Engineered FGF10 polypeptides, compositions, and uses thereof

Abstract

The present disclosure provides engineered fibroblast growth factor 10 (FGF10) polypeptides and compositions comprising engineered FGF10 polypeptides. The engineered FGF10 polypeptides of the present disclosure include at least one amino acid substitution, deletion, and / or addition compared to wild-type FGF10, and exhibit increased stability and / or activity.

Description

BIOTN-44681.601ENGINEERED EGE10 POLYPEPTIDES, COMPOSITIONS, AND USES THEREOFFIELD100011 The present disclosure provides engineered fibroblast growth factor 10 (FGF10) polypeptides and compositions comprising engineered FGF10 polypeptides. The engineered FGF10 polypeptides of the present disclosure include at least one amino acid substitution, deletion, and / or addition compared to wild-type FGF10, and exhibit increased stability and / or activity. CROSS REFERENCE TO RELATED APPLICATIONSI0002J This application claims the benefit of U.S. Provisional Application No. 63 / 762,467, filed February 24, 2025, the content of which is herein incorporated by reference in its entirety.SEQUENCE LISTING STATEMENT[0 03J The content of the electronic sequence listing titled BIOTN_44681_601_SequenceListing.xml (Size: 64,135 bytes; and Date of Creation: February 24, 2026) is herein incorporated by reference in its entirety.BACKGROUND10004} Fibroblast Growth Factors (FGFs) are a large family of cell signaling proteins involved in many aspects of development including cell proliferation, growth, and differentiation. They act on several cell types to regulate diverse physiological functions including angiogenesis, cell growth, pattern formation, embryonic development, metabolic regulation, cell migration, neurotrophic effects, and tissue repair. FGF10 is in the subgroup of FGFs that also includes FGF 3, 7, and 22. FGF10 is a paracrine growth factor that has essential functions during development and tissue homeostasis. Recombinant FGF10 is used in a wide variety of organoid protocols but is also relevant in many regenerative medicine applications. Depending on the context, FGF10 can promote cell proliferation, differentiation, cell survival, tissue repair and tissue regeneration.

[0005] The FGF family of proteins is well known to be thermally unstable. When used in cell cultures these proteins often lose activity quickly and require frequent replacement. This creates spikes in activity and requirements for daily cell culture feeding. Thus, thermally stable FGF family members are needed to reduce signaling spikes and less frequent cell culture maintenance.BIOTN-44681.601SUMMARY

[0006] Embodiments of the present disclosure include engineered FGF10 polypeptides. In some embodiments, the engineered FGF10 polypeptides comprise less than 100% amino acid sequence identity with SEQ ID NO: 1 and at least one amino acid substitution, deletion, or addition compared to SEQ ID NO: 1 or a fragment thereof. In some embodiments, the engineered FGF10 polypeptides have increased stability as compared to wild-type FGF-10.

[0007] In some embodiments, the at least one amino acid substitution is at amino acid position 42, 43, 49, 54, 62, 66, 78, 81, 88, 145, 148, 150, 160, or any combination thereof, as compared to SEQ ID NO: 1. In some embodiments, the engineered FGF10 polypeptides comprise at least two amino acid substitutions at amino acid positions selected from positions 42, 43, 49, 54, 62, 66, 78, 81, 88, 145, 148, 150, and 160. In some embodiments, the at least one amino acid substitution comprises W42I, R43V, T49H, T49N, K54A, K54C, S62F, K66D, S78W, 18 IP, A88C, G145K, A148K, R150M, R150L, T160P, or any combinations thereof.[0008[ In some embodiments, the engineered FGF10 polypeptide comprises at least 2 amino acid substitutions, at least 3 amino acid substitutions, at least 4 amino acid substitutions, at least 5 amino acid substitutions, or at least 6 amino acid substitutions. In some embodiments, the engineered FGF10 polypeptide comprises 4 to 6 amino acid substitutions.[0009J In some embodiments, the engineered FGF10 polypeptide comprises an amino acid substitution at amino acid positions 42 and 150. In some embodiments, the engineered FGF10 polypeptide comprises amino acid substitution W42I; and amino acid substitution R150M or R150L.

[0010] In some embodiments, the engineered FGF10 polypeptide comprises an amino acid substitution at amino acid position 62. In some embodiments, the engineered FGF10 polypeptide comprises amino acid substitution S62F.

[0011] In some embodiments, the engineered FGF10 polypeptide further comprises an amino acid substitution at amino acid position 43, 49, 54, 66, 78, 145, 148, 160, or a combination thereof. In some embodiments, the engineered FGF10 polypeptide comprises amino acid substitution R43V; amino acid substitution T49N or T49H; amino acid substitution K54A or K54C; amino acid substitution K66D; amino acid substitution S78W; amino acid substitution G145K; amino acid substitution A148K; amino acid substitution T160P; or a combination thereof. In some embodiments, the engineered FGF10 polypeptide comprises amino acid substitution R43V; aminoBIOTN-44681.601acid substitution T49H; amino acid substitution K54A; amino acid substitution K66D; amino acid substitution S78W; amino acid substitution G145K; amino acid substitution A148K; amino acid substitution T160P; or a combination thereof. In some embodiments, the engineered FGF10 polypeptide comprises amino acid substitution T49H; amino acid substitution K66D; or a combination thereof.

[0012] In some embodiments, the engineered FGF10 polypeptide comprises amino acid substitution W42I; amino acid substitution S62F; amino acid substitution R150M; and one or both of amino acid substitutions T49H and K66D.[00131 In some embodiments, the engineered FGF10 polypeptide comprises at least 70% amino acid sequence identity to any one of SEQ ID NOs: 5, 8-16, and 33-61, or a fragment thereof. In some embodiments, the engineered FGF10 polypeptide comprises at least 80% amino acid sequence identity to any one of SEQ ID NOs: 5, 8-16, and 33-61, or a fragment thereof. In some embodiments, the engineered FGF10 polypeptide comprises at least 90% amino acid sequence identity to any one of SEQ ID NOs: 5, 8-16, and 33-61, or a fragment thereof. In some embodiments, the engineered FGF10 polypeptide comprises at least 95% amino acid sequence identity to any one of SEQ ID NOs: 5, 8-16, and 33-61, or a fragment thereof. In some embodiments, the engineered FGF10 polypeptide comprises an amino acid sequence of any one of SEQ ID NOs: 5, 8-16, and 33-61, or a fragment thereof.[0014| In some embodiments, the engineered FGF10 polypeptide has an increased melting temperature as compared to wild-type FGF10. In some embodiments, the engineered FGF10 polypeptide maintains activity level following incubation at 37° C for at least 4 days.[0015J Additional embodiments include compositions comprising an engineered FGF10 polypeptide as disclosed herein and a carrier. In some embodiments, the composition is a cell culture medium.

[0016] Further embodiments include nucleic acids encoding an engineered FGF10 polypeptide as disclosed herein, cells configured to express an engineered FGF10 polypeptide as disclosed herein, and cells comprising a nucleic acid encoding the engineered FGF10 polypeptide.

[0017] Other embodiments include methods for promoting cell proliferation, cell differentiation, cell survival, tissue repair and tissue regeneration. In some embodiments, the methods comprise contacting a cell, a precursor or progenitor cell, a tissue, a spheroid, or an organoid with an engineered FGF10 polypeptide as disclosed herein, or a composition comprisingBIOTN-44681.601thereof. In some embodiments, the organoid is a lung, stomach, pancreas, heart, bladder, dental epithelium, salivary gland, prostate, or liver organoid. In some embodiments, the cell is pancreatic progenitor cell. In some embodiments, the cell, precursor or progenitor cell, tissue, spheroid, or organoid is or comprises a cancer cell, a cancer precursor or progenitor cell, a cancerous tissue, a cancer spheroid, or a cancer organoid.

[0018] Other aspects and embodiments of the disclosure will be apparent in light of the following detailed description.BRIEF DESCRIPTION OF THE DRAWINGS[0019J FIG. 1 is a graph of melting temperatures of the indicated engineered FGF10 polypeptides and wild-type FGF10 measured using SYPRO orange dye and a thermal ramp.[0020| FIG. 2 is a graph of activity for WT FGF10 and the indicated engineered FGF10 polypeptides measured by stimulating proliferation of BA / F3 cells expressing FGFR2b(IIIB). FGF10 V3 proteins had comparable activity to FGF10-WT except for FGF10 V3-15.[0021 J FIG. 3 is a graph of relative stability measured by comparing activity with and without incubation of WT FGF10 and the indicated engineered FGF10 polypeptides in growth media at 1 ug / ml at 37 C for 96h. Activity was measured by stimulating proliferation of BA / F3 cells expressing FGFR2b(IIIB), as in FIG. 2.[0022J FIG. 4 is a graph of melting temperatures of WT FGF10 and the indicated engineered FGF10 polypeptides measured using intrinsic fluorescence and a thermal ramp.

[0023] FIGS. 5A and 5B are graphs of relative stability measured by comparing activity with and without incubation of WT FGF10 and the indicated engineered FGF10 polypeptides in growth media at 1 ug / ml at 37 C for 96h (FIG. 5A) or 8 days (FIG. 5B). Activity was measured by stimulating proliferation of BA / F3 cells expressing FGFR2b(IIIB), as in FIG. 2. Error bars represent standard deviation (no error was reported for those which did not include replicates).

[0024] FIG. 6 is a graph of melting temperatures of WT FGF10 and the indicated engineered FGF10 polypeptides measured using SYPRO orange dye and a thermal ramp.

