# Patent

## Title
Cargo carrying nanoparticles

## Abstract

Disclosed herein are cargo carrying nanoparticles and pharmaceutical formulations thereof. The cargo carrying nanoparticles can self-assemble from cargo molecule subunits and/or targeting subunits. The cargo carrying molecules can be configured to induce macropinocytosis in a cell when a targeting moiety specifically binds a binding partner on the surface of a cell. The pharmaceutical formulations containing the cargo carrying nanoparticles disclosed herein can be administered to a patient for the treatment or prevention of cancer.

## Background
CROSS-REFERENCE TO RELATED APPLICATIONS
This application claims the benefit of U.S. provisional application Ser. No. 61/882,892 filed on Sep. 26, 2013, having the title “A Platform for Selective Intracellular Delivery by the Growth Factor Mediated Macropinocytosis Pathway,” the disclosure of which is incorporated by reference herein in its entirety. This application also claims the benefit of U.S. provisional application Ser. No. 61/884,417 filed on Sep. 30, 2013, having the title “Targeted Enhancement of Macropinocytosis for the Intracellular Delivery of Lytic Peptides to Lung Tumors,” the disclosure of which is incorporated by reference herein in its entirety.


SEQUENCE LISTING
This application contains a sequence listing filed in electronic form as an ASCII.txt file entitled 02086823.txt, created on Sep. 26, 2014 and having a size of 68,500 bytes. The content of the sequence listing is incorporated herein in its entirety.
BACKGROUND
According to the Centers for Disease Control and Prevention (CDC), more than 1 million people are diagnosed with cancer and about a half million people will die from cancer in the United States each year. About half of all men and one-third of all women will develop cancer during their lifetime. According to the American Cancer Society, there are about 14.5 million people living in the United States that have had or currently suffer from cancer. Current traditional treatments include surgery, chemotherapy, and radiation. A person with cancer may have any or all of these treatments. Traditional chemotherapy treatments can have deleterious side effects which often lead to their intolerance by patients. These side effects are mainly due to the toxicity of the chemotherapy treatments and their systemic effects. Further, many current chemotherapy treatments become ineffective after continued use as the cancer adapts and becomes resistant to the chemotherapy treatment. As such, there is an extended need for improved therapeutic treatments for cancer.
SUMMARY
Described herein are nanoparticles (referred to also herein as cargo carrying nanoparticles) containing a first fusion polypeptide containing a first elastin-like peptide (ELP) and a targeting moiety configured to specifically bind a binding partner, where the targeting moiety induces macropinocytosis when the targeting moiety specifically binds to the binding partner, and where the first ELP is operatively linked to the targeting moiety and a second fusion polypeptide containing a second ELP and a first cargo molecule, where the second ELP is operatively linked to the first cargo molecule, where the nanoparticle self assembles at a temperature that is greater than a phase transition temperature.
In some aspects, the targeting moiety is selected from keratinocyte growth factor, epidermal growth factor, and platelet derived growth factor. In further aspects, the first cargo molecule is a lytic peptide or a glycolysis inhibitor. In other aspects, the nanoparticle further comprises a cargo binding peptide sequence, the cargo binding peptide sequence configured to deliver small molecules. In further aspects, the second cargo molecule is a lytic peptide or a glycolysis inhibitor. In some aspects, the binding partner is located on the surface of a cell. In some aspects the cell is a cancer cell. In further aspects, the binding partner is selected from keratinocyte growth receptor, epidermal growth factor receptor, and platelet derived growth factor. In some aspects, the phase transition temperature ranges from about 5° C. to about 40° C.
Also described herein are vectors containing a first nucleotide sequence encoding a first fusion polypeptide, where the first fusion polypeptide contains a first elastin-like peptide (ELP) operatively linked to a targeting moiety configured to specifically bind a binding partner, where the targeting moiety induces macropinocytosis when the targeting moiety specifically binds to the binding partner and a second nucleotide sequence encoding a second fusion polypeptide, where the second fusion polypeptide contains a second ELP operatively linked to a first cargo molecule, where the second nucleotide sequence is operatively linked to the first nucleotide sequence. In some aspects, the vector further contains a third nucleotide sequence encoding a third fusion polypeptide, where the third fusion polypeptide contains a third ELP fused to a second cargo molecule, wherein the third nucleotide sequence is operatively linked to the first nucleotide sequence or the second nucleotide sequence. In some aspects, the first cargo molecule is selected from keratinocyte growth factor, epidermal growth factor, and platelet derived growth factor. In further aspects, the he first cargo molecule is a mitochondrial lytic peptide or a glycolysis inhibitor.
Also described are pharmaceutical formulations containing an effective amount of a nanoparticle, the nanoparticle containing a first fusion polypeptide comprising a first elastin-like peptide (ELP) and a targeting moiety configured to specifically bind a binding partner, where the targeting moiety induces macropinocytosis when the targeting moiety specifically binds to the binding partner, and where the first ELP is operatively linked to the targeting moiety, and a second fusion polypeptide comprising a second ELP and a first cargo molecule, where the second ELP is operatively linked to the first cargo molecule, where the nanoparticle self assembles above a phase transition temperature; and a pharmaceutically acceptable carrier, where the nanoparticle self assembles at a temperature greater than a phase transition temperature. In other aspects, the pharmaceutical formulation contains a nanoparticle having third fusion polypeptide comprising a third ELP and a second cargo molecule, wherein the third ELP is operatively linked to the second cargo molecule. In additional aspects, the first cargo molecule is a mitochondrial lytic peptide and wherein the second cargo molecule is a glycolysis inhibitor. In further aspects the targeting moiety is a growth factor selected from keratinocyte growth factor, epidermal growth factor, and platelet derived growth factor. In other aspects, the first cargo molecule is a mitochondrial lytic peptide or a glycolysis inhibitor.



BRIEF DESCRIPTION OF THE DRAWINGS
Further aspects of the present disclosure will be readily appreciated upon review of the detailed description of its various embodiments, described below, when taken in conjunction with the accompanying drawings.
FIG. 1 shows one embodiment of a cargo carrying nanoparticle having two subunits: a targeting subunit and a cargo subunit.
FIG. 2 shows one embodiment of a cargo carrying nanoparticle having three subunits: a targeting subunit and a first cargo subunit and a second cargo subunit.
FIG. 3 demonstrates macropiocytosis mediated internalization of the cargo carrying nanoparticles in a cell.
FIGS. 4A and 4B demonstrate cell death resulting from exposure to a lytic peptide fused to an elastin-like peptide (ELP).
FIGS. 5A through 5D demonstrate the results from flow cytometry of cells after treatment with the lytic peptide fused to an ELP of FIGS. 4A and 4B. Treatment with the lytic peptide fused to an ELP resulted in depolarization of mitochondria.
FIGS. 6A and 6B shown transmission electron microscope (TEM) pictures of control cells (FIG. 6A) and cells treated with the lytic peptide fused to an ELP (FIG. 6B) taken at 8600× that demonstrate swelling of mitochondria in response to the lytic peptide fused to an ELP, which is a consequence of mitochondrial depolarization.
FIG. 7 demonstrates the uptake of the lytic peptide fused to an ELP in cells treated with blockers of receptor mediated endocytosis (chlopromazine) or macropinocytosis (amiloride).
FIG. 8 demonstrates the uptake of the lytic peptide fused to an ELP in cells treated with surfen, which is an antagonist of heparan sulfate.
FIGS. 9A and 9B demonstrate the relative gene expression (FIG. 9A) of keratinocyte growth factor receptor (KGFR) in HCC827, H23, A549m, H1650, fibroblasts, and H292 cells. The no template (NT) is the negative control. qPCR products were confirmed by agarose gel electrophoresis. Results demonstrate the expected 135 bp PCR product (FIG. 9B).
FIG. 10 demonstrates the effect of a targeting subunit on cellular uptake of cargo carrying nanoparticles.
FIGS. 11A-11D demonstrate the effect of a growth factor targeting moiety in improving the cytotoxic effect of a lytic peptide. Cells were treated with a control (FIG. 11A), 50 μM of a lytic-ELP fusion polypeptide (FIG. 11B), 50 μM of a lytic-ELP fusion polypeptide and 100 μM of a growth factor-ELP fusion polypeptide (FIG. 11C), or 50 μM of a lytic-ELP fusion polypeptide and 200 μM of a growth factor-ELP fusion polypeptide (FIG. 11D).
FIG. 12 shows a TEM image of an assembled cargo carrying nanoparticle.
FIG. 13 demonstrates the effect of hydrophobicity of the ELP in the nanoparticle subunits on nanoparticle size.
FIGS. 14A and 14B demonstrate the effect of increasing concentrations of either (KLAKLAK)2-ELP nanoparticles (FIG. 14A) or (KLAKLAK)2 peptides (FIG. 14B) on cancer cell viability in vitro. KLAKLAK2 is also referred to herein as KLAK.
FIG. 15 demonstrates the effect of increasing concentrations of either (KLAKLAK)2-ELP nanoparticles or (KLAKLAK)2 peptides on lung cancer cell (A549) viability in vitro.
FIGS. 16A-16C demonstrate the effect of (KLAKLAK)2-ELP on mitochondrial depolarization. Cells were untreated (FIG. 16A), or treated with positive control (FIG. 16B), or with 25 μM (KLAKLAK)2-ELP nanoparticles (FIG. 16C).
FIG. 17 shows a TEM image of a cell treated with KLAKLAK2-ELP nanoparticles demonstrating mitochondrial swelling (arrows).
FIGS. 18A and 18B demonstrate the results from a JC-1 mitochondrial depolarization assay in untreated cells (FIG. 18A) or cells treated with (KLAKLAK)2-ELP nanoparticles. All cells were treated with a JC-1 dye prior to analysis. Non-apoptotic and apoptotic regions are marked.
FIG. 19 demonstrates the effect of cargo carrying nanoparticle (k) uptake in cells treated with a blocker of receptor mediated endocytosis (RME) or a blocker or macropinocytosis (MAC).
FIG. 20 demonstrates the quantitation of heterogeneous cargo carrying nanoparticle uptake per cell for H292 or fibroblasts (fb). Cells were treated with nanoparticles made of (KLAKLAK)2-ELP nanoparticles (homogeneous control), nanoparticles containing (KLAKLAK)2-ELP subunits and Growth factor (GF)-ELP subunits (heterogeneous treatment), or nanoparticles containing (KLAKLAK)2-ELP subunits and ELP alone (heterogeneous control).
FIG. 21 demonstrates the effect of cell surface protein blockers (Surfen, Heparinase iii, or lactose) on the uptake of the cargo carrying nanoparticles.
FIG. 22 demonstrates the effect of MAC or RME blockers on the uptake of cargo carrying nanoparticles.
FIGS. 23A-23D demonstrate the killing efficiency of cargo carrying nanoparticles containing a targeting subunit and a cargo subunit as compared to a control (FIG. 23A). Nanoparticles were formed from 50 μM of a cargo subunit: (KLAKLAK)2-ELP and varying concentrations of a target subunit (keratinocyte growth factor-ELP (“KGF”): at 0 μM (FIG. 23B), 100 μM (FIG. 23C), or 200 μM (FIG. 23D). Cell death was measured by a live (green) dead (red) assay. Bar=400 μM.
FIG. 24 demonstrates the results from quantifying the live cells after the cells were treated as in FIG. 21.
FIG. 25 demonstrates the relative gene expression of keratinocyte growth factor receptor IIIb in various cancer cell lines or fibroblast cells (fb).
FIG. 26 demonstrates the effect of a targeting subunit containing a keratinocyte growth factor on uptake of cargo carrying nanoparticles in A549 lung cancer cells.
FIG. 27 demonstrates the targeting selectivity of the targeting subunit containing a keratinocyte growth factor between cells that express the keratinocyte growth factor receptor (A549 cells) and fibroblasts (fb).
FIG. 28 demonstrates the selective killing of cells that express a keratinocyte growth factor receptor (A549) by cargo carrying nanoparticles having a targeting subunit that contains a keratinocyte growth factor as compared to cells that do not express a keratinocyte growth factor receptor (MiaPaCa-2). * indicates values that are significantly different.
FIG. 29 demonstrates the dose-dependent effect of a targeting subunit that contains a keratinocyte growth factor on macropinosome formation in cells (A549) expressing increased levels of keratinocyte growth factor receptor iiib (KGFR).
FIG. 30 demonstrates the effect of the a targeting subunit that contains a keratinocyte growth factor on macropinosome formation in cells (fb) that do not express keratinocyte growth factor receptor iiib.



