Modified porcine pancreatic elastase protein

Abstract

The present invention provides a modified porcine pancreatic elastase (PPE) protein containing a proprotein that includes at least one amino acid change that reduces binding to serine protease inhibitors such as alpha-1 antitrypsin, thereby increasing cancer cell death activity, as well as related pharmaceutical compositions and methods of use for treating diseases such as cancer. [Solution] A modified PPE protein comprising at least one amino acid change from the wild-type PPE protein. In some embodiments, the modified PPE protein and a pharmaceutical composition comprising the modified PPE protein are provided, wherein at least one amino acid change is selected from one or more of Q211F, T55A, D74A, R75A, R75E, Q211A, S214A, R237A, N241A, and N241Y.

Description

Technical Field

[0001] Cross - reference to Related Applications This application claims the benefit under 35 U.S.C. § 119(e) of U.S. Patent Application No. 63 / 067,058, filed Aug. 18, 2020, which is hereby incorporated by reference in its entirety.

[0002] Description of the Sequence Listing The sequence listing associated with this application is provided in text format instead of a paper copy and is hereby incorporated by reference into this specification. The name of the text file containing the sequence listing is OPNI_001_01WO_ST25.txt. The text file is approximately 29 KB, was created on Aug. 16, 2021, and was electronically filed via EFS - Web.

Background Art

[0003] Technical Field The present disclosure relates to modified porcine pancreatic elastase (PPE) proteins, including a proprotein, that contain at least one amino acid change that reduces binding to serine protease inhibitors such as alpha - 1 - antitrypsin (A1AT), thereby increasing its cancer cell killing activity, and related pharmaceutical compositions and methods of use for treating diseases such as cancer.

[0004] Description of Related Art Precision medicine designed to optimize efficiency or therapeutic benefit for specific patient populations by using gene or molecular profiling has had great success in cancer treatment. Identifying specific genomic abnormalities that (i) confer cancer risk, (ii) affect tumor growth, and (iii) regulate metastasis defines how cancer is diagnosed, determines how targeted therapies are developed and implemented, and shapes cancer prevention strategies.

[0005] The need for precision medicine in cancer is primarily based on the inability to identify targetable characteristics of tumor cells that distinguish them from healthy, non-cancerous cells. Indeed, while radiation and / or chemotherapy have the ability to effectively kill a large number of cancer cells, if not the majority, their effectiveness is significantly limited by their cytotoxic effects on non-cancerous cells. These findings suggest that rapid cell division, a targetable characteristic of radiation and chemotherapy, is not sufficiently specific to cancer cells to achieve the specificity required to limit widespread side effects.

[0006] Certain elastase enzymes have been shown to be selectively toxic to cancer cells but relatively non-toxic to normal or other healthy cells (see, for example, WO2018 / 232273). However, there is a need in the art to identify the optimal enzymes capable of such selective cancer cytotoxicity and to improve the clinical utility of such enzymes. [Overview of the Initiative]

[0007] Embodiments of the present disclosure include a modified porcine pancreatic elastase (PPE) protein comprising at least one amino acid change from the wild-type PPE protein (SEQ ID NO: 4), wherein the at least one change is in a residue selected from one or more of Q211, T55, D74, R75, S214, R237, and N241, and the residue numbering is defined by SEQ ID NO: 1 (wild-type PPE proprotein). In some embodiments, the at least one amino acid change is selected from one or more of Q211F, T55A, D74A, R75A, R75E, Q211A, S214A, R237A, N241A, and N241Y, and the residue numbering is defined by SEQ ID NO: 1.

[0008] In some embodiments, the modified PPE protein contains, consists of, or is essentially composed of, an amino acid sequence that is at least 80, 85, 90, 95, 98, or 100% identical to a sequence selected from Table S2 and retains at least one amino acid change. In some embodiments, the modified PPE protein, A modified PPE protein comprising, consisting of, or essentially comprising an amino acid sequence that is at least 80, 85, 90, 95, 98, or 99% identical to SEQ ID NO: 5 and retains the Q211F amino acid substitution, A modified PPE protein comprising, consisting of, or essentially comprising an amino acid sequence that is at least 80, 85, 90, 95, 98, or 99% identical to SEQ ID NO: 6 and retains the T55A amino acid substitution, A modified PPE protein containing, consisting of, or essentially comprising an amino acid sequence that is at least 80, 85, 90, 95, 98, or 99% identical to SEQ ID NO: 7 and retains the N241A amino acid substitution, A modified PPE protein containing, consisting of, or essentially comprising an amino acid sequence that is at least 80, 85, 90, 95, 98, or 99% identical to SEQ ID NO: 8 and retains the N241Y amino acid substitution, A modified PPE protein comprising, consisting of, or essentially comprising an amino acid sequence that is at least 80, 85, 90, 95, 98, or 99% identical to SEQ ID NO: 9 and retains the R75A amino acid substitution, A modified PPE protein comprising, consisting of, or essentially comprising, an amino acid sequence that is at least 80, 85, 90, 95, 98, or 99% identical to SEQ ID NO: 10 and retains the R75E amino acid substitution, A modified PPE protein containing, consisting of, or essentially comprising an amino acid sequence that is at least 80, 85, 90, 95, 98, or 99% identical to SEQ ID NO: 11 and retains the Q211A amino acid substitution, A modified PPE protein comprising, consisting of, or essentially comprising an amino acid sequence that is at least 80, 85, 90, 95, 98, or 99% identical to SEQ ID NO: 12 and retains the R237A amino acid substitution, A modified PPE protein containing, consisting of, or essentially comprising an amino acid sequence that is at least 80, 85, 90, 95, 98, or 99% identical to SEQ ID NO: 13 and retains the S214A amino acid substitution, Selected from: SEQ ID NO: 14, or a modified PPE protein containing, consisting of, or essentially derived from, an amino acid sequence that is at least 80, 85, 90, 95, 98, or 99% identical to SEQ ID NO: 14 and retains the D74A amino acid substitution.

[0009] In some embodiments, the modified PPE protein has increased cancer cell killing activity compared to that of the wild-type PPE protein (SEQ ID NO: 4). In some embodiments, the modified PPE protein has increased cancer cell killing activity of approximately or at least approximately 2x, 5x, 10x, 50x, 100x, 500x, or 1000x or more compared to the cancer cell killing activity of the wild-type PPE protein (SEQ ID NO: 4). In some embodiments, the increased cancer cell killing activity is observed in vitro or in vivo in the absence of human A1AT protein. In some embodiments, the increased cancer cell killing activity is observed in vitro or in vivo in the presence of human A1AT protein.

[0010] In some embodiments, the modified PPE protein has reduced binding to or interaction with the human alpha-1 antitrypsin (A1AT) protein compared to the wild-type PPE protein (SEQ ID NO: 4). In some embodiments, the modified PPE protein has approximately or at least approximately 2x, 5x, 10x, 50x, 100x, 500x, or 1000x or more reduced binding to the human A1AT protein compared to the binding of the wild-type PPE protein to the human A1AT protein. In some embodiments, the modified PPE protein has approximately or at least approximately 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700, 800, 900, or 1000% or more serine protease activity of the wild-type PPE.

[0011] In some embodiments, the modified PPE protein has increased serine protease activity compared to the wild-type PPE protein. In some embodiments, the serine protease activity, when measured in the absence of human A1AT protein, is about or at least about 2 times, 5 times, 10 times, 50 times, 100 times, 500 times, or 1000 times higher than the serine protease activity of wild-type PPE. In some embodiments, the serine protease activity, when measured in the presence of human A1AT protein, is about or at least about 2 times, 5 times, 10 times, 50 times, 100 times, 500 times, or 1000 times higher than the serine protease activity of wild-type PPE.

[0012] A particular embodiment includes a modified PPE proprotein comprising, in an N-terminal to C-terminal orientation, a signal peptide (optionally SEQ ID NO: 2), an activating peptide (optionally SEQ ID NO: 3), and the modified PPE proprotein described herein, wherein the modified PPE proprotein is activatable via protease cleavage of the activating peptide to produce an enzymatically active modified PPE proprotein.

[0013] Also included herein are recombinant nucleic acid molecules encoding the modified PPE protein or proprotein, vectors containing recombinant nucleic acid molecules, or host cells containing recombinant nucleic acid molecules or vectors. Specific embodiments include methods for producing the modified PPE protein or proprotein described herein, comprising culturing host cells under culture conditions suitable for the expression of the modified PPE protein or proprotein, and isolating the modified PPE protein or proprotein from the culture.

[0014] Certain embodiments include a pharmaceutical composition comprising a modified PPE protein or proprotein as described herein, or an expressible polynucleotide encoding a modified PPE protein or proprotein, and a pharmaceutically acceptable carrier. Also included are methods for treating cancer, improving its symptoms, and / or reducing its progression in subjects requiring such treatment, comprising administering the pharmaceutical composition to a subject. In some embodiments, the cancer is primary or metastatic cancer, and includes melanoma (optionally metastatic melanoma), breast cancer (optionally triple-negative breast cancer, TNBC), kidney cancer (optionally renal cell carcinoma), pancreatic cancer, bone cancer, prostate cancer, small cell lung cancer, non-small cell lung cancer (NSCLC), mesothelioma, leukemia (optionally lymphocytic leukemia, chronic myeloid leukemia, acute myeloid leukemia, or recurrent leukemia). One or more of the following are selected: acute myeloid leukemia, multiple myeloma, lymphoma, hepatocellular carcinoma, sarcoma, B-cell malignancy, ovarian cancer, colorectal cancer, glioma, glioblastoma multiforme, meningioma, pituitary adenoma, vestibular schwannoma, primary CNS lymphoma, primitive neuroectodermal tumor (medulloblastoma), bladder cancer, uterine cancer, esophageal cancer, brain cancer, head and neck cancer, cervical cancer, testicular cancer, thyroid cancer, and gastric cancer.

[0015] In some embodiments, the pharmaceutical composition comprises a modified PPE proprotein, which is activated by protease cleavage of an activating peptide in the cancerous tissue or tumor site of the subject requiring the pharmaceutical composition, thereby generating an enzymatically active modified PPE protein. In some embodiments, administration of the pharmaceutical composition increases cancer cell death in the subject by about or at least about 2x, 5x, 10x, 50x, 100x, 500x, or 1000x or more compared to a control or reference. In some embodiments, administration of the pharmaceutical composition results in tumor regression in the subject, as indicated by a statistically significant reduction in the amount of surviving tumor or tumor volume, or by a tumor volume reduction of at least about 10%, 20%, 30%, 40%, or 50% or more, as indicated by an optional reduction in tumor volume.

[0016] Certain embodiments involve administering the pharmaceutical composition to a target by parenteral administration or intratumor administration. In some embodiments, parenteral administration is intravenous administration. [Brief explanation of the drawing]

[0017] [Figure 1A] This demonstrates the effective trypsin cleavage of wild-type and modified PPE proproteins. As shown in Figure 1A, incubation of native PPE (active PPE peptidase domain) with trypsin did not result in the appearance of a low molecular weight band, suggesting that trypsin does not further cleave the PPE protein morphology after the initial conversion to active PPE. [Figure 1B] This demonstrates the effective trypsin cleavage of wild-type and modified PPE proproteins. As shown in Figure 1A, incubation of native PPE (active PPE peptidase domain) with trypsin did not result in the appearance of a low molecular weight band, suggesting that trypsin does not further cleave the PPE protein morphology after the initial conversion to active PPE. [Figure 1C]Shows effective trypsin cleavage of wild-type and modified PPE proteins. As shown in Figure 1A, incubation of native PPE (active PPE peptidase domain) with trypsin did not result in the appearance of low molecular weight bands, suggesting that trypsin does not further cleave the PPE protein form after the initial conversion to active PPE. [Figure 1D] Shows effective trypsin cleavage of wild-type and modified PPE proteins. As shown in Figure 1A, incubation of native PPE (active PPE peptidase domain) with trypsin did not result in the appearance of low molecular weight bands, suggesting that trypsin does not further cleave the PPE protein form after the initial conversion to active PPE. [Figure 2A] Shows increased enzyme activity of wild-type and modified PPE proteins after trypsin cleavage and conversion to active PPE. [Figure 2B] Shows increased enzyme activity of wild-type and modified PPE proteins after trypsin cleavage and conversion to active PPE. [Figure 3A] Shows cancer cell activity of the active forms of wild-type and modified PPE proteins. Figure 3A shows the increasing dose activity of the test protein. [Figure 3B] Shows cancer cell activity of the active forms of wild-type and modified PPE proteins. Figure 3B shows the activity of the test protein in the absence or presence of increasing amounts of the A1AT serine protease inhibitor.

Mode for Carrying Out the Invention

[0018] Detailed Description Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those commonly understood by those skilled in the art to which this disclosure belongs. Any methods, materials, compositions, reagents, and cells similar or equivalent to those described herein may be used in carrying out or testing the subject matter of this disclosure, but preferred methods and materials are described. All publications and references, including but not limited to patents and patent applications cited herein, are incorporated herein by reference in their entirety, as if each individual publication or reference were to be incorporated herein by reference in its entirety. Any patent application for which this application claims priority is also incorporated herein by reference in its entirety, in the manner described above for publications and references.

[0019] Standard techniques may be used for recombinant DNA, oligonucleotide synthesis, and tissue culture and transformation (e.g., electroporation, lipofection). Enzyme reactions and purification techniques may be performed according to the manufacturer's specifications, or as commonly achieved in the art, or as described herein. These and related techniques and procedures may generally be performed according to conventional methods well known in the art and as described in the various general and more specific references cited and discussed throughout this specification. Unless otherwise specified, the nomenclature used in connection with molecular biology, analytical chemistry, synthetic organic chemistry, and medicinal and pharmaceutical chemistry, as well as their laboratory procedures and techniques, are well known and commonly used in the art. Standard techniques may be used for recombinant techniques, molecular biological, microbiological, chemical synthesis, chemical analysis, pharmaceutical preparation, formulation, and delivery, and for the treatment of patients.

[0020] For the purposes of this disclosure, the following terms are defined below.