[0025] FIG. 7 is a graph showing bioactivity of the indicated engineered FGF10 polypeptides. FGF10 activity measured by stimulating proliferation of BA / F3 cells expressing FGFR2b(IIIB), as in FIG. 2.

[0026] FIG. 8 is a graph showing FGF receptor 2 binding of the indicated engineered FGF10 polypeptides. Recombinant Human FGF R2 beta (Illb) was captured on Biacore Sensor Chip CM5BIOTN-44681.601via Recombinant Protein A / G / L and binding to the indicated engineered FGF10 polypeptide was measured at a concentration range between 0.0488 nM and 12.5 nM. The double-referenced sensorgram was fit to a two-state reaction binding model to determine the binding kinetics and affinity.

[0027] FIG. 9 shows engineered FGF10 polypeptides preserve bioactivity. FGF-10 protein induces proliferation of Ba / F3 mouse pro-B cells transfected with human FGFR2b. BA / F3 cells expressing FGFR2b(IIIB) were incubated with the indicated engineered FGF10 polypeptide and WT FGF10 and were either untreated or heat stressed at 37°C for 15 or 30 days in media.[0028[ FIG. 10 shows that an exemplary engineered FGF10 polypeptide with increased thermostability retains bioactivity after brief heat stress, whereas WT FGF10 loses activity. To evaluate the signal transduction response triggered by FGF-10, a HEK293 cell line that expresses a reporter for SEAP controlled by the Serum Response Element was utilized. WT FGF10 and FGF10 v5-7 were incubated in DMEM media at 37°C for 48 or 72 hrs at concentrations of 600 ng / mL. Specific activity was assessed through a SEAP bioassay in HEK293 cells, wherein cells were treated with respective FGF-lOs. The specific activity % of control was determined, with the control comprising FGF-10 stored at 4°C without any incubation.

[0029] FIG. 11 is growth curves of adult stem cell-derived lung organoids. Area measured with image quantification for treatment with FGFlO v5-7 orWT FGF10, using either Cultrex UltiMatrix or Cultrex Synthetic Hydrogel.

[0030] FIG. 12 is brightfield images showing morphology of adult stem cell-derived lung organoids grown in Cultrex Synthetic Hydrogel with FGFlO v5-7 orWT FGF10. Scale bar indicates 1 mm.[00311 FIG. 13 is images of sections of adult stem cell-derived lung organoid (cultured for 24 days withFGFlO v5-7) in Cultrex Synthetic Hydrogel stained for SFTPC (AT2 cell marker), SCGB1A1 (club cell marker), FOXJ1 (ciliated cell marker), and DAPI. Scale bar indicates 50 pm. DETAILED DESCRIPTION

[0032] Disclosed herein are engineered FGF10 polypeptides and compositions comprising thereof.[0033 [ The members of the FGF family of proteins are well known to be thermally unstable. When used in culture media these proteins often quickly lose activity and require frequent replacement or supplementation, creating spikes in activity. As shown herein, wild-type FGF10BIOTN-44681.601can lose over 50% of its activity in 96 hours. Disclosed herein are engineered FGF 10 polypeptides which are more stable at lower concentrations and higher temperatures. Thus, the disclosed engineered FGF 10 polypeptides would decrease the large fluctuations in protein concentration and maintain consistent stimulation as compared to wild-type FGF10.[0034| Section headings as used in this section and the entire disclosure herein are merely for organizational purposes and are not intended to be limiting.Definitions|0035] Unless otherwise defined herein, scientific, and technical terms used in connection with the present disclosure shall have the meanings that are commonly understood by those of ordinary skill in the art. For example, any nomenclature used in connection with, and techniques of cell and tissue culture, molecular biology, microbiology, genetics and protein and nucleic acid chemistry and hybridization described herein are those that are well known and commonly used in the art. The meaning and scope of the terms should be clear; in the event, however of any latent ambiguity, definitions provided herein take precedent over any dictionary or extrinsic definition. Further, unless otherwise required by context, singular terms shall include pluralities and plural terms shall include the singular.

[0036] The terms “comprise(s),” “include(s),” “having,” “has,” “can,” “contain(s),” and variants thereof, as used herein, are intended to be open-ended transitional phrases, terms, or words that do not preclude the possibility of additional acts or structures. As used herein, comprising a certain sequence or a certain SEQ ID NO usually implies that at least one copy of said sequence is present in recited peptide or polynucleotide. However, two or more copies are also contemplated. The singular forms “a,” “and,” and “the” include plural references unless the context clearly dictates otherwise. The present disclosure also contemplates other embodiments “comprising,” “consisting of,” and “consisting essentially of,” the embodiments or elements presented herein, whether explicitly set forth or not.|0037] For the recitation of numeric ranges herein, each intervening number there between with the same degree of precision is explicitly contemplated. For example, for the range of 6-9, the numbers 7 and 8 are contemplated in addition to 6 and 9, and for the range 6.0-7.0, the number 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, and 7.0 are explicitly contemplated.

[0038] The term “contacting” as used herein refers to bring or put in contact, to be in or come into contact. The term “contact” as used herein refers to a state or condition of touching or ofBIOTN-44681.601immediate or local proximity. Contacting a composition to a target may occur by any means known to the skilled artisan.

[0039] The term “ex vivo” as used herein refers to cells that have been removed from a living organism (e.g., a human) and propagated outside the organism (e.g., in a culture dish, test tube, or bioreactor).

[0040] As used herein, the term “melting temperature” or “Tm” refers to the temperature at which 50% of the protein is unfolded. The melting temperature is a measure of thermal stability. Tm can be determined by any suitable method known in the art such as circular dichroism (CD) spectroscopy, differential scanning calorimetry (DSC) or differential scanning fluorimetry (DSF) DSF monitors changes in the overall tertiary structure of proteins as a function of temperature by following the changes in the intrinsic fluorescence or by following changes of dye interactions with the hydrophobic core of proteins which results in an increase in fluorescence. As described in the Examples below, melting temperature is defined as the temperature at half max fluorescence, either intrinsic or as a result of dye interactions.

[0041] As used herein, “nucleic acid” or “nucleic acid sequence” refers to a polymer or oligomer of pyrimidine and / or purine bases, preferably cytosine, thymine, and uracil, and adenine and guanine, respectively (See Albert L. Lehninger, Principles of Biochemistry, at 793-800 (Worth Pub. 1982)). The present technology contemplates any deoxyribonucleotide, ribonucleotide, or peptide nucleic acid component, and any chemical variants thereof, such as methylated, hydroxymethylated, or glycosylated forms of these bases, and the like. The polymers or oligomers may be heterogenous or homogenous in composition and may be isolated from naturally occurring sources or may be artificially or synthetically produced. In addition, the nucleic acids may be DNA or RNA, or a mixture thereof, and may exist permanently or transitionally in single-stranded or double-stranded form, including homoduplex, heteroduplex, and hybrid states. In some embodiments, a nucleic acid or nucleic acid sequence comprises other kinds of nucleic acid structures such as, for instance, a DNA / RNA helix, peptide nucleic acid (PNA), morpholino nucleic acid (see, e.g., Braasch and Corey, Biochemistry, 41(14): 4503-4510 (2002)) and U.S. Pat. No. 5,034,506), locked nucleic acid (LNA; see Wahlestedt et al., Proc. Natl. Acad. Sci. U.S.A., 97: 5633-5638 (2000)), cyclohexenyl nucleic acids (see Wang, J. Am. Chem. Soc., 122: 8595-8602 (2000)), and / or a ribozyme. Hence, the term “nucleic acid” or “nucleic acid sequence” may also encompass a chain comprising non-natural nucleotides, modified nucleotides, and / or non-BIOTN-44681.601nucleotide building blocks that can exhibit the same function as natural nucleotides (e.g., “nucleotide analogs”); further, the term “nucleic acid sequence” as used herein refers to an oligonucleotide, nucleotide or polynucleotide, and fragments or portions thereof, and to DNA or RNA of genomic or synthetic origin, which may be single or double-stranded, and represent the sense or antisense strand. The terms “nucleic acid,” “polynucleotide,” “nucleotide sequence,” and “oligonucleotide” are used interchangeably. They refer to a polymeric form of nucleotides of any length, either deoxyribonucleotides or ribonucleotides, or analogs thereof.

[0042] A “peptide” or “polypeptide” is a linked sequence of two or more amino acids linked by peptide bonds. The polypeptide can be natural, synthetic, or a modification or combination of natural and synthetic. The peptide or polypeptide may be modified by the addition of sugars, lipids or other moieties not included in the amino acid chain. The terms “polypeptide,” “oligopeptide,” “protein,” and “peptide” are used interchangeably herein. The peptide may be produced by recombinant genetic technology or chemical synthesis. The peptide may be isolated and purified by any number of standard methods including, but not limited to, differential solubility (e.g., precipitation), centrifugation, chromatography (e.g., affinity, ion exchange, and size exclusion), or by any other standard techniques known in the art.