DETAILED DESCRIPTION
Before the present disclosure is described in greater detail, it is to be understood that this disclosure is not limited to particular embodiments described, and 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.
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 disclosure. The upper and lower limits of these smaller ranges may independently be included in the smaller ranges and are also encompassed within the disclosure, 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 disclosure.
Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. Although any methods and materials similar or equivalent to those described herein can also be used in the practice or testing of the present disclosure, the preferred methods and materials 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 methods and/or materials 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 disclosure is not entitled to antedate such publication by virtue of prior disclosure. Further, the dates of publication provided could be different from the actual publication dates that may need to be independently confirmed.
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 disclosure. Any recited method can be carried out in the order of events recited or in any other order that is logically possible.
Embodiments of the present disclosure will employ, unless otherwise indicated, techniques of molecular biology, microbiology, nanotechnology, organic chemistry, biochemistry, botany and the like, which are within the skill of the art. Such techniques are explained fully in the literature.
DEFINITIONS
As used herein, “about,” “approximately,” and the like, when used in connection with a numerical variable, generally refers to the value of the variable and to all values of the variable that are within the experimental error (e.g., within the 95% confidence interval for the mean) or within .+−.10% of the indicated value, whichever is greater.
As used herein, “control” is an alternative subject or sample used in an experiment for comparison purposes and included to minimize or distinguish the effect of variables other than an independent variable. A “control” can be positive or negative.
As used herein, “effective amount” is an amount sufficient to effect beneficial or desired results. An effective amount can be administered in one or more administrations, applications, or dosages.
As used herein, “concentrated” used in reference to an amount of a molecule, compound, or composition, including, but not limited to, a chemical compound, polynucleotide, peptide, polypeptide, protein, antibody, or fragments thereof, that indicates that the sample is distinguishable from its naturally occurring counterpart in that the concentration or number of molecules per volume is greater than that of its naturally occurring counterpart.
As used herein, “diluted” used in reference to a an amount of a molecule, compound, or composition including but not limited to, a chemical compound, polynucleotide, peptide, polypeptide, protein, antibody, or fragments thereof, that indicates that the sample is distinguishable from its naturally occurring counterpart in that the concentration or number of molecules per volume is less than that of its naturally occurring counterpart.
As used herein, “purified” is used in reference to a nucleic acid sequence, peptide, or polypeptide or other compound that has increased purity relative to the natural environment or the environment in which it was produced in.
As used herein, “pharmaceutical formulation” refers to the combination of an active agent, compound, or ingredient with a pharmaceutically acceptable carrier or excipient, making the composition suitable for diagnostic, therapeutic, or preventive use in vitro, in vivo, or ex vivo.
As used herein, “pharmaceutically acceptable carrier or excipient” refers to a carrier or excipient that is useful in preparing a pharmaceutical formulation that is generally safe, non-toxic, and is neither biologically or otherwise undesirable, and includes a carrier or excipient that is acceptable for veterinary use as well as human pharmaceutical use. A “pharmaceutically acceptable carrier or excipient” as used in the specification and claims includes both one and more than one such carrier or excipient.
As used herein, “pharmaceutically acceptable salt” refers to any acid or base addition salt whose counter-ions are non-toxic to the subject to which they are administered in pharmaceutical doses of the salts.
As used interchangeably herein, “subject,” “individual,” or “patient,” refers to a vertebrate, preferably a mammal, more preferably a human. Mammals include, but are not limited to, murines, simians, humans, farm animals, sport animals, and pets. The term “pet” includes a dog, cat, guinea pig, mouse, rat, rabbit, ferret, and the like. The term farm animal includes a horse, sheep, goat, chicken, pig, cow, donkey, llama, alpaca, turkey, and the like.
As used herein, “biocompatible” or “biocompatibility” refers to the ability of a material to be used by a patient without eliciting an adverse or otherwise inappropriate host response in the patient to the material or a derivative thereof, such as a metabolite, as compared to the host response in a normal or control patient.
As used herein, “therapeutic” refers to treating or curing a disease or condition.
As used herein, “preventative” refers to hindering or stopping a disease or condition before it occurs or while the disease or condition is still in the sub-clinical phase.
As used herein, “separated” refers to the state of being physically divided from the original source or population such that the separated compound, agent, particle, chemical compound, or molecule can no longer be considered part of the original source or population.
As used herein, “active agent” or “active ingredient” refers to a component or components of a composition to which the whole or part of the effect of the composition is attributed.
As used herein, “tangible medium of expression” refers to a medium that is physically tangible and is not a mere abstract thought or an unrecorded spoken word. Tangible medium of expression includes, but is not limited to, words on a cellulosic or plastic material or data in stored on a suitable device such as a flash memory or CD-ROM.
As used herein, “chemotherapeutic agent” or “chemotherapeutic” refer to a therapeutic agent utilized to prevent or treat cancer.
As used herein, “nanoparticle” refers to any molecule, particle, or entity having a greatest dimension (diameter or length, width, or height) of less than 600 μm. Nanoparticles having a spherical shape are also generally referred to as “nanospheres.” Nanoparticles can be heterogeneous or homogeneous.
As used herein, “dose,” “unit dose,” or “dosage” refers to physically discrete units suitable for use in a subject, each unit containing a predetermined quantity of the nanoparticle composition or formulation calculated to produce the desired response or responses in association with its administration.
As used herein, “plasmid” as used herein refers to a non-chromosomal double-stranded DNA sequence including an intact “replicon” such that the plasmid is replicated in a host cell.
As used herein, the term “vector” or is used in reference to a vehicle used to introduce an exogenous nucleic acid sequence into a cell. A vector may include a DNA molecule, linear or circular (e.g. plasmids), which includes a segment encoding a polypeptide of interest operatively linked to additional segments that provide for its transcription and translation upon introduction into a host cell or host cell organelles. Such additional segments may include promoter and terminator sequences, and may also include one or more origins of replication, one or more selectable markers, an enhancer, a polyadenylation signal, etc. Expression vectors are generally derived from yeast or bacterial genomic or plasmid DNA or viral DNA, or may contain elements of both.
As used herein, “antibody” refers to a protein produced by B cells that is used by the immune system to identify and neutralize foreign compounds, which are also known as antigens. Antibodies are glycoproteins belonging to the immunoglobulin superfamily. Antibodies, recognize and bind to specific epitopes on an antigen.
As used herein, “aptamer” refers to single-stranded DNA or RNA molecules that can bind to pre-selected targets including proteins with high affinity and specificity. Their specificity and characteristics are not directly determined by their primary sequence, but instead by their tertiary structure.
As used herein, “specific binding partner” or “binding partner” is a compound or molecule to which a second compound or molecule binds with a higher affinity than all other molecules or compounds.
As used herein, “specifically binds” or “specific binding” refers to binding that occurs between such paired species such as enzyme/substrate, receptor/agonist or antagonist, antibody/antigen, lectin/carbohydrate, oligo DNA primers/DNA, enzyme or protein/DNA, and/or RNA molecule to other nucleic acid (DNA or RNA) or amino acid, which may be mediated by covalent or non-covalent interactions or a combination of covalent and non-covalent interactions. When the interaction of the two species produces a non-covalently bound complex, the binding that occurs is typically electrostatic, hydrogen-bonding, or the result of lipophilic interactions. Accordingly, “specific binding” occurs between a paired species where there is interaction between the two which produces a bound complex having the characteristics of an antibody/antigen, enzyme/substrate, DNA/DNA, DNA/RNA, DNA/protein, RNA/protein, RNA/amino acid, receptor/substrate interaction. In particular, the specific binding is characterized by the binding of one member of a pair to a particular species and to no other species within the family of compounds to which the corresponding member of the binding member belongs. Thus, for example, an antibody preferably binds to a single epitope and to no other epitope within the family of proteins.
As used herein “immunomodulator,” refers to an agent, such as a therapeutic agent, which is capable of modulating or regulating one or more immune function or response.
As used herein, “targeting moiety” refers to a moiety or molecule that localizes to or away from a specific local, cell, and/or other molecule.
As used herein, “differentially expressed,” refers to the differential production of RNA, including but not limited to mRNA, tRNA, miRNA, siRNA, snRNA, and piRNA transcribed from a gene or regulatory region of a genome or the protein product encoded by a gene as compared to the level of production of RNA by the same gene or regulator region in a normal or a control cell. In another context, “differentially expressed,” also refers to nucleotide sequences or proteins in a cell or tissue which have different temporal and/or spatial expression profiles as compared to a normal or control cell.
As used herein, “overexpressed” or “overexpression” refers to an increased expression level of an RNA or protein product encoded by a gene as compared to the level of expression of the RNA or protein product in a normal or control cell.
As used herein, “underexpressed” or “underexpression” refers to decreased expression level of an RNA or protein product encoded by a gene as compared to the level of expression of the RNA or protein product in a normal or control cell.
As used herein, “expression” refers to the process by which polynucleotides are transcribed into RNA transcripts. In the context of mRNA and other translated RNA species, “expression” also refers to the process or processes by which the transcribed RNA is subsequently translated into peptides, polypeptides, or proteins.
As used herein, “isolated” means separated from constituents, cellular and otherwise, in which the polynucleotide, peptide, polypeptide, protein, antibody, or fragments thereof, are normally associated with in nature. A non-naturally occurring polynucleotide, peptide, polypeptide, protein, antibody, or fragments thereof, do not require “isolation” to distinguish it from its naturally occurring counterpart.
As used herein, “operatively linked” indicates that the regulatory sequences useful for expression of the coding sequences of a nucleic acid are placed in the nucleic acid molecule in the appropriate positions relative to the coding sequence so as to effect expression of the coding sequence. This same definition is sometimes applied to the arrangement of coding sequences and transcription control elements (e.g. promoters, enhancers, and termination elements), and/or selectable markers in an expression vector. The same definition is sometimes applied to the specific arrangement of multiple coding sequences within a vector such that the sequences are expressed from the same vector in such a way that each coding sequence produces its desired product.
As used herein, “promoter” includes all sequences capable of driving transcription of a coding sequence. In particular, the term “promoter” as used herein refers to a DNA sequence generally described as the 5′ regulator region of a gene, located proximal to the start codon. The transcription of an adjacent coding sequence(s) is initiated at the promoter region. The term “promoter” also includes fragments of a promoter that are functional in initiating transcription of the gene.
As used herein, “identity,” is a relationship between two or more polypeptide sequences, as determined by comparing the sequences. In the art, “identity” also refers to the degree of sequence relatedness between polypeptide as determined by the match between strings of such sequences. “Identity” can be readily calculated by known methods, including, but not limited to, those described in (Computational Molecular Biology, Lesk, A. M., Ed., Oxford University Press, New York, 1988; Biocomputing: Informatics and Genome Projects, Smith, D. W., Ed., Academic Press, New York, 1993; Computer Analysis of Sequence Data, Part I, Griffin, A. M., and Griffin, H. G., Eds., Humana Press, New Jersey, 1994; Sequence Analysis in Molecular Biology, von Heinje, G., Academic Press, 1987; and Sequence Analysis Primer, Gribskov, M. and Devereux, J., Eds., M Stockton Press, New York, 1991; and Carillo, H., and Lipman, D., SIAM J. Applied Math. 1988, 48: 1073. Preferred methods to determine identity are designed to give the largest match between the sequences tested. Methods to determine identity are codified in publicly available computer programs. The percent identity between two sequences can be determined by using analysis software (e.g., Sequence Analysis Software Package of the Genetics Computer Group, Madison Wis.) that incorporates the Needelman and Wunsch, (J. Mol. Biol., 1970, 48: 443-453,) algorithm (e.g., NBLAST, and XBLAST). The default parameters are used to determine the identity for the polypeptides of the present disclosure.
As used herein, “polypeptides” or “proteins” are as amino acid residue sequences. Those sequences are written left to right in the direction from the amino to the carboxy terminus. In accordance with standard nomenclature, amino acid residue sequences are denominated by either a three letter or a single letter code as indicated as follows: Alanine (Ala, A), Arginine (Arg, R), Asparagine (Asn, N), Aspartic Acid (Asp, D), Cysteine (Cys, C), Glutamine (Gln, Q), Glutamic Acid (Glu, E), Glycine (Gly, G), Histidine (His, H), Isoleucine (Ile, I), Leucine (Leu, L), Lysine (Lys, K), Methionine (Met, M), Phenylalanine (Phe, F), Proline (Pro, P), Serine (Ser, S), Threonine (Thr, T), Tryptophan (Trp, W), Tyrosine (Tyr, Y), and Valine (Val, V).
As used herein, the term “transfection” refers to the introduction of an exogenous and/or recombinant nucleic acid sequence into the interior of a membrane enclosed space of a living cell, including introduction of the nucleic acid sequence into the cytosol of a cell as well as the interior space of a mitochondria, nucleus, or chloroplast. The nucleic acid may be in the form of naked DNA or RNA, it may be associated with various proteins or regulatory elements (e.g., a promoter and/or signal element), or the nucleic acid may be incorporated into a vector or a chromosome.
As used herein, “transformation” or “transformed” refers to the introduction of a nucleic acid (e.g., DNA or RNA) into cells in such a way as to allow expression of the coding portions of the introduced nucleic acid.
As used herein, the term “recombinant” generally refers to a non-naturally occurring nucleic acid, nucleic acid construct, or polypeptide. Such non-naturally occurring nucleic acids may include natural nucleic acids that have been modified, for example that have deletions, substitutions, inversions, insertions, etc., and/or combinations of nucleic acid sequences of different origin that are joined using molecular biology technologies (e.g., a nucleic acid sequences encoding a “fusion protein.” Recombinant also refers to the polypeptide encoded by the recombinant nucleic acid. Non-naturally occurring nucleic acids or polypeptides include nucleic acids and polypeptides modified by man.
As used herein, “expression cassette” refers to the part of a DNA expression vector or plasmid that is capable of directing the cell to make RNA and protein. An expression cassette contains of one or more DNA sequences that code for a polypeptide, the sequences controlling expression of the coding DNA sequence(s) and other regulatory elements that otherwise influence expression of the coding DNA sequence(s) contained within the expression cassette.
As used herein, the term “fusion protein” or “fusion polypeptide” refers to a protein or polypeptide formed from the protein or polypeptide formed from the combination of two or more different proteins, polypeptides, or peptides. “Fusion polypeptide” or “fusion protein” also refers to the combination of a nucleic acid encoding a polypeptide to a promoter sequence, where the coding sequence and promoter sequence are from different sources or otherwise do not typically occur together naturally (e.g, a nucleic acid and a constitutive promoter),
As used herein, “gene” refers to a hereditary unit corresponding to a sequence of DNA that occupies a specific location on a chromosome and that contains the genetic instruction for a characteristic(s) or trait(s) in an organism.
As used herein, “nucleic acid” and “polynucleotide” generally refer to a string of at least two base-sugar-phosphate combinations and refers to, among others, single- and double-stranded DNA, DNA that is a mixture of single- and double-stranded regions, single- and double-stranded RNA, and RNA that is mixture of single- and double-stranded regions, hybrid molecules comprising DNA and RNA that may be single-stranded or, more typically, double-stranded or a mixture of single- and double-stranded regions. In addition, polynucleotide as used herein refers to triple-stranded regions comprising RNA or DNA or both RNA and DNA. The strands in such regions may be from the same molecule or from different molecules. The regions may include all of one or more of the molecules, but more typically involve only a region of some of the molecules. One of the molecules of a triple-helical region often is an oligonucleotide. “Polynucleotide” and “nucleic acids” also encompasses such chemically, enzymatically or metabolically modified forms of polynucleotides, as well as the chemical forms of DNA and RNA characteristic of viruses and cells, including simple and complex cells, inter alia. For instance, the term polynucleotide includes DNAs or RNAs as described above that contain one or more modified bases. Thus, DNAs or RNAs comprising unusual bases, such as inosine, or modified bases, such as tritylated bases, to name just two examples, are polynucleotides as the term is used herein. “Polynucleotide” and “nucleic acids” also includes PNAs (peptide nucleic acids), phosphorothioates, and other variants of the phosphate backbone of native nucleic acids. Natural nucleic acids have a phosphate backbone, artificial nucleic acids may contain other types of backbones, but contain the same bases. Thus, DNAs or RNAs with backbones modified for stability or for other reasons are “nucleic acids” or “polynucleotide” as that term is intended herein.