[0021] The articles "a" and "an" are used herein to refer to one or more (i.e., at least one) grammatical objects of an article. For example, "element" includes "one element," "one or more elements," and / or "at least one element."

[0022] "Approximately" means a quantity, level, value, number, frequency, percentage, dimension, size, volume, weight, or length that varies by approximately 20, 15, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1% relative to the reference quantity, level, value, number, frequency, percentage, dimension, size, volume, weight, or length.

[0023] An "antagonist" refers to a biological structure or chemical agent that interferes with or otherwise reduces the physiological action of another drug or molecule. In some cases, antagonists specifically bind to other drugs or molecules. This includes complete and partial antagonists.

[0024] An "agonist" refers to a biological structure or chemical agent that increases or enhances the physiological effect of another drug or molecule. In some cases, agonists specifically bind to other drugs or molecules. This includes full and partial agonists.

[0025] As used herein, the term “amino acid” is intended to mean both naturally occurring and unnaturally occurring amino acids, as well as both amino acid analogs and mimics. Naturally occurring amino acids include the 20(L) amino acids utilized in protein biosynthesis, as well as others such as 4-hydroxyproline, hydroxylysine, desmosine, isodesmosine, homocysteine, citrulline, and ornithine. Unnaturally occurring amino acids include, for example, (D)-amino acids, norleucine, norvaline, p-fluorophenylalanine, and ethionine, which are known to those skilled in the art. Amino acid analogs include modified forms of naturally occurring and unnaturally occurring amino acids. Such modifications may include, for example, substitution or exchange of chemical groups and parts of an amino acid, or by derivatization of an amino acid. Amino acid mimics include organic structures that exhibit functionally similar properties, such as the charge and charge spacing characteristics of a reference amino acid. For example, an organic structure mimicking arginine (Arg or R) would have a positively charged moiety located in a similar molecular space and possessing the same degree of mobility as the e-amino group of the side chain of the naturally occurring Arg amino acid. The mimetic also includes a constrained structure to maintain optimal spacing and charge interactions between amino acids or amino acid functional groups. Those skilled in the art know or can determine which structures construct functionally equivalent amino acid analogs and amino acid mimetic compounds.

[0026] As used herein, subjects “at risk” of developing a disease or adverse reaction may or may not have a detectable disease or symptoms of a disease, and may or may not exhibit a detectable disease or symptoms of a disease prior to the treatment methods described herein. “At risk” means that a subject has one or more risk factors, which are measurable parameters that correlate with the development of diseases described herein and known in the art. Subjects having one or more of these risk factors have a higher probability of developing the disease or of having an adverse reaction than subjects not having one or more of these risk factors.

[0027] "Biocompatibility" refers to a material or compound that is generally not harmful to the biological function of cells or objects and does not result in any unacceptable degree of toxicity, including allergenicity and pathological conditions.

[0028] The term "bond" refers to a direct relationship between two molecules resulting from covalent, electrostatic, hydrophobic, and ionic and / or hydrogen bonding interactions, including, for example, salt and water bridges.

[0029] A "coding sequence" refers to any nucleic acid sequence that contributes to coding the polypeptide product of a gene. In contrast, the term "non-coding sequence" refers to any nucleic acid sequence that does not directly contribute to coding the polypeptide product of a gene.

[0030] Throughout this disclosure, unless otherwise required by context, the words “comprise,” “comprises,” and “comprising” are understood to imply the inclusion of the described step or element or group of steps or elements, but not the exclusion of other steps or elements or groups of steps or elements.

[0031] "Consists of" means including and being limited to what follows the phrase "consists of." Thus, the phrase "consists of" indicates that the enumerated elements are necessary or essential, and the other elements are not required. "Essentially consists of" means including any elements enumerated after the phrase, and being limited to other elements that do not interfere with or contribute to the activity or action of the enumerated elements as expressed in this disclosure. Thus, the phrase "essentially consists of" indicates that the enumerated elements are necessary or essential, but the other elements are optional and may or may not be present, depending on whether they substantially affect the activity or action of the enumerated elements.

[0032] The terms “endotoxin-free” or “substantially endotoxin-free” generally refer to compositions, solvents, and / or blood vessels containing the maximum trace amount of endotoxin (e.g., an amount that does not have clinically harmful physiological effects on the subject), and preferably an undetectable amount of endotoxin. Endotoxins are toxins associated with certain microorganisms, such as bacteria, typically Gram-negative bacteria, although endotoxins can be found in Gram-positive bacteria such as Listeria monocytogenes. The most commonly found endotoxins are lipopolysaccharides (LPS) or lipooligosaccharides (LOS) found on the outer membranes of various Gram-negative bacteria, representing a central pathogenic feature in the disease-causing ability of these bacteria. Small amounts of endotoxin in humans can produce harmful physiological effects, including fever, decreased blood pressure, and activation of inflammation and coagulation.

[0033] Therefore, in pharmaceutical production, it is often desirable to remove most or all traces of endotoxins from drug products and / or drug containers, as even small amounts can cause adverse effects on humans. Since temperatures above 300°C are typically required to decompose most endotoxins, depyrogenation ovens can be used for this purpose. For example, based on the primary packaging material such as a syringe or vial, a combination of a glass temperature of 250°C and a holding time of 30 minutes is often sufficient to achieve a 3-log reduction in endotoxin levels. Other methods for removing endotoxins are being considered, for example, including chromatography and filtration methods described herein and known in the art.

[0034] Endotoxins can be detected using conventional techniques known in the art. For example, the Limulus Amoebocyte Lysate assay, which utilizes horseshoe crab blood, is a highly sensitive assay for detecting the presence of endotoxins. In this test, very low levels of LPS can cause detectable coagulation of Limulus lysate due to a potent enzyme cascade that amplifies the reaction. Endotoxins can also be quantified by enzyme-linked immunosorbent assay (ELISA). Since they are substantially endotoxin-free, endotoxin levels may be approximately 0.001, 0.005, 0.01, 0.02, 0.03, 0.04, 0.05, 0.06, 0.08, 0.09, 0.1, 0.5, 1.0, 1.5, 2, 2.5, 3, 4, 5, 6, 7, 8, 9, or less than 10 EU / mg of active compounds. Typically, 1 ng of lipopolysaccharide (LPS) is equivalent to approximately 1 to 10 EU.

[0035] "Median effective concentration" or "EC 50 The term "EC" refers to the concentration of the agent described herein (e.g., modified PPE) that induces a response midway between baseline and maximum after several specified exposure times, and thus the EC of the graded dose-response curve. 50 EC50 represents the concentration of the compound at which 50% of its maximum effect is observed. EC50 also represents the plasma concentration required to obtain 50% of the maximum effect in vivo. Similarly, EC50 represents the concentration at which 50% of the maximum effect is observed. 90 "EC" refers to the concentration of a drug or composition at which 90% of its maximum effect is observed. 90 The EC50 can be calculated from the Hill gradient or determined directly from the data using conventional knowledge in the art. In some embodiments, the EC50 of a drug (e.g., modified PPE) 50These values ​​are approximately 0.01, 0.05, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 40, 50, 60, 70, 80, 90, 100, 200, or less than 500 nM. In some embodiments, the drug will have an EC50 value of approximately 1 nM or less.

[0036] The “half-life” of a drug, such as modified PPE, may refer to the time it takes for the drug to lose half of its pharmacological, physiological, or other activity compared to such activity at the time of administration to the serum or tissue of an organism, or at any other defined point in time. “Half-life” may also refer to the time it takes for the amount or concentration of the drug to be reduced by half of the initial dose administered to the serum or tissue of an organism, compared to such amount or concentration at the time of administration to the serum or tissue of an organism, or at any other defined point in time. Half-life may be measured in serum and / or any one or more selected tissues.

[0037] The term "heterogeneous" refers to features or elements (e.g., protease cleavage sites) of a polypeptide or polynucleotide that originate from a source different from that of the wild-type polypeptide or the polynucleotide it encodes, such as features from a species different from the wild-type, or manipulated features of non-natural origin.

[0038] The terms “modify” and “change” typically include “increase,” “enhance,” or “stimulate,” as well as “decrease,” or “reduce,” by a statistically significant or physiologically significant amount or degree compared to a control. The “increase,” “stimulate,” or “enhance” is typically a “statistically significant” amount and may include an increase of about or at least about 1.1, 1.2, 1.5, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, or 1000 times greater than the amount produced without the composition (e.g., in the absence of the drug) or by the control composition. The amount “reduced” or “reduced” is typically a “statistically significant” amount and may include reductions of about or at least less than about 1.1, 1.2, 1.5, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, or 1000 times the amount produced without the composition (e.g., in the absence of the drug) or with the control composition. Examples of comparisons and “statistically significant” amounts are described herein.

[0039] The terms “polypeptide,” “protein,” and “peptide” are used interchangeably and refer to polymers of amino acids, not limited to any particular length. The term “enzyme” includes polypeptide or protein catalysts. As used herein, “proprotein,” “proenzyme,” or “enzyme precursor” refers to an inactive (or substantially inactive) protein or enzyme that is typically activated by protease cleavage of an activated peptide to produce an active protein or enzyme. The term includes modifications such as myristoylation, sulfation, glycosylation, phosphorylation, and addition or deletion of signal sequences. The term “polypeptide” or “protein” means one or more chains of amino acids, each chain containing amino acids covalently linked by peptide bonds, and such polypeptide or protein may contain multiple chains non-covalently and / or covalently linked together by peptide bonds, including native proteins, i.e., proteins of natural origin, particularly those produced by non-recombinant cells, or molecules having sequences of genetically engineered or recombinant cells and possessing the amino acid sequence of a native protein, or molecules having deletions, additions to, and / or substitutions of one or more amino acids from a native sequence. In certain embodiments, the polypeptide is a “recombinant” polypeptide produced by a recombinant cell containing one or more recombinant DNA molecules, and is typically made from heterogeneous polynucleotide sequences or combinations of polynucleotide sequences not otherwise found in the cell.

[0040] The terms “polynucleotide” and “nucleic acid” include mRNA, RNA, cRNA, cDNA, and DNA. The terms typically refer to macromolecular forms of nucleotides, ribonucleotides, or deoxynucleotides, or modified forms of either type of nucleotide, that are at least 10 bases long. The terms include single-stranded and double-stranded forms of DNA. The terms “isolated DNA,” “isolated polynucleotide,” and “isolated nucleic acid” refer to molecules isolated without containing the total genomic DNA of a particular species. Therefore, an isolated DNA segment encoding a polypeptide refers to a DNA segment containing one or more coding sequences, and further isolated substantially from or freely purified from the total genomic DNA of the species from which the DNA segment is obtained. It also includes non-coding polynucleotides that do not encode polypeptides (e.g., primers, probes, oligonucleotides). Furthermore, it includes recombinant vectors, such as expression vectors, viral vectors, plasmids, cosmids, phagemids, phages, and viruses.

[0041] Additional coding or non-coding sequences may be present within the polynucleotides described herein, although this is not essential, and polynucleotides may be bound to other molecules and / or supporting materials, although this is not essential. Therefore, polynucleotides or expressible polynucleotides can be combined with other sequences, such as expression regulatory sequences, regardless of the length of the coding sequence itself.

[0042] "Expression regulatory sequences" include corresponding amino acid regulatory sequences for nucleic acids, or for promoters, leaders, enhancers, introns, recognition motifs for RNA or DNA-binding proteins, polyadenylation signals, terminators, intra-sequence ribosome entry sites (IRESs), secretory signals, and intracellular localization signals, which have the ability to influence the transcription or translation of coding sequences within host cells, or their location within or within cells. Exemplary expression regulatory sequences are described in Goeddel; Gene Expression Technology: Methods in Enzymology 185, Academic Press, San Diego, Calif. (1990).

[0043] A "promoter" is a DNA regulatory region capable of binding to an intracellular RNA polymerase and initiating the transcription of a downstream (3' direction) coding sequence. As used herein, a promoter sequence is bound at its 3' end by a transcription start site, extends upstream (5' direction), and contains the minimum number of bases or elements necessary to initiate transcription at a detectable level above the background. The transcription start site (conveniently defined by mapping to nuclease S1) may be found within the promoter sequence and the protein-binding domain (consensus sequence) involved in RNA polymerase binding. Eukaryotic promoters may, often but not always, contain "TATA" and "CAT" boxes. Prokaryotic promoters contain the Shine-Dalgano sequence in addition to the -10 and -35 consensus sequences.

[0044] Numerous promoters, including constitutive, inducible, and repressive promoters from various different sources, are well known in the art. Typical sources include, for example, viruses, mammals, insects, plants, yeasts, and bacterial cell types, and suitable promoters from these sources can be readily available or constructed based on sequences published online, or synthetically from deposits such as ATCC and other commercial or individual sources. Promoters may be unidirectional (i.e., initiating transcription in one direction) or bidirectional (i.e., initiating transcription in either the 3' or 5' direction). Non-limiting examples of promoters include, for example, the T7 bacterial expression system, the pBAD(araA) bacterial expression system, the cytomegalovirus (CMV) promoter, the SV40 promoter, and the RSV promoter. Examples of inducible promoters include the Tet system (US Patent Nos. 5,464,758 and 5,814,618), the Ecdysone-inducible system (No et al., Proc. Natl. Acad. Sci. (1996) 93(8):3346-3351), the T-REx™ system (Invitrogen Carlsbad, CA), LacSwitch® (Stratagene, San Diego, CA), and the Cre-ERT tamoxifen-inducible recombinase system (Indra et al. Nuc. Acid. Res. (1999) 27(22):4324-4327; Nuc. Acid. Res. (2000) 28(23):e99, US Patent No. 7,112,715, and Kramer & Fussenegger, Methods) Examples include Mol. Biol. (2005) 308:123-144), or any promoter known in the art that is suitable for expression in the desired cells.

[0045] "Expressable polynucleotides" include cDNA, RNA, mRNA, or other polynucleotides comprising at least one coding sequence and optionally at least one expression regulatory sequence, such as transcriptional and / or translational regulatory elements, which can express encoded polypeptides (e.g., modified PPE proproteins) upon introduction into cells, e.g., cells of interest.