[0043] The term “amino acid” or “any amino acid” as used here refers to any and all amino acids, including naturally occurring amino acids (e.g., a-amino acids), unnatural amino acids, modified amino acids, and non-natural amino acids. It includes both D- and L-amino acids. Natural amino acids include those found in nature, such as, e.g., the 23 amino acids that combine into peptide chains to form the building-blocks of a vast array of proteins. These are primarily L stereoisomers, although a few D-amino acids occur in bacterial envelopes and some antibiotics. The “non-standard,” natural amino acids include, for example, pyrolysine (found in methanogenic organisms and other eukaryotes), selenocysteine (present in many non-eukaryotes as well as most eukaryotes), norvaline (found in the antifungal peptide of Bacillus subtilis and N-formylmethionine (encoded by the start codon AUG in bacteria, mitochondria, and chloroplasts). “Unnatural” or “non-natural” amino acids are non-proteinogenic amino acids (e.g., those not naturally encoded or found in the genetic code) that either occur naturally or are chemically synthesized. Over 140 unnatural amino acids are known and thousands of more combinations are possible. Examples of “unnatural” amino acids include P-amino acids (3and p2), homo-amino acids, proline and pyruvic acid derivatives, -substituted alanine derivatives, glycine derivatives,BIOTN-44681.601ring-substituted phenylalanine and tyrosine derivatives, linear core amino acids, diamino acids, D-amino acids, alpha-methyl amino acids, and N-methyl amino acids. Unnatural or non-natural amino acids also include modified amino acids. “Modified” amino acids include amino acids (e.g., natural amino acids) that have been chemically modified to include a group, groups, or chemical moiety not naturally present on the amino acid. According to certain embodiments, a peptide inhibitor comprises an intramolecular bond between two amino acid residues present in the peptide inhibitor. It is understood that the amino acid residues that form the bond will be altered somewhat when bonded to each other as compared to when not bonded to each other. Reference to a particular amino acid is meant to encompass that amino acid in both its unbonded and bonded state. For example, the amino acid residue homoSerine (hSer) in its unbonded form may take the form of 2-aminobutyric acid (Abu) when participating in an intramolecular bond according to the present invention.

[0044] For the most part, the names of naturally occurring and non-naturally occurring aminoacyl residues used herein follow the naming conventions suggested by the IUPAC Commission on the Nomenclature of Organic Chemistry and the IUPAC -IUB Commission on Biochemical Nomenclature as set out in “Nomenclature of a-Amino Acids (Recommendations, 1974)” Biochemistry, 14(2), (1975). To the extent that the names and abbreviations of amino acids and aminoacyl residues employed in this specification and appended claims differ from those suggestions, they will be made clear to the reader.

[0045] Throughout the present specification, unless naturally occurring amino acids are referred to by their full name (e.g., alanine, arginine, etc.), they are designated by their conventional three-letter or single-letter abbreviations (e.g., Ala or A for alanine, Arg or R for arginine, etc.). The term “L-amino acid,” as used herein, refers to the “L” isomeric form of a peptide, and conversely the term “D-amino acid” refers to the “D” isomeric form of a peptide (e.g., Dphe, (D)Phe, D-Phe, orDF for the D isomeric form of Phenylalanine). Amino acid residues in the D isomeric form can be substituted for any L-amino acid residue, as long as the desired function is retained by the peptide.[0046| In the case of less common or non-naturally occurring amino acids, unless they are referred to by their full name (e.g. sarcosine, ornithine, etc.), frequently employed three- or four-character codes are employed for residues thereof, including, Sar or Sarc (sarcosine, i.e. N-methylglycine), Aib (a-aminoisobutyric acid), Dab (2,4-diaminobutanoic acid), Dapa (2,3-BIOTN-44681.601di aminopropanoic acid), y-Glu (y-glutamic acid), Gaba (y-aminobutanoic acid), [3-Pro (pyrrolidine-3 -carboxylic acid), and 8 Ado (8-amino-3,6-dioxaoctanoic acid), Abu (2-amino butyric acid), hPro (P-homoproline), phPhe (P-homophenylalanine) and Bip (P,P diphenylalanine), and Ida (Iminodiacetic acid).[0047J Natural amino acids can be described by their chemical properties (e.g., charged, nonpolar, polar, hydrophobic, hydrophilic). For example, naturally occurring amino acids classified as positively charged include lysine, arginine, and histidine whereas those classified as negatively charged include aspartic acid (aspartate) and glutamic acid (glutamate). Polar, uncharged naturally occurring amino acids include, for example, serine, proline, threonine, tyrosine, asparagine, and glutamine. Non-polar naturally occurring amino acids include, for example, glycine, cysteine, alanine, valine, leucine, isoleucine, methionine, tryptophan, and phenylalanine.

[0048] As used herein, the terms “percent sequence identity” or “percent identity” refer to the percentage of amino acids in an amino acid sequence, that is identical with the corresponding amino acids in a reference sequence after aligning the two sequences and introducing gaps, if necessary, to achieve the maximum percent identity. A number of mathematical algorithms for obtaining the optimal alignment and calculating identity between two or more sequences are known and incorporated into a number of available software programs. Examples of such programs include CLUSTAL-W, T-Coffee, and ALIGN (for alignment of nucleic acid and amino acid sequences), BLAST programs (e g., BLAST 2.1, BL2SEQ, and later versions thereof) and FASTA programs (e g., FASTA3x, FAS™, and SSEARCH) (for sequence alignment and sequence similarity searches). Sequence alignment algorithms also are disclosed in, for example, Altschul et al., J. Molecular Biol., 215(3): 403-410 (1990), Beigert et al., Proc. Natl. Acad. Sci. USA, 106(10): 3770-3775 (2009), Durbin et al., eds., Biological Sequence Analysis: Probabilistic Models of Proteins and Nucleic Acids, Cambridge University Press, Cambridge, UK (2009), Soding, Bioinformatics, 21(7): 951-960 (2005), Altschul et al., Nucleic Acids Res., 25(17): 3389-3402 (1997), and Gusfield, Algorithms on Strings, Trees and Sequences, Cambridge University Press, Cambridge UK (1997)).]0049| As used herein, the terms “thermostability” and “thermal stability” in reference to an engineered polypeptide disclosed herein encompass thermodynamic and kinetic stabilities. Thermodynamic stability is associated with the equilibrium between a folded and unfolded or partially-unfolded state of the protein and is quantitatively represented by the difference in GibbsBIOTN-44681.601free energy between these two protein states. Kinetic stability reflects the rate (e g., kinetics) of protein folding and unfolding and is quantitatively represented by the free-energy barrier between the folded, functional state of the polypeptide from non-functional states (e.g., unfolded or partially unfolded states, irreversibly-denatured protein, aggregated protein).[0050| The terms “non-naturally occurring,” “engineered,” and “synthetic” are used interchangeably and indicate the involvement of the hand of man. The terms, when referring to macromolecules (e.g., nucleic acid molecules or polypeptides) mean that the macromolecule is at least substantially free from at least one other component with which it is naturally associated in nature and as found in nature, and / or the macromolecule is associated with at least one other component with which it is not naturally associated in nature and / or that there is one or more changes in, for example, the nucleic acid or amino acid sequence as compared with such sequence as it is found in nature.[0051 [ A “subject” may be human or non-human and may include, for example, animal strains or species used as “model systems” for research purposes, such as a mouse model. Likewise, the subject may include either adults or juveniles (e.g., children). Moreover, subject may mean any living organism, preferably a mammal (e.g., humans and non-humans). Examples of mammals include, but are not limited to, any member of the Mammalian class: humans, non-human primates such as chimpanzees, and other apes and monkey species; farm animals such as cattle, horses, sheep, goats, swine; domestic animals such as rabbits, dogs, and cats; laboratory animals including rodents, such as rats, mice and guinea pigs, and the like. Examples of non-mammals include, but are not limited to, birds, fish, and the like.[0052J Preferred methods and materials are described below, although methods and materials similar or equivalent to those described herein can be used in practice or testing of the present disclosure. All publications, patent applications, patents and other references mentioned herein are incorporated by reference in their entirety. The materials, methods, and examples disclosed herein are illustrative only and not intended to be limiting.Engineered Polypeptides[0053J Disclosed herein are engineered fibroblast growth factor 10 (FGF10) polypeptides. The engineered FGF10 polypeptides of the present disclosure have less than 100% amino acid sequence identity with wild-type FGF10 (SEQ ID NO: 1) and at least one amino acid substitution, deletion, or addition as compared to SEQ ID NO: 1.BIOTN-44681.601

[0054] The engineered FGF10 polypeptides of the present disclosure exhibit various structural and functional enhancements over other FGF10 polypeptides. In some embodiments, the engineered FGF 10 polypeptides of the present disclosure exhibit an increased melting temperature as compared to wild-type FGF10. In some embodiments, the engineered FGF10 polypeptides maintain an activity level following incubation at 37° C for at least 4 days. For example, the disclosed engineered FGF10 polypeptides following incubation at 37° C for at least 4 days have the same, or substantially the same, activity as before the incubation. Activity may be measured as described in the example, e.g., measurement of proliferation of cells expressing cognate receptors for FGF10. In some embodiments, the engineered FGF10 polypeptides exhibit an activity level following incubation at 37° C for at least 4 days higher than wildtype FGF 10.

[0055] In some embodiments, an engineered FGF10 polypeptide of the present disclosure (also referred to herein as an FGF 10 variant, an FGF 10 polypeptide variant, an engineered FGF 10 variant, or an engineered FGF10 polypeptide variant) comprises at least one amino acid substitution, deletion, or addition as compared to SEQ ID NO: 1. SEQ ID NO: 1 corresponds to a portion of the wild-type FGF 10 sequence, the portion following the signal sequence in the mature protein. All amino acid substitutions or mutations recited herein are based on their positions relative to SEQ ID NO: 1.

[0056] In accordance with these embodiments, an amino acid substitution or mutation can be present at one or more of the following amino acid positions of the engineered FGF 10 polypeptide: 42, 43, 49, 54, 62, 66, 78, 81, 88, 145, 148, 150, 160, or any combination thereof, as compared to SEQ ID NO: 1. In some embodiments, an engineered FGF 10 polypeptide of the present disclosure comprises at least two amino acid substitutions at amino acid positions selected from positions 42, 43, 49, 54, 62, 66, 78, 81, 88, 145, 148, 150, and 160.