As used herein, “deoxyribonucleic acid (DNA)” and “ribonucleic acid (RNA)” generally refer to any polyribonucleotide or polydeoxribonucleotide, which may be unmodified RNA or DNA or modified RNA or DNA. RNA may be in the form of a tRNA (transfer RNA), snRNA (small nuclear RNA), rRNA (ribosomal RNA), mRNA (messenger RNA), anti-sense RNA, RNAi (RNA interference construct), siRNA (short interfering RNA), or ribozymes.
As used herein, “nucleic acid sequence” and “oligonucleotide” also encompasses a nucleic acid and polynucleotide as defined above.
As used herein, “DNA molecule” includes nucleic acids/polynucleotides that are made of DNA.
As used herein, “variant” refers to a polypeptide that differs from a reference polypeptide, but retains essential properties. A typical variant of a polypeptide differs in amino acid sequence from another, reference polypeptide. Generally, differences are limited so that the sequences of the reference polypeptide and the variant are closely similar overall and, in many regions, identical. A variant and reference polypeptide may differ in amino acid sequence by one or more modifications (e.g., substitutions, additions, and/or deletions). A substituted or inserted amino acid residue may or may not be one encoded by the genetic code. A variant of a polypeptide may be naturally occurring such as an allelic variant, or it may be a variant that is not known to occur naturally.
As used herein, “functional variant” refers to a variant of a protein or polypeptide (e.g., a variant of a KGF protein) that can perform the same functions or activities as the original protein or polypeptide, although not necessarily at the same level (e.g., the variant may have enhanced, reduced or changed functionality, so long as it retains the basic function).
As used herein, “inhibit” or “inhibiting” expression of a gene indicates that something (e.g., antisense nucleotide, suppressor, antagonist, etc.) acts to reduce or prevent (completely or partially) the transcription, translation and/or other processing step in the expression of a gene, thereby down-regulating the gene expression so that a reduced amount of the active protein encoded by the gene is produced as compared to wild type.
As used herein, “selectable marker” refers to a gene whose expression allows one to identify cells that have been transformed or transfected with a vector containing the marker gene. For instance, a recombinant nucleic acid may include a selectable marker operatively linked to a gene of interest and a promoter, such that expression of the selectable marker indicates the successful transformation of the cell with the gene of interest.
As used herein, “constitutive promoter” is a promoter that allows for continual or ubiquitous transcription of its associated gene or polynucleotide. Constitutive promoters are generally are unregulated by cell or tissue type, time, or environment.
As used herein, “inducible promoter” is a promoter that allows transcription of its associated gene or polynucleotide in response to a substance or compound (e.g. an antibiotic, or metal), an environmental condition (e.g. temperature), developmental stage, or tissue type.
As used herein, “wild-type” is the typical form of an organism, variety, strain, gene, protein, or characteristic as it occurs in nature, as distinguished from mutant forms that may result from selective breeding or transformation with a transgene.
As used herein, “cDNA” refers to a DNA sequence that is complementary to a RNA transcript in a cell. It is a man-made molecule. Typically, cDNA is made in vitro by an enzyme called reverse-transcriptase using RNA transcripts as templates.
As used herein “induces,” “inducing,” or “induced” refers to activating or stimulating a process or pathway within a cell, such as macropinocytosis.
As used herein “heterogeneous” refers to a population of molecules, including nanoparticles, proteins, and polypeptides, or a population of subunits of a molecule that contains at least 2 molecules or subunits that are different from one another.
As used herein “homogenous” refers to a population of molecules, including nanoparticles, proteins, and polypeptides, or a population of subunits of a molecule in which all the molecules or subunits are identical to one another.
As used herein “small molecule” refers to a compound or molecule that is so dimensioned such that it can be internalized by a single cell.
DISCUSSION
According to the CDC, cancer is the second leading cause of death in the United States. Lung cancer remains the leading cause of death among all cancers in the United States. In 2008, an estimated 1.61 million new cases of lung cancer, which represents 12.7% of all new cancer diagnosis, were diagnosed worldwide. Further, in 2008 an estimated 1.38 million deaths were attributed to lung cancer.
Lung cancers are typically treated with chemotherapy to improve quality of life, palliate symptoms, and improve survival. Several chemotherapeutic drugs have been developed for treatment of lung cancer. These chemotherapeutic drugs include DNA damaging drugs, such as cisplatin and mitotic inhibitors, such as paclitaxel or doxorubicin. However, all these agents come with significant side effects such as nephrotoxicity, which may result from its nonspecific systemic organ distribution and inadequate intratumor concentrations.
Efforts have been expended to develop chemotherapeutic drugs or agents that that selectively affect tumor cells. Typically, these selective chemotherapeutic drugs or agents are configured to target molecules that are uniquely expressed, overexpressed, or mutated in cancer tumors. One such target is the epidermal growth factor receptor (EGFR), which is overexpressed in about 50 to about 90 percent of lung cancers. Monoclonal antibodies against EGFR (cetuximab) and tyrosine kinase inhibitors (TKI) that inhibit the tyrosine kinase activity of EGFR (erlotinib or gefitinib) have been developed and have only been marginally successful in clinical trials for the treatment of lung cancer.
Further, acquired resistance remains a problem with chemotherapy treatments, particularly with TKI treatment, with chemotherapeutic treatment. For example, although TKIs show promising response rates in lung cancer patients, acquired resistance to the TKI always develops about 10 months (on average) after beginning treatment. The acquired resistance can be attributed to several factors such as secondary mutation of the EGFR gene, amplification of the hepatocyte growth factor receptor (MET) gene, and/or overexpression of hepatocyte growth factor (HGF). As such, patient prognosis for lung cancer remains poor. Indeed, the 5-year survival rate of less than about 5% and a median survival of about 1 year.
With this in mind, described herein are compositions and formulations that include polypeptides that can selectively target cancer cells and induce cell death via mechanisms that are difficult for the cancer cell to adapt to and reduce or eliminate acquired resistance by cancer cells. In one embodiment, a cargo carrying nanoparticle contains a first fusion polypeptide that contains a growth factor targeting moiety operatively linked to an elastin-like peptide (ELP) and a second fusion polypeptide that contains a cargo molecule, such as a mitochondrial lytic peptide or glycolysis inhibitor, operatively linked to an (ELP). The growth factor can specifically bind to a binding partner located on the surface of a cell such that the macropinocytosis pathway is induced. The cargo carrying nanoparticles are internalized by a cell by macropinocytosis, which increases the efficiency of drug uptake and reduces systemic circulation of the cargo carrying nanoparticles.
By operatively linking the targeting moiety or cargo molecule to an ELP, the cargo carrying nanoparticles can self-assemble from their constituent subunit polypeptides and be produced at a low cost. Moreover, as disclosed herein, ELP interacts with cell surface heparan sulfates, which ensures that the cargo carrying nanoparticles are close to the cell surface and increases the efficiency of uptake. Indeed, for every one cargo carrying nanoparticle that has a targeting moiety specifically binds a binding partner on the cell surface that induces macropinocytosis, multiple cargo carrying nanoparticles can be internalized. Thus, the efficiency of uptake of the cargo carrying nanoparticles disclosed herein is greater than what can be achieved by other chemotherapeutics that rely on receptor mediated endocytosis, in which only one molecule (the molecule that is bound to the receptor) is internalized at a time.
Additionally, lytic peptides and glycolysis inhibitors do not target any specific pathway or receptor within a cell. As such, it is difficult for the cell to adapt or mutate to develop resistance to the cargo carrying nanoparticle. Insofar as the cargo carrying nanoparticles described herein can target cancer, particularly lung cancer, cells and do not target any specific cellular pathway or receptor, these cargo carrying nanoparticles can reduce tumor volume and burden with minimal side effects and minimize or eliminate acquired resistance.
Other compositions, compounds, methods, features, and advantages of the present disclosure will be or become apparent to one having ordinary skill in the art upon examination of the following drawings, detailed description, and examples. It is intended that all such additional compositions, compounds, methods, features, and advantages be included within this description, and be within the scope of the present disclosure.
Cargo Carrying Nano Particles
Provided herein are embodiments of cargo carrying nanoparticles that contain a targeting moiety fused to a first ELP (ELP1), which forms a targeting subunit, and a cargo molecule that is also fused to a second ELP (ELP2), which forms a cargo subunit. In other embodiments, the cargo carrying nanoparticles contain one or more additional cargo molecules that are each fused to a separate ELP (ELPn), which forms additional cargo subunits. In yet further embodiments, the cargo carrying nanoparticles contain one or more transport moieties that are each fused to a separate ELP, which form additional transport subunits.
In some embodiments, ELP1 . . . n are substantially (95% or greater sequence identity) the same ELP In other embodiments ELP1 . . . n are substantially different (less than 95% sequence identity) from at least one other ELP in the assembled cargo carrying nanoparticle In some embodiments, there are equal numbers of targeting subunits and cargo subunits in the assembled cargo carrying nanoparticles. In other embodiments, the number of targeting subunits in the assembled cargo carrying nanoparticles is unequal to the number of cargo subunits in the assembled cargo carrying nanoparticles. In embodiments where there is more than one type of targeting subunits in the assembled cargo carrying nanoparticle, the number of one type targeting subunit can be equal to the number of the other types of targeting subunits in the assembled nanoparticle. In other of these embodiments, the number of one type of targeting subunits in the assembled cargo carrying nanoparticle is different from the number of at least one other type of targeting subunit present in the assembled nanoparticle.
The cargo carrying nanoparticles are spontaneously formed or self-assembled when the temperature of a solution containing the nanoparticle subunits is above the transition temperature. FIG. 1 shows one embodiment of a cargo carrying nanoparticle 100 that contains two different subunits: 1) a targeting subunit 101 that contains at least a targeting moiety 102 fused to a first ELP 103a and 2) a cargo subunit 104 that contains a cargo molecule 105 fused to a second ELP 103b. FIG. 2 shows one embodiment of a cargo carrying nanoparticle 200 that contains three different subunits: 1) a targeting subunit 101 that contains a targeting moiety 102 fused to a first ELP 103a, 2) a first cargo subunit 104 that contains a first cargo molecule 105 fused to a second ELP 103b, and 3) a second cargo subunit 201 that contains a second cargo molecule 202 fused to a third ELP 103c. As is shown in FIGS. 1 and 2, the cargo transporting nanoparticles (100 and 200) self-assemble from the subunits at a temperature (T) above a phase transition temperature of the nanoparticle (Ttm) to form the cargo carrying nanoparticles (100 and 200). One of ordinary skill in the art will instantly appreciate that the transition temperature is dependent on the chemical composition of the individual units of the nanoparticle discussed above. In some embodiments, the T can range between about 5° C. and about 40° C.
The size of the cargo carrying nanoparticles is less than about 1000 nm. In some embodiments, the size of the cargo carrying nanoparticles ranges from about 1000 nm to about 500 nm. In further embodiments, the size of the nanoparticles range from about 250 nm to about 500 nm. In other embodiments, the size of the nanoparticles range from about 100 nm to about 250 nm. In yet other embodiments, the size of the nanoparticles ranges from about 0.001 nm to about 100 nm. In some embodiments, the size of the cargo carrying nanoparticle is minimized so as to increase the efficacy of the nanoparticles in vivo. In these embodiments, the size of the nanoparticle is about 500 nm or less. Size of the nanoparticles can be manipulated by, inter alia, by increasing the hydrophobicity of the nanoparticle subunits.
Elastin-Like Polypeptides (ELPs)
ELPs are a class of stimulus responsive biopolymers that are soluble macromolecules at temperatures below a phase transition temperature of the ELP (TtELP) and self-assemble into nanoparticles such as micelles, micron-scale cacervates, or viscous gels, at temperatures above the TtELP. This phase transition is reversible and the self-assembly can be controlled by manipulating the TtELP. The TtELP can be manipulated by altering the ELP. Manipulation of the ELP can be by altering the polypeptide chain length or by altering the polypeptide sequence.
ELPs are repetitive polypeptides that evolved from the hydrophobic domain of elastin. The repeat polypeptide unit in ELPs is a pentapeptide of VPGXG (SEQ ID NO: 1), where “X” can be any amino acid other than P. The number of repeat units in the ELP can vary. The ELPs described herein can have about 1 to about 180 repeat units per ELP. The exact sequence of the ELP depends on a variety of factors including properties (e.g. hydrophobicity and molecular weight) of the cargo molecules and targeting moieties to be fused to the ELP, the desired TtELP and/or Ttm, the desired nanoparticle to be formed, the application in which the nanoparticle will be used. The ELP can be homogenous (having the same repeat units) or can be heterogeneous (having different repeat units). In some embodiments, X is C, V, M, I, L, F, or W. In some embodiments, the ELP is made of five repeat units. In some of these embodiments, the X in the repeat units 1, 2, 4, and 5 is V and the X in repeat unit 3 is C, M, I, L, F, or W. In these embodiments, the sequence of the ELP corresponds to SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, or SEQ ID NO: 7. The ELPs can interact with cell surface heparan sulfates, which attract and keep the cargo carrying nanoparticles near the cell surface to increase uptake of more cargo carrying nanoparticles during macropinocytosis. In some embodiments, the ELP has a cDNA sequence according to SEQ ID NO: 8, which encodes a polypeptide sequence according to SEQ ID NO: 9.
In other embodiments, the ELP contains strings of hydrophobic amino acids, e.g. poly leucine repeat units. In these embodiments, the fusion polypeptide takes to form of Cargo Molecule or Targeting moiety-[poly leucine]n-ELP. The number of leucine residues in the poly leucine repeat unit can range from 2-50. The number of poly leucine repeat units (n) can range from 1 to 50. In some embodiments the number of ELP repeat units ranges from about 50 to about 250 repeat units. In other embodiments, the ELP has a cDNA sequence according to SEQ ID NO: 8, which encodes a polypeptide sequence according to SEQ ID NO: 9.
Targeting Moieties and Binding Partners
The cargo carrying nanoparticles contain at least one targeting subunit that made of a targeting moiety fused to an ELP. The targeting moiety is configured to specifically bind a binding partner, which can be any receptor or protein present on the cell surface that enhances the macropinocytotic pathway. In some embodiments, the specific binding of the targeting moiety to its binding partner results in enhanced uptake of the nanoparticles. In this way, toxicity and other side effects can be decreased. Toxicity and other side effects are reduced because the cargo carrying nanoparticles are targeted to only the desired cells. The binding partner is located on the surface of a cell (i.e. associated with, attached to, or integrated within the cell membrane), such as a cancer cell.
Macropinocytosis is a form of bulk uptake of fluid and solid cargo into cytoplasmic vacuoles, called micropiosomes. As shown in FIG. 3, specific binding of the targeting moiety 101 of the cargo carrying nanoparticle 200 to its binding partner 301 located on the plasma membrane of a cell 302 activates the macropinocytosis pathway and the cargo carrying nanoparticle is internalized. Briefly, after specific binding of the targeting moiety 101 to its binding partner 301, actin-mediated bending of a single surface lamellipodia gives rise to a curved “ruffles” 303 during a process referred to as “ruffling.” The ruffle 303 then fuses back with the plasma membrane of the cell during the “fusion” step and encapsulates the extracellular fluid and cargo carrying nanoparticles 200 thus forming the a macropinosome 304. The macropinosomes are inherently leaky. Thus, the cargo carrying nanoparticle 200 can leak out of the macropinosomes and escapes lysosomal degradation and is delivered to the cell. Once delivered, cargo carrying nanoparticles are lysed into their individual subunits (101, 102, and 201). In some instances lysing of the cargo carrying nanoparticles 200 renders the cargo molecule biologically active.
Macropinocytosis is beneficial over the commonly described receptor mediated internalization approaches that typically involve clathrin coated endocytosis. First, macropinosomes are relatively large (>about 1 μm), thereby efficiently internalizing several cargo carrying nanoparticles in one macropinosome. As the macropinosomes are inherently leaky, macropinocytosis results in rapid release of the cargo carrying nanoparticles into the cytoplasm of the cell, thus escaping lysosomal degradation. Further, typical receptor mediated internalization results in the uptake of one nanoparticle per receptor, whereas in internalization by macropinocytosis one cargo carrying nanoparticle specifically binds to the receptor but, as shown in FIG. 3, several cargo carrying nanoparticles that are in the vicinity of the cell surface are internalized. Moreover, not only does the targeting moiety allow targeted delivery of the cargo carrying nanoparticles to specific cells, the specific binding of the targeting moiety to its binding partner on the surface of the cell can increase macropinocytosis in these cells.