[0046] In some embodiments, the expressible polynucleotide is a modified RNA or modified mRNA polynucleotide, e.g., a non-natural RNA analog. In certain embodiments, the modified RNA or mRNA polypeptide contains one or more modified or non-natural bases, e.g., a nucleotide base other than adenine (A), guanine (G), cytosine (C), thymine (T), and / or uracil (U). In some embodiments, the modified mRNA contains one or more modified or non-natural nucleotide interlinks. Expressible RNA polynucleotides for delivering the encoded therapeutic polypeptide are described, for example, in Kormann et al., Nat Biotechnol. 29:154-7, 2011, and U.S. Patent Applications 2015 / 0111248, 2014 / 0243399, 2014 / 0147454, and 2013 / 0245104, which are incorporated in their entirety by reference.

[0047] In some embodiments, various viral vectors that can be used to deliver expressible polynucleotides include adenovirus vectors, herpesvirus vectors, vaccinia virus vectors, adeno-associated virus (AAV) vectors, and retrovirus vectors. In some cases, the retrovirus vector is a derivative of a mouse or avian retrovirus, or a lentivirus vector. Examples of retrovirus vectors into which a single foreign gene may be inserted include, but are not limited to, Moloney mouse leukemia virus (MoMuLV), Harvey mouse sarcoma virus (HaMuSV), mouse mammary tumor virus (MuMTV), SIV, BIV, HIV, and Rous sarcoma virus (RSV). Some additional retrovirus vectors may incorporate multiple genes. All of these vectors may transmit or incorporate genes for selectable markers so that transduced cells can be identified and produced. By inserting the polypeptide sequence of interest into the viral vector, along with another gene encoding a ligand for a receptor on a specific target cell, for example, the vector can be made target-specific. Retroviral vectors can be made target-specific, for example, by inserting polynucleotides that encode proteins. Exemplary targeting can be achieved by targeting retroviral vectors using antibodies. Those skilled in the art know, or can readily confirm without excessive experimentation, specific polynucleotide sequences that can be inserted into the retroviral genome to enable target-specific delivery of retroviral vectors.

[0048] In certain cases, the expressible polynucleotides described herein are manipulated for localization within a specific compartment of the cell, potentially such as the nucleus, or for secretion from the cell or translocation to the cell's plasma membrane. In an exemplary embodiment, the expressible polynucleotide is manipulated for nuclear localization.

[0049] The term “isolated” polypeptide or protein as used herein means that the protein in question (1) does not contain at least some other proteins that would typically be found in nature, (2) does not essentially contain other proteins from the same source, e.g., from the same species, (3) is expressed by cells of a different species, (4) is isolated from at least about 50% polynucleotides, lipids, carbohydrates, or other substances associated in nature, (5) the “isolated protein” is not associated (by covalent or non-covalent interactions) with any part of a protein associated in nature, (6) is manipulably associated (by covalent or non-covalent interactions) with a polypeptide not associated in nature, or (7) is not of natural origin. Such isolated proteins may be encoded by genomic DNA, cDNA, mRNA, or other RNA, or any combination thereof, which may be of synthetic origin. In certain embodiments, the isolated protein is substantially free of proteins or polypeptides or other contaminants found in its natural environment that would interfere with its use (therapeutic, diagnostic, prophylactic, research, or otherwise).

[0050] In certain embodiments, the "purity" of any given agent (e.g., modified PPE) in a composition may be defined. For example, certain compositions may include, but are not limited to, agents such as polypeptide agents that, when measured by high-performance liquid chromatography (HPLC) or well-known forms of column chromatography frequently used in biochemistry and analytical chemistry for separating, identifying, and quantifying compounds, have a purity of at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% on a protein-based or weight-based basis, and include all minorities and ranges in between.

[0051] The term "reference sequence" generally refers to a nucleic acid coding sequence or amino acid sequence that another sequence is being compared to. All polypeptide and polynucleotide sequences described herein are included as reference sequences, including those described by name, as well as those described in tables and sequence lists.

[0052] Certain embodiments include biologically active “mutants” and “fragments” of the proteins / polypeptides described herein, as well as polynucleotides encoding them. A “mutant” contains one or more substitutions, additions, deletions, and / or insertions to a reference polypeptide or polynucleotide (see, for example, the Table and Sequence List). A mutant polypeptide or polynucleotide contains an amino acid or nucleotide sequence that has at least about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or more of sequence identity, similarity, or homology with a reference sequence, as described herein, and substantially retains the activity of the reference sequence. Furthermore, the sequence may consist of a reference sequence formed by the addition, deletion, insertion, or substitution of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, or 150 or more amino acids or nucleotides, or may differ from such a sequence but substantially retains the activity of at least one of the reference sequences. In certain embodiments, the addition or deletion may include C-terminal and / or N-terminal additions and / or deletions.

[0053] As used herein, the term “sequence identity,” or for example, “50% identical sequences,” refers to the extent to which sequences are identical at the nucleotide or amino acid level across a comparison window. Thus, the “percentage of sequence identity” can be calculated by comparing two optimally aligned sequences across a comparison window, determining the number of positions in which identical nucleic acid bases (e.g., A, T, C, G, I) or identical amino acid residues (e.g., Ala, Pro, Ser, Thr, Gly, Val, Leu, Ile, Phe, Tyr, Trp, Lys, Arg, His, Asp, Glu, Asn, Gln, Cys, and Met) occur in both sequences to obtain the number of matching positions, dividing the number of matching positions by the total number of positions within the comparison window (i.e., the window size), and multiplying the result by 100 to obtain the percentage of sequence identity. The optimal alignment of sequences for aligning the comparison window can be achieved by a computerized implementation of the algorithm (GAP, BESTFIT, FASTA, and TFASTA from Wisconsin Genetics Software Package Release 7.0, Genetics Computer Group, 575 Science Drive Madison, Wis., USA) or by one of a variety of selected methods, through inspection and best alignment (i.e., resulting in the highest percentage of homology across the comparison window). For example, the BLAST family of programs can also be referenced, as disclosed by Altschul et al., Nucl. Acids Res. 25:3389, 1997.

[0054] The term "solubility" refers to the property of the agents described herein (e.g., modified PPEs) to dissolve in a liquid solvent and form a homogeneous solution. Solubility is typically expressed as concentration, by any of the following: mass of solute per unit volume of solvent (e.g., g, g / dL (100 mL), mg / mL of solute per 1 kg of solvent), molar concentration, molar concentration fraction, or any other similar description of concentration. The maximum equilibrium amount of solute that can dissolve per unit volume of solvent is the solubility of that solute in that solvent under specified conditions, including temperature, pressure, pH, and the properties of the solvent. In certain embodiments, solubility is measured at physiological pH or other pH, e.g., pH 5.0, pH 6.0, pH 7.0, pH 7.4, pH 7.6, pH 7.8, or pH 8.0 (e.g., approximately pH 5–8). In certain embodiments, solubility is measured in water or a physiological buffer such as PBS or NaCl (with or without NaPO4). In certain embodiments, solubility is measured at relatively low pH (e.g., pH 6.0) and relatively high salt (e.g., 500 mM NaCl and 10 mM NaPO4). In certain embodiments, solubility is measured in a biological fluid (solvent) such as blood or serum. In certain embodiments, the temperature can be about room temperature (e.g., about 20, 21, 22, 23, 24, 25°C) or about body temperature (37°C). In certain embodiments, the drug has a solubility of at least about 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 40, 50, 60, 70, 80, 90, or 100 mg / ml at room temperature or 37°C.

[0055] "Subject" or "Subject requiring it" or "Patient" or "Patient requiring it" includes mammalian subjects such as human subjects.

[0056] "Substantially" or "essentially" means, for example, nearly all or completely of a given quantity, such as 95%, 96%, 97%, 98%, 99%, or more.

[0057] "Statistically significant" means that the result was unlikely to have occurred by chance. Statistical significance can be determined by any method known in the art. A commonly used measure of significance is the p-value, which is the frequency or probability that the observed event would occur if the null hypothesis were true. If the obtained p-value is less than the significance level, the null hypothesis is rejected. In simple terms, the significance level is defined as a p-value of 0.05 or less.

[0058] "Therapeutic response" refers to the improvement (whether or not it is sustained) of symptoms based on the administration of one or more medications.

[0059] As used herein, “therapeutic dose,” “therapeutic dose,” “preventive dose,” and “diagnostic dose” refer to the amount of drug (e.g., modified PPE protein) required to elicit a desired biological response after administration.

[0060] As used herein, “treatment” of an object (e.g., a mammal such as a human) or cells is any type of intervention used in an attempt to alter the natural course of an individual or cell. Treatment includes, but is not limited to, the administration of a pharmaceutical composition and may be carried out either prophylactically or after the onset of a pathological event or contact with a pathogen. It also includes “prophylactic” treatment, which may be directed to reduce the rate of progression of the disease or condition being treated, delay the onset of the disease or condition, or reduce the severity of its onset. “Treatment” or “prevention” does not necessarily imply the complete eradication, cure, or prevention of the disease or condition or its associated symptoms.

[0061] The term "wild-type" refers to a gene or gene product (e.g., polypeptide) that is most frequently observed in a population and is therefore arbitrarily designed to have a "normal" or "wild-type" form of the gene.

[0062] Each embodiment described herein applies to all other embodiments unless otherwise expressly stated.

[0063] Modified PPE protein Embodiments of the present disclosure include modified porcine pancreatic elastase (PPE) proteins comprising at least one amino acid change from the wild-type PPE protein (e.g., SEQ ID NO: 4), including a change in one or more of the residues Q211, T55, D74, R75, S214, R237, and / or N241, with numbering defined by SEQ ID NO: 1 (wild-type PPE proprotein). Pancreatic elastases such as PPE are a class of serine proteases produced as inactive enzyme precursors (or proproteins, proenzymes) consisting of a signal peptide, an activating peptide, and a peptidase domain. The wild-type PPE proprotein is activated by trypsin cleavage of the activating peptide to release an enzymatically active PPE peptidase domain, or PPE protein. The amino acid sequence and its domain of wild-type PPE are provided in Table S1. [Table 1]

[0064] As described herein, PPEs can kill cancer cells regardless of their genetic abnormalities and are relatively harmless to non-cancerous or healthy cells. However, one barrier to the antitumor efficacy of PPEs is the presence of serine protease inhibitors, such as alpha-1 antitrypsin (A1AT, UniProtKB-P01009), in the blood and tumor microenvironment. In some instances, A1AT binds to wild-type PPE, inhibiting its catalytic activity and thereby impairing the cancer cell-killing activity of wild-type PPEs.

[0065] Accordingly, embodiments of the present disclosure relate to modified PPE having increased cancer cell killing activity and / or reduced binding to and / or interaction with the human A1AT protein compared to that of the wild-type PPE protein. In certain embodiments, at least one amino acid change is one or more of the residues Q211, T55, D74, R75, S214, R237, and / or N241 within the wild-type PPE peptidase domain (SEQ ID NO: 4), and the numbering is defined by SEQ ID NO: 1. In some embodiments, at least one amino acid change is an amino acid substitution, deletion, and / or addition. In some embodiments, at least one amino acid change is selected from one or more of Q211F, T55A, D74A, R75A, R75E, Q211A, S214A, R237A, N241A, and N241Y, and the numbering is defined by SEQ ID NO: 1. Exemplary amino acid sequences of the modified PPE protein (active PPE peptidase domain) are provided in Table S2. [Table 2]

[0066] Therefore, in some embodiments, the modified PPE protein contains, consists of, or is essentially composed of, an amino acid sequence that is at least 80, 85, 90, 95, 98, or 99% identical to an amino acid sequence selected from Table S2, or an amino acid sequence that retains at least one amino acid change relative to the wild-type PPE protein. In certain embodiments, the modified PPE protein from Table S2, or its variant, contains a signal peptide (e.g., SEQ ID NO: 2), an activation peptide (e.g., SEQ ID NO: 3), or both at its N-terminus. Certain modified PPE proteins are in an enzymatically inactive protein form (i.e., the modified PPE protein) and contain the N-terminal signal peptide and activation peptide, as well as an amino acid sequence that is at least 80, 85, 90, 95, 98, or 99% identical to an amino acid sequence selected from Table S2, or an amino acid sequence selected from Table S2 that retains at least one amino acid change relative to the wild-type PPE. In some cases, the modified PPE protein in its inactive PPE proprotein form is activated by protease cleavage of the activating peptide (e.g., by trypsin cleavage of SEQ ID NO: 3, or by cleavage of an alternative protease in the modified activating peptide), generating an enzymatically active modified PPE protein. Activation may occur in vitro or in vivo, for example, in cancerous tissue or tumor sites.

[0067] For example, in certain embodiments, the modified PPE protein contains, consists of, or essentially consists of an amino acid sequence that is at least 80, 85, 90, 95, 98, or 99% identical to SEQ ID NO: 5 and retains the Q211F amino acid substitution, and in some cases is in a proprotein form (modified PPE proprotein) that includes an N-terminal signal peptide (e.g., SEQ ID NO: 2) and an activation peptide (e.g., SEQ ID NO: 3). In some embodiments, the modified PPE protein contains, consists of, or essentially consists of an amino acid sequence that is at least 80, 85, 90, 95, 98, or 99% identical to SEQ ID NO: 6 and retains the T55A amino acid substitution, and in some cases is in a proprotein form (modified PPE proprotein) that includes an N-terminal signal peptide (e.g., SEQ ID NO: 2) and an activation peptide (e.g., SEQ ID NO: 3). In certain embodiments, the modified PPE protein contains, consists of, or essentially consists of an amino acid sequence that is at least 80, 85, 90, 95, 98, or 99% identical to SEQ ID NO: 7 and retains the N241A amino acid substitution, and in some cases is in a proprotein form (modified PPE proprotein) containing an N-terminal signal peptide (e.g., SEQ ID NO: 2) and an activation peptide (e.g., SEQ ID NO: 3).