[0057] In some embodiments, the engineered FGF10 polypeptide comprises at least 2 amino acid substitutions, at least 3 amino acid substitutions, at least 4 amino acid substitutions, at least 5 amino acid substitutions, or at least 6 amino acid substitutions. In some embodiments, the engineered FGF10 polypeptide comprises 4 to 6 amino acid substitutions.[0058| In some embodiments, the engineered FGF10 polypeptide comprises at least one amino acid substitution, including but not limited to, W42I, R43V, T49H, T49N, K54A, K54C, S62F, K66D, S78W, I81P, A88C, G145K, A148K,R150M, R150L, T160P, or any combinations thereof.BIOTN-44681.601[0059J In some embodiments, the engineered FGF10 polypeptide comprises at least one amino acid substitution at amino acid positions 42 and 150. In some embodiments, the engineered FGF10 polypeptide comprises amino acid substitution W42I. In some embodiments, the engineered FGF10 polypeptide comprises amino acid substitution R150M or R150L. In some embodiments, the engineered FGF10 polypeptide comprises amino acid substitution W42I and amino acid substitution R150M or R150L. The engineered polypeptide may comprise one or more amino acid substitutions in addition to W42I and R150M or R150L.

[0060] In some embodiments, the engineered FGF10 polypeptide comprises an amino acid substitution at amino acid position 62. In some embodiments, the engineered FGF10 polypeptide comprises amino acid substitution S62F.

[0061] In some embodiments, the engineered FGF10 polypeptide comprises an amino acid substitution at amino acid position 42, 62, and 150. In some embodiments, the engineered FGF10 polypeptide comprises amino acid substitution W42I, amino acid substitution S62F, and amino acid substitution R150M or R150L. The engineered polypeptide may comprise one or more amino acid substitutions in addition to W42I, S62F, and R150M or R150L.

[0062] In some embodiments, the engineered FGF10 polypeptide further comprises an amino acid substitution at amino acid position 43, 49, 54, 66, 78, 145, 148, 160, or a combination thereof. In some embodiments, the engineered FGF10 polypeptide comprises amino acid substitution R43V; amino acid substitution T49N or T49H; amino acid substitution K54A or K54C; amino acid substitution K66D; amino acid substitution S78W; amino acid substitution G145K; amino acid substitution A148K; amino acid substitution T160P; or a combination thereof. In some embodiments, the engineered FGF10 polypeptide comprises amino acid substitution R43V; amino acid substitution T49H; amino acid substitution K54A; amino acid substitution K66D; amino acid substitution S78W; amino acid substitution G145K; amino acid substitution A148K; amino acid substitution T160P; or a combination thereof.

[0063] In some embodiments, the engineered FGF10 polypeptide further comprises an amino acid substitution at amino acid position 57, 81, 88, or a combination thereof. In some embodiments, the engineered FGF10 polypeptide comprises amino acid substitution K57P; amino acid substitution 18 IP; amino acid substitution A88C; or a combination thereof.|0064] In some embodiments, the engineered FGF10 polypeptide comprises an amino acid substitution at amino acid position 42, 62, and 150, and one or more substitutions at amino acidBIOTN-44681.601positions 43, 49, 54, 66, 78, 145, 148, and 160. In some embodiments, the engineered FGF10 polypeptide comprises amino acid substitution W42I, amino acid substitution S62F, and amino acid substitution R150M orR150L, and one or more of: amino acid substitution R43V; amino acid substitution T49N or T49H; amino acid substitution K54A or K54C; amino acid substitution K66D; amino acid substitution S78W; amino acid substitution G145K; amino acid substitution A148K; and amino acid substitution T160P.

[0065] In some embodiments, the engineered FGF10 polypeptide comprises amino acid substitution W42I, amino acid substitution S62F, and amino acid substitution R150M or R150L, and one or more of: amino acid substitution R43V; amino acid substitution T49H; amino acid substitution K54A; amino acid substitution K66D; amino acid substitution S78W; amino acid substitution G145K; amino acid substitution A148K; amino acid substitution T160P; or a combination thereof. In some embodiments, the engineered FGF10 polypeptide comprises amino acid substitution T49H; amino acid substitution K66D; or a combination thereof.[0066| In some embodiments, the engineered FGF10 polypeptide comprises amino acid substitution W42I, amino acid substitution R43V, amino acid substitution S62F, and amino acid substitution R150M or R150L. In some embodiments, the engineered FGF10 polypeptide comprises amino acid substitution W42I, amino acid substitution T49H or T49N, amino acid substitution S62F, and amino acid substitution R150M or R150L. In some embodiments, the engineered FGF10 polypeptide comprises amino acid substitution W42I, amino acid substitution T49H, amino acid substitution S62F, and amino acid substitution R150M or R150L. In some embodiments, the engineered FGF10 polypeptide comprises amino acid substitution W42I, amino acid substitution K54A or K54C, amino acid substitution S62F, and amino acid substitution R150M or R150L. In some embodiments, the engineered FGF10 polypeptide comprises amino acid substitution W42I, amino acid substitution K54A, amino acid substitution S62F, and amino acid substitution R150M or R150L. In some embodiments, the engineered FGF10 polypeptide comprises amino acid substitution W42I, amino acid substitution S62F, amino acid substitution K66D, and amino acid substitution R150M or R150L. In some embodiments, the engineered FGF 10 polypeptide comprises amino acid substitution W42I, amino acid substitution S62F, amino acid substitution S78W, and amino acid substitution R150M or R150L. In some embodiments, the engineered FGF 10 polypeptide comprises amino acid substitution W42I, amino acid substitution S62F, amino acid substitution G145K, and amino acid substitution R150M or R150L. In someBIOTN-44681.601embodiments, the engineered FGF 10 polypeptide comprises amino acid substitution W42I, amino acid substitution S62F, amino acid substitution A148K, and amino acid substitution R150M or R150L. In some embodiments, the engineered FGF 10 polypeptide comprises amino acid substitution W42I, amino acid substitution S62F, amino acid substitution R150M or R150L, and amino acid substitution T160P.

[0067] In some embodiments, the engineered FGF 10 polypeptide comprises amino acid substitution W42I, amino acid substitution S62F, amino acid substitution R150M; and one or both of amino acid substitutions T49H and K66D. In select embodiments, the engineered FGF10 polypeptide comprises amino acid substitution W42I, amino acid substitution T49H amino acid substitution S62F, amino acid substitution K66D, and amino acid substitution R150M.

[0068] Any of the engineered polypeptides described herein may comprise one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25) additional amino acid substitutions as compared to those mutations specifically contemplated and disclosed herein.

[0069] An amino acid “replacement” or “substitution” refers to the replacement of one amino acid at a given position or residue by another amino acid at the same position or residue within a polypeptide sequence. Amino acids are broadly grouped as “aromatic” or “aliphatic.” An aromatic amino acid includes an aromatic ring. Examples of “aromatic” amino acids include histidine (H or His), phenylalanine (F or Phe), tyrosine (Y or Tyr), and tryptophan (W or Trp). Non- aromatic amino acids are broadly grouped as “aliphatic.” Examples of “aliphatic” amino acids include glycine (G or Gly), alanine (A or Ala), valine (V or Vai), leucine (L or Leu), isoleucine (I or He ), methionine (M or Met), serine (S or Ser), threonine (T or Thr), cysteine (C or Cys), proline (P or Pro), glutamic acid (E or Glu), aspartic acid (D or Asp), asparagine (N or Asn), glutamine (Q or Gin), lysine (K or Lys), and arginine (R or Arg).

[0070] The amino acid replacement or substitution can be conservative, semi-conservative, or non-conservative. The phrase “conservative amino acid substitution” or “conservative mutation” refers to the replacement of one amino acid by another amino acid with a common property. A functional way to define common properties between individual amino acids is to analyze the normalized frequencies of amino acid changes between corresponding proteins of homologous organisms (Schulz and Schirmer, Principles of Protein Structure, Springer-Verlag, New York (1979)). According to such analyses, groups of amino acids may be defined where amino acids within a group exchange preferentially with each other, and therefore resemble each other most inBIOTN-44681.601their impact on the overall protein structure (Schulz and Schirmer, supra). Examples of conservative amino acid substitutions include substitutions of amino acids within the sub-groups described above, for example, lysine for arginine and vice versa such that a positive charge may be maintained, glutamic acid for aspartic acid and vice versa such that a negative charge may be maintained, serine for threonine such that a free -OH can be maintained, and glutamine for asparagine such that a free -NH2 can be maintained. “Semi-conservative mutations” include amino acid substitutions of amino acids within the same groups listed above, but not within the same subgroup. For example, the substitution of aspartic acid for asparagine, or asparagine for lysine, involves amino acids within the same group, but different sub-groups. “Non-conservative mutations” involve amino acid substitutions between different groups, for example, lysine for tryptophan, or phenylalanine for serine, etc.

[0071] In some embodiments, the engineered FGF10 polypeptide further comprises a localization or signal sequence, a sequence tag (e.g., a tag for detection, purification, and / or monitoring expression), a protein transduction domain sequence, or a combination thereof. Sequence tags may include, but are not limited to: 3xFLAG tag, an HA tag, a Myc tag, a polyhistidine tag, a SNAP-tag, a CLIP -tag, and the like. The tags may be at the N-terminus, a C-terminus, or embedded in the sequence of the engineered FGF10 polypeptide.