As discussed above, the targeting moiety is configured to induce the micropinocytosis pathway when it specifically binds to its binding partner. In some embodiments the targeting moiety is a growth factor. In some of these embodiments, the targeting moiety is epidermal growth factor, platelet derived growth factor, keratinocyte growth factor, or combinations thereof. In other embodiments, the targeting moiety is not a growth factor but otherwise induces the macropinocytotic pathway upon specifically binding to a binding partner.
In some embodiments, the growth factor is a human EGF. The cDNA sequence of the human EGF can correspond to SEQ ID NO: 10. The human EGF polypeptide can have a sequence according to SEQ ID NO: 11. In some embodiments, the human EGF is a variant of SEQ ID NO: 10 or SEQ ID NO: 11. In further embodiments, the EGF cDNA sequence is about 75% to about 99% identical to SEQ ID NO: 10. In other embodiments, the EGF polypeptide sequence is about 75% to about 99% identical to SEQ ID NO: 11.
In other embodiments, the growth factor is a human KGF. The cDNA sequence of the human KGF can correspond to SEQ ID NO: 12. The human KGF polypeptide can have a sequence according to SEQ ID NO. 13. In further embodiments, the KGF cDNA sequence is about 75% to about 99% identical to SEQ ID NO: 12. In other embodiments, the KGF polypeptide sequence is about 75% to about 99% identical to SEQ ID NO: 13.
The cDNA sequence of the human platelet derived growth factor can correspond to SEQ ID NO: 14. In further embodiments, the platelet derived cDNA sequence is about 75% to about 99% identical to SEQ ID NO: 14. In some embodiments, a platelet derived growth factor polypeptide is encoded by a cDNA according to SEQ ID NO: 14. In other embodiments, the platelet derived growth factor polypeptide is encoded by a cDNA that is about 75% to about 99% identical to SEQ ID NO: 14.
As previously discussed, the targeting moiety specifically binds a binding partner that is located on the cell surface. This specific binding triggers macropinocytosis and internalization of the cargo carrying nanoparticles. The binding partner can be associated with, attached to, or integrated with a membrane of a cell. In some embodiments, the binding partner is associated with, attached to, or integrated with the plasma membrane. The binding partner is configured to activate the macropinocytosis pathway upon specific binding of a targeting moiety.
In some embodiments, the binding partner is a growth factor receptor. In some of these embodiments, the growth factor receptor is epidermal growth factor receptor, platelet derived growth factor receptor, keratinocyte growth factor receptor, or combinations thereof. The epidermal growth factor receptor can be a human epidermal growth factor receptor. In some embodiments the epidermal growth factor receptor has a cDNA sequence according to SEQ ID NO: 15. In other embodiments, the epidermal growth factor receptor has a cDNA sequence that is about 75% to about 99% identical to SEQ ID NO: 15. In some of embodiments, the epidermal growth factor receptor has a polypeptide sequence according to SEQ ID NO: 16. In other embodiments, the sequence of the epidermal growth factor receptor is at least about 75% to about 99% identical to SEQ ID NO: 16.
The keratinocyte growth factor receptor can be a human keratinocyte growth factor receptor. In some embodiments the keratinocyte growth factor receptor has a cDNA sequence according to SEQ ID NO: 17. In other embodiments, the keratinocyte growth factor receptor has a cDNA sequence that is about 75% to about 99% identical to SEQ ID NO: 17. In some embodiments, the human keratinocyte growth factor receptor has a polypeptide sequence according to SEQ ID NO: 18. In other embodiments, the sequence of the keratinocyte growth factor receptor is at least about 75% to about 99% identical to SEQ ID NO: 18.
The platelet derived growth factor receptor can be a human platelet derived growth factor receptor. In some of embodiments, the human platelet derived growth factor receptor has a cDNA sequence according to SEQ ID NO: 19. In other embodiments, the cDNA sequence of the platelet-derived growth factor receptor is at least about 75% to about 99% identical to SEQ ID NO: 19. In some embodiments, a platelet derived growth factor receptor polypeptide is encoded by a cDNA corresponding to SEQ ID NO: 19. In other embodiments, the platelet derived growth factor receptor is encoded by a cDNA sequence that is about 75% to about 99% identical to SEQ ID NO: 19. In other embodiments, the binding partner is not a growth factor receptor.
Cargo Molecules
The cargo carrying nanoparticle contains one or more cargo subunits that each contain a cargo molecule fused to an ELP. A single cargo carrying nanoparticle can contain a single type of cargo molecule, as in FIG. 1. In other embodiments, a single cargo carrying nanoparticle contains more than one type of cargo molecules. An example of this embodiment is shown in FIG. 2. The cargo molecule can be any polypeptide that can be fused to an ELP according to the methods described herein. In some embodiments, the cargo molecule is a polypeptide that can suppress, stimulate, or otherwise modify a cellular process, including but not limited to, glycolysis, ATP production, transcription, translation, growth, metabolism, catabolism, endocytosis, phagocytosis, pinocytosis, macropinocytosis, migration, malignant transformation, signal transduction, DNA modification, histone modification, mitosis, meiosis, DNA repair, senescense, and apoptosis.
The cargo molecule can be a lytic peptide, including but not limited to a mitochondrial lytic peptide. In an embodiment where the cargo molecule is a mitochondrial lytic peptide, the mitochondrial lytic peptide has a cDNA sequence according to SEQ ID NO: 22, which encodes a polypeptide having a sequence according to SEQ ID NO: 23. In some embodiments, the cDNA sequence of the mitochondrial lytic peptide is about 75% to about 99% identical to SEQ ID NO: 22. In other embodiments, the mitochondrial lytic peptide is about 75% to about 99% identical to SEQ ID NO: 23. Once internalized, the mitochondrial lytic peptides lyse the mitochondrial membrane, which results in cell death. Lytic peptides do not target specific pathways with in the cells. Therefore, this will not result in acquired resistance, unlike typical cancer therapies.
In other embodiments, the cargo molecule can be an inhibitor of glycolysis. In some embodiments, the glycolysis inhibitor inhibits hexokinase. In an embodiment, the hexokinase inhibitor has a cDNA sequence according to SEQ ID NO: 24, which encodes a polypeptide according to SEQ ID NO: 25. In some embodiments, the cDNA sequence of the hexokinase inhibitor is about 75% to about 99% identical to SEQ ID NO: 24. In other embodiments, the hexokinase inhibitor polypeptide is about 75% to about 99% identical to SEQ ID NO: 25.
In other embodiments, the glycolysis inhibitor inhibits phosphoglycerate mutase. In an embodiment the cDNA of the phosphoglycerate mutase inhibitor corresponds to SEQ ID NO: 26, which encodes a polypeptide according to SEQ ID NO: 27. In some embodiments, the cDNA sequence of the phosphoglycerate mutase inhibitor is about 75% to about 99% identical to SEQ ID NO: 26. In other embodiments, the phosphoglycerate mutase inhibitor polypeptide is about 75% to about 99% identical to SEQ ID NO: 27.
In other embodiments, the glycolysis inhibitor inhibits phosphofructokinase. In an embodiment the cDNA of the phosphofructokinase inhibitor corresponds to SEQ ID NO: 28, which encodes a polypeptide according to SEQ ID NO: 29. In some embodiments, the cDNA sequence of the phosphofructokinase inhibitor is about 75% to about 99% identical to SEQ ID NO: 28. In other embodiments, the phosphofructokinase inhibitor polypeptide is about 75% to about 99% identical to SEQ ID NO: 29.
In further embodiments, the cargo molecule can be DNA, RNA, virus particles and complete viruses, lipids (e.g. all forms of cholesterol, including but not limited to LDL cholesterol), bacteria, amino acids, peptides, polypeptides, antibodies, aptamers, ribozymes, guide sequences for ribozymes that inhibit translation or transcription of essential tumor proteins and genes, hormones, immunomodulators, antipyretics, anxiolytics, antipsychotics, analgesics, antispasmodics, anti-inflammatories, anti-histamines, anti-infectives, and chemotherapeutics.
Suitable hormones include, but are not limited to, amino-acid derived hormones (e.g. melatonin and thyroxine), small peptide hormones and protein hormones (e.g. thyrotropin-releasing hormone, vasopressin, insulin, growth hormone, luteinizing hormone, follicle-stimulating hormone, and thyroid-stimulating hormone), eiconsanoids (e.g. arachidonic acid, lipoxins, and prostaglandins), and steroid hormones (e.g. estradiol, testosterone, tetrahydro testosteron cortisol).
Suitable immunomodulators include, but are not limited to, prednisone, azathioprine, 6-MP, cyclosporine, tacrolimus, methotrexate, interleukins (e.g. IL-2, IL-7, and IL-12), cytokines (e.g. interferons (e.g. IFN-α, IFN-β, IFN-ε, IFN-κ, IFN-ω, and IFN-γ), granulocyte colony-stimulating factor, and imiquimod), chemokines (e.g. CCL3, CCL26 and CXCL7), cytosine phosphate-guanosine, oligodeoxynucleotides, glucans, antibodies, and aptamers).
Suitable antipyretics include, but are not limited to, non-steroidal anti-inflammants (e.g. ibuprofen, naproxen, ketoprofen, and nimesulide), aspirin and related salicylates (e.g. choline salicylate, magnesium salicylae, and sodium salicaylate), paracetamol/acetaminophen, metamizole, nabumetone, phenazone, and quinine.
Suitable anxiolytics include, but are not limited to, benzodiazepines (e.g. alprazolam, bromazepam, chlordiazepoxide, clonazepam, clorazepate, diazepam, flurazepam, lorazepam, oxazepam, temazepam, triazolam, and tofisopam), serotenergic antidepressants (e.g. selective serotonin reuptake inhibitors, tricyclic antidepressants, and monoamine oxidase inhibitors), mebicar, afobazole, selank, bromantane, emoxypine, azapirones, barbituates, hyxdroxyzine, pregabalin, validol, and beta blockers.
Suitable antipsychotics include, but are not limited to, benperidol, bromoperidol, droperidol, haloperidol, moperone, pipaperone, timiperone, fluspirilene, penfluridol, pimozide, acepromazine, chlorpromazine, cyamemazine, dizyrazine, fluphenazine, levomepromazine, mesoridazine, perazine, pericyazine, perphenazine, pipotiazine, prochlorperazine, promazine, promethazine, prothipendyl, thioproperazine, thioridazine, trifluoperazine, triflupromazine, chlorprothixene, clopenthixol, flupentixol, tiotixene, zuclopenthixol, clotiapine, loxapine, prothipendyl, carpipramine, clocapramine, molindone, mosapramine, sulpiride, veralipride, amisulpride, amoxapine, aripiprazole, asenapine, clozapine, blonanserin, iloperidone, lurasidone, melperone, nemonapride, olanzaprine, paliperidone, perospirone, quetiapine, remoxipride, risperidone, sertindole, trimipramine, ziprasidone, zotepine, alstonie, befeprunox, bitopertin, brexpiprazole, cannabidiol, cariprazine, pimavanserin, pomaglumetad methionil, vabicaserin, xanomeline, and zicronapine.
Suitable analgesics include, but are not limited to, paracetamol/acetaminophen, non-steroidal anti-inflammants (e.g. ibuprofen, naproxen, ketoprofen, and nimesulide), COX-2 inhibitors (e.g. rofecoxib, celecoxib, and etoricoxib), opioids (e.g. morphine, codeine, oxycodone, hydrocodone, dihydromorphine, pethidine, buprenorphine), tramadol, norepinephrine, flupiretine, nefopam, orphenadrine, pregabalin, gabapentin, cyclobenzaprine, scopolamine, methadone, ketobemidone, piritramide, and aspirin and related salicylates (e.g. choline salicylate, magnesium salicylae, and sodium salicaylate).
Suitable antispasmodics include, but are not limited to, mebeverine, papverine, cyclobenzaprine, carisoprodol, orphenadrine, tizanidine, metaxalone, methodcarbamol, chlorzoxazone, baclofen, dantrolene, baclofen, tizanidine, and dantrolene.
Suitable anti-inflammatories include, but are not limited to, prednisone, non-steroidal anti-inflammants (e.g. ibuprofen, naproxen, ketoprofen, and nimesulide), COX-2 inhibitors (e.g. rofecoxib, celecoxib, and etoricoxib), and immune selective anti-inflammatory derivatives (e.g. submandibular gland peptide-T and its derivatives).
Suitable anti-histamines include, but are not limited to, H1-receptor antagonists (e.g. acrivastine, azelastine, bilastine, brompheniramine, buclizine, bromodiphenhydramine, carbinoxamine, cetirizine, chlorpromazine, cyclizine, chlorpheniramine, clemastine, cyproheptadine, desloratadine, dexbromapheniramine, dexchlorpheniramine, dimenhydrinate, dimetindene, diphenhydramine, doxylamine, ebasine, embramine, fexofenadine, hydroxyzine, levocetirzine, loratadine, meclozine, mirtazapine, olopatadine, orphenadrine, phenindamine, pheniramine, phenyltoloxamine, promethazine, pyrilamine, quetiapine, rupatadine, tripelennamine, and triprolidine), H2-receptor antagonists (e.g. cimetidine, famotidine, lafutidine, nizatidine, rafitidine, and roxatidine), tritoqualine, catechin, cromoglicate, nedocromil, and β2-adrenergic agonists.
Suitable anti-infectives include, but are not limited to, amebicides (e.g. nitazoxanide, paromomycin, metronidazole, tnidazole, chloroquine, and iodoquinol), aminoglycosides (e.g. paromomycin, tobramycin, gentamicin, amikacin, kanamycin, and neomycin), anthelmintics (e.g. pyrantel, mebendazole, ivermectin, praziquantel, abendazole, miltefosine, thiabendazole, oxamniquine), antifungals (e.g. azole antifungals (e.g. itraconazole, fluconazole, posaconazole, ketoconazole, clotrimazole, miconazole, and voriconazole), echinocandins (e.g. caspofungin, anidulafungin, and micafungin), griseofulvin, terbinafine, flucytosine, and polyenes (e.g. nystatin, and amphotericin b), antimalarial agents (e.g. pyrimethamine/sulfadoxine, artemether/lumefantrine, atovaquone/proquanil, quinine, hydroxychloroquine, mefloquine, chloroquine, doxycycline, pyrimethamine, and halofantrine), antituberculosis agents (e.g. aminosalicylates (e.g. aminosalicylic acid), isoniazid/rifampin, isoniazid/pyrazinamide/rifampin, bedaquiline, isoniazid, ethanmbutol, rifampin, rifabutin, rifapentine, capreomycin, and cycloserine), antivirals (e.g. amantadine, rimantadine, abacavir/lamivudine, emtricitabine/tenofovir, cobicistat/elvitegravir/emtricitabine/tenofovir, efavirenz/emtricitabine/tenofovir, avacavir/lamivudine/zidovudine, lamivudine/zidovudine, emtricitabine/tenofovir, emtricitabine/opinavir/ritonavir/tenofovir, interferon alfa-2v/ribavirin, peginterferon alfa-2b, maraviroc, raltegravir, dolutegravir, enfuvirtide, foscarnet, fomivirsen, oseltamivir, zanamivir, nevirapine, efavirenz, etravirine, rilpiviirine, delaviridine, nevirapine, entecavir, lamivudine, adefovir, sofosbuvir, didanosine, tenofovir, avacivr, zidovudine, stavudine, emtricitabine, xalcitabine, telbivudine, simeprevir, boceprevir, telaprevir, lopinavir/ritonavir, fosamprenvir, dranuavir, ritonavir, tipranavir, atazanavir, nelfinavir, amprenavir, indinavir, sawuinavir, ribavirin, valcyclovir, acyclovir, famciclovir, ganciclovir, and valganciclovir), carbapenems (e.g. doripenem, meropenem, ertapenem, and cilastatin/imipenem), cephalosporins (e.g. cefadroxil, cephradine, cefazolin, cephalexin, cefepime, ceflaroline, loracarbef, cefotetan, cefuroxime, cefprozil, loracarbef, cefoxitin, cefaclor, ceftibuten, ceftriaxone, cefotaxime, cefpodoxime, cefdinir, cefixime, cefditoren, cefizoxime, and ceftazidime), glycopeptide antibiotics (e.g. vancomycin, dalbavancin, oritavancin, and telvancin), glycylcyclines (e.g. tigecycline), leprostatics (e.g. clofazimine and thalidomide), lincomycin and derivatives thereof (e.g. clindamycin and lincomycin), macrolides and derivatives thereof (e.g. telithromycin, fidaxomicin, erthromycin, azithromycin, clarithromycin, dirithromycin, and troleandomycin), linezolid, sulfamethoxazole/trimethoprim, rifaximin, chloramphenicol, fosfomycin, metronidazole, aztreonam, bacitracin, penicillins (amoxicillin, ampicillin, bacampicillin, carbenicillin, piperacillin, ticarcillin, amoxicillin/clavulanate, ampicillin/sulbactam, piperacillin/tazobactam, clavulanate/ticarcillin, penicillin, procaine penicillin, oxaxillin, dicloxacillin, and nafcillin), quinolones (e.g. lomefloxacin, norfloxacin, ofloxacin, qatifloxacin, moxifloxacin, ciprofloxacin, levofloxacin, gemifloxacin, moxifloxacin, cinoxacin, nalidixic acid, enoxacin, grepafloxacin, gatifloxacin, trovafloxacin, and sparfloxacin), sulfonamides (e.g. sulfamethoxazole/trimethoprim, sulfasalazine, and sulfasoxazole), tetracyclines (e.g. doxycycline, demeclocycline, minocycline, doxycycline/salicyclic acid, doxycycline/omega-3 polyunsaturated fatty acids, and tetracycline), and urinary anti-infectives (e.g. nitrofurantoin, methenamine, fosfomycin, cinoxacin, nalidixic acid, trimethoprim, and methylene blue).
Suitable chemotherapeutics include, but are not limited to, paclitaxel, brentuximab vedotin, doxorubicin, 5-FU (fluorouracil), everolimus, pemetrexed, melphalan, pamidronate, anastrozole, exemestane, nelarabine, ofatumumab, bevacizumab, belinostat, tositumomab, carmustine, bleomycin, bosutinib, busulfan, alemtuzumab, irinotecan, vandetanib, bicalutamide, lomustine, daunorubicin, clofarabine, cabozantinib, dactinomycin, ramucirumab, cytarabine, cytoxan, cyclophosphamide, decitabine, dexamethasone, docetaxel, hydroxyurea, decarbazine, leuprolide, epirubicin, oxaliplatin, asparaginase, estramustine, cetuximab, vismodegib, aspargainase erwinia chyrsanthemi, amifostine, etoposide, flutamide, toremifene, fulvestrant, letrozole, degarelix, pralatrexate, methotrexate, floxuridine, obinutuzumab, gemcitabine, afatinib, imatinib mesylatem, carmustine, eribulin, trastuzumab, altretamine, topotecan, ponatinib, idarubicin, ifosfamide, ibrutinib, axitinib, interferon alfa-2a, gefitinib, romidepsin, ixabepilone, ruxolitinib, cabazitaxel, ado-trastuzumab emtansine, carfilzomib, chlorambucil, sargramostim, cladribine, mitotane, vincristine, procarbazine, megestrol, trametinib, mesna, strontium-89 chloride, mechlorethamine, mitomycin, busulfan, gemtuzumab ozogamicin, vinorelbine, filgrastim, pegfilgrastim, sorafenib, nilutamide, pentostatin, tamoxifen, mitoxantrone, pegaspargase, denileukin diftitox, alitretinoin, carboplatin, pertuzumab, cisplatin, pomalidomide, prednisone, aldesleukin, mercaptopurine, zoledronic acid, lenalidomide, rituximab, octretide, dasatinib, regorafenib, histrelin, sunitinib, siltuximab, omacetaxine, thioguanine (tioguanine), dabrafenib, erlotinib, bexarotene, temozolomide, thiotepa, thalidomide, BCG, temsirolimus, bendamustine hydrochloride, triptorelin, aresnic trioxide, lapatinib, valrubicin, panitumumab, vinblastine, bortezomib, tretinoin, azacitidine, pazopanib, teniposide, leucovorin, crizotinib, capecitabine, enzalutamide, ipilimumab, goserelin, vorinostat, idelalisib, ceritinib, abiraterone, epothilone, tafluposide, azathioprine, doxifluridine, vindesine, and all-trans retinoic acid.
In other embodiments, the cargo carrying nanoparticles contain one or more cargo binding peptide sequences. The cargo binding peptide sequences can bind to the cargo molecule and assist in its cellular internalization. For example, the cholesterol binding sequence, LDLR-3, can be fused or operatively linked with ELP and thus cholesterol molecules can be loaded in the nanoparticles via binding to the LDLR-3 sequence leading to the uptake of cholesterol. In some embodiments, LDL-R 3 has a cDNA sequence according to SEQ ID NO: 20. In other embodiments, LDL-3 has a cDNA sequence that is about 90 to about 99% identical to SEQ ID NO: 20. In some embodiments, LDL-3 has a polypeptide sequence according to SEQ ID NO: 21. In other embodiments, LDL-3 has a polypeptide sequences that is about 90% to about 99% identical to SEQ ID NO: 21.