[0068] In certain embodiments, the modified PPE protein contains, consists of, or essentially consists of, an amino acid sequence that is at least 80, 85, 90, 95, 98, or 99% identical to SEQ ID NO: 8 and retains the N241Y amino acid substitution, and in some cases is in a proprotein form (modified PPE proprotein) that includes an N-terminal signal peptide (e.g., SEQ ID NO: 2) and an activation peptide (e.g., SEQ ID NO: 3). In certain embodiments, the modified PPE protein contains, consists of, or essentially consists of, an amino acid sequence that is at least 80, 85, 90, 95, 98, or 99% identical to SEQ ID NO: 9 and retains the R75A amino acid substitution, and in some cases is in a proprotein form (modified PPE proprotein) that includes an N-terminal signal peptide (e.g., SEQ ID NO: 2) and an activation peptide (e.g., SEQ ID NO: 3). In some embodiments, the modified PPE protein contains, consists of, or essentially consists of, an amino acid sequence that is at least 80, 85, 90, 95, 98, or 99% identical to SEQ ID NO: 10 and retains the R75E amino acid substitution, and in some cases is in a proprotein form (modified PPE proprotein) that includes an N-terminal signal peptide (e.g., SEQ ID NO: 2) and an activation peptide (e.g., SEQ ID NO: 3). In some embodiments, the modified PPE protein contains, consists of, or essentially consists of, an amino acid sequence that is at least 80, 85, 90, 95, 98, or 99% identical to SEQ ID NO: 11 and retains the Q211A amino acid substitution, and in some cases is in a proprotein form (modified PPE proprotein) that includes an N-terminal signal peptide (e.g., SEQ ID NO: 2) and an activation peptide (e.g., SEQ ID NO: 3).

[0069] In certain embodiments, the modified PPE protein contains, consists of, or essentially consists of, an amino acid sequence that is at least 80, 85, 90, 95, 98, or 99% identical to SEQ ID NO: 12 and retains the R237A amino acid substitution, and in some cases is in a proprotein form (modified PPE proprotein) that includes an N-terminal signal peptide (e.g., SEQ ID NO: 2) and an activation peptide (e.g., SEQ ID NO: 3). In some embodiments, the modified PPE protein contains, consists of, or essentially consists of, an amino acid sequence that is at least 80, 85, 90, 95, 98, or 99% identical to SEQ ID NO: 13 and retains the S214A amino acid substitution, and in some cases is in a proprotein form (modified PPE proprotein) that includes an N-terminal signal peptide (e.g., SEQ ID NO: 2) and an activation peptide (e.g., SEQ ID NO: 3). In some embodiments, the modified PPE protein contains, consists of, or essentially consists of an amino acid sequence that is at least 80, 85, 90, 95, 98, or 99% identical to SEQ ID NO: 14 and retains the D74A amino acid substitution, and in some cases is in a proprotein form (modified PPE proprotein) that includes an N-terminal signal peptide (e.g., SEQ ID NO: 2) and an activation peptide (e.g., SEQ ID NO: 3).

[0070] In some embodiments, the modified PPE protein or proprotein comprises a modified activating peptide relative to the wild-type activating peptide sequence of SEQ ID NO: 3. In these and related embodiments, the modified activating peptide comprises a heteroprotease cleavage site that is not cleavable by trypsin (as is the wild-type activating peptide), but instead cleavable by a protease selected from metalloproteases, asparagine proteases, and cysteine ​​proteases. The incorporation of such non-trypsin, heteroprotease cleavage sites can be used, for example, to improve or otherwise facilitate the selective cleavage and activation of the modified PPE protein in cancer tissue or tumor sites in vivo, compared to systemic activation via the wild-type activating peptide by a trypsin protease. In some embodiments, the heteroprotease cleavage site is cleavable by a protease selected from MMP12, cathepsin D (CTSD), cathepsin C (CTSC), and cathepsin L (CTSL). Specific examples of heteroprotease cleavage sites include MMP12 cleavage sites such as GAAG / LGGA (SEQ ID NO: 15), GAAG / VVGG (SEQ ID NO: 16), and GAAG / LVGG (SEQ ID NO: 17), CTSD cleavage sites such as LLVL / VVLG (SEQ ID NO: 18) and LLVL / VVGG (SEQ ID NO: 19), CTSC cleavage sites such as ASEI / VGGR (SEQ ID NO: 20), and CTSL cleavage sites such as ALLG / AAGG (SEQ ID NO: 21), ALLG / VVGG (SEQ ID NO: 22), and ALLG / AVGG (SEQ ID NO: 23).

[0071] In some embodiments, the modified PPE protein has increased cancer cell killing activity compared to the wild-type PPE protein (SEQ ID NO: 4). In some embodiments, the increased cancer cell killing activity is in vitro or in vivo, in the absence of human A1AT protein. For example, in some embodiments, the modified PPE protein has increased cancer cell killing activity in the absence of human A1AT protein, approximately or at least approximately 2x, 5x, 10x, 50x, 100x, 500x, or 1000x compared to the cancer cell killing activity of the wild-type PPE protein. In certain embodiments, the increased cancer cell killing activity is in vitro or in vivo, in the presence of human A1AT protein. For example, in some embodiments, the modified PPE protein has increased cancer cell killing activity in the presence of human A1AT protein, approximately or at least approximately 2x, 5x, 10x, 50x, 100x, 500x, or 1000x compared to the cancer cell killing activity of the wild-type PPE protein.

[0072] In certain embodiments, the modified PPE protein has reduced binding to or interaction with the human A1AT protein compared to that of the wild-type PPE protein (SEQ ID NO: 4). In some embodiments, the modified PPE has approximately or at least approximately 2x, 5x, 10x, 50x, 100x, 500x, or 1000x or more reduced binding to the human A1AT protein compared to the binding of the wild-type PPE protein to the human A1AT protein. Binding can be measured in vivo or in vitro.

[0073] In certain embodiments, the modified PPE has serine protease activity such that, for example, the modified PPE has the same or substantially the same serine protease activity as the wild-type PPE (SEQ ID NO: 4). In some embodiments, the modified PPE has serine protease activity of about or at least about 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700, 800, 900, or 1000% or more of that of the wild-type PPE. In some embodiments, the modified PPE has increased serine protease activity compared to the wild-type PPE, for example, when measured in the presence or absence of human A1AT protein. In some embodiments, the serine protease activity of modified PPE in the presence of human A1AT protein (e.g., in vivo, in vitro) is about or at least about 2 times, 5 times, 10 times, 50 times, 100 times, 500 times, or 1000 times higher than the serine protease activity of wild-type PPE under the same or equivalent conditions. In some embodiments, the serine protease activity of modified PPE in the absence of human A1AT protein (e.g., in vivo, in vitro) is about or at least about 2 times, 5 times, 10 times, 50 times, 100 times, 500 times, or 1000 times higher than the serine protease activity of wild-type PPE under the same or equivalent conditions.

[0074] Serine protease activity and cancer cell killing activity can be measured according to conventional techniques in the art. For example, serine protease activity can be monitored using a chromogenic substrate activity assay (N-methoxysuccinyl-Ala-Ala-Pro-Val p-nitroanilide), and cancer cell killing activity can be measured in vitro or in vivo.

[0075] Method of use and pharmaceutical composition Certain embodiments include a method for treating, improving the symptoms of, and / or reducing the progression of a disease or condition in a subject that requires such treatment, improvement, and / or reduction of its progression, the method comprising administering to the subject a composition comprising at least one modified PPE protein or proprotein as described herein. In certain embodiments, the disease is cancer, i.e., the subject requiring it has cancer, is suspected of having cancer, or is at risk of having cancer. In some embodiments, the composition comprises a modified PPE proprotein (inactive form), which is activated by protease cleavage of an activating peptide in cancerous tissue or tumor site in the subject requiring the composition to produce an active modified PPE protein.

[0076] In certain embodiments, the cancer is either a primary cancer or a metastatic cancer. In some embodiments, cancer is selected from one or more of the following: melanoma (optionally metastatic melanoma), breast cancer (optionally triple-negative breast cancer, TNBC), kidney cancer (optionally renal cell carcinoma), pancreatic cancer, bone cancer, prostate cancer, small cell lung cancer, non-small cell lung cancer (NSCLC), mesothelioma, leukemia (optionally lymphocytic leukemia, chronic myeloid leukemia, acute myeloid leukemia, or relapsed acute myeloid leukemia), multiple myeloma, lymphoma, hepatocellular carcinoma, sarcoma, B-cell malignancy, ovarian cancer, colorectal cancer, glioma, glioblastoma multiforme, meningioma, pituitary adenoma, vestibular schwannoma, primary CNS lymphoma, primitive neuroectodermal tumor (medulloblastoma), bladder cancer, uterine cancer, esophageal cancer, brain cancer, head and neck cancer, cervical cancer, testicular cancer, thyroid cancer, and gastric cancer.

[0077] In some embodiments, as described above, the cancer is metastatic cancer. In addition to the cancers described above, exemplary metastatic cancers include, but are not limited to, bladder cancer with metastasis to the bone, liver, and / or lungs; breast cancer with metastasis to the bone, brain, liver, and / or lungs; colorectal cancer with metastasis to the liver, lungs, and / or peritoneum; kidney cancer with metastasis to the adrenal glands, bone, brain, liver, and / or lungs; lung cancer with metastasis to the adrenal glands, bone, brain, liver, and / or other parts of the lungs; melanoma with metastasis to the bone, brain, liver, lungs, and / or skin / muscles; ovarian cancer with metastasis to the liver, lungs, and / or peritoneum; pancreatic cancer with metastasis to the liver, lungs, and / or peritoneum; prostate cancer with metastasis to the adrenal glands, bone, liver, and / or lungs; stomach cancer with metastasis to the liver, lungs, and / or peritoneum; thyroid cancer with metastasis to the bone, liver, and / or lungs; and uterine cancer with metastasis to the bone, liver, lungs, peritoneum, and / or vagina.

[0078] Methods for treating cancer may be combined with other treatments. For example, the combination therapies described herein may be administered to a subject before, during, or after other therapeutic interventions, including symptomatic treatment, radiotherapy, surgery, transplantation, hormone therapy, photodynamic therapy, antibiotic therapy, or any combination thereof. Symptomatic treatment includes the administration of corticosteroids to reduce cerebral edema, headache, cognitive impairment, and vomiting, as well as the administration of anticonvulsants to reduce seizures. Radiotherapy includes radiosurgery such as whole-brain irradiation, fractionated radiotherapy, and stereotactic radiosurgery, which may be further combined with conventional surgery.

[0079] Certain embodiments include combination therapies for treating cancer, which include methods for treating cancer, improving its symptoms, or inhibiting its progression in subjects that require treatment of cancer, improvement of its symptoms, or inhibition of its progression, comprising administering the modified PPP protein or proprotein described herein to a subject in combination with at least one additional agent, e.g., an immunotherapy agent, a chemotherapeutic agent, a hormonal therapy, and / or a kinase inhibitor. In some embodiments, administration of the modified PPE protein or proprotein enhances the sensitivity of the cancer to the additional agent (e.g., an immunotherapy agent, a chemotherapeutic agent, a hormonal therapy, and / or a kinase inhibitor) by about or at least about 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, 100, 2000, 300, 400, 500, 600, 700, 800, 900, 1000, 2000% or more compared to the additional agent alone.

[0080] Certain combination therapies employ one or more cancer immunotherapy agents, or “immunotherapy agents.” In certain cases, the immunotherapy agent modifies the target immune response, for example, to increase or maintain a cancer-related or cancer-specific immune response, thereby resulting in increased immune cell inhibition or reduction of cancer cells. Exemplary immunotherapy agents include polypeptides, e.g., antibodies and their antigen-binding fragments, ligands, and small peptides, as well as mixtures thereof. Immunotherapy agents may also include small molecules, cells (e.g., immune cells such as T cells), various cancer vaccines, gene therapies, or other polynucleotide agents including viral agents such as oncolytic viruses, and others known in the art. Thus, in certain embodiments, the cancer immunotherapy agent is selected from one or more of immune checkpoint modifiers, cancer vaccines, oncolytic viruses, cytokines, and cell-system immunotherapies.

[0081] In certain embodiments, the cancer immunotherapy agent is an immune checkpoint modifier. Specific examples include “antagonists” of one or more inhibitory immune checkpoint molecules and “agonists” of one or more stimulating immune checkpoint molecules. Generally, immune checkpoint molecules are components of the immune system that either signal upward (co-stimulatory molecules) or signal downward, and their targeting has therapeutic potential in cancer because cancer cells can interfere with the innate function of immune checkpoint molecules (see, e.g., Sharma and Allison, Science. 348:56-61, 2015; Topalian et al., Cancer Cell. 27:450-461, 2015; Pardoll, Nature Reviews Cancer. 12:252-264, 2012). In some embodiments, the immune checkpoint modifier (e.g., antagonists, agonists) “binds” or “specifically binds” to one or more immune checkpoint molecules as described herein.

[0082] In some embodiments, an immune checkpoint modifier is an antagonist or inhibitor of one or more inhibitory immune checkpoint molecules. Exemplary inhibitory immune checkpoint molecules include programmed cell death ligand 1 (PD-L1), programmed cell death ligand 2 (PD-L2), programmed cell death 1 (PD-1), V-domain Ig inhibitor of T cell activation (VISTA), cytotoxic T lymphocyte-associated protein 4 (CTLA-4), indoleamine 2,3-dioxygenase (IDO), tryptophan 2,3-dioxygenase (TDO), T cell immunoglobulin domain and mucin domain 3 (TIM-3), lymphocyte activation gene 3 (LAG-3), B and T lymphocyte attenuation factor (BTLA), CD160, and T cell immune receptor having Ig and ITIM domains (TIGIT).