[0072] In some embodiments, the engineered FGF10 polypeptide comprises an amino acid sequence having at least 70% identity (e.g., at least 80% identity, at least 90% identity, at least 95% identity, at least 98% identity) to one of the sequences listed in the table below. In some embodiments, the engineered FGF10 polypeptide comprises an amino acid sequence having at least 70% identity (e.g., at least 80% identity, at least 90% identity, at least 95% identity, at least 98% identity) to one of SEQ ID NOs: 5, 8-16, and 33-61. In some embodiments, the engineered FGF10 polypeptide comprises an amino acid sequence of any of SEQ ID NOs: 5, 8-16, and 33-61.Nucleic Acids

[0073] Also disclosed herein are nucleic acids encoding the engineered FGF10 polypeptides as described herein. The nucleic acids may be DNA, RNA, or combinations thereof. In some embodiments, the nucleic acids comprise one or more vectors.[0074| In certain embodiments, the nucleic acids are engineered for codon-optimization. It will be appreciated altering codons to those most frequently used in the cells or subject of interest allows for maximum expression. Such modified nucleic acid sequences are commonly describedBIOTN-44681.601in the art as “codon-optimized.” Tn some embodiments, the nucleic acid sequence is considered codon-optimized if at least about 60% (e.g., about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, or about 98%) of the codons encoded therein are preferred codons to the subject of interest.

[0075] The present disclosure also provides for DNA segments encoding the engineered polypeptides disclosed herein, vectors containing these segments, and cells containing the vectors. The vectors may be used to propagate the DNA segment in an appropriate cell and / or to allow expression from the segment (e.g., an expression vector). The person of ordinary skill in the art would be aware of the various vectors available for propagation and expression of a nucleic acid sequence.

[0076] The present disclosure further provides engineered, non-naturally occurring vectors and vector systems, which can encode the engineered polypeptides as disclosed herein. The vector(s) can be introduced into a cell that is capable of expressing the polypeptide encoded thereby, including any suitable prokaryotic or eukaryotic cell.

[0077] Viral and non-viral based gene transfer methods can be used to introduce nucleic acids encoding components of the present system into cells. Such methods can be used to administer nucleic acids encoding components of the present system to cells in culture. Non-viral vector delivery systems include DNA plasmids, cosmids, RNA (e.g., a transcript of a vector described herein), nucleic acids, and nucleic acids complexed with a delivery vehicle. Viral vector delivery systems include DNA and RNA viruses, which have either episomal or integrated genomes after delivery to the cell. Viral vectors include, for example, retroviral, lentiviral, adenoviral, adeno-associated and herpes simplex viral vectors.[0078[ In certain embodiments, plasmids that are non-replicative, or plasmids that can be cured by high temperature may be used, such that any or all of the synthetic polypeptides or protein conjugates or fusion proteins thereof may be removed from the cells under certain conditions. For example, this may allow for DNA integration by transforming bacteria of interest, but then being left with engineered strains that have no memory of the plasmids or vectors used for the integration.[0079| Drug selection strategies may be adopted by positively selecting for cells that underwent DNA integration. A donor nucleic acid may contain one or more drug-selectable markers within the cargo. Then presuming that the original donor plasmid is removed, drugBIOTN-44681.601selection may be used to enrich for integrated clones. Colony screenings may be used to isolate clonal events.

[0080] A variety of viral constructs may be used to deliver the engineered polypeptides or compositions thereof to the targeted cells. Nonlimiting examples of such recombinant viruses include recombinant adeno-associated virus (AAV), recombinant adenoviruses, recombinant lentiviruses, recombinant retroviruses, recombinant herpes simplex viruses, recombinant poxviruses, phages, etc. The present disclosure provides vectors capable of integration in the host genome, such as retrovirus or lentivirus. See, e.g., Ausubel et al., Current Protocols in Molecular Biology, John Wiley & Sons, New York, 1989; Kay, M. A., et al., 2001 Nat. Medic. 7(l):33-40; and Walther W. and Stein U., 2000 Drugs, 60(2): 249-71, incorporated herein by reference.

[0081] In one embodiment, a DNA segment encoding an engineered polypeptide is contained in a plasmid vector that allows expression of the protein(s) and subsequent isolation and purification produced by the recombinant vector. Accordingly, the proteins disclosed herein can be purified following expression, obtained by chemical synthesis, or obtained by recombinant methods.

[0082] To construct cells that express an engineered polypeptide, expression vectors for stable or transient expression may be constructed via conventional methods as described herein and introduced into cells. For example, nucleic acids encoding engineered polypeptides may be cloned into a suitable expression vector, such as a plasmid or a viral vector in operable linkage to a suitable promoter. The selection of expression vectors / plasmids / viral vectors should be suitable for integration and replication in eukaryotic cells.

[0083] In certain embodiments, vectors of the present disclosure can drive the expression of one or more sequences in prokaryotic cells. Promoters that may be used include T7 RNA polymerase promoters, constitutive E. coli promoters, and promoters that could be broadly recognized by transcriptional machinery in a wide range of bacterial organisms.

[0084] In certain embodiments, vectors of the present disclosure can drive the expression of one or more sequences in mammalian cells using a mammalian expression vector. Examples of mammalian expression vectors include pCDM8 (Seed, Nature (1987) 329:840, incorporated herein by reference) and pMT2PC (Kaufman, et al., EMBO J. (1987) 6:187, incorporated herein by reference). When used in mammalian cells, the expression vector's control functions are typically provided by one or more regulatory elements. For example, commonly used promoters are derivedBIOTN-44681.601from polyoma, adenovirus 2, cytomegalovirus, simian virus 40, and others disclosed herein and known in the art. For other suitable expression systems for both prokaryotic and eukaryotic cells see, e.g., Chapters 16 and 17 of Sambrook, et al., MOLECULAR CLONING: A LABORATORY MANUAL. 2nd eds., Cold Spring Harbor Laboratory, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1989, incorporated herein by reference.

[0085] Vectors of the present disclosure can comprise any of a number of promoters known to the art, wherein the promoter is constitutive, regulatable or inducible, cell type specific, tissuespecific, or species specific. In addition to the sequence sufficient to direct transcription, a promoter sequence of the invention can also include sequences of other regulatory elements that are involved in modulating transcription (e.g., enhancers, Kozak sequences and introns). Many promoter / regulatory sequences useful for driving constitutive expression of a gene are available in the art and include, but are not limited to, for example, CMV (cytomegalovirus promoter), EFla (human elongation factor 1 alpha promoter), SV40 (simian vacuolating virus 40 promoter), PGK (mammalian phosphoglycerate kinase promoter), Ubc (human ubiquitin C promoter), human betaactin promoter, rodent beta-actin promoter, CBh (chicken beta-actin promoter), CAG (hybrid promoter contains CMV enhancer, chicken beta actin promoter, and rabbit beta-globin splice acceptor), TRE (Tetracycline response element promoter), Hl (human polymerase III RNA promoter), U6 (human U6 small nuclear promoter), and the like. Additional promoters that can be used for expression of the components of the present system, include, without limitation, cytomegalovirus (CMV) intermediate early promoter, a viral LTR such as the Rous sarcoma virus LTR, HIV-LTR, HTLV-1 LTR, Maloney murine leukemia virus (MMLV) LTR, myeoloproliferative sarcoma virus (MPSV) LTR, spleen focus-forming virus (SFFV) LTR, the simian virus 40 (SV40) early promoter, herpes simplex tk virus promoter, elongation factor 1-alpha (EFl -a) promoter with or without the EFl -a intron. Additional promoters include any constitutively active promoter. Alternatively, any regulatable promoter may be used, such that its expression can be modulated within a cell.[0086| Moreover, inducible and tissue specific expression can be accomplished by placing the nucleic acid encoding such a molecule under the control of an inducible or tissue specific promoter / regulatory sequence. Examples of tissue specific or inducible promoter / regulatory sequences which are useful for this purpose include, but are not limited to, the rhodopsin promoter, the MMTV LTR inducible promoter, the SV40 late enhancer / promoter, synapsin 1 promoter, ETBIOTN-44681.601hepatocyte promoter, GS glutamine synthase promoter and many others. In addition, promoters which are well known in the art can be induced in response to inducing agents such as metals, glucocorticoids, tetracycline, hormones, and the like, are also contemplated for use with the invention. Thus, it will be appreciated that the present disclosure includes the use of any promoter / regulatory sequence capable of driving expression of the desired protein operably linked thereto.

[0087] The vectors of the present disclosure may direct expression of the nucleic acid in a particular cell type (e.g., tissue-specific regulatory elements are used to express the nucleic acid). Such regulatory elements include promoters that may be tissue specific or cell specific. The term “tissue specific” as it applies to a promoter refers to a promoter that is capable of directing selective expression of a nucleotide sequence of interest to a specific type of tissue (e.g., seeds) in the relative absence of expression of the same nucleotide sequence of interest in a different type of tissue. The term “cell type specific” as applied to a promoter refers to a promoter that is capable of directing selective expression of a nucleotide sequence of interest in a specific type of cell in the relative absence of expression of the same nucleotide sequence of interest in a different type of cell within the same tissue. The term “cell type specific” when applied to a promoter also means a promoter capable of promoting selective expression of a nucleotide sequence of interest in a region within a single tissue. Cell type specificity of a promoter may be assessed using methods well known in the art, e.g., immunohistochemical staining.