Methods of Making Cargo Carrying Nano Particles
The cargo subunits and the targeting subunits can be made using recombinant DNA techniques that result in fusion of the ELP polypeptide to a cargo molecule polypeptide or a targeting moiety. In other embodiments, the cargo subunits and targeting subunits are chemically synthesized de novo, using techniques and methods generally known to those skilled in the art.
Vectors
In some embodiments, a nucleotide sequence encoding a cargo molecule or a targeting moiety is operatively linked (e.g. cloned in frame) with a nucleotide sequence encoding an ELP in a vector. In some embodiments, a linker molecule is inserted between the nucleotide sequence encoding the targeting moiety or cargo molecule and the ELP. In some embodiments, the nucleotide sequence also includes regulatory sequences (e.g. promoters) in addition to those already present in the vector. In some embodiments, the nucleotide sequence is chemically synthesized de novo using techniques generally known to those skilled in the art. In other embodiments, recombinant DNA engineering techniques are used to generate the nucleotide sequence. In some embodiments, the nucleotide sequence encoding a cargo molecule operatively linked to an ELP corresponds to SEQ ID NO: 30, which encodes a polypeptide according to SEQ ID NO: 31. In other embodiments, the nucleotide sequence encoding a cargo molecule operatively linked to an ELP is about 75% to about 99% identical to SEQ ID NO: 30. In other embodiments, the cargo molecule cDNA corresponds to any of SEQ ID NOs: 24, 26, or 28, or cDNA sequences about 75% to about 99% identical to SEQ ID NOs: 24, 26, or 28.
In some embodiments, the nucleotide encoding a targeting moiety operatively linked to an ELP corresponds to SEQ ID NO: 32, which encodes a polypeptide according to SEQ ID NO: 33. In other embodiments, the nucleotide encoding a targeting moiety operatively linked to an ELP corresponds to SEQ ID NO: 34, which encodes a polypeptide according to SEQ ID NO: 35. In other embodiments, the nucleotide sequence encoding a targeting moiety operatively linked to an ELP is about 75% to about 99% identical to SEQ ID NO: 34. In other embodiments, the targeting moiety cDNA corresponds SEQ ID NO: 14, or cDNA sequences about 75% to about 99% identical to SEQ ID NO: 14.
In some embodiments, a single DNA vector contains multiple nucleotide sequences that each encode a different polypeptide subunit of the cargo carrying nanoparticle. For example, a single plasmid can contain a first nucleotide sequence that encodes a targeting subunit and a second nucleotide sequence that is operatively linked to the first nucleotide sequence, wherein the second nucleotide sequence encodes a first cargo subunit. In other embodiments, the vector can contain a third nucleotide sequence encoding a second cargo subunit, wherein the third nucleotide sequence is operatively linked to the first nucleotide sequence and/or the second nucleotide sequence. In further embodiments, a single vector can contain 1 to 50 more additional nucleotide sequences encoding additional targeting and/or cargo subunits
In some embodiments the DNA vector is a suitable bacterial expression vector. In other embodiments, the plasmid vector is a suitable eukaryotic expression vector. Suitable bacterial and eukaryotic expression vectors are generally known in the art. In other embodiments, the nucleotides encoding the cargo subunit or targeting subunit can be synthetically generated using de novo nucleotide synthesis techniques generally known in the art. The de novo generated subunits can be inserted into a plasmid vector using techniques generally known in the art. In some embodiments the plasmid vector is a suitable bacterial expression vector. In other embodiments, the plasmid vector is a suitable eukaryotic expression vector. Suitable bacterial and eukaryotic expression vectors are generally known in the art. Suitable bacterial expression cells include, but are not limited to, PET25b+, BLR, and BLR(DE3) cells.
The nucleotides encoding the cargo subunit and/or the targeting subunit, if not already present in an expression vector, can be cloned into a suitable expression plasmid vector. In some embodiments the plasmid vector is a suitable bacterial expression vector. In other embodiments, the plasmid vector is a suitable eukaryotic expression vector. Suitable bacterial and eukaryotic expression vectors are generally known in the art.
The vectors can include any suitable regulatory elements (e.g. promoter sequences) or other nucleotide sequences which allow for, inter alia, replication, increased stability, selection (such as antibiotic resistance genes), and inducible expression.
Vector Expression
In some embodiments, bacterial cells are transformed with the plasmid expression vectors containing the nucleotides encoding the cargo subunit and/or the targeting subunit using techniques generally known in the art, such as but not limited to electroporation. Transformed bacteria can be selected for using generally known techniques and grown for an amount of time in a media at a suitable temperature to allow expression of the cargo subunit and/or targeting subunit in the bacterial cells. The amount of time the transformed bacteria are grown for can range from about 4 to about 24 hours. One of ordinary skill in the art will appreciate that the time for growth will vary, inter alia, according to the bacterial strain used, temperature of growth, the subunit being expressed, the expression vector used, and the media employed. The temperature can vary from about 25° C. to about 37° C. In an embodiment, the bacteria are cultured at 37° C. One of ordinary skill in the art will appreciate that temperature will vary, inter alia, according to the bacterial strain used, the desired growth rate, the subunit being expressed, the expression vector used, and the media employed. Suitable bacterial strains include, but are not limited to, variants of Escherichia coli that have been optimized for protein expression and purification and transformation. These are commercially available. In this way, the transformed bacteria express and nucleotide sequence in the plasmid expression vector and generate the cargo subunit and/or targeting subunit polypeptides. Suitable bacterial strains have previously been described herein.
In other embodiments, the cargo subunit and/or targeting subunit polypeptides are produced in a eukaryotic expression system. In these embodiments, the plasmid expression vector containing the nucleotide sequence encoding the cargo subunit/and or the targeting subunit is introduced using generally know techniques, such as transformation using retroviral particles or transfection using cationic liposomes, into an appropriate eukaryotic cell, such as but not limited to yeast. Other cell types will be appreciated by one of ordinary skill in the art. Suitable expression systems are generally known in the art, are commercially available, and include but are not limited to, Chinese hamster ovary cells. Transformed/transfected eukaryotic cells are grown using techniques generally known in the art to express the peptide sequence in the plasmid expression vector and generate the cargo subunit and/or targeting subunit polypeptides.
Polypeptide Purification and Evaluation
Once the cells have generated the subunit polypeptides, the subunit polypeptides are purified from the cell culture. Suitable polypeptide purification methods are generally known in the art. In some embodiments, the subunit polypeptides include a molecular tag, such as antibody epitope or metal binding site, which allows for affinity purification of the subunits. Such molecular tags are generally known in the art (Terpe, K. 2003. Appl. Microbiol. Biotechnol. 60:523-533) and include but are not limited to, FLAG tag, poly-His tag, and a poly-Arg tag. In these embodiments, the subunits can be affinity purified. In other embodiments, the cells in the cell culture are lysed using an appropriate method. Lysis can be performed using a physical method, such as sonication or using chemical lysis process, such as by using detergents. One of ordinary skill will appreciate that optimization of cell lysis will depend on, inter alia, the culture system used and the polypeptide to be purified. After cells have been lysed, cells can be fractionated and organelles can be isolated using techniques generally known in the art. Fractionation and/or organelle isolation can be obtained using physical disruption techniques and/or detergent-buffer solutions and density gradient methods. Optimization and utilization of tools generally known in the art can be used to obtain membrane bound proteins, cytosolic proteins, intact cell nuclei, mitochondria and other organelles.
In some embodiments, protease inhibitors, phosphatase inhibitors and protein stabilizers are added during the purification process to prevent and limit subunit degradation during the process. Such inhibitors are generally known in the art and are commercially available. In some embodiments, lysis and purification is conducted at cold temperatures (about 4° C. to about 8° C.) to minimize polypeptide degradation. In some instances, particularly where correct folding of a portion or all of the subunit are important reagents used during the lysis/fractionation or other compounds, such as salt, can be removed via dialysis. In some cases, dialysis of these reagents and/or compounds results in protein re-folding. In other instances, other buffers or reagents can be added to the purified protein to stimulate proper folding of the purified polypeptide.
After the subunit polypeptides are purified, physical properties of the purified polypeptides can be evaluated using suitable methods, such as but not limited to, spectrophotometric analysis or 1-D or 2-D gel electrophoresis followed by protein detection (gel staining (e.g. Coomassie staining, Western blotting, or mass-spectrometry). The function of the purified subunit polypeptides can be evaluated by a suitable method. For example, if the polypeptide contains a cargo molecule that is a mitochondrial lytic peptide, the functional activity of the purified subunit polypeptide can be evaluated by contacting a cell with the purified subunit polypeptide and evaluate cell death. After the subunits are allowed to spontaneously form the cargo-carrying nanoparticles, uptake of the cargo carrying nanoparticles can be analyzed using a suitable technique, including but not limited to flow cytometry and transmission electron microscopy.
Pharmaceutical Formulations Containing Cargo Carrying Nanoparticles
The cargo carrying nanoparticles described herein can be provided to a subject as an ingredient, such as an active ingredient, in a pharmaceutical formulation. As such, also described are pharmaceutical formulations containing one or more of the cargo carrying nanoparticles described herein. In some embodiments, the pharmaceutical formulations contain an effective amount of the cargo carrying nanoparticles described herein. The pharmaceutical formulations can be administered to a subject in need thereof. In some embodiments, the subject has cancer. In some embodiments, the cancer is a lung cancer. In further embodiments, the cargo carrying nanoparticles described herein are used in the manufacture of a medicament for the treatment of cancer.
The pharmaceutical formulations can contain a homogenous population of cargo carrying nanoparticles. In these embodiments, the cargo carrying nanoparticles are made of the same type of subunits as one another. For example, all cargo carrying nanoparticles in the pharmaceutical formulation contains one or more targeting subunits growth factor targeting moiety and one or more cargo subunits containing a glycolysis inhibitor peptide.
In other embodiments, the pharmaceutical formulations can contain a heterogeneous population of cargo carrying nanoparticles. In some of these embodiments, the cargo molecule can vary from nanoparticle to nanoparticle. For example, some cargo carrying nanoparticles in the pharmaceutical formulation contain one or more targeting subunits containing a growth factor targeting moiety and one or more cargo subunits containing a glycolysis inhibitor peptide and other cargo carrying nanoparticles contain one or more targeting subunits containing a growth factor targeting moiety and one or more cargo subunits containing a mitochondrial lytic peptide. In other embodiments, the targeting moiety varies between nanoparticles. For example, some cargo carrying nanoparticles contain one or more targeting subunits containing an epidermal growth factor targeting moiety and one or more cargo subunits containing a glycolysis inhibitor, while other cargo carrying nanoparticles in the population contain one or more targeting subunits having a platelet derived growth factor targeting moiety and one or more cargo subunits containing a glycolysis inhibitor. In yet further embodiments, the cargo carrying some of the cargo carrying nanoparticles contain different cargo subunits and targeting subunits than at least one other cargo carrying nanoparticles in the population.
Pharmaceutically Acceptable Carriers and Auxiliary Ingredients and Agents
The pharmaceutical formulations containing an effective amount of the cargo carrying nanoparticles described herein can further include a pharmaceutically acceptable carrier. Suitable pharmaceutically acceptable carriers include, but are not limited to water, salt solutions, alcohols, gum arabic, vegetable oils, benzyl alcohols, polyethylene glycols, gelatin, carbohydrates such as lactose, amylose or starch, magnesium stearate, talc, silicic acid, viscous paraffin, perfume oil, fatty acid esters, hydroxy methylcellulose, and polyvinyl pyrrolidone, which do not deleteriously react with the active composition.
The pharmaceutical formulations can be sterilized, and if desired, mixed with auxiliary agents, such as lubricants, preservatives, stabilizers, wetting agents, emulsifiers, salts for influencing osmotic pressure, buffers, coloring, flavoring and/or aromatic substances, and the like which do not deleteriously react with the active composition.
In addition to the effective amount of the cargo carrying nanoparticles, the pharmaceutical formulation can also include an effective amount of auxiliary active agents, including but not limited to, antisense or RNA interference molecules, traditional chemotherapeutics, antineoplasic agents, immunomodulating compounds, hormones, antibiotics, antivirals, and/or antibodies or fragments thereof
Effective Amounts of the Cargo Carrying Nanoparticles and Auxiliary Agents
As the subunits spontaneously form into nanoparticles, the effective amount is based on an amount or concentration of the subunits present. In some embodiments, the effective amount is based on the amount or concentration of a cargo subunit present in the formulation. In some of these embodiments, the effective amount ranges from about 0.001 pg of cargo subunit to about 1,000 μg of cargo subunit. In other embodiments, the concentration of the cargo subunit in the effective amount ranges from about 1 μM to about 1,000 μM.
In other embodiments, the effective amount is based on the amount or concentration of a targeting subunit present. In some of these embodiments, the effective amount ranges from about 0.001 pg of cargo subunit to about 1,000 μg of targeting subunit. In other embodiments, the concentration of the targeting subunit in the effective amount ranges from about 1 μM to about 1,000 μM.
In further embodiments, the effective amount is based on the amount or concentration of all subunits present in the formulation. In some of these embodiments, the effective amount ranges from about 0.001 pg of the total subunits to about 1,000 μg of the total subunits. In other embodiments, the concentration of the subunits in the effective amount ranges from about 1 μM to about 1,000 μM.
In embodiments where there is an auxiliary active agent contained in the pharmaceutical formulation in addition to the cargo carrying nanoparticles, the effective amount of the auxiliary active agent will vary depending on the auxiliary active agent. In some embodiments, the effective amount of the auxiliary active agent ranges from 0.001 micrograms to about 1000 grams. In other embodiments, the effective amount of the auxiliary active agent ranges from about 0.01 IU to about 1000 IU. In further embodiments, the effective amount of the auxiliary active agent ranges from 0.001 mL to about 1000 mL. In yet other embodiments, the effective amount of the auxiliary active agent ranges from about 1% w/w to about 50% w/w of the total pharmaceutical formulation. In additional embodiments, the effective amount of the auxiliary active agent ranges from about 1% v/v to about 50% v/v of the total pharmaceutical formulation. In still other embodiments, the effective amount of the auxiliary active agent ranges from about 1% w/v to about 50% w/v of the total pharmaceutical formulation.
The auxiliary active agent can be included in the pharmaceutical formulation or can exist as a stand-alone compound or pharmaceutical formulation that is administered contemporaneously or sequentially with the conjugate compound, derivative thereof or pharmaceutical formulation thereof. In embodiments where the auxiliary active agent is a stand-alone compound or pharmaceutical formulation, the effective amount of the auxiliary active agent can vary depending on the auxiliary active agent used. In some of these embodiments, the effective amount of the auxiliary active agent ranges from 0.001 micrograms to about 1000 grams. In other embodiments, the effective amount of the auxiliary active agent ranges from about 0.01 IU to about 1000 IU. In further embodiments, the effective amount of the auxiliary active agent ranges from 0.001 mL to about 1000 mL. In yet other embodiments, the effective amount of the auxiliary active agent ranges from about 1% w/w to about 50% w/w of the total auxiliary active agent pharmaceutical formulation. In additional embodiments, the effective amount of the auxiliary active agent ranges from about 1% v/v to about 50% v/v of the total pharmaceutical formulation. In still other embodiments, the effective amount of the auxiliary active agent ranges from about 1% w/v to about 50% w/v of the total auxiliary agent pharmaceutical formulation.
Dosage Forms
In some embodiments, the pharmaceutical formulations described herein may be in a dosage form. The dosage forms can be adapted for administration by any appropriate route. Appropriate routes include, but are not limited to, oral (including buccal or sublingual), rectal, intraocular, inhaled, intranasal, topical (including buccal, sublingual, or transdermal), vaginal, parenteral, subcutaneous, intramuscular, intravenous, and intradermal. Such formulations may be prepared by any method known in the art.
Dosage forms adapted for oral administration can discrete dosage units such as capsules, pellets or tablets, powders or granules, solutions, or suspensions in aqueous or non-aqueous liquids; edible foams or whips, or in oil-in-water liquid emulsions or water-in-oil liquid emulsions. In some embodiments, the pharmaceutical formulations adapted for oral administration also include one or more agents which flavor, preserve, color, or help disperse the pharmaceutical formulation. Dosage forms prepared for oral administration can also be in the form of a liquid solution that can be delivered as a foam, spray, or liquid solution. The oral dosage form can be administered to a subject in need thereof. In some embodiments, this is a subject having cancer. In some embodiments, the cancer is a lung cancer.