[0083] In certain embodiments, the agent is a PD-1 (receptor) antagonist or inhibitor, and its targeting has been shown to reclaim immune function in the tumor environment (see, for example, Phillips et al., Int Immunol. 27:39-46, 2015). PD-1 is a cell surface receptor belonging to the immunoglobulin superfamily and expressed on T cells and pro-B cells. PD-1 interacts with two ligands, PD-L1 and PD-L2. PD-1 functions as an inhibitory immune checkpoint molecule, for example, by reducing or preventing T cell activation, thereby reducing autoimmunity and promoting autoimmune tolerance. The inhibitory effect of PD-1 is achieved at least partially through a dual mechanism that promotes apoptosis of antigen-specific T cells in lymph nodes while also reducing apoptosis in regulatory T cells (suppressive T cells). Some examples of PD-1 antagonists or inhibitors include antibodies, antigen-binding fragments, or small molecules that specifically bind to PD-1 and reduce one or more of its immunosuppressive activities, such as its downstream signaling or its interaction with PD-L1. Specific examples of PD-1 antagonists or inhibitors include the antibodies nivolumab, pembrolizumab, PDR001, MK-3475, AMP-224, AMP-514, and pizilizumab, as well as their antigen-binding fragments (e.g., U.S. Patent Nos. 8,008,449, 8,993,731, 9,073,994, and 9,073,994). See Patent Nos. 9,084,776, 9,102,727, 9,102,728, 9,181,342, 9,217,034, 9,387,247, 9,492,539, 9,492,540, and U.S. Patent Applications Nos. 2012 / 0039906 and 2015 / 0203579.

[0084] In some embodiments, the drug is a PD-L1 antagonist or inhibitor. As described above, PD-L1 is one of the natural ligands for the PD-1 receptor. Common examples of PD-L1 antagonists or inhibitors include antibodies, antigen-binding fragments, or small molecules that specifically bind to PD-L1 and reduce one or more of its immunosuppressive activities, such as its binding to the PD-1 receptor. Specific examples of PD-L1 antagonists include the antibodies atezolizumab (MPDL3280A), avelumab (MSB0010718C), and durvalumab (MEDI4736), as well as their antigen-binding fragments (see, for example, U.S. Patents 9,102,725, 9,393,301, 9,402,899, and 9,439,962).

[0085] In some embodiments, the agent is a PD-L2 antagonist or inhibitor. As described above, PD-L2 is one of the natural ligands for the PD-1 receptor. Common examples of PD-L2 antagonists or inhibitors include antibodies, antigen-binding fragments, or small molecules that specifically bind to PD-L2 and reduce one or more of its immunosuppressive activities, such as its binding to the PD-1 receptor.

[0086] In certain embodiments, the agent is a VISTA antagonist or inhibitor. VISTA is approximately 50 kDa in size and belongs to the immunoglobulin superfamily (having one IgV domain) and the B7 family. It is primarily expressed in leukocytes, and its transcription is partially regulated by p53. There is evidence that VISTA can act as both a ligand and receptor on T cells to inhibit T cell effector function and maintain peripheral immune tolerance. VISTA is produced at high levels in tumor-infiltrating lymphocytes such as myeloid suppressor cells and regulatory T cells, and antibody inhibition results in delayed tumor growth in mouse models of melanoma and squamous cell carcinoma. An exemplary anti-VISTA antagonist antibody is, for example, the antibody described in WO2018 / 237287, which is incorporated in its entirety by reference.

[0087] In some embodiments, the agent is a CTLA-4 antagonist or inhibitor. CTLA4 or CTLA-4 (cytotoxic T lymphocyte-associated protein 4), also known as CD152 (cluster of differentiated 152), is a protein receptor that functions as an inhibitory immune checkpoint molecule by transmitting inhibitory signals to T cells when bound to CD80 or CD86 on the surface of antigen-presenting cells, for example. Common examples of CTLA-4 antagonists or inhibitors include antibodies, antigen-binding fragments, or small molecules that specifically bind to CTLA-4. Specific examples include the antibodies ipilimumab and tremelimumab, as well as their antigen-binding fragments. At least part of the activity of ipilimumab is thought to be mediated by antibody-dependent cell-mediated cytotoxicity (ADCC) death of CTLA-4-expressing repressor Tregs.

[0088] In some embodiments, the agent is an IDO antagonist or inhibitor, or a TDO antagonist or inhibitor. IDO and TDO are tryptophan-degrading enzymes with immunosuppressive properties. For example, IDO is known to suppress T cells and NK cells, generate and activate Treg and myeloid-derived suppressor cells, and promote tumor angiogenesis. Common examples of IDO and TDO antagonists or inhibitors include antibodies, antigen-binding fragments, or small molecules that specifically bind to IDO or TDO (see, e.g., Platten et al., Front Immunol. 5:673, 2014) and reduce or inhibit one or more immunosuppressive activities. Specific examples of IDO antagonists or inhibitors include indoximod (NLG-8189), 1-methyltryptophan (1MT), β-carboline (norharmane, 9H-pyrido[3,4-b]indole), rosmarinic acid, and epacadostat (see, e.g., Sheridan, Nature Biotechnology. 33:321-322, 2015). Specific examples of TDO antagonists or inhibitors include 680C91 and LM10 (see, e.g., Pilotte et al., PNAS USA. 109:2497-2502, 2012).

[0089] In some embodiments, the agent is a TIM-3 antagonist or inhibitor. T cell immunoglobulin domain and mucin domain 3 (TIM-3) are expressed on activated human CD4+ T cells and regulate Th1 and Th17 cytokines. TIM-3 also acts as a negative regulator of Th1 / Tc1 function by inducing cell death through interaction with its ligand, galectin-9. TIM-3 contributes to the suppressive tumor microenvironment, and its overexpression is associated with poor prognosis in various cancers (see, e.g., Li et al., Acta Oncol. 54:1706-13, 2015). Common examples of TIM-3 antagonists or inhibitors include antibodies, antigen-binding fragments, or small molecules that specifically bind to TIM-3 and reduce or inhibit one or more of its immunosuppressive activities.

[0090] In some embodiments, the agent is a LAG-3 antagonist or inhibitor. Lymphocyte activation gene 3 (LAG-3) is expressed on activated T cells, natural killer cells, B cells, and plasmacytoid dendritic cells. It has been reported to negatively regulate T cell proliferation, activation, and homeostasis in a manner similar to CTLA-4 and PD-1 (see, e.g., Workman and Vignali, European Journal of Immun. 33:970-9, 2003, and Workman et al., Journal of Immun. 172:5450-5, 2004), and to play a role in Treg suppression (see, e.g., Huang et al., Immunity. 21:503-13, 2004). LAG3 also maintains CD8+ T cells in an immunotolerative state and, in combination with PD-1, maintains CD8 T cell exhaustion. Common examples of LAG-3 antagonists or inhibitors include antibodies, antigen-binding fragments, or small molecules that specifically bind to LAG-3 and inhibit one or more of its immunosuppressive activities. Specific examples include the antibody BMS-986016 and its antigen-binding fragment.

[0091] In some embodiments, the agent is a BTLA antagonist or inhibitor. Expression of B- and T-lymphocyte attenuation factor (BTLA, CD272) is induced during T cell activation and inhibits T cells through interactions with tumor necrosis family receptors (TNF-R) and B7 family cell surface receptors. BTLA is a ligand for tumor necrosis factor (receptor) superfamily member 14 (TNFRSF14), also known as herpesvirus entry mediator (HVEM). The BTLA-HVEM complex negatively modulates the T cell immune response, for example, by inhibiting the function of human CD8+ cancer-specific T cells (see, e.g., Derre et al., J Clin Invest 120:157-67, 2009). Common examples of BTLA antagonists or inhibitors include antibodies, antigen-binding fragments, or small molecules that specifically bind to BTLA-4 and reduce one or more of its immunosuppressive activities.

[0092] In some embodiments, the agent is an HVEM antagonist or inhibitor, for example, an antagonist or inhibitor that specifically binds to HVEM and interferes with its interaction with BTLA or CD160. Common examples of HVEM antagonists or inhibitors include antibodies, antigen-binding fragments, or small molecules that specifically bind to HVEM and selectively reduce the HVEM / BTLA and / or HVEM / CD160 interaction, thereby reducing one or more of the immunosuppressive activities of HVEM.

[0093] In some embodiments, the agent is a CD160 antagonist or inhibitor, for example, an antagonist or inhibitor that specifically binds to CD160 and interferes with its interaction with HVEM. Common examples of CD160 antagonists or inhibitors include antibodies, antigen-binding fragments, or small molecules that specifically bind to CD160 and selectively reduce the CD160 / HVEM interaction, thereby reducing or inhibiting one or more of its immunosuppressive activities.

[0094] In some embodiments, the drug is a TIGIT antagonist or inhibitor. T cell Ig and the ITIM domain (TIGIT) are co-inhibitory receptors found on the surface of various lymphocytes, suppressing antitumor immunity, for example, via Tregs (Kurtulus et al., J Clin Invest. 125:4053-4062, 2015). Common examples of TIGIT antagonists or inhibitors include antibodies, antigen-binding fragments, or small molecules that specifically bind to TIGIT and reduce one or more of its immunosuppressive activities (see, for example, Johnston et al., Cancer Cell. 26:923-37, 2014).

[0095] In certain embodiments, an immune checkpoint modifier is an agonist of one or more stimulating immune checkpoint molecules. Exemplary stimulating immune checkpoint molecules include CD40, OX40, glucocorticoid-inducible TNFR family-related genes (GITR), CD137(4-1BB), CD27, CD28, CD226, and herpesvirus entry mediator (HVEM).

[0096] In some embodiments, the agent is a CD40 agonist. CD40 is expressed in antigen-presenting cells (APCs) and in some malignancies. Its ligand is CD40L (CD154). In APCs, ligation results in upward regulation of costimulatory molecules, potentially bypassing the need for T cell support in the antitumor immune response. CD40 agonist therapy plays a crucial role in APC maturation and their migration from tumors to lymph nodes, resulting in high antigen presentation and T cell activation. Anti-CD40 agonist antibodies produce substantial responses and sustained anti-cancer immunity in animal models, at least partially mediated by cytotoxic T cells (see, e.g., Johnson et al. Clin Cancer Res. 21:1321-1328, 2015, and Vonderheide and Glennie, Clin Cancer Res. 19:1035-43, 2013). Common examples of CD40 agonists include antibodies, antigen-binding fragments, small molecules, or ligands that specifically bind to CD40 and increase one or more of its immunostimulatory activities. Specific examples include CP-870, 893, dacetuzumab, Chi Lob7 / 4, ADC-1013, CD40L, rhCD40L, and their antigen-binding fragments. Specific examples of CD40 agonists include, but are not limited to, APX005 (see, e.g., US2012 / 0301488) and APX005M (see, e.g., US2014 / 0120103).

[0097] In some embodiments, the agent is an OX40 agonist. OX40 (CD134) promotes the proliferation of effector and memory T cells and suppresses the differentiation and activity of regulatory T cells (see, e.g., Croft et al., Immunol Rev. 229:173-91, 2009). Its ligand is OX40L (CD252). Because OX40 signaling affects both T cell activation and survival, it plays a crucial role in the initiation of the antitumor immune response in lymph nodes and the maintenance of the antitumor immune response in the tumor microenvironment. Common examples of OX40 agonists include antibodies or antigen-binding fragments or small molecules or ligands that specifically bind to OX40 and increase one or more of its immunostimulatory activities. Specific examples include OX86, OX-40L, Fc-OX40L, GSK3174998, MEDI0562 (humanized OX40 agonist), MEDI6469 (mouse OX4 agonist), and MEDI6383 (OX40 agonist), as well as their antigen-binding fragments.

[0098] In some embodiments, the drug is a GITR agonist. Glucocorticoid-inducible TNFR family-related genes (GITRs) increase T cell proliferation, inhibit Treg suppressive activity, and prolong T effector cell survival. GITR agonists have been shown to promote antitumor responses through loss of Treg lineage stability (see, e.g., Schaer et al., Cancer Immunol Res. 1:320-31, 2013). These diverse mechanisms indicate that GITR plays a crucial role in initiating immune responses in lymph nodes and maintaining immune responses in tumor tissue. Its ligand is GITRL. Common examples of GITR agonists include antibodies or antigen-binding fragments or small molecules or ligands that specifically bind to GITR and increase one or more of its immunostimulatory activities. Specific examples include GITRL, INCAGN01876, DTA-1, MEDI1873, and their antigen-binding fragments.

[0099] In some embodiments, the agent is a CD137 agonist. CD137 (4-1BB) is a member of the tumor necrosis factor (TNF) receptor family, and crosslinking of CD137 enhances T cell proliferation, IL-2 secretion, survival, and cytolytic activity. CD137-mediated signaling also protects T cells, such as CD8+ T cells, from activation-induced cell death. Common examples of CD137 agonists include antibodies or antigen-binding fragments or small molecules or ligands that specifically bind to CD137 and increase one or more of its immunostimulatory activities. Specific examples include CD137 (or 4-1BB) ligands (see, e.g., Shao and Schwarz, J Leukoc Biol. 89:21-9, 2011) and antibodies such as utomilumab, including their antigen-binding fragments.

[0100] In some embodiments, the agent is a CD27 agonist. Stimulation of CD27 increases antigen-specific proliferation of naive T cells, contributing to T cell memory and the long-term maintenance of T cell immunity. Its ligand is CD70. Targeting of human CD27 with agonist antibodies stimulates T cell activation and anti-tumor immunity (see, e.g., Thomas et al., Oncoimmunology. 2014;3:e27255.doi:10.4161 / onci.27255, and He et al., J Immunol. 191:4174-83, 2013). Common examples of CD27 agonists include antibodies or antigen-binding fragments or small molecules or ligands that specifically bind to CD27 and increase one or more of its immunostimulatory activities. Specific examples include CD70, as well as the antibodies varlilumab and CDX-1127(1F5), and their antigen-binding fragments.

[0101] In some embodiments, the drug is a CD28 agonist. CD28 is constitutively expressed in CD4+ T cells and some CD8+ T cells. Its ligands include CD80 and CD86, and their stimulation increases T cell proliferation. Common examples of CD28 agonists include antibodies or antigen-binding fragments or small molecules or ligands that specifically bind to CD28 and increase one or more of its immunostimulatory activities. Specific examples include CD80, CD86, the antibody TAB08, and its antigen-binding fragments.