[0088] Additionally, the vector may contain, for example, some or all of the following: a selectable marker gene, such as the neomycin gene for selection of stable or transient transfectants in host cells; enhancer / promoter sequences from the immediate early gene of human CMV for high levels of transcription; transcription termination and RNA processing signals from SV40 for mRNA stability; 5 ’-and 3 ’-untranslated regions for mRNA stability and translation efficiency from highly-expressed genes like a-globin or -globin; SV40 polyoma origins of replication and ColEl for proper episomal replication; internal ribosome binding sites (IRESes), versatile multiple cloning sites; T7 and SP6 RNA promoters for in vitro transcription of sense and antisense RNA; a “suicide switch” or “suicide gene” which when triggered causes cells carrying the vector to die (e.g., HSV thymidine kinase, an inducible caspase such as iCasp9), and reporter gene for assessing expression of the chimeric receptor. Suitable vectors and methods for producing vectors containing transgenes are well known and available in the art. Selectable markers also includeBIOTN-44681.601chloramphenicol resistance, tetracycline resistance, spectinomycin resistance, streptomycin resistance, erythromycin resistance, rifampicin resistance, bleomycin resistance, thermally adapted kanamycin resistance, gentamycin resistance, hygromycin resistance, trimethoprim resistance, dihydrofolate reductase (DHFR), GPT; the URA3, HIS4, LEU2, and TRP1 genes of S. cerevisiae.[0089| When introduced into the cell, the vectors may be maintained as an autonomously replicating sequence or extrachromosomal element or may be integrated into host DNA.

[6090] Vectors according to the present disclosure can be transformed, transfected, or otherwise introduced into a wide variety of cells. Transfection refers to the taking up of a vector by a cell whether or not any coding sequences are in fact expressed. Numerous methods of transfection are known to the ordinarily skilled artisan, for example, lipofectamine, calcium phosphate coprecipitation, electroporation, DEAE-dextran treatment, microinjection, viral infection, and other methods known in the art. Transduction refers to entry of a virus into the cell and expression (e.g., transcription and / or translation) of sequences delivered by the viral vector genome. In the case of a recombinant vector, “transduction” generally refers to entry of the recombinant viral vector into the cell and expression of a nucleic acid of interest delivered by the vector genome.

[0091] Any of the vectors comprising a nucleic acid sequence that encodes an engineered polypeptide is also within the scope of the present disclosure. Such a vector may be delivered into host cells by a suitable method. Methods of delivering vectors to cells are well known in the art and may include DNA or RNA electroporation, transfection reagents such as liposomes or nanoparticles to delivery DNA or RNA; delivery of DNA, RNA, or protein by mechanical deformation (see, e.g., Sharei et al. Proc. Natl. Acad. Sci. USA 110(6): 2082-2087(2013) incorporated herein by reference); or viral transduction. Nucleic acids can be delivered as part of a larger construct, such as a plasmid or viral vector, or directly, e.g., by electroporation, lipid vesicles, viral transporters, microinjection, and biolistics (high-speed particle bombardment). Similarly, the construct containing the one or more transgenes can be delivered by any method appropriate for introducing nucleic acids into a cell. In some embodiments, the construct or the nucleic acid encoding the engineered polypeptide is a DNA molecule. In some embodiments, the nucleic acid encoding the engineered polypeptide is a DNA vector and may be electroporated to cells. In some embodiments, the nucleic acid encoding the engineered polypeptide is an RNA molecule, which may be electroporated to cells.BIOTN-44681.601

[0092] Additionally, delivery vehicles such as nanoparticle- and lipid-based mRNA or protein delivery systems can be used. Further examples of delivery vehicles include lentiviral vectors, ribonucleoprotein (RNP) complexes, lipid-based delivery system, gene gun, hydrodynamic, electroporation or nucleofection microinjection, and biolistics. Various gene delivery methods are discussed in detail by Nayerossadat et al. (Adv Biomed Res. 2012; 1 : 27) and Ibraheem et al. (Int J Pharm. 2014 Jan 1 ;459(1 -2):70-83), incorporated herein by reference.

[0093] Accordingly, also disclosed herein are cells configured to express an engineered FGF10 polypeptide. In some embodiments, the cell comprises a nucleic acid encoding the engineered FGF10 polypeptide.Compositions[0094| Also disclosed herein are compositions comprising an engineered FGF10 polypeptide as described herein or a nucleic acid molecule comprising a sequence encoding the engineered FGF10 polypeptide.[0095J In some embodiments, the composition is a cell culture medium. Cell culture medium refers to any media for culturing cells containing nutrients that maintain cell viability and support proliferation. The cell culture medium may contain any of the following in an appropriate combination: salt(s), buffer(s), amino acids, glucose or other sugar(s), antibiotics, serum or serum replacement, and other components such as peptide growth factors, etc.[0096| The compositions may further comprise excipients or pharmaceutically acceptable carriers. The choice of excipients or pharmaceutically acceptable carriers will depend on factors including, but not limited to, the particular mode of administration, the effect of the excipient on solubility and stability, and the nature of the dosage form.

[0097] Excipients and carriers may include any and all solvents, dispersion media, antibacterial and antifungal agents, isotonic and absorption delaying agents. Some examples of materials which can serve as excipients and / or carriers are sugars including, but not limited to, lactose, glucose and sucrose; starches including, but not limited to, com starch and potato starch; cellulose and its derivatives including, but not limited to, sodium carboxymethyl cellulose, ethyl cellulose and cellulose acetate; powdered tragacanth; malt; gelatin; talc; excipients including, but not limited to, cocoa butter and suppository waxes; oils including, but not limited to, peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, com oil and soybean oil; glycols; including propylene glycol; esters including, but not limited to, ethyl oleate and ethyl laurate; agar; buffering agents including,BIOTN-44681.601but not limited to, magnesium hydroxide and aluminum hydroxide; alginic acid; pyrogen-free water; isotonic saline; Ringer's solution; ethyl alcohol, and phosphate buffer solutions, as well as other non-toxic compatible lubricants including, but not limited to, sodium lauryl sulfate and magnesium stearate, as well as coloring agents, releasing agents, preservatives, and antioxidants. The compositions of the present invention and methods for their preparation will be readily apparent to those skilled in the art. Techniques and formulations may be found, for example, in Remington's Pharmaceutical Sciences, 19th Edition (Mack Publishing Company, 1995).

[0098] The compositions may also comprise buffers (e.g., neutral buffered saline or phosphate buffered saline), carbohydrates (e.g., glucose, mannose, sucrose or dextrans), mannitol, antioxidants, bacteriostats, chelating agents such as EDTA or glutathione, solutes that render the formulation isotonic, hypotonic, or weakly hypertonic with the blood of a recipient, suspending agents, thickening agents and / or preservatives, commonly found in proteinaceous compositions.

[0099] The disclosed engineered polypeptide may be entrapped in microcapsules (for example, liposome, albumin microspheres, microemulsions, nanoparticles and nanocapsules), in macroemulsions, or in sustained-release preparation. The disclosed compositions or components thereof may be in a liposome and combined with amphipathic agents such as lipids which exist in aggregated form as micelles, insoluble monolayers, liquid crystals, or lamellar layers in aqueous solution. Suitable lipids for liposomal formulations include, without limitation, monoglycerides, diglycerides, sulfatides, lysolecithin, phospholipids, saponin, and bile acids. Preparation of such liposomal formulations is within the level of skill in the art.

[0100] The disclosed compositions or components thereof may be individually or as a group prepared in a hydrogel. The term “hydrogel” herein refers to a specific type of gel in which water-swellable polymeric matrices that can absorb a substantial amount of water in a three-dimensional network of macromolecules held together by covalent or noncovalent crosslinks.Methods10101] The disclosure also provides methods for promoting cell proliferation, cell differentiation, cell survival, tissue repair and tissue regeneration in vitro or ex vivo. The methods described herein can be used to support stable organoid or spheroid growth e.g., (lung, liver, pancreas, etc.) for reliable disease modeling and high-throughput screening applications, enhance cell proliferation and survival, such as for use with tissue repair, regenerative therapies, and clinical-scale protocols, and maintain protein activity throughout extended cultivation in large-BIOTN-44681.601scale biomanufacturing, thereby streamlining production processes and reducing costs for cellbased manufacturing.

[0102] “ Differentiation” refers to a process by which cells differentiate from one cell type (e.g., a multipotent, totipotent, or pluripotent differentiable cell) to another cell type such as a target differentiated cell. Oftentimes, as a result of differentiation, the potency or proliferation of a cell is decrease or the cell is moved to a more developmentally restricted state. “Proliferation” refers to increasing cell division, either symmetric or asymmetric division of cells. “Expansion” refers to increasing the number of cells as compared to the number originally present, for example, as the outcome of cell division and cell death. “Stimulation” or “activation” refers to inducing a change in the biologic state of a cell which can result in expression of activation markers, production of cytokines, increases in autophagy, proliferation, and / or cytotoxicity to target cells.

[0103] The methods comprise contacting a cell, a precursor or progenitor cell, a tissue, a spheroid, or an organoid with an engineered FGF10 polypeptide or a composition comprising thereof, as described herein. The cell, precursor or progenitor cell, tissue, or cells in the tissue, spheroid, or organoid may be genetically modified.

[0104] In some embodiments, the methods comprise adding the engineered FGF10 polypeptide, or composition comprising thereof, to a buffer or cell culture medium containing the cell, precursor or progenitor cell, tissue, spheroid, or organoid. Alternatively, or in addition, in some embodiments, the methods comprise adding a buffer or cell culture medium comprising the engineered FGF10 polypeptide to a population of cells, a population of precursor or progenitor cells, a tissue, a spheroid or an organoid.