Where appropriate, the dosage forms described herein can be microencapsulated. The dosage form can also be prepared to prolong or sustain the release of any ingredient. In some embodiments, the conjugate compound or derivative thereof is the ingredient whose release is delayed. In other embodiments, the release of an auxiliary ingredient is delayed. Suitable methods for delaying the release of an ingredient include, but are not limited to, coating or embedding the ingredients in material in polymers, wax, gels, and the like. Delayed release dosage formulations can be prepared as described in standard references such as “Pharmaceutical dosage form tablets,” eds. Liberman et. al. (New York, Marcel Dekker, Inc., 1989), “Remington—The science and practice of pharmacy”, 20th ed., Lippincott Williams & Wilkins, Baltimore, Md., 2000, and “Pharmaceutical dosage forms and drug delivery systems”, 6th Edition, Ansel et al., (Media, Pa.: Williams and Wilkins, 1995). These references provide information on excipients, materials, equipment, and processes for preparing tablets and capsules and delayed release dosage forms of tablets and pellets, capsules, and granules. The delayed release can be anywhere from about an hour to about 3 months or more.
Examples of suitable coating materials include, but are not limited to, cellulose polymers such as cellulose acetate phthalate, hydroxypropyl cellulose, hydroxypropyl methylcellulose, hydroxypropyl methylcellulose phthalate, and hydroxypropyl methylcellulose acetate succinate; polyvinyl acetate phthalate, acrylic acid polymers and copolymers, and methacrylic resins that are commercially available under the trade name EUDRAGIT® (Roth Pharma, Westerstadt, Germany), zein, shellac, and polysaccharides
Coatings may be formed with a different ratio of water soluble polymer, water insoluble polymers, and/or pH dependent polymers, with or without water insoluble/water soluble non polymeric excipient, to produce the desired release profile. The coating is either performed on the dosage form (matrix or simple) which includes, but is not limited to, tablets (compressed with or without coated beads), capsules (with or without coated beads), beads, particle compositions, “ingredient as is” formulated as, but not limited to, suspension form or as a sprinkle dosage form.
Dosage forms adapted for topical administration can be formulated as ointments, creams, suspensions, lotions, powders, solutions, pastes, gels, sprays, aerosols, or oils. In some embodiments for treatments of the eye or other external tissues, for example the mouth or the skin, the pharmaceutical formulations are applied as a topical ointment or cream. When formulated in an ointment, the conjugate compound, derivative thereof, auxiliary active ingredient, and/or pharmaceutically acceptable salt thereof can be formulated with a parafinnic or water-misicible ointment base. In other embodiments, the active ingredient can be formulated in a cream with an oil-in-water cream base or a water-in-oil base. Dosage forms adapted for topical administration in the mouth include lozenges, pastilles, and mouth washes.
Dosage forms adapted for nasal or inhalation administration include aerosols, solutions, suspension drops, gels, or dry powders. In some embodiments, the conjugate compound, derivative thereof, auxiliary active ingredient, and/or pharmaceutically acceptable salt thereof in a dosage form adapted for inhalation is in a particle-size-reduced form that is obtained or obtainable by micronization. In some embodiments, the particle size of the size reduced (e.g. micronized) compound or salt or solvate thereof, is defined by a D50 value of about 0.5 to about 10 microns as measured by an appropriate method known in the art. Dosage forms adapted for administration by inhalation also include particle dusts or mists. Suitable dosage forms wherein the carrier or excipient is a liquid for administration as a nasal spray or drops include aqueous or oil solutions/suspensions of an active ingredient, which may be generated by various types of metered dose pressurized aerosols, nebulizers, or insufflators.
In some embodiments, the dosage forms are aerosol formulations suitable for administration by inhalation. In some of these embodiments, the aerosol formulation contains a solution or fine suspension of a conjugate compound, derivative thereof, auxiliary active ingredient, and/or pharmaceutically acceptable salt thereof a pharmaceutically acceptable aqueous or non-aqueous solvent. Aerosol formulations can be presented in single or multi-dose quantities in sterile form in a sealed container. For some of these embodiments, the sealed container is a single dose or multi-dose nasal or an aerosol dispenser fitted with a metering valve (e.g. metered dose inhaler), which is intended for disposal once the contents of the container have been exhausted.
Where the aerosol dosage form is contained in an aerosol dispenser, the dispenser contains a suitable propellant under pressure, such as compressed air, carbon dioxide, or an organic propellant, including but not limited to a hydrofluorocarbon. The aerosol formulation dosage forms in other embodiments are contained in a pump-atomizer. The pressurized aerosol formulation can also contain a solution or a suspension of a conjugate compound, derivative thereof, auxiliary active ingredient, and/or pharmaceutically acceptable salt thereof. In further embodiments, the aerosol formulation also contains co-solvents and/or modifiers incorporated to improve, for example, the stability and/or taste and/or fine particle mass characteristics (amount and/or profile) of the formulation. Administration of the aerosol formulation can be once daily or several times daily, for example 2, 3, 4, or 8 times daily, in which 1, 2, or 3 doses are delivered each time.
For some dosage forms suitable and/or adapted for inhaled administration, the pharmaceutical formulation is a dry powder inhalable formulations. In addition to the conjugate compound, derivative thereof, auxiliary active ingredient, and/or pharmaceutically acceptable salt thereof, such a dosage form can contain a powder base such as lactose, glucose, trehalose, manitol, and/or starch. In some of these embodiments, the conjugate compound, derivative thereof, auxiliary active ingredient, and/or pharmaceutically acceptable salt thereof is in a particle-size reduced form. In further embodiments, a performance modifier, such as L-leucine or another amino acid, cellobiose octaacetate, and/or metals salts of stearic acid, such as magnesium or calcium stearate.
In some embodiments, the aerosol formulations are arranged so that each metered dose of aerosol contains a predetermined amount of an active ingredient, such as the one or more of the compounds described herein.
Dosage forms adapted for vaginal administration can be presented as pessaries, tampons, creams, gels, pastes, foams, or spray formulations. Dosage forms adapted for rectal administration include suppositories or enemas.
Dosage forms adapted for parenteral administration and/or adapted for injection can include aqueous and/or non-aqueous sterile injection solutions, which can contain anti-oxidants, buffers, bacteriostats, solutes that render the composition isotonic with the blood of the subject, and aqueous and non-aqueous sterile suspensions, which can include suspending agents and thickening agents. The dosage forms adapted for parenteral administration can be presented in a single-unit dose or multi-unit dose containers, including but not limited to sealed ampoules or vials. The doses can be lyophilized and resuspended in a sterile carrier to reconstitute the dose prior to administration. Extemporaneous injection solutions and suspensions can be prepared in some embodiments, from sterile powders, granules, and tablets.
For some embodiments, the dosage form contains a predetermined amount of a conjugate compound per unit dose. In an embodiment, the predetermined amount of the conjugate compound is an effective amount of the conjugate compound to treat, prevent, or mitigate the symptoms of multiple myeloma. In other embodiments, the predetermined amount of the conjugate compound is an appropriate fraction of the effective amount of the active ingredient. Such unit doses may therefore be administered once or more than once a day. Such pharmaceutical formulations may be prepared by any of the methods well known in the art.
Use of Cargo Carrying Nanoparticles
The cargo carrying nanoparticles described herein can be used for intracellular delivery of a polypeptide or other small molecules, including but not limited to, drugs, plasmids, genes etc. In some embodiments, a cell is contacted with one or more of the cargo carrying gene nanoparticles having a cargo molecule corresponding to a polypeptide desired to be delivered to a cell or containing a polypeptide that binds to specific cargo molecules such as plasmids or small molecules, including but not limited to cholesterol. In this way, targeted intracellular delivery of a polypeptide or other cargo molecules can be achieved.
Any amount of the cargo carrying nanoparticles or pharmaceutical formulations thereof, described herein can be administered to a subject in need thereof one or more times per day, week, month, or year. In some embodiments, the subject has one or more symptoms of a disease, condition, or syndrome. In some of these embodiments, the disease, condition, or syndrome is cancer. In some embodiments, the cancer is lung cancer. In some embodiments, the amount administered is the effective amount of the cargo carrying nanoparticles or pharmaceutical formulations thereof. For example, the cargo carrying nanoparticles or pharmaceutical formulations thereof, can be administered in a daily dose. This amount may be given in a single dose per day. In other embodiments, the daily dose may be administered over multiple doses per day, in which each containing a fraction of the total daily dose to be administered (sub-doses). In some embodiments, the amount of doses delivered per day is 2, 3, 4, 5, or 6. In further embodiments, the compounds, formulations, or salts thereof are administered one or more times per week, such as 1, 2, 3, 4, 5, or 6 times per week. In other embodiments, the cargo carrying nanoparticles or pharmaceutical formulations thereof are administered one or more times per month, such as 1 to 5 times per month. In still further embodiments, the cargo carrying nanoparticles or pharmaceutical formulations thereof are administered one or more times per year, such as 1 to 11 times per year.
In embodiments where more than one of the cargo carrying nanoparticles or pharmaceutical formulations thereof, and/or auxiliary agents are administered sequentially; the sequential administration may be close in time or remote in time. For example, administration of the second the cargo carrying nanoparticles or pharmaceutical formulations thereof can occur within seconds or minutes (up to about 1 hour) after administration of the first agent (close in time). In other embodiments, administration of the second the cargo carrying nanoparticles or pharmaceutical formulations thereof occurs at some other time that is more than an hour after administration of the first the cargo carrying nanoparticles or pharmaceutical formulations thereof.
The amount of the cargo carrying nanoparticles or pharmaceutical formulations thereof described herein can be administered in an amount ranging from about 0.01 mg to about 1000 mg per day, as calculated as the free or unsalted pharmaceutical formulations. The amount of the cargo carrying nanoparticles or pharmaceutical formulations thereof described herein can be administered in an amount ranging from about 0.01 uM to about 1000 uM per day, as calculated as free unassembled polypeptide subunits.
The cargo carrying nanoparticles or pharmaceutical formulations thereof described herein can be administered in combination with one or more other auxiliary agents that are independent of the pharmaceutical formulation. Suitable auxiliary agents include, but are not limited to DNA, RNA, amino acids, peptides, polypeptides, antibodies, aptamers, ribozymes, guide sequences for ribozymes that inhibit translation or transcription of essential tumor proteins and genes, hormones, immunomodulators, antipyretics, anxiolytics, antipsychotics, analgesics, antispasmodics, anti-inflammatories, anti-histamines, anti-infectives, and chemotherapeutics. Suitable compounds and molecules that can be auxiliary agents are described elsewhere herein in connection with the description of cargo molecules.
Kits
The cargo carrying nanoparticles, expression vector(s) containing nucleotide sequences encoding cargo subunit(s) and/or targeting subunit(s), cells containing one or more expression vector, and pharmaceutical formulations described herein can be presented as a combination kit. As used herein, the terms “combination kit” or “kit of parts” refers to the cargo carrying nanoparticles, expression vectors containing nucleotide sequences encoding cargo subunit(s) and/or targeting subunit(s), cells containing one or more expression vector, and pharmaceutical formulations described herein and additional components that are used to package, sell, market, deliver, and/or administer the combination of elements or a single element, such as the active ingredient, contained therein. Such additional components include but are not limited to, packaging, syringes, blister packages, bottles, and the like. When one or more of the components (e.g. active agents) contained in the kit are administered simultaneously, the combination kit can contain the active agents in a single pharmaceutical formulation (e.g. a tablet) or in separate pharmaceutical formulations.
When the cargo carrying nanoparticles, pharmaceutical formulations thereof, or other auxiliary agent are not administered simultaneously, the combination kit can contain each agent, compound, nanoparticle, or formulation in separate compositions or pharmaceutical formulations. The separate compositions or pharmaceutical formulations can be contained in a single package or in separate packages within the kit. Also provided in some embodiments, are buffers, diluents, solubilization reagents, cell culture media and other reagents. These additional components can be contained in a single package or in separate packages within the kit.
In some embodiments, the combination kit also includes instructions printed on or otherwise contained in a tangible medium of expression. The instructions can provide information regarding the content of the cargo carrying nanoparticles, expression vectors containing nucleotide sequences encoding cargo subunit(s) and/or targeting subunit(s), cells containing one or more expression vectors, and pharmaceutical formulations contained therein, safety information regarding the content of the cargo carrying nanoparticles, expression vector(s) containing nucleotide sequences encoding cargo subunit(s) and/or targeting subunit(s), cells containing one or more expression vector, and pharmaceutical formulation(s) contained therein, information regarding the dosages, indications for use, and/or recommended treatment regimen(s) for the cargo carrying nanoparticles, expression vectors containing nucleotide sequences encoding cargo subunit(s) and/or targeting subunit(s), cells containing one or more expression vectors, and pharmaceutical formulations contained therein. In some embodiments, the instructions provide directions for administering the compounds, pharmaceutical formulations, or salts thereof to a subject having a cancer. In some embodiments, the cancer is lung cancer.
EXAMPLES
Now having described the embodiments of the present disclosure, in general, the following Examples describe some additional embodiments of the present disclosure. While embodiments of the present disclosure are described in connection with the following examples and the corresponding text and figures, there is no intent to limit embodiments of the present disclosure to this description. On the contrary, the intent is to cover all alternatives, modifications, and equivalents included within the spirit and scope of embodiments of the present disclosure.
Example 1
Induction of Cell Death by a Lytic Peptide-ELP Fusion Polypeptide
The lytic peptide (KLAKLAK)2 (SEQ ID NO: 23) was fused to an ELP to produce a lytic peptide-ELP fusion polypeptide according to methods described herein. 105 cells H292 cells were seeded in a 48 well plate, serum starved, and treated with comparable concentrations of the lytic peptide-ELP fusion polypeptide, which self-assembled into nanoparticles containing lytic peptide-ELP subunits, or the lytic peptide alone (i.e., (KLAKLAK)2 only) for about 24 hours. The lytic peptide alone does not self-assemble into nanoparticles. A MTT cell proliferation assay was then performed to determine cell viability. The results from this experiment are shown in FIGS. 4A and 4B.
FIG. 4A demonstrates the effect of varying concentrations of the lytic peptide alone on cell viability. FIG. 4B demonstrates the effect of varying concentrations of the lytic peptide-ELP fusion polypeptide on cell viability. The lytic peptide alone did not result in a significant reduction in cell viability. In contrast, treatment of cells with nanoparticles made from lytic peptide-ELP fusion polypeptides resulted in a dose-dependent reduction of cell viability. At the greatest concentration, the nanoparticles containing the lytic peptide-ELP fusion polypeptides killed up to 68% of the cells.
Example 2
Depolarization of Mitochondria by Lytic Fusion
2×105 H292 cells were treated with a positive control (Carbonylcyanide m-chlorophenylhydrazone (CCCP)), negative control (JC-1), assay control (untreated cells) or 50 μM of the lytic peptide-ELP fusion polypeptides (See Example 1), which self-assembled into nanoparticles, for about 24 hours. After treatment, a JC-1 assay was performed to determine the mitochondrial membrane potential and detect mitochondrial membrane depolarization. Samples were then analyzed using flow cytometry and fluorescent microscopy.
The results are shown in FIGS. 5A to 5D. FIG. 5A shows the results from the assay control. FIG. 5B shows the results from treatment of the cells with the negative control. FIG. 5C shows the results from treatment of the cells with the positive control. FIG. 5D shows the results from treatment with the lytic peptide-ELP fusion polypeptides. As shown in FIGS. 5A-5D, treatment with the lytic peptide-ELP fusion polypeptides resulted in apoptosis of a portion of the cell population.
Additionally TEM pictures were taken at 8600× of the cells. FIG. 6A shows a TEM image of an untreated cell Arrows indicate regular shaped mitochondria. FIG. 6B shows a TEM image of a cell treated with lytic peptide-ELP fusion polypeptides. Arrows indicate swelled mitochondria, which is a consequence of mitochondrial depolarization.
FIGS. 16A-16C show representative images of HCC827 cells treated with a negative control (FIG. 16A), positive control (FIG. 16B), or 25 μM of (KLAKLAK)2-ELP fusion polypeptides analyzed using fluorescent microscopy demonstrating mitochondrial depolarization. Red fluorescence indicates intact mitochondria and green fluorescence shows depolarized mitochondria.
Example 3
Uptake of Lytic Peptide-ELP is Via Macropinocytosis