[0102] In some embodiments, the agent is a CD226 agonist. CD226 is a stimulatory receptor that shares a ligand with TIGIT, and, contrary to TIGIT, CD226 involvement enhances T cell activation (see, e.g., Kurtulus et al., J Clin Invest. 125:4053-4062, 2015, Bottino et al., J Exp Med. 1984:557-567, 2003, and Tahara-Hanaoka et al., Int Immunol. 16, 533-538, 2004). Common examples of CD226 agonists include antibodies or antigen-binding fragments or small molecules or ligands (e.g., CD112, CD155) that specifically bind to CD226 and increase one or more of its immunostimulatory activities.

[0103] In some embodiments, the drug is an HVEM agonist. Herpesvirus entry mediator (HVEM), also known as tumor necrosis factor receptor superfamily member 14 (TNFRSF14), is a human cell surface receptor of the TNF receptor superfamily. HVEM is found on a variety of cells, including T cells, APCs, and other immune cells. Unlike other receptors, HVEM is highly expressed on resting T cells and is downwardly regulated upon activation. HVEM signaling has been shown to play a crucial role in the early stages of T cell activation and during the proliferation of tumor-specific lymphocyte populations in lymph nodes. Common examples of HVEM agonists include antibodies or antigen-binding fragments or small molecules or ligands that specifically bind to HVEM and increase one or more of its immunostimulatory activities.

[0104] In certain embodiments, the immunotherapy agent is a bispecific or multispecific antibody. For example, a particular bispecific or multispecific antibody may (i) bind to one or more inhibitory immune checkpoint molecules to inhibit them, and (ii) bind to one or more stimulating immune checkpoint molecules to cause them to act. In certain embodiments, the bispecific or multispecific antibody may (i) bind to one or more of PD-L1, PD-L2, PD-1, CTLA-4, IDO, TDO, TIM-3, LAG-3, BTLA, CD160, and / or TIGIT to inhibit them, and (ii) bind to one or more of CD40, OX40 glucocorticoid-inducible TNFR family-related genes (GITR), CD137 (4-1BB), CD27, CD28, CD226, and / or herpesvirus entry mediator (HVEM) to cause them to act.

[0105] In some embodiments, the immunotherapy agent is a cancer vaccine. In certain embodiments, the cancer vaccine is selected from one or more of the following: Oncophage, human papillomavirus (HPV) vaccine, optionally Gardasil or Cervarix, hepatitis B vaccine, optionally Engerix-B, Recombivax HB or Twinrix, and sipuleucel-T (Provenge); or human Her2 / neu, Her1 / EGF receptor (EGFR), Her3, A33 antigen, B7H3, CD5, CD19, CD20, CD22, CD23 (IgE receptor), MAGE-3, C242 antigen, 5T4, IL-6, IL-13, vascular endothelial growth factor (VEGF) (e.g., VEGF-A), VEGFR-1, VEGFR-2, CD30, CD33 CD37, CD40, CD44, CD51, CD52, CD56, CD74, CD80, CD152, CD200, CD221, CCR4, HLA-DR, CTLA-4, NPC-1C, Tenacin, Vimentin, Insulin-like growth factor 1 receptor (IGF-1R), Alpha-fetoprotein, Insulin-like growth factor 1 (IGF-1), Carbonic anhydrase 9 (CA-IX), Carcinoembryonic antigen (CEA), Guanylyl cyclase C, NY-ESO-1, p53, Survivin, Inte Glyn αvβ3, integrin α5β1, folate receptor 1, transmembrane glycoprotein NMB, fibroblast-activating protein alpha (FAP), glycoprotein 75, TAG-72, MUC1, MUC16 (or CA-125), phosphatidylserine, prostate-specific membrane antigen (PMSA), NR-LU-13 antigen, TRAIL-R1, tumor necrosis factor receptor superfamily member 10b (TNFRSF10B or TRAIL-R2), SLAM family member 7 (S The cancer antigen is selected from one or more of the following: LAMF7, EGP40 pan-cancer antigen, B-cell activator (BAFF), platelet-derived growth factor receptor, glycoprotein EpCAM (17-1A), programmed death 1, protein disulfide isomerase (PDI), regenerating liver phosphatase 3 (PRL-3), prostatic acid phosphatase, Lewis-Y antigen, GD2 (a disialoganglioside expressed in neuroectoderm-derived tumors), glypican-3 (GPC3), and mesothelin.

[0106] In some embodiments, the immunotherapy agent is an oncolytic virus. In some embodiments, the oncolytic virus was selected from one or more of the following: talimogene laherparepvec (T-VEC), coxsackievirus A21 (CAVATAK®), Oncorine (H101), pelareorep (REOLYSIN®), Seneca Valley virus (NTX-010), Senecavirus SVV-001, ColoAd1, SEPREHVIR (HSV-1716), CGTG-102 (Ad5 / 3-D24-GMCSF), GL-ONC1, MV-NIS, and DNX-2401.

[0107] In certain embodiments, the cancer immunotherapy agent is a cytokine. Exemplary cytokines include interferon (IFN)-α, IL-2, IL-12, IL-7, IL-21, and granulocyte-macrophage colony-stimulating factor (GM-CSF).

[0108] In certain embodiments, cancer immunotherapy agents are immune cell-based therapies, including cell-system immunotherapy, such as ex vivo-derived immune cells, including lymphocytes, natural killer (NK) cells, macrophages, and / or dendritic cells (DCs). In some embodiments, lymphocytes include T cells, such as cytotoxic T lymphocytes (CTLs). See, for example, June, J Clin Invest. 117:1466-1476, 2007; Rosenberg and Restifo, Science. 348:62-68, 2015; Cooley et al., Biol. of Blood and Marrow Transplant. 13:33-42, 2007; and Li and Sun, Chin J Cancer Res. 30:173-196, 2018. In some embodiments, T cells include cancer antigen-specific T cells directed towards at least one cancer antigen. In some embodiments, cancer antigen-specific T cells are selected from one or more of the following: chimeric antigen receptor (CAR) modified T cells, T cell receptor (TCR) modified T cells, tumor-infiltrating lymphocytes (TILs), and peptide-induced T cells. In certain embodiments, CAR modified T cells are targeted toward CD-19 (see, e.g., Maude et al., Blood. 125:4017-4023, 2015). In some cases, ex vivo-derived immune cells are autologous cells obtained from the patient being treated.

[0109] Certain combination therapies involve the use of one or more chemotherapeutic agents, such as small molecule chemotherapeutic agents. Non-exclusive examples of chemotherapeutic agents include, among others, alkylating agents, antimetabolites, cytotoxic antibiotics, topoisomerase inhibitors (type 1 or type II), and microtubule inhibitors.

[0110] Examples of alkylating agents include nitrogen mustards (e.g., mechloretamine, cyclophosphamide, mustine, melphalan, chlorambucil, ifosfamide, and busulfan), nitrosoureas (e.g., N-nitroso-N-methylurea (MNU), carmustine (BCNU), lomustine (CCNU), semustine (MeCCNU), fotemustine, and streptozotocin), tetrazine (e.g., dacarbazine, mitozolomide, and temozolomide), aziridines (e.g., thiotepa, mitomycin, diazicone (AZQ)), cisplatin and its derivatives (e.g., carboplatin and oxaliplatin), and non-classical alkylating agents (optionally, procarbazine and hexamethylmelamine).

[0111] Examples of antimetabolites include antifolic acid agents (e.g., methotrexate and pemetrexed), fluoropyrimidines (e.g., 5-fluorouracil and capecitabine), deoxynucleoside analogs (e.g., ancitabine, enokitabine, cytarabine, gemcitabine, decitabine, azacitidine, fludarabine, nerarabine, cladribine, clofarabine, fludarabine, and pentostatin), and thiopurines (e.g., thioguanine and mercaptopurine).

[0112] Examples of cytotoxic antibiotics include anthracyclines (e.g., doxorubicin, daunorubicin, epirubicin, idarubicin, pirarubicin, acralubicin, and mitoxantrone), bleomycin, mitomycin C, mitoxantrone, and actinomycin. Examples of topoisomerase inhibitors include camptothecin, irinotecan, topotecan, etoposide, doxorubicin, mitoxantrone, teniposide, novobiocin, melbaron, and acralubicin.

[0113] Examples of microtubule inhibitors include taxanes (e.g., paclitaxel and docetaxel) and vinca alkaloids (e.g., vinblastine, vincristine, vindesine, and vinorelbine).

[0114] The various chemotherapeutic agents described herein may be combined with one or more of the modified PPE proteins or proproteins described herein and used according to one or more of the methods or compositions described herein.

[0115] Certain combination therapies involve the use of at least one hormonal therapy drug. Common examples of hormonal therapy drugs include hormonal agonists and hormone antagonists. Specific examples of hormonal agonists include progestins, corticosteroids (e.g., prednisolone, methylprednisolone, dexamethasone), insulin-like growth factor, VEGF-derived angiogenic and lymphangiogenic factors (e.g., VEGF-A, VEGF-A145, VEGF-A165, VEGF-C, VEGF-D, PIGF-2), fibroblast growth factor (FGF), galectin, hepatocyte growth factor (HGF), platelet-derived growth factor (PDGF), transforming growth factor (TGF)-beta, androgens, estrogens, and somatostatin analogs. Examples of hormone antagonists include hormone synthesis inhibitors such as aromatase inhibitors, gonadotropin-releasing hormone (GnRH) agonists (e.g., leuprolide, goserelin, triptorelin, histrelin), and their analogues. Also included are hormone receptor antagonists such as selective estrogen receptor modifiers (SERMs, e.g., tamoxifen, raloxifene, toremifene) and antiandrogens (e.g., flutamide, bicalutamide, nilutamide).

[0116] This also includes hormone pathway inhibitors such as antibodies directed at hormone receptors. Examples include IGF receptor inhibitors (e.g., IGF-IR1) such as cictumumab, darotuzumab, figtumumab, ganitumumab, istiratumumab, and lobatumumab; vascular endothelial growth factor receptor 1, 2, or 3 (VEGFR1, VEGFR2, or VEGFR3) inhibitors such as aracizumab pegol, bevacizumab, iclusukumab, and ramucirumab; TGF-beta receptor R1, R2, and R3 inhibitors such as fresolimmumab and metelimumab; and Naxita. Examples include c-Met inhibitors such as mab, EGF receptor inhibitors such as cetuximab, depatuxizumab mafodotin, futuximab, imugatuzumab, laprituximab emtansine, matsuzumab, modotuximab, necitumumab, nimotuzumab, panitumumab, tomzotuximab, and zaltumumab, FGF receptor inhibitors such as aplutamab ixadotin and bemarituzumab, and PDGF receptor inhibitors such as olaratumab and tobetumab.

[0117] The various hormonal therapeutic agents described herein may be combined with one or more of the modified PPE proteins or proproteins described herein and may be used according to one or more of the methods or compositions described herein.

[0118] Certain combination therapies involve the use of at least one kinase inhibitor, including tyrosine kinase inhibitors. Examples of kinase inhibitors include, without limitation, adavocertib, afanitib, aflibercept, axitinib, bevacizumab, bosutinib, cabozantinib, cetuximab, cobimetinib, crizotinib, dasatinib, antrecutinib, erdafitinib, erlotinib, fostamitinib, gefitinib, ibrutinib, imatinib, lapatinib, lenvatinib, mbritinib, nilotinib, panitumumab, pazopanib, pegaptanib, ponatinib, ranibisumab, regorafenib, ruxolitinib, sorafenib, sunitinib, SU6656, tofatinib, trastezumab, vandetanib, and bemafenib.

[0119] The various kinase inhibitors described herein may be combined with one or more of the modified PPE proteins or proproteins described herein and used according to one or more of the methods or compositions described herein.

[0120] In some embodiments, the methods and compositions described herein increase cancer cell death in subjects by about or at least about 2 times, 5 times, 10 times, 50 times, 100 times, 500 times, or 1000 times or more compared to a control or reference. In some embodiments, the methods and compositions described herein increase the median survival time of subjects by 4 weeks, 5 weeks, 6 weeks, 7 weeks, 8 weeks, 9 weeks, 10 weeks, 15 weeks, 20 weeks, 25 weeks, 30 weeks, or 40 weeks or more. In certain embodiments, the methods and compositions described herein increase the median survival time of subjects by 1 year, 2 years, or 3 years or more. In some embodiments, the methods and pharmaceutical compositions increase progression-free survival by 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 7 weeks, 8 weeks, 9 weeks, or 10 weeks or more. In certain embodiments, the methods and compositions described herein increase progression-free survival by 1 year, 2 years, or 3 years or more.

[0121] In certain embodiments, the methods and compositions described herein are sufficient to result in tumor regression, for example, a statistically significant reduction in viable tumor volume, e.g., a reduction of at least 10%, 20%, 30%, 40%, or 50% or more in tumor volume, or as indicated by a changed (e.g., statistically significant decrease) scan dimension. In certain embodiments, the methods and compositions described herein are sufficient to result in stability. In certain embodiments, the methods and compositions described herein are sufficient to result in a clinically relevant reduction in the symptoms of a particular disease indication known to clinicians skilled in the art.

[0122] As described above, for in vivo applications for the treatment or testing of human or non-human mammalian diseases, the modified PPE proteins or proproteins described herein are generally incorporated into one or more therapeutic or pharmaceutical compositions, including animal therapeutic compositions, prior to administration.

[0123] Accordingly, certain embodiments relate to pharmaceutical or therapeutic compositions comprising modified PPE proteins or proproteins as described herein. In some cases, the pharmaceutical or therapeutic composition comprises one or more of the modified PPE proteins or proproteins described herein, combined with a pharmaceutically or physiologically acceptable carrier or excipient. Certain pharmaceutical or therapeutic compositions further comprise at least one additional agent as described herein, e.g., an immunotherapy agent, a chemotherapeutic agent, a hormonal therapeutic agent, and / or a kinase inhibitor.

[0124] Some therapeutic compositions contain only one modified PPE protein or proprotein (and utilize specific methods). Certain therapeutic compositions contain a mixture of at least two, three, four, or five different modified PPE proteins or proproteins (and utilize specific methods).

[0125] In certain embodiments, a pharmaceutical or therapeutic composition comprising a modified PPE protein or proprotein is substantially pure on a protein-based or weight-based basis, for example, the composition having a purity of at least about 80%, 85%, 90%, 95%, 98%, or 99% on a protein-based or weight-based basis.