[0105] A vessel used for the contacting and subsequent culturing can include, but is particularly not limited to: flask, flask for tissue culture, dish, petri dish, dish for tissue culture, multi dish, micro plate, micro-well plate, multi plate, multi-well plate, micro slide, chamber slide, tube, tray, CellSTACK® Chambers, G-Rex® culture vessels, culture bag, and roller bottle, as long as it is capable of culturing the cells therein. The cells may be cultured in any volume (e.g., at least or about 0.2, 0.5, 1, 2, 5, 10, 20, 30, 40, 50 ml, 100 ml, 150 ml, 200 ml, 250 ml, 300 ml, 350 ml, 400 ml, 450 ml, 500 ml, 550 ml, 600 ml, 800 ml, 1000 ml, 1500 ml, or any range derivable therein) depending on the needs of the culture. The vessel may be a bioreactor, which may refer to any device or system that supports a biologically active environment. The vessel may be part of an open or closed system. The length of time in culture, as well as other components of the cell cultureBIOTN-44681.601medium and cell culture conditions (e.g., temperature, CO2) are those which promote cell growth and allow for the desired degree of differentiation, stimulation, and / or expansion.

[0106] Precursor or progenitor cells are less differentiated cells or undifferentiated cells that have the potential to differentiate into the cell type of interest. Depending on cell differentiation, precursor cells could be multipotent, pluripotent, and totipotent, whereas progenitor cells can be unipotent or oligopotent. In some embodiments, the cell is pancreatic progenitor cell.

[0107] Precursor or progenitor cells include stem cells (e.g., embryonic stem cells (ESCs), adult stem cells (ASCs), induced pluripotent stem cells (iPSCs), cells which are more differentiated and specified as compared to stem cells (e.g., descendants of stem cells). In certain embodiments, the stem or progenitor cells may be selected from embryonic stem cells, hematopoietic stem or progenitor cells, cells isolated from bone marrow, cord blood, peripheral blood, thymus, or the progenitor cells may have been differentiated from embryonic stem cells (ESC) or induced pluripotent stem cells (iPSC) in vitro. Stem or progenitor cells herein may be, but are not limited to, ESCs, induced pluripotent stem cells or tissue stem cells (also called tissue-specific stem cell, or somatic stem cell). Stem or progenitor cells from primary tissue or ESC or iPSC may be from human or non-human animals (e.g., mouse) in origin.

[0108] Embryonic stem (ES) cells are pluripotent stem cells derived from early embryos. Tissue stem cells are present at particular locations in tissues and have an undifferentiated intracellular structure. Therefore, the pluripotency of tissue stem cells is typically low. Tissue stem cells are separated into categories, based on the sites from which the cells are derived, such as the dermal system, the digestive system, the bone marrow system, the nervous system, and the like. Tissue stem cells in the bone marrow system include hematopoietic stem cells, mesenchymal stem cells, and the like.

[0109] Hematopoietic stem or progenitor cells are cells that are committed to a hematopoietic lineage but are capable of further hematopoietic differentiation and include hematopoietic stem cells, multipotential hematopoietic stem cells (hematoblasts), myeloid progenitors, megakaryocyte progenitors, erythrocyte progenitors, and lymphoid progenitors. Hematopoietic stem cells (HSCs) are multipotent stem cells that give rise to all the blood cell types including myeloid (monocytes and macrophages, neutrophils, basophils, eosinophils, erythrocytes, megakaryocytes / platelets, dendritic cells), and lymphoid lineages (T-cells, B-cells, NK-cells).BIOTN-44681.601

[0110] Induced pluripotent stem cells (iPS cells or iPSCs) are pluripotent stem cells artificially prepared from a non-pluripotent cell, typically an adult somatic cell, or terminally differentiated cell, such as fibroblast, a hematopoietic cell, a myocyte, a neuron, an epidermal cell, or the like, by introducing certain factors, referred to as reprogramming factors.

[0111] The term “organoid” refers to a three-dimensional organ grown in vitro and in isolation from an intact organism. An organoid contains the variety of cell types found in the corresponding organ and closely recapitulates many aspects of the structure, organization, and functions of the corresponding in vivo tissue. Organoids are derived from either pluripotent or tissue-resident stem (embryonic or adult) or progenitor or differentiated cells from healthy or diseased tissues, such as tumors. Organoids made from a tumor cells have histologic, genomic, molecular, and other features that are similar to those found in the original tumor. In some embodiments, the organoid is a lung, stomach, pancreas, heart, bladder, dental epithelium, salivary gland, prostate, or liver organoid.[0U2| Spheroids, like organoids, are culture models with a three-dimensional conformation. However, spheroids are spherical cellular units that are generally cultured as free-floating aggregates and are generally of lower complexity in mirroring tissue or organ organization as compared to an organoid. Spheroids can be derived from primary cells, cell lines, multicellular mixtures, tissues, and tumor cells. Generally, spheroids contain a representative sampling of the tissue’s cell types, usually without selection or curation for specific cells. In some embodiments, the spheroid is a lung, stomach, pancreas, heart, bladder, dental epithelium, salivary gland, prostate, or liver organoid.

[0113] The methods described herein can be used for disease modeling. Accordingly, the cell, precursor or progenitor cell, tissue, spheroid, or organoid may be a diseased cell, a diseased precursor or progenitor cell, a diseased tissue, a diseased spheroid, or a diseased organoid or may be derived from or include cells derived from a diseased tissue, a subject having or suspected of having a disease or disorder, or genetically engineered to have features similar to those found with the disease. In some embodiments, the cell, precursor or progenitor cell, tissue, spheroid, or organoid is a cancer cell, a cancer precursor or progenitor cell, a cancerous tissue, a cancer spheroid, or a cancer organoid.BIOTN-44681.601Kits

[0114] Also within the scope of the present disclosure are kits that include the engineered FGF10 polypeptides and compositions thereof as disclosed herein.

[0115] For example, the kits may contain one or more reagents or components useful, necessary, or sufficient for practicing any of the methods described herein, such as, administration reagents, cell culture media or components thereof, negative and positive control samples (e.g., cells, template DNA), cells, containers (e.g., cell culture vessels), detection and analysis instruments, software, instructions, and the like.

[0116] The kit may include instructions for use in any of the methods described herein. The instructions can comprise a description of methods for cell proliferation, cell differentiation, cell survival, tissue repair and tissue regeneration. The instructions generally include information as to effective quantity of the engineered polypeptides and compositions disclosed herein and conditions for contacting the cells, tissues, spheroids and organoids with the disclosed engineered polypeptides and compositions. Instructions supplied in the kits of the disclosure are typically written instructions on a label or package insert.[01 J 7] The kits provided herein are in suitable packaging. Suitable packaging includes, but is not limited to, vials, bottles, jars, flexible packaging, and the like. The packaging may be singleuse or multi-use packages. As described above, kits optionally may provide additional components such as buffers and interpretive information. Normally, the kit comprises a container and a label or package insert(s) on or associated with the container. In some embodiments, the disclosure provides articles of manufacture comprising contents of the kits described above.

[0118] The kit will typically be provided with its various components in one or more packages, e.g., a fiber-based, a cardboard, polymeric, or a Styrofoam box. The enclosure(s) can be configured so as to maintain a temperature differential between the interior and the exterior, for example, to provide insulating properties to keep the reagents at a preselected temperature for a preselected time. The packaging can be air-tight, waterproof (e.g., impermeable to changes in moisture or evaporation), and / or light-tight.Sequences|0119] The various polypeptides referenced in the present disclosure include the following.BIOTN-44681.601<<<<BIOTN-44681.601BIOTN-44681.601BIOTN-44681.601BIOTN-44681.601BIOTN-44681.601Examples

[0120] The following are examples of the present invention and are not to be construed as limiting.Example 1

[0121] To identify the FGF10 variants of the present disclosure, amino acid mutations in the context of wild-type FGF10 were identified using multiple different Al models, such as protein stability machine learning models, and iteratively combined to find variants with high melting temperature. After reach round of engineering melting temperatures were determined and activity for select variants was evaluated in a bioassay.

[0122] Melting temperatures were determined using a thermal ramp while monitoring SYPRO orange dye fluorescence or intrinsic fluorescence. For measurements with SYPRO orange dye, as the temperature is increased the dye interacts with the hydrophobic core and increases in fluorescence. For the intrinsic fluorescence measurements, the fluorescence increases as the aromatic amino acids are exposed during unfolding. The melting temperature, in both assays, was defined as the temperature at half max fluorescence.

[0123] Select FGF10 variants were also evaluated in a bioassay for activity. The bioassay measured the proliferation of BA / F3 cells expressing FGFR2b(IIIB) following incubation in the presence of wild-type FGF10 or the FGF variants. Initial activity was measured as well as activity following incubation of the wild-type FGF10 or the FGF variants in growth media at 1 ug / ml at 37° C for 4 or 8 days. EC50 values were determined as the concentration that resulted in 50% max cell count. Lower EC50 value indicates less protein needed for the same response and therefore stronger activity.[0124| FGF 10 round 1 variants each included three mutations from a random selection of seven mutations predicted to affect thermal stability. The contribution of the mutations to melting temperature was inferred from averaging values for each individual mutation. FGF 10 round 2 variants combined three high performing mutations while also adding other mutations from round 1 variants one at a time. FGF 10 round 3 variants probed other mutations potentially implicated in thermal stability which could increase melting temperature. FGF 10 round 4 combined mutations from round 3.

[0125] As shown in FIG. 1, the first four rounds of engineering resulted in variable results in terms of melting temperature, with round four resulting in the most consistently high-performingBIOTN-44681.601variants, variants with increased melting temperatures of more than 5° C compared to a corresponding wild-type FGF10. Thermal stability paralleled increases in melting temperature, except in the case of FGF10 V3-15, which showed a much-reduced activity level not consistent with a melting temperature similar to that of wild-type FGF10 (FIG. 2). Wild-type FGF10 lost 3-fold activity over the course of the 4-day incubation.