2×105 H292 cells per well were seeded in a 24 well plate. Cells were blocked prior to treatment with no blocking agent, chlorpromazine, or amiloride, which block the two major methods of uptake mechanisms. Chlorpromazine blocks receptor mediated endocytosis (RME) and amiloride blocks macropinocytosis (MAC). After blocking, cells were treated with 20 μM of (KLAKLAK)2 peptide-ELP fusion polypeptides (See Example 2). Uptake of the (KLAKLAK)2 peptide was measured using flow cytometry. The results from this experiment are shown in FIG. 7, which shows the normalized KLAK-ELP uptake in cells pretreated with chlopromazine or amiloride. Uptake was most effected by pretreatment with amiloride, which indicates that macropinocytosis is a major mechanism of uptake of the KLAK-ELP observed.
It was also determined that heparan sulfate (HS), which is a polysaccharide found on the surface of cells, interacts with ELPs. To determine if an interaction between ELP and HS assists in the uptake mechanism of the fusion polypeptides and nanoparticles disclosed herein, cells were pre-blocked with surfen, which is an antagonist of HS, or a negative control (KLAK-ELP only). As shown in FIG. 8, pre-blocking with surfen reduces uptake of the fusion polypeptide by 80%. This indicates that HS plays a role in the uptake of the fusion polypeptides.
Example 4
Differential Expression of Keratinocyte Growth Factor Receptor in Cell Lines
mRNA expression of the human keratinocyte growth factor receptor was examined in lung cancer cell lines (hcc827, h23, a549, h1650, and h292). Expression of human keratinocyte growth factor receptor was analyzed using qPCR. The forward primer sequence for detecting KGFRiiib in all cells except fibroblasts is according to SEQ ID NO: 39. The forward primer for detecting KGFRiiic in fibroblasts is according to SEQ ID NO: 40. The reverse primer used in all cells to detect KGFRiiib and KGFRiiic is according to SEQ ID NO: 41. FIG. 9A shows the fold increase in RNA expression of the human keratinocyte growth factor receptor in various lung cancer cell lines. Data was normalized to RNA expression observed in the h23 cell line. qPCR products were confirmed using agarose gel electrophoresis. As shown in FIG. 9B, a 135 bp PCR product corresponds to a human keratinocyte growth factor receptor.
Example 5
Keratinocyte Growth Factor Increases Uptake of Cargo Carrying Nanoparticles
To determine if inclusion of keratinocyte growth factor increase uptake of the cargo carrying nanoparticles, cells H292 cells were treated with nanoparticles made from (KLAKLAK)2-ELP fusion polypeptides, nanoparticles assembled from (KLAKLAK)2-ELP fusion polypeptides and keratinocyte growth factor (GF)-ELP fusion polypeptides, or nanoparticles assembled from ELP and (KLAKLAK)2-ELP fusion polypeptides. GF-ELP fusion polypeptides were generated from a nucleic acid encoding an keratinocyte growth factor and a nucleic acid encoding an ELP according to methods described herein.
After the fusion polypeptides were generated, 2×105 cells H292 cells per well were seeded onto cell culture plates and treated with formulations containing 50 μM of the (KLAKLAK)2-ELP fusion polypeptides, 50 μM of GF-ELP fusion polypeptides, or 50 μM of ELP. Additionally, each formulation was mixed with 1.5 μM of FITC labeled-(KLAKLAK)2-ELP fusion polypeptides. Cells were treated with the formulations for about 24 hours. After treatment, cells were treated with trypan blue prior to analysis by flow cytometry in order to capture only internalized particles.
FIG. 10 demonstrates the results from the flow cytometry analysis. The results in FIG. 10 indicate that the presence of the keratinocyte growth factor in the cargo carrying nanoparticles increases the uptake of the cargo carrying nanoparticles.
Example 6
Growth Factor-ELP Fusion Polypeptides Increase Cell Death
To determine if the increase in uptake of the cargo carrying nanoparticles by inclusion of a growth factor observed in Example 5 would translate in an increase in cell death, H292 cells were treated with three formulations containing cargo carrying nanoparticles or a control for about 24 hours. A live/dead cell viability assay where green fluorescence indicates a live cell and red fluorescence indicates a dead cell was performed to assess cell death. Results were analyzed by fluorescent microscopy.
FIGS. 11A-11D show representative images generated by fluorescent microscopy of the treated cells. FIG. 11A shows a representative image of cells treated with a negative control (Untreated cells) FIG. 11B shows a representative image of cells treated with 50 μM of (KLAKLAK)2-ELP fusion polypeptides. FIG. 11C shows a representative image from cells treated with 50 μM of (KLAKLAK)2-ELP fusion polypeptides and 100 μM of GF-ELP fusion polypeptides (See Example 5). FIG. 11D shows a representative image from cells treated with 50 μM (KLAKLAK)2-ELP and 200 μM of GF-ELP fusion polypeptides. As shown in FIGS. 11A-11D, a greater death rate was observed when the cargo carrying nanoparticles contained GF-ELP fusion polypeptides.
Example 7
Self-Assembly of Fusion ELP Polypeptides into Cargo Carrying Nanoparticles and Induction of Cell Death by Cargo Carrying Nanoparticles
Epidermal growth factor receptor (EGFR) is a target for development therapeutics for treatment of lung cancer. EGFR is ubiquitously expressed and thus may not address issues with systemic treatments as treatment may also stimulate EGFRs expressed in normal tissue. EGFR specifically binds epidermal growth factor (EGF). However, EGFR can offer an advantage in instances where systemic delivery of a cargo molecule is desired.
Keratinocyte growth factor receptor (KGFR) is up-regulated in lung cancer tumors. As demonstrated in Example 4, KGFR is differentially expressed across several lung cancer cell lines. Compared to EGFR, KGFR is not ubiquitously expressed. Targeting the KGFR can result in reduced systemic toxicity resulting from ubiquitously delivered therapeutic agents, particularly chemotherapeutics. KGFR can offer an advantage in instances where systemic delivery of a cargo molecule is undesirable.
Fusion polypeptides have been generated that contain KGF fused to an ELP. Also, fusion polypeptides have been generated that contain EGF fused to an ELP. In solution, the fusion polypeptides can self-assemble into nanoparticles at the Ttm. FIG. 12 shows a TEM image of an assembled nanoparticle made from KGF-ELP fusion polypeptides. The nanoparticle shown in FIG. 12 has a diameter of about 500 nm.
Smaller nanoparticles, particularly those less than about 100 nm in diameter are more efficacious for in vivo delivery of cargo molecules. The hydrophobicity of the ELP domain of the fusion polypeptides can be manipulated. To determine if the hydrophobicity of the ELP domain effects the size of the resulting nanoparticles, KGF-ELP fusion polypeptides were generated using different ELPs (V40C2, V120, and V180) that vary in their hydrophobicity. Hydrophobicity of V40C2 is less than V120, which is less than the hydrophobicity of V180. The cDNA sequence of V180 is according to SEQ ID NO: 36, which encodes the V180 polypeptide having a sequence according to SEQ ID NO: 37. The cDNA sequence of V120 is according to SEQ ID NO: 38. As shown in FIG. 13, increasing the hydrophobicity of the ELP domain of the fusion polypeptides resulted in smaller nanoparticles. Nanoparticle size was assessed by dynamic light scattering.
2×105 cancer cells (H292 cells) were treated for about 24 hours with nanoparticles assembled from (KLAKLAK)2-ELP fusion polypeptides or (KLAKLAK)2 peptides and fraction of live cells were quantified. FIGS. 14A and 14B demonstrates the percent cell viability of cancer cells treated with nanoparticles assembled from (KLAKLAK)2-ELP fusion polypeptides (FIG. 14A) or (KLAKLAK)2 peptides (FIG. 14B). As demonstrated in FIG. 14A, it was observed that nanoparticles assembled from (KLAKLAK)2-ELP fusion polypeptides induced cell death in a dose dependent manner. In contrast, (KLAKLAK)2 peptides by themselves, when used at comparable concentrations, were not observed to induce cell death. Thus, the data suggest that not only the bioactivity of (KLAKLAK)2 is maintained in the fusion polypeptide, but also the assembly of (KLAKLAK)2-ELP nanoparticles increases the internalization of (KLAKLAK)2, which likely led to the observed increase in cell death.
Supporting the previously discussed data, FIG. 15 demonstrates a dose dependent increase in cell death in lung cancer cells (A549 cells) treated with increasing concentrations (10 μM “KLAK(10),” 20 μM “KLAK(20),” 50 μM “KLAK(50),” and 100 μM “KLAK(100)”) of (KLAKLAK)2-ELP nanoparticles or (KLAKLAK)2 peptides alone.
Additionally, the (KLAKLAK)2-ELP nanoparticles were observed to induce mitochondrial depolarization as shown in FIGS. 16A-16C. Lung cancer cells (A549) were treated with a negative control (cells treated with JC-1 only) a positive control (CCCP), or 25 μM of (KLAKLAK)2-ELP peptides. Red shows intact mitochondria while green shows depolarized mitochondria. About all of the cells treated with the negative control had intact mitochondria whereas at least about 90% of the cells treated with the positive control had depolarized mitochondria. About 70% of the cells treated with the (KLAKLAK)2-ELP peptides had depolarized mitochondria. FIG. 17 shows a TEM image of a cell treated with 50 μM (KLAKLAK)2-ELP peptides. Arrows indicate swollen mitochondria, which suggest that these mitochondria were depolarized and demonstrate damage to the mitochondrial membranes.
To further quantify the damage to the mitochondrial membranes, a JC-1 assay was used to measure mitochondrial membrane potential. JC-1 dye exhibits membrane-potential dependent accumulation in mitochondria, indicated by a fluorescence emission shift from green (−529 nm) to red (−590 nm). A decrease in the red/green fluorescence intensity ratio, as measured by flow cytometry, indicates mitochondrial depolarization. FIGS. 18A-18B shows the results of the JC-1 assay, which support the visual indications of mitochondrial depolarization. FIG. 18A shows the results of a JC-1 assay performed on cells treated with a negative control. FIG. 18B shows the results of a JAC-1 assay performed on cells treated with (KLAKLAK)2-ELP peptides. Non-apoptotic and apoptotic regions are marked.
Example 8
Uptake of (KLAKLAK)2-ELP Cargo Carrying Nanoparticles is Via Macropinocytosis and Involves Heparan Sulfate
Using inhibitors of receptor mediated endocytosis (RME, chloropromazine) and macropinocytosis (MAC, amilioride) it was observed that (KLAKLAK)2-ELP (k) cargo carrying nanoparticles were taken up by cells predominantly by macropinocytosis (FIG. 19). Briefly, c H292 cells were treated with (KLAKLAK)2-ELP peptides, which self-assembled into cargo carrying nanoparticles, cargo carrying nanoparticles and an RME inhibitor (k+RME), or cargo carrying nanoparticles and a MAC inhibitor (k+MAC). Uptake/cell was then measured and normalized. Uptake was measured by flow cytometry and the results were normalized to the k treatment.
To determine if the addition of a growth factor to the cargo carrying nanoparticles, KGF-ELP fusion peptides as well as (KLAKLAK)2-ELP peptides were used as the building blocks for the cargo carrying nanoparticles and uptake was evaluated. Briefly, lung cancer cells (H292) or fibroblasts (Fb) were treated with cargo carrying nanoparticles made from (KLAKLAK)2-ELP peptides alone (Homogenous control), cargo carrying nanoparticles made from (KLAKLAK)2-ELP peptides and about KGF-ELP peptides (Heterogeneous treatment), or cargo carrying nanoparticles made from (KLAKLAK)2-ELP peptides and about 50 μM ELP peptides alone (Heterogeneous control). The concentrations of peptides used was about 2.5 uM (KLAKLAK)2, about 50 μM of KGF-ELP, and about 50 μM of ELP. Uptake was measured using flow cytometry and the results were normalized to the homogeneous treatment. As shown in FIG. 20, inclusion of the KGF-ELP peptide increased uptake in the H292 cells, which have a greater expression of the KGF receptor than the Fb cells. These data suggest that inclusion of a targeting moiety, such as KGF, can increase uptake of the cargo carrying nanoparticles in a selective and specific manner.
To determine which molecule(s) on the cell surface was interacting with the (KLAKLAK)2-ELP peptides, H292 cells were treated with cargo carrying nanoparticles containing (KLAKLAK)2-ELP peptides after pretreating cells with a control (cells that were treated with KLAKLAK)2-ELP but not pretreated with any of the blockers), surfen, heparinase iii, or lactose. Surfen specifically binds to heparan sulfate on the cell surface, heparinase iii cleaves off heparan sulfate on the cell surface, and lactose blocks the elastin receptor complex (ERC). As shown in FIG. 21, the results suggest that heparan sulfate interacts with the (KLAKLAK)2-ELP peptide at the cell surface.
Supporting previously discussed results, FIG. 22 demonstrates that macropinocytosis is the main pathway of internalization of the cargo carrying nanoparticles. Briefly, (H292 cells were pretreated with amiloride (blocks MAC) or chlorpromazine (blocks RME) and uptake of (KLAKLAK)2-ELP cargo carrying nanoparticles was measured, normalized, and compared to uptake of (KLAKLAK)2-ELP cargo carrying nanoparticles in un-treated cells. Uptake was measured using flow cytometry. The control were the cells that were treated with (KLAKLAK)2-ELP but were not pre-blocked with amiloride or chlorpromazine
Killing efficiency of the cargo carrying nanoparticles made from (KLAKLAK)2-ELP peptides (klak) and KGF-ELP (KGF) peptides was evaluated. Briefly, H292 were treated with a control (FIG. 23A) Untreated cells served as a control. 50 μM of klak (FIG. 23B), 50 μM of klak and 100 μM of KGF (FIG. 23C), or 50 μM of klak and 200 μM of KGF (FIG. 23D). Cell death was measured by a live (green fluorescence)/dead (red fluorescence) assay. Bar=400 μm in FIGS. 23A-23D. As shown in FIG. 23A, about all of the cells were alive after treatment with the control. As shown in FIGS. 23B through 23C, the number of dead cells increased with the increasing concentration of KGF. As such, the results suggest that cargo carrying nanoparticles containing growth factors induce killing more effectively than cargo carrying nanoparticles containing only lytic fusion peptides.
The results demonstrated in FIGS. 23A-23D are supported by the results shown in FIG. 24, which demonstrate the quantitation of live H292 cells after treatment with or 50 μM of klak, 50 μM of klak and 100 μM of KGF, or 200 μM of KGF. As shown in FIG. 24, cell viability decreased as the concentration of KGF was increased.
Example 9
Expression of KGFR iiib in Various Cells
Gene expression of KGFR iiib in various cell lines (SAEC, fibroblasts (fb), H292, H1650, A549, HCC827, h23, A431, MIA PaCa-2) was determined. As demonstrated in FIG. 25, KGFR iiib was differentially expressed among the various cell lines. A549 cells, a lung cancer cell line, had the greatest expression.
Example 10
Increased and Selective Internalization of Cargo Carrying Nanoparticles by KGF
As shown in Example 9, A549 cells have greatest expression of KGFR iiib. A549 cells were treated with 10 μM (KLAKLAK)2-ELP (KLAK), 10 μM KGF-ELP (KGF) and 10 μM KLAK, 20 μM KLAK, or 20 μM ELP and 20 μM KLAK. Uptake was measured and normalized. Uptake was measured using flow cytometry and the results were normalized to (KLAKLAK)2-ELP 10 uM treatment. As shown in FIG. 26, inclusion of KGF in the cargo carrying nanoparticles increases the uptake of the cargo carrying nanoparticles by the cells.
To determine if the uptake induced by KGF is selective, A549 or fibroblasts, were treated with 10 μM KLAK or 10 μM KGF and 10 μM KLAK. A549 express KGFR IIIb whereas fibroblasts do not express KGFE iiib (See FIG. 25). After treatment, uptake was measured and normalized. Uptake was measured using flow cytometry and the normalization was to the KLAK(10) treatment. As shown in FIG. 27, the KFG increased uptake only in the A549 cells.
This suggests that KGF induced uptake of the cargo carrying nanoparticles is selective.
Example 11
Selective Killing of Cells by Cargo Carrying Nanoparticles Containing KGF
To determine if killing induced by the cargo carrying nanoparticles containing KGF is selective, cells expressing high levels (A549) or low levels (MiaPaCa-2), were treated with a control Untreated cells served as a control. 10 μM KLAK, 10 μM KLAK and 10 μM KGF, 10 μM KLAK and 20 μM KGF, 10 μM KLAK and 50 μM KGF, or 50 μM KGF. The percent of live cells was measured. The percent of live cells was normalized to the percent of live cells remaining in the control. As suggested by the results shown in FIG. 28, cells expressing high levels (as compared to other cell types) KGFR iiib are killed by the combination of (KLAKLAK)2-ELP and KGF-ELP, while cells having low expression (if any), are not killed by the combination of (KLAKLAK)2-ELP and KGF-ELP.
Example 12
Enhancement of Macropinosome Formation by Cargo Carrying Nanoparticles Containing KGF
To determine the effect of KGF on the macropinocytosis pathway, macropinosome formation was evaluated in cells after treatment with a control (untreated cells) 1 μM KGF-ELP peptides, 10 μM KGF-ELP peptides, or 50 μM KGF-ELP peptides. The number of macropinosomes per cell was determined and the average number was normalized to the number of macropinosomes in the control cells, which was set to 1.0. As demonstrated by the results shown in FIG. 29, KGF-ELP peptides enhance macropinosome formation in a dose-dependent manner in A549 cells, which express KGFR iiib. As shown in FIG. 30, the enhancement in macropinosome formation was not observed in fibroblast cells, which doe not express KGFR iiib.
Example 13
Production of Heterogeneous Cargo Carrying Nanoparticles
Genes encoding recombinant growth factors (KGF or EGF) were excised from a PUC57 plasmid and cloned in frame with the gene encoding the ELP cassette V40C2. In the ELP cassette V40C2, V=VPGVG and C=(VPGVGVPGVGVPGCGVPGVGVPGVG). The “V” repeat subunit is repeated 40 times and the C repeat unit is repeated twice in this ELP. This yielded a gene encoding the building blocks (subunits) of the cargo carrying nanoparticles. These nucleic acids formed here encoded the growth factor (GF)-ELP subunits. The gene encoding the GF-ELP was then cloned into an expression plasmid (pET25b+) and was used to transform bacteria (BRL cells). The bacteria were grown at about 37° C. for about 12 h to allow for expression of the genes. The expression of the fusion protein was verified using western blots.
For purification, transformed bacteria were grown overnight and then lysed using two twelve-minute cycles in a sonicator. Each cycle was made of alternating on/off minutes for the sonicator. The GF-ELP was transitioned using salt and incubating the solution at about 40° C. The GF-ELP was then pelleted by a hot spin with the supernatant being discarded. The GF-ELP in the pellet was solubilized at about 4° C. using a buffer containing dithiotheritol (DTT). The solution was then centrifuged at about 4° C. to pellet the impurities with the GF-ELP in the supernatant. This completed one full cycle of the purification. This was repeated twice for a total for three cycles. After the last cycle the purified GF-ELP was dialyzed against water overnight to remove salts. The dialyzed GF-ELP was then lyophilized and stored until further use.
The physical properties of the purified GF-ELPs were characterized using a UV spectrophotometer and dynamic light scattering. The biological activity of the GF-ELPs was evaluated using h292, a431, and hcc287 cell lines. The uptake of the nanoparticles formed from GF-ELPs was evaluated using flow cytometry and transmission electron microscopy. These results are shown in FIGS. 6A-6B, 9A, and 10.