[0126] In some embodiments, the modified PPE proteins or proproteins described herein have an immunogenicity profile that does not form aggregates, has desired solubility, and / or is suitable for use in humans, as is known in the art. Thus, in some embodiments, therapeutic compositions comprising modified PPE proteins or proproteins are substantially aggregate-free. For example, certain compositions contain less than about 10% (protein-based) of high molecular weight aggregated protein, or less than about 5% of high molecular weight aggregated protein, or less than about 4% of high molecular weight aggregated protein, or less than about 3% of high molecular weight aggregated protein, or less than 2% of high molecular weight aggregated protein, or less than 1% of high molecular weight aggregated protein. Some compositions contain modified PPE proteins or proproteins that are monodisperse at least about 50%, about 60%, about 70%, about 80%, about 90%, or about 95% of their apparent molecular weight.

[0127] In some embodiments, the modified PPE protein or proprotein is concentrated to about or at least about 0.1 mg / ml, 0.2 mg / ml, 0.3 mg / ml, 0.4 mg / ml, 0.5 mg / ml, 0.6, 0.7, 0.8, 0.9, 1 mg / ml, 2 mg / ml, 3 mg / ml, 4 mg / ml, 5 mg / ml, 6 mg / ml, 7 mg / ml, 8 mg / ml, 9 mg / ml, 10 mg / ml, 11, 12, 13, 14, or 15 mg / ml and formulated for biological use.

[0128] To prepare therapeutic or pharmaceutical compositions, one or more modified PPE proteins or proproteins in effective or desired amounts are mixed with any pharmaceutical carrier or excipient known to those skilled in the art to be suitable for a particular drug and / or mode of administration. The pharmaceutical carrier may be liquid, semi-liquid, or solid. Solutions or suspensions used for parenteral, intradermal, subcutaneous, or topical application may include, for example, sterile diluents (such as water), saline solutions (e.g., phosphate-buffered saline, PBS), fixative oils, polyethylene glycol, glycerin, propylene glycol, or other synthetic solvents, antimicrobial agents (e.g., benzyl alcohol, methylparaben), antioxidants (e.g., ascorbic acid and sodium bisulfite), and chelating agents (e.g., ethylenediaminetetraacetic acid (EDTA)), and buffers (e.g., acetates, citrates, and phosphates). For intravenous administration (e.g., IV infusion), suitable carriers include saline or phosphate-buffered saline (PBS), and solutions containing thickeners and solubilizers such as glucose, polyethylene glycol, polypropylene glycol, and mixtures thereof.

[0129] Administration of the modified PPE proteins or proproteins described herein, either in their pure form or in appropriate therapeutic or pharmaceutical compositions, may be carried out via any acceptable mode of administration of the agent for delivering similar utility. Therapeutic or pharmaceutical compositions may be prepared by combining the composition with an appropriate physiologically acceptable carrier, diluent, or excipient, and may be formulated into preparations in solid, semi-solid, liquid, or gaseous forms such as tablets, capsules, powders, granules, ointments, solutions, suppositories, injections, inhalants, gels, microparticles, and aerosols. In addition, other pharmaceutically active ingredients (including other small molecules as described elsewhere herein), and / or suitable excipients such as salts, buffers, and stabilizers may be present in the composition, although these are not essential.

[0130] Administration can be achieved by various routes, including oral, parenteral, nasal, intravenous, intradermal, intramuscular, subcutaneous, or topical. The preferred mode of administration depends on the nature of the condition being treated or prevented. Specific embodiments include administration by IV infusion.

[0131] The carrier may include, for example, a pharmaceutically or physiologically acceptable carrier, excipient, or stabilizer that is nontoxic to cells or mammals to which it is exposed at the dose and concentration used. In many cases, the physiologically acceptable carrier is an aqueous pH buffer solution. Examples of physiologically acceptable carriers include buffers such as phosphates, citrate, and other organic acids; antioxidants including ascorbic acid; low molecular weight (less than about 10 residues) polypeptides; proteins such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, arginine, or lysine; monosaccharides, disaccharides, or other carbohydrates including glucose, mannose, or dextrin; chelating agents such as EDTA; sugar alcohols such as mannitol or sorbitol; salt-forming counterions such as sodium; and / or nonionic surfactants such as polysorbate 20 (TWEEN®), polyethylene glycol (PEG), and polyoxamers (PLURONICS®).

[0132] In some embodiments, one or more agents may be encapsulated in microcapsules (e.g., hydroxymethylcellulose or gelatin-microcapsules and poly-(methyl metasylate) microcapsules, respectively) prepared by coacervation techniques or interfacial polymerization, in colloidal drug delivery systems (e.g., liposomes, albumin microspheres, microemulsions, nanoparticles, and nanocapsules), or in macroemulsions. Such techniques are disclosed in Remington's Pharmaceutical Sciences, 16th edition, Oslo, A., Ed., (1980). The particles or liposomes may further comprise other therapeutic or diagnostic agents.

[0133] The precise dose and duration of treatment are functional to the disease being treated and can be determined empirically using known test protocols or by testing the composition in a model system known in the art and extrapolating therefrom. Controlled clinical trials may also be conducted. The dose may also vary depending on the severity of the condition being alleviated. Pharmaceutical compositions are generally formulated and administered to produce therapeutically beneficial effects while minimizing undesirable side effects. The composition may be administered as a single dose or divided into several smaller doses administered at time intervals. For any particular subject, a specific dosing regimen may be adjusted over time according to individual needs.

[0134] Accordingly, typical routes for administering these and related therapeutic or pharmaceutical compositions include, without limitation, oral, topical, transdermal, inhalation, parenteral, sublingual, oral cavity, rectal, vaginal, and nasal cavity. As used herein, the term parenteral includes subcutaneous, intravenous, intramuscular, intrasternal injection, or infusion techniques. The therapeutic or pharmaceutical compositions according to specific embodiments of this disclosure are formulated such that the active ingredients contained therein become bioavailable when the composition is administered to a subject or patient. The composition to be administered to a subject or patient may take the form of one or more dosage units; for example, a tablet may be a single dosage unit, and a container of a drug described herein in aerosol form may hold multiple dosage units. Practical methods for preparing such dosage forms are known or obvious to those skilled in the art; see, for example, Remington: The Science and Practice of Pharmacy, 20th Edition (Philadelphia College of Pharmacy and Science, 2000). The administered composition will typically contain a therapeutically effective amount of the agent described herein for the treatment of the disease or condition of interest.

[0135] Therapeutic or pharmaceutical compositions may be in solid or liquid form. In some embodiments, the carrier is fine particles, and as a result, the composition is, for example, in tablet or powder form. The carrier may also be liquid, and the composition is, for example, an oral oil, an injectable liquid, or an aerosol, which is useful, for example, for inhalation administration. When intended for oral administration, the pharmaceutical composition is preferably either in solid or liquid form, and semi-solid, semi-liquid, suspension, and gel forms are included within the forms considered as either solid or liquid form herein. Certain embodiments include sterile injectable solutions.

[0136] As a solid composition for oral administration, the pharmaceutical composition may be formulated into powder, granules, compressed tablets, pills, capsules, chewing gum, wafers, etc. Such solid compositions typically contain one or more inert diluents or food carriers. In addition, one or more of the following may be present: binders such as carboxymethylcellulose, ethylcellulose, microcrystalline cellulose, tragacanth gum, or gelatin; excipients such as starch, lactose, or dextrin; disintegrants such as alginic acid, sodium alginate, Primogel, or corn starch; lubricants such as magnesium stearate or Sterotex; lubricants such as colloidal silicon dioxide; sweeteners such as sucrose or saccharin; flavorings such as peppermint, methyl salicylate, or orange flavoring; and colorants. When the pharmaceutical composition is in the form of a capsule, for example, a gelatin capsule, it may contain a liquid carrier such as polyethylene glycol or oil in addition to the above types of materials.

[0137] Therapeutic or pharmaceutical compositions may be in the form of liquids, such as elixirs, syrups, solutions, emulsions, or suspensions. Liquids may, as two examples, be intended for oral administration or delivery by injection. When intended for oral administration, preferred compositions contain, in addition to the compound, one or more of the following: sweeteners, preservatives, dyes / colorants, and flavorings. Compositions intended for administration by injection may contain one or more of the following: surfactants, preservatives, wetting agents, dispersants, suspending agents, buffers, stabilizers, and isotonic agents.

[0138] Liquid therapeutic or pharmaceutical compositions, whether they are solutions, suspensions, or other similar forms, may contain one or more of the following adjuvants: water for injection, saline, preferably physiological saline, Ringer's solution, isotonic sodium chloride, fixing oils such as synthetic mono or diglycerides that can function as a solvent or suspension medium, sterile diluents such as polyethylene glycol, glycerin, propylene glycol, or other solvents, antimicrobial agents such as benzyl alcohol or methylparaben, antioxidants such as ascorbic acid or sodium bisulfite, chelating agents such as ethylenediaminetetraacetic acid, buffers such as acetates, citrates, or phosphates, and isotonic agents such as sodium chloride or glucose. Parenteral preparations may be sealed in ampoules made of glass or plastic, disposable syringes, or multi-dose vials. Physiological saline is a preferred adjuvant. Injectable pharmaceutical compositions are preferably sterile.

[0139] Liquid therapeutic or pharmaceutical compositions intended for either parenteral or oral administration should contain a certain amount of the drug so that a suitable dose is obtained. Typically, this amount is at least 0.01% of the drug of interest in the composition. When intended for oral administration, this amount can vary to 0.1% to about 70% of the weight of the composition. Certain oral therapeutic or pharmaceutical compositions contain about 4% to about 75% of the drug of interest. In certain embodiments, therapeutic or pharmaceutical compositions and preparations are prepared so that parenteral dose units contain 0.01% to 10% by weight of the drug of interest before dilution.

[0140] Therapeutic or pharmaceutical compositions may be intended for topical administration, in which case the carrier may preferably comprise a solution, emulsion, ointment, or gel base. The base may comprise, for example, diluents such as petrolatum, lanolin, polyethylene glycol, beeswax, mineral oil, water, and alcohol, as well as one or more emulsifiers and stabilizers. Thickeners may be present in the therapeutic or pharmaceutical composition for topical administration. When intended for transdermal administration, the composition may comprise a transdermal patch or an iontophoresis device.

[0141] Therapeutic or pharmaceutical compositions may be intended for rectal administration, for example, in the form of suppositories that dissolve in the rectum and release the drug. Compositions for rectal administration may contain oily bases as suitable non-irritating excipients. Such bases include, without limitation, lanolin, cocoa butter, and polyethylene glycol.

[0142] Therapeutic or pharmaceutical compositions may contain various materials that modify the physical form of solid or liquid dose units. For example, a composition may contain a material that forms a coating shell around an active ingredient. The material forming the coating shell is typically inert and can be selected from, for example, sugars, shellac, and other enteric coating agents. Alternatively, the active ingredient may be encapsulated in a gelatin capsule. Therapeutic or pharmaceutical compositions in solid or liquid form may contain components that bind to a drug and thereby assist in the delivery of the compound. Suitable components that can act with this ability include monoclonal or polyclonal antibodies, one or more proteins, or liposomes.

[0143] Therapeutic or pharmaceutical compositions may essentially consist of dosing units that can be administered as aerosols. The term aerosol is used to describe a variety of systems, ranging from colloidal in nature to systems consisting of pressurized packages. Delivery may be by liquefaction or compressed gas, or by a suitable pump system for dispensing the active ingredient. Aerosols may be delivered in single-phase, two-phase, or three-phase systems to deliver the active ingredient. Aerosol delivery may involve necessary containers, activators, valves, sub-containers, etc., which together may form a kit. Without excessive experimentation, those skilled in the art can determine a preferred aerosol.

[0144] The compositions described herein may be prepared using carriers that protect the drug from rapid removal from the body, such as time-release formulations or coatings. Such carriers include, but are not limited to, controlled-release formulations such as implants and microcapsule delivery systems, as well as biodegradable, biocompatible polymers such as ethylene vinyl acetate, polyanhydride, polyglycolic acid, polyorthoester, polylactic acid, and others known to those skilled in the art.

[0145] Therapeutic or pharmaceutical compositions may be prepared by methodologies well known in the pharmaceutical art. For example, a therapeutic or pharmaceutical composition intended to be administered by injection may contain one or more salts, buffers, and / or stabilizers, with sterile distilled water to form a solution. Surfactants may be added to facilitate the formation of a homogeneous solution or suspension. Surfactants are compounds that interact non-covalently with a drug to facilitate its dissolution or homogeneous suspension in an aqueous delivery system.

[0146] Therapeutic or pharmaceutical compositions may be administered in therapeutically effective doses, which will vary depending on various factors, including the activity of the specific compound used, the metabolic stability and duration of the compound's action, age, weight, overall health, sex, and the subject's diet, mode and timing of administration, excretion rate, drug combinations, the severity of a particular disorder or condition, and the subject receiving treatment. In some cases, the therapeutically effective daily dose is approximately 0.001 mg / kg (i.e., approximately 0.07 mg) to approximately 100 mg / kg (i.e., approximately 7.0 g) (for a 70 kg mammal), preferably approximately 0.01 mg / kg (i.e., approximately 0.7 mg) to approximately 50 mg / kg (i.e., approximately 3.5 g) (for a 70 kg mammal), and more preferably approximately 1 mg / kg (i.e., approximately 70 mg) to approximately 25 mg / kg (i.e., approximately 1.75 g) (for a 70 kg mammal). In some embodiments, the therapeutically effective dose is administered weekly, bi-weekly, or monthly. In certain embodiments, the therapeutically effective dose is administered weekly, bi-weekly, or monthly at doses of, for example, about 1 to 10 or 1 to 5 mg / kg, or about 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 mg / kg.