[0126] Variants from the fourth round of engineering yielded FGF10 polypeptides with improved stability over wild-type FGF10, though a minimal loss of activity was observed after 4 days (FIG. 3). Select FGF10 round 4 variant, V4-7, had a 1.17-fold decrease in activity when stored in cell culture media at 37° C for 4 days as compared to 2.65-fold decrease in activity for wild-type FGF10.

[0127] FGF10 round 5 expanded on round 4 variant V4-7 with more predicted mutations. Generally, the variants from the fifth round had at least a 5° C increase in melting temperature (FIG. 4). When evaluated in the proliferation bioassay, the polypeptides from the round 5 had improved activity over round 4 showing little to no change in activity after 4 days (FIG. 5A) and 8 days (FIG. 5B). FGF10 V5-7 maintained the greatest activity in cell culture media at 37° C for 4 and 8 days, while the wild-type protein decreased 2- and 3 -fold over the same time periods, respectively. Two high performing variants from the fifth round were used to generate a subsequent combination polypeptide (V6-1) with an even further increased melting temperature (FIG. 6). These high performing variants show similar bioactivity and FGFR2b binding as compared to WT FGF10 (FIGS. 7 and 8) but preserved bioactivity over long durations and periods of heat stress (FIGS. 9 and 10).Example 2[0128| FGF10 V5-7 was used in organoid culture. Adult lung stem cells were embedded at a density of 2 * 104cells per 20 pL dome in either Cultrex UltiMatrix or Cultrex Synthetic Hydrogel within 24-well plates. Organoids were cultured in 500 pL of Advanced DMEM / F12 media with 100 ng / mL of either WT FGF10 or FGF10 V5-7 (rhFGF-10 v5-7), IX N21-MAX Supplement, 2 mM GlutaMAX, lOmMHEPES, IX Penicillin / Streptomycin, 5 mM Nicotinamide, 0.5 pMA 83-01, 0.5 pM SB 2020190, 1.25 mM N-Acetylcysteine, 100 ng / mL rhNoggin, 25 ng / mL FGF-7, and 0.5 pg / mL rhR-Spondin 1. Organoid growth was monitored using an Incucyte SX5 system, with images acquired every 12 hours and subsequently processed by MATLAB-based image segmentation and quantification.BIOTN-44681.601[0129J FGF10 V5-7 yielded larger adult stem cell-derived lung organoids than WT FGF10, while retaining expected morphology and expression of lung organoid markers (FIGS. 10-13).[0130j The scope of the present invention is not limited by what has been specifically shown and described hereinabove. Those skilled in the art will recognize that there are suitable alternatives to the depicted examples of materials, configurations, constructions, and dimensions. Variations, modifications, and other implementations of what is described herein will occur to those of ordinary skill in the art without departing from the spirit and scope of the invention.[0131| Numerous references, including patents and various publications, are cited and discussed in the description of this invention. The citation and discussion of such references is provided merely to clarify the description of the present invention and is not an admission that any reference is prior art to the invention described herein. All references cited and discussed in this specification are incorporated herein by reference in their entirety.

Claims

BIOTN-44681.601CLAIMSWhat is claimed is:

1. An engineered FGF10 polypeptide comprising less than 100% amino acid sequence identity with SEQ ID NO: 1 and at least one amino acid substitution, deletion, or addition compared to SEQ ID NO: 1 or a fragment thereof,wherein the engineered FGF10 polypeptide has increased stability as compared to wildtype FGF-10.

2. The engineered FGF10 polypeptide of claim 1, wherein the at least one amino acid substitution is at amino acid position 42, 43, 49, 54, 62, 66, 78, 81, 88, 145, 148, 150, 160, or any combination thereof.

3. The engineered FGF10 polypeptide of claim 1 or 2, wherein the engineered FGF10 polypeptide comprises at least two amino acid substitutions at amino acid positions selected from positions 42, 43, 49, 54, 62, 66, 78, 81, 88, 145, 148, 150, 160, or any combination thereof.

4. The engineered FGF10 polypeptide of any one of claims 1-3, wherein the engineered FGF10 polypeptide comprises at least 2 amino acid substitutions, at least 3 amino acid substitutions, at least 4 amino acid substitutions, at least 5 amino acid substitutions, or at least 6 amino acid substitutions.

5. The engineered FGF10 polypeptide of any one of claims 1-4, wherein the engineered FGF10 polypeptide comprises 4 to 6 amino acid substitutions.

6. The engineered FGF10 polypeptide of any one of claims 1-5, wherein the at least one amino acid substitution comprises W42I, R43V, T49H, T49N, K54A, K54C, S62F, K66D, S78W, I81P, A88C, G145K, A148K, R150M, R150L, T160P, or any combinations thereof.

7. The engineered FGF10 polypeptide of any one of claims 1-6, wherein the engineered FGF10 polypeptide comprises an amino acid substitution at amino acid positions 42 and 150.

8. The engineered FGF10 polypeptide of any one of claims 1-7, wherein the engineered FGF10 polypeptide comprises amino acid substitution W42I; and amino acid substitution R150M orR150L.BIOTN-44681.6019. The engineered FGF10 polypeptide of any one of claims 1-8, wherein the engineered FGF10 polypeptide comprises an amino acid substitution at amino acid position 62.

10. The engineered FGF10 polypeptide of any one of claims 1-9, wherein the engineered FGF10 polypeptide comprises amino acid substitution S62F.

11. The engineered FGF10 polypeptide of any one of claims 7-10, wherein the engineered FGF10 polypeptide further comprises an amino acid substitution at amino acid position 43, 49, 54, 66, 78, 145, 148, 160, or a combination thereof.

12. The engineered FGF10 polypeptide of claim 11, wherein the engineered FGF10 polypeptide comprises amino acid substitution R43V; amino acid substitution T49N or T49H; amino acid substitution K54A or K54C; amino acid substitution K66D; amino acid substitution S78W; amino acid substitution G145K; amino acid substitution A148K; amino acid substitution T160P; or a combination thereof.

13. The engineered FGF10 polypeptide of claim 11 or 12, wherein the engineered FGF10 polypeptide comprises amino acid substitution R43V; amino acid substitution T49H; amino acid substitution K54A; amino acid substitution K66D; amino acid substitution S78W; amino acid substitution G145K; amino acid substitution A148K; amino acid substitution T160P; or a combination thereof.

14. The engineered FGF10 polypeptide of any of claims 11-13, wherein the engineered FGF10 polypeptide comprises amino acid substitution T49H; amino acid substitution K66D; or a combination thereof.

15. The engineered FGF10 polypeptide of any of claims 1-14, wherein the engineered FGF10 polypeptide comprises amino acid substitution W42I; amino acid substitution S62F; amino acid substitution R150M; and one or both of amino acid substitutions T49H and K66D.

16. The engineered FGF10 polypeptide of any one of claims 1-15, wherein the engineered FGF10 polypeptide comprises at least 70% amino acid sequence identity to any one of SEQ ID NOs: 5, 8-16, and 33-61, or a fragment thereof.BIOTN-44681.60117. The engineered FGF10 polypeptide of any one of claims 1-16, wherein the engineered FGF10 polypeptide comprises at least 80% amino acid sequence identity to any one of SEQ ID NOs: 5, 8-16, and 33-61, or a fragment thereof.

18. The engineered FGF10 polypeptide of any one of claims 1-17, wherein the engineered FGF10 polypeptide comprises at least 90% amino acid sequence identity to any one of SEQ ID NOs: 5, 8-16, and 33-61, or a fragment thereof.

19. The engineered FGF10 polypeptide of any one of claims 1-18, wherein the engineered FGF10 polypeptide comprises at least 95% amino acid sequence identity to any one of SEQ ID NOs: 5, 8-16, and 33-61, or a fragment thereof.

20. The engineered FGF10 polypeptide of any one of claims 1-19, wherein the engineered FGF10 polypeptide comprises an amino acid sequence of any one of SEQ ID NOs: 5, 8-16, and 33-61, or a fragment thereof.

21. The engineered FGF10 polypeptide of any of claims 1-20, wherein the engineered FGF10 polypeptide has an increased melting temperature as compared to wild-type FGF10.

22. The engineered FGF10 polypeptide of any of claims 1-21, wherein the engineered FGF10 polypeptide maintains activity level following incubation at 37° C for at least 4 days.

23. A composition comprising an engineered FGF10 polypeptide of any one of claims 1-22 and a carrier.

24. The composition of claim 23, wherein the composition is a cell culture medium.

25. A cell configured to express an engineered FGF10 polypeptide of any one of claims 1-22.

26. The cell of claim 25, wherein the cell comprises a nucleic acid encoding the engineered FGF10 polypeptide.

27. A nucleic acid encoding an engineered FGF10 polypeptide of any one of claims 1-22.

28. A method for promoting cell proliferation, cell differentiation, cell survival, tissue repair and tissue regeneration, the method comprising contacting a cell, a precursor or progenitor cell, a tissue, a spheroid, or an organoid with an engineered FGF10 polypeptide of any one of claims 1-22, or a composition comprising thereof.BIOTN-44681.60129. The method of claim 28, herein the organoid is a lung, stomach, pancreas, heart, bladder, dental epithelium, salivary gland, prostate, or liver organoid.

30. The method of claim 29, wherein the cell is pancreatic progenitor cell.

31. The method of any one of claims 28-30, wherein the cell, precursor or progenitor cell, tissue, spheroid, or organoid is a cancer cell, a cancer precursor or progenitor cell, a cancerous tissue, a cancer spheroid, or a cancer organoid.

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