site is cleavable by a cancer-associated protease.

22. The pro-polypeptide of claim 21 , wherein the cancer-associated protease is selected from the group consisting of: urokinase-type plasminogen activator (uPA), cathepsin B, cathepsin D, cathepsin E, cathepsin G, cathepsin K, cathepsin L, cathepsin S, matrix metalloprotease 1 (MMP1 ), MMP2, MMP3, MMP9, granzyme B, a kallikrein, ADAM10, ADAM17, FAP, and a caipain.

23. The pro-polypeptide of any one of claims 1 to 19, wherein the protease-cleavable site is cleavable by a protease of the coagulation cascade.

24. The pro-polypeptide of claim 23, wherein the protease-cleavable site is cleavable by thrombin.

25. The pro-polypeptide of claim 23 or claim 24, wherein the protease-cleavable site is cleavable by a protease present at the site of a thrombus.

26. The pro-polypeptide of any one of claims 1 to 14, wherein the protease-cleavable site is cleavable by a protease present at the site of a bacterial infection.

27. The pro-polypeptide of any one of claims 1 to 14 or 26, wherein the protease-cleavable site is cleavable by a bacterial protease.

28. The pro-polypeptide of claim 27, wherein the bacterial protease is elastase, V8, gelatinase, proteinase K, aureolysin, staphopain A, staphopain B, or Vibrio cholerae hemagglutinin/protease (HapA).

29. The pro-polypeptide of claim 26, wherein the protease-cleavable site is cleavable by a protease from an immune cell.

30. The pro-polypeptide of claim 29, wherein the protease is neutrophil elastase, neutrophil- derived serine proteases (NSPs), a metalloprotease, a serine protease, or a cysteine protease.

31 . A computer-implemented method of designing the pro-polypeptide of any one of claims 1 to 30, the method comprising:

(a) identifying a polypeptide scaffold comprising a folded tertiary structure for masking the POI;

(b) substituting a portion of the polypeptide scaffold with the POI;

(c) including one or more protease-cleavable sites through which the POI is linked to the masking structure; and (d) modifying the sequence of the masking structure, the POI, the one or more protease-cleavable sites, or any combination thereof, thereby designing the propolypeptide.

32. The method of claim 31 , wherein the substituting at step (b) is based on substitution scores for the amino acids of the POI and corresponding amino acids of the substituted portion of the polypeptide scaffold.

33. The method of claim 31 or 32, wherein the one or more protease-cleavable sites are linked to, or part of, the POI, such that steps (b) and (c) occur at the same time.

34. The computer-implemented method of any one of claims 31 to 33, wherein the modifying comprises modifying the sequence of the masking structure after step (b), after step (c), or both.

35. The computer-implemented method of any one of claims 31 to 34, wherein the modifying comprises modifying the sequence of the POI after step (b), after step (c), or both.

36. The computer-implemented method of any one of claims 31 to 35, wherein the modifying is performed to stabilize the folded tertiary structure.

37. The computer-implemented method according to any one of claims 31 to 36, wherein the polypeptide scaffold comprises a naturally occurring polypeptide or fragment thereof, a de novo designed polypeptide, or a combination thereof.

38. A computer-implemented method of designing the pro-polypeptide of any one of claims 1 to 30, the method comprising:

(a) defining a backbone conformation of the POI;

(b) identifying a polypeptide scaffold comprising a folded tertiary structure for masking the POI in its defined backbone conformation;

(c) linking the masking structure to the POI via one or more linkages;

(d) converting the one or more linkages to one or more protease-cleavable sites; and

(e) modifying the sequence of the masking structure, the POI, the one or more flexible protease-cleavable sites, or any combination thereof, thereby designing the pro- polypeptide.

39. The computer-implemented method of claim 38, wherein the modifying comprises modifying the sequence of the masking structure after step (c), after step (d), or both.

40. The computer-implemented method of claim 39, wherein the modifying comprises modifying the sequence of the POI after step (c), after step (d), or both.

41 . The computer-implemented method according to any one of claims 38 to 40, wherein defining the backbone conformation of the POI comprises selecting a set of phi/psi angles that define the backbone conformation.

42. The computer-implemented method according to claim 38 or 41 , wherein step (a) is performed using a protein structure prediction algorithm.