## Claims

1. A nanoparticle comprising:
a first fusion polypeptide comprising a first elastin-like peptide (ELP) and a targeting moiety configured to specifically bind a binding partner, wherein the targeting moiety induces macropinocytosis when the targeting moiety specifically binds to the binding partner, wherein the targeting moiety is keratinocyte growth factor and the binding partner is keratinocyte growth factor receptor, and wherein the first ELP is operatively linked to the targeting moiety;
a second fusion polypeptide comprising a second ELP and a first cargo molecule, wherein the second ELP is operatively linked to the first cargo molecule, and wherein the nanoparticle self assembles at a temperature that is greater than a phase transition temperature,
wherein the first fusion polypeptide and the second fusion polypeptide are present in the nanoparticle at a molar concentration ratio ranging from about 1:1 to 2:1, and
wherein the nanoparticle further comprises a cargo binding peptide, and wherein the cargo binding peptide comprises the amino acid sequence of SEQ ID NO: 21.

2. The nanoparticle of claim 1, wherein the first cargo molecule is a mitochondrial lytic peptide.

3. The nanoparticle of claim 2, wherein the mitochondrial lytic peptide comprises the amino acid of SEQ ID NO: 23.

4. The nanoparticle of claim 1, wherein the nanoparticle further comprises a third fusion polypeptide comprising a third ELP and a second cargo molecule, wherein the third ELP is operatively linked to the second cargo molecule, and wherein the second cargo molecule is a glycolysis inhibitor.

5. The nanoparticle of claim 1, wherein the binding partner is located on the surface of a cell.

6. The nanoparticle of claim 5, wherein the cell is a cancer cell.

7. The nanoparticle of claim 1, wherein the phase transition temperature ranges from about 5° C. to about 40° C.

8. The nanoparticle of claim 1, wherein the first or the second ELP comprises the amino acid sequence of any one of SEQ ID NOs: 9, 37, or the amino acid sequence encoded by SEQ ID NO: 38.

9. The nanoparticle of claim 1, wherein the first fusion polypeptide comprises the amino acid of SEQ ID NO: 33.

10. A pharmaceutical formulation comprising:
an effective amount of a nanoparticle, wherein the nanoparticle comprises:
a first fusion polypeptide comprising a first elastin-like peptide (ELP) and a targeting moiety configured to specifically bind a binding partner, wherein the targeting moiety induces macropinocytosis when the targeting moiety specifically binds to the binding partner, wherein the targeting moiety is keratinocyte growth factor and the binding partner is keratinocyte growth factor receptor, and wherein the first ELP is operatively linked to the targeting moiety;
a second fusion polypeptide comprising a second ELP and a first cargo molecule, wherein the second ELP is operatively linked to the first cargo molecule, wherein the nanoparticle self assembles above a phase transition temperature,
wherein the first fusion polypeptide and the second fusion polypeptide are present in the nanoparticle at a molar concentration ratio ranging from about 1:1 to 2:1;
wherein the nanoparticle further comprises a cargo binding peptide, and wherein the cargo binding peptide comprises the amino acid sequence of SEQ ID NO: 21; and
a pharmaceutically acceptable carrier.

11. The pharmaceutical formulation of claim 10, wherein the nanoparticle further comprising a third fusion polypeptide comprising a third ELP and a second cargo molecule, wherein the third ELP is operatively linked to the second cargo molecule.

12. The pharmaceutical formulation of claim 11, wherein the first cargo molecule is a mitochondrial lytic peptide and wherein the second cargo molecule is a glycolysis inhibitor.

13. The pharmaceutical formulation of claim 11, wherein the second fusion polypeptide comprises the amino acid sequence of SEQ ID NO: 31.

14. The pharmaceutical formulation of claim 10, wherein the first cargo molecule is a mitochondrial lytic peptide.

15. The pharmaceutical formulation of claim 10, wherein the amount is effective for killing a lung cancer cell or population thereof.

16. The pharmaceutical formulation of claim 10, wherein the first or the second ELP comprises the amino acid sequence of any one of SEQ ID NOs: 9, 37, or the amino acid sequence encoded by SEQ ID NO: 38.

17. The pharmaceutical formulation of claim 10, wherein the first fusion polypeptide comprises the amino acid sequence of SEQ ID NO: 33.

18. A nanoparticle comprising:
a first fusion polypeptide comprising a first elastin-like peptide (ELP) and a targeting moiety configured to specifically bind a binding partner, wherein the targeting moiety induces macropinocytosis when the targeting moiety specifically binds to the binding partner, wherein the targeting moiety is keratinocyte growth factor and the binding partner is keratinocyte growth factor receptor, and wherein the first ELP is operatively linked to the targeting moiety;
a second fusion polypeptide comprising a second ELP and a first cargo molecule, wherein the second ELP is operatively linked to the first cargo molecule, and wherein the nanoparticle self assembles at a temperature that is greater than a phase transition temperature; and
a cargo binding peptide, wherein the cargo binding peptide is low-density lipoprotein receptor 3 (LDLR3).