43. The computer-implemented method according to claim 42, wherein the protein structure prediction algorithm is a Chou Fasman method, Alphafold3, or ESMFold3.

44. The computer-implemented method according to any one of claims 38 to 43, wherein a generative model is used to design the stably-folded tertiary structure to mask the POI.

45. The computer-implemented method according to any one of claims 38 to 43, wherein a diffusion-based algorithm is used to design the stably-folded tertiary structure to mask the POI.

46. The computer-implemented method according to any one of claims 38 to 45, wherein the modifying is performed to stabilize the folded tertiary structure.

47. The computer-implemented method according to any one of claims 31 to 46, wherein the modifying comprises substituting one or more amino acids of the masking structure, the POI, the flexible protease-cleavable sites, or any combination thereof.

48. The computer-implemented method according to any one of claims 31 to 47, wherein the modifying comprises substituting one or more amino acids of the masking structure.

49. The computer-implemented method according to any one of claims 31 to 48, wherein the modifying comprises substituting one or more amino acids of the one or more protease- cleavable sites.

50. The computer-implemented method according to any one of claims 31 to 49, wherein the modifying comprises increasing the length of one or more secondary structures of the masking structure.

51 . The computer-implemented method according to any one of claims 31 to 50, wherein the modifying comprises increasing the length of one or more of the protease-cleavable sites.

52. The computer-implemented method according to any one of claims 31 to 51 , wherein the modifying comprises decreasing the length of one or more secondary structures of the masking structure.

53. The computer-implemented method according to any one of claims 31 to 52, wherein the modifying comprises decreasing the length of one or more of the one or more protease- cleavable sites.

54. The computer-implemented method according to any one of claims 31 to 53, wherein the sequence of the POI is fixed during the modifying.

55. The computer-implemented method according to any one of claims 31 to 54, further comprising producing the designed pro-polypeptide.

56. The computer-implemented method according to claim 55, further comprising administering the produced pro-polypeptide to a subject in need thereof, thereby delivering the POI to the biological target in the subject.

57. A nucleic acid encoding the pro-polypeptide of any one of claims 1 to 30.

58. The nucleic acid of claim 57 operably linked to a promoter.

59. An expression vector comprising the nucleic acid of claim 58.

60. A cell comprising the expression vector of claim 59.

61 . A method of producing a pro-polypeptide, the method comprising culturing the cell of claim 60 under conditions suitable for the cell to express the pro-polypeptide, wherein the pro- polypeptide is produced.

62. A method of producing the pro-polypeptide of any one of claims 1 to 30, the method comprising synthesizing the pro-polypeptide by solid-phase synthesis.

63. The method according to claim 61 or claim 62, further comprising conjugating an agent to the POI.

64. The method according to claim 63, wherein the agent is a ligand for radioimaging or therapy.

65. The method according to claim 63, wherein the agent is a detectable label.

66. The method according to claim 65, wherein the detectable label is an in vivo imaging agent.

67. The method according to claim 63, wherein the agent is a therapeutic agent.

68. The method according to any one of claims 63 to 67, wherein the agent is a radioisotope.

69. A pharmaceutical composition comprising the pro-polypeptide of any one of claims 1 to 30, or a pro-polypeptide produced according to the method of any one of claims 61 to 68.

70. A method of delivering a POI to a biological target in a subject in need thereof, the method comprising administering the pharmaceutical composition of claim 69 to the subject, wherein a protease that cleaves the protease-cleavable site is present on the biological target, secreted by the biological target, within the biological target, or in the microenvironment of the biological target.

71 . The method according to claim 70, wherein the biological target is a cell.

72. The method according to claim 71 , wherein the cell is a cancer cell.

73. The method according to any one of claims 70 to 72, wherein the subject has cancer, and wherein the protease-cleavable site is cleavable by a cancer-associated protease.

74. The method according to claim 71 , wherein the cell is a bacterial cell.

75. The method according to any one of claims 70 to 72, wherein the subject has a bacterial infection, and wherein the protease-cleavable site is cleavable by a bacterial protease.

76. The method according to any one of claims 70 to 72, wherein the subject has a bacterial infection, and wherein the protease-cleavable site is cleavable by a host protease.