[0147] The combination therapies described herein may include the administration of a single pharmaceutical dosage form of a modified PPE protein or proprotein and an additional therapeutic agent (e.g., an immunotherapy agent, a chemotherapeutic agent, a hormonal agent, a kinase inhibitor), as well as the administration of a composition comprising a modified PPE protein or proprotein and an additional therapeutic agent in a separate pharmaceutical dosage form of itself. For example, the modified PPE protein or proprotein and the additional therapeutic agent may be administered together to a subject in a single parenteral dose composition such as saline solution or other physiologically acceptable solution, or each agent may be administered in a separate parenteral dosage form. When separate dosage forms are used, the compositions may be administered essentially simultaneously, i.e., at the same time, or separately at time-staggered intervals, i.e., sequentially and in any order, and the combination therapy is understood to include all of these regimens.

[0148] Also included are patient care kits comprising (a) at least one modified PPE protein or proprotein as described herein, and optionally, (b) at least one additional therapeutic agent (e.g., an immunotherapy agent, a chemotherapy agent, a hormonal therapy agent, a kinase inhibitor). In certain kits, (a) and (b) are in separate therapeutic compositions. In some kits, (a) and (b) are in the same therapeutic composition.

[0149] The kits described herein may also include one or more additional therapeutic agents or other components that are suitable or desired for the indication being treated or for the desired diagnostic use. The kits described herein may also include one or more syringes or other components that are necessary or desired to facilitate the intended mode of delivery (e.g., stents, implantable depots).

[0150] In some embodiments, the patient care kit includes separate containers, dividers, or compartments for the composition and informational material. For example, the composition may be contained in a bottle, vial, or syringe, and the informational material may be contained in association with the container. In some embodiments, the separate elements of the kit are contained in a single, unseparated container. For example, the composition is contained in a bottle, vial, or syringe, with the informational material attached therein in the form of labeling. In some embodiments, the kit comprises a plurality of individual containers (e.g., packs), each containing one or more unit dosage forms (e.g., dosage forms described herein) of modified PPE protein or proprotein, and optionally, at least one additional therapeutic agent. For example, the kit comprises a plurality of syringes, ampoules, foil packets, or blister packs, each containing a single unit dose of modified PPE protein or proprotein, and optionally, at least one additional therapeutic agent. The containers of the kit may be airtight, waterproof (e.g., impermeable to changes or evaporation of moisture), and / or lightfast.

[0151] The patient care kit optionally includes a device suitable for administering the composition, such as a syringe, inhalant, infusion device (e.g., eye dropper), swab (e.g., cotton swab or wooden swab), or any such delivery device. In some embodiments, the device is a portable device for dispensing a quantitative dose of the drug. Methods of providing the kit also include, for example, combining the components described herein.

[0152] Expression and purification system Specific embodiments include methods and related compositions for expressing and purifying the recombinant modified PPE proteins or proproteins described herein. Such recombinant modified PPE proteins or proproteins can be readily prepared using standard protocols such as those described, for example, in Sambrook, et al., (1989, above), particularly sections 16 and 17; Ausubel et al., (1994, above), particularly chapters 10 and 16; and Coligan et al., Current Protocols in Protein Science (John Wiley & Sons, Inc. 1995–1997), particularly chapters 1, 5 and 6. As a general example, a modified PPE protein or proprotein may be prepared by a procedure comprising one or more of the following steps: (a) preparing a vector or construct containing a polynucleotide sequence encoding the modified PPE protein or proprotein described herein, which is operably linked to one or more regulatory elements (see, for example, Table S2); (b) introducing the vector or construct into host cells; (c) culturing the host cells to express the modified PPE protein or proprotein; and (d) isolating the modified PPE protein or proprotein from the host cells.

[0153] To express a desired polypeptide, the nucleotide sequence encoding the modified PPE protein or proprotein can be inserted into a suitable expression vector, i.e., a vector containing the elements necessary for the transcription and translation of the inserted coding sequence. Methods well known to those skilled in the art can be used to construct an expression vector containing the polypeptide of interest and the sequence encoding the appropriate transcription and translation regulatory elements. These methods include in vitro recombinant DNA techniques, synthetic techniques, and in vivo genetic recombination. Such techniques are described in Sambrook et al., Molecular Cloning, A Laboratory Manual (1989), and Ausubel et al., Current Protocols in Molecular Biology (1989).

[0154] Various expression vectors / host systems are known and can be used to contain and express polynucleotide sequences. These include, but are not limited to, microorganisms such as bacteria transformed with recombinant bacteriophages, plasmids, or cosmid DNA expression vectors; yeast transformed with yeast expression vectors; insect cell systems infected with viral expression vectors (e.g., baculovirus); plant cell systems transformed with viral expression vectors (e.g., cauliflower mosaic virus, CaMV, tobacco mosaic virus, TMV) or bacterial expression vectors (e.g., Ti or pBR322 plasmids); or animal cell systems, including mammalian cells, more specifically human cell systems.

[0155] The “regulatory elements” or “regulatory sequences” present in an expression vector are the untranslated regions of the vector—enhancers, promoters, and the 5' and 3' untranslated regions—that interact with host cell proteins to carry out transcription and translation. Such elements can vary in their intensity and specificity. Depending on the vector system and host used, any number of suitable transcription and translation elements, including constitutive and inducible promoters, may be used. For example, when cloning in bacterial systems, inducible promoters such as the hybrid lacZ promoter of the PBLUESCRIPT phagemide (Stratagene, La Jolla, Calif.) or PSPORT1 plasmid (Gibco BRL, Gaithersburg, Md.) may be used. In mammalian cell systems, promoters from mammalian genes or mammalian viruses are generally preferred. When it is necessary to generate cell lines containing multiple copies of the polypeptide-encoding sequence, SV40 or EBV-based vectors may be advantageously used with appropriate selectable markers.

[0156] In bacterial systems, several expression vectors may be selected depending on the intended use of the expressed polypeptide. For example, when large quantities are required, vectors that direct high levels of expression of readily purifiable fusion proteins may be used. Such vectors, though not limited to them, include polyfunctional E. coli cloning and expression vectors such as BLUESCRIPT (Stratagene), and pIN vectors (Van Heeke & Schuster, J. Biol. Chem. 264:5503 5509 (1989)), in which the sequence encoding the polypeptide of interest can be linked to the vector in a frame with an amino-terminal Met sequence and seven subsequent residues of β-galactosidase so that a hybrid protein is produced. pGEX vectors (Promega, Madison, Wis.) can also be used to express exogenous polypeptides as fusion proteins using glutathione S-transferase (GST). Generally, such fusion proteins are soluble and can be readily purified from lysed cells by adsorption to glutathione-agarose beads, followed by elution in the presence of free glutathione. Proteins produced in such systems may be designed to include heparin, thrombin, or factor XA protease cleavage sites so that the cloned polypeptide of interest can be freely released from the GST moiety.

[0157] Certain embodiments utilize an E. coli-based expression system (see, e.g., Structural Genomics Consortium et al., Nature Methods. 5:135-146, 2008). These and related embodiments may rely on partially or entirely ligation-independent cloning (LIC) to generate suitable expression vectors. In certain embodiments, protein expression may be controlled by T7 RNA polymerase (e.g., the pET vector series). These and related embodiments may utilize the expression host strain BL21(DE3), a BL21 λDE3 lysogen that supports T7-mediated expression and lacks lon and ompT proteases for improved target protein stability. Also included are expression host strains carrying plasmids encoding tRNAs rarely used in E. coli, such as the ROSETTA(DE3) strain and the Rosetta 2(DE3) strain. Cell lysis and sample processing can also be improved using reagents marketed under the trademarks BENZONASE® nuclease and BUGBUSTER® Protein Extraction Reagent. For cell culture, automated induction media can improve the efficiency of many expression systems, including high-throughput expression systems. This type of medium (e.g., OVERNIGHT EXPRESS® Autoinduction System) gradually induces protein expression via metabolic shifts without the addition of artificial inducers such as IPTG. Certain embodiments utilize hexahistidine tags (e.g., those marketed under the trademark HIS·TAG® fusion), followed by immobilized metal affinity chromatography (IMAC) purification, or related techniques. However, in certain embodiments, clinical-grade proteins can be isolated from E. coli-containing organisms without or without the use of affinity tags (see, e.g., Shimp et al., Protein Expr Purif. 50:58-67, 2006).As a further example, certain embodiments may employ a cold shock-induced high-yield production system for Escherichia coli, where protein overexpression in Escherichia coli at low temperatures improves their solubility and stability (see, for example, Qing et al., Nature Biotechnology. 22:877-882, 2004).

[0158] This also includes high-density bacterial fermentation systems. For example, culturing Ralstonia eutropha at high cell density enables protein production at cell densities exceeding 150 g / L and recombinant protein expression at titers exceeding 10 g / L.

[0159] In the yeast *Saccharomyces cerevisiae*, several vectors containing constitutive or inducible promoters such as alpha factor, alcohol oxidase, and PGH may be used. For reviews, see Ausubel et al. (above) and Grant et al., *Methods Enzymol.* 153:516-544 (1987). This also includes the Pichia pandoris expression system (see, for example, Li et al., *Nature Biotechnology.* 24,210-215,2006, and Hamilton et al., *Science* 301:1244,2003). Specific embodiments include, among other things, yeast systems manipulated to selectively glycosylate proteins, including yeast having a humanized N-glycosylation pathway (see, for example, Hamilton et al., Science. 313:1441-1443, 2006; Wildt et al., Nature Reviews Microbiol. 3:119-28, 2005; and Gerngross et al., Nature-Biotechnology. 22:1409-1414, 2004; U.S. Patents 7,629,163, 7,326,681, and 7,029,872). Simply as an example, recombinant yeast cultures can be grown, among other things, in Fernbach Flasks or 15 L, 50 L, 100 L, and 200 L fermentation apparatus.

[0160] When plant expression vectors are used, the expression of the polypeptide-encoding sequence can be driven by one of several promoters. For example, viral promoters such as the 35S and 19S promoters of CaMV can be used alone or in combination with the omega-leader sequence from TMV (Takamatsu, EMBO J.6:307-311 (1987)). Alternatively, plant promoters or heat shock promoters such as the small subunit of RUBISCO can be used (Coruzzi et al., EMBO J.3:1671-1680 (1984), Broglie et al., Science 224:838-843 (1984), and Winter et al., Results Probl. Cell Differ. 17:85-105 (1991)). These constructs can be introduced into plant cells by direct DNA transformation or pathogen-mediated transfection. Such techniques are described in several publicly available reviews (see, for example, Hobbs in McGraw Hill, Yearbook of Science and Technology, pp. 191–196 (1992)).

[0161] Insect systems can also be used to express polypeptides of interest. For example, in one such system, Autographa californica nuclear polyhedrosis virus (AcNPV) is used as a vector for expressing exogenous genes in Spodoptera frugiperda or Trichoplusia cells. The polypeptide-coding sequence can be cloned into a non-essential region of the virus, such as the polyhedrin gene, and placed under the control of the polyhedrin promoter. Successful insertion of the polypeptide-coding sequence inactivates the polyhedrin gene and produces a recombinant virus lacking the coat protein. The recombinant virus can then be used to infect S. frugiperda or Trichoplusia cells, for example, in which the polypeptide of interest can be expressed (Engelhard et al., Proc. Natl. Acad. Sci. USA 91:3224-3227 (1994)). This also includes baculovirus expression systems, including those utilizing SF9, SF21, and T.ni cells (see, for example, Murphy and Piwnica-Worms, Curr Protoc Protein Sci. Chapter 5: Unit 5.4, 2001). Insect systems may offer post-translational modifications similar to those found in mammalian systems.

[0162] In mammalian host cells, several virus-based expression systems are commonly available. For example, when adenovirus is used as an expression vector, the sequence encoding the polypeptide of interest can be ligated to an adenovirus transcription / translation complex consisting of a late promoter and a three-part reader sequence. Insertions into non-essential E1 or E3 regions of the viral genome can be used to obtain viable viruses capable of expressing polypeptides in infected host cells (Logan & Shenk, Proc. Natl. Acad. Sci. USA 81:3655-3659 (1984)). In addition, transcriptional enhancers, such as the Rous sarcoma virus (RSV) enhancer, can be used to increase expression in mammalian host cells.

[0163] Examples of useful mammalian host cell lines include the CV1 monkey kidney cell line transformed with SV40 (COS-7, ATCC CRL1651), human embryonic kidney cell line (293 or 293 cells sub-cloned for growth in suspension culture, Graham et al., J. Gen Virol. 36:59 (1977)), baby hamster kidney cells (BHK, ATCC CCL10), mouse Sertoli cells (TM4, Mather, Biol. Reprod. 23:243-251 (1980)), monkey kidney cells (CV1 ATCC CCL70), African green monkey kidney cells (VERO-76, ATCC CRL-1587), human cervical cancer cells (HELA, ATCC CCL2), canine kidney cells (MDCK, ATCC CCL34), and buffalo rat liver cells (BRL3A, ATCC Examples of useful mammalian host cell lines include CRL1442), human lung cells (W138, ATCC CCL75), human liver cells (Hep G2, HB8065), mouse mammary tumor cells (MMT 060562, ATCC CCL51), TR1 cells (Mather et al., Annals NYAcad.Sci.383:44-68(1982)), MRC5 cells, FS4 cells, and human hepatocellular carcinoma cell lines (Hep G2). Other useful mammalian host cell lines include Chinese hamster ovary (CHO) cells, including DHFR-CHO cells (Urlaub et al., PNAS USA77:4216(1980)), and myeloma cell lines such as NSO and Sp2 / 0. For a review of specific mammalian host cell lines suitable for protein production, see, for example, Yazaki and Wu, Methods in Molecular Biology, Vol. 248 (BKC Lo, ed., Humana Press, Totowa, NJ, 2003), pp. 255-268. Specific preferred mammalian cell expression systems include CHO and HEK293 cell-based expression systems.Mammalian expression systems can utilize, among those known in the art, for example, T-flasks, roller bottles, or attached cell lines in cell factories, or suspension cultures in, for example, 1 L and 5 L spinners, 5 L, 14 L, 40 L, 100 L and 200 L stirred tank bioreactors, or 20 / 50 L and 100 / 200 L WAVE bioreactors.

[0164] This also includes cell-free expression of proteins. These and related embodiments typically utilize purified RNA polymerase, ribosomes, tRNA, and ribonucleotides, which can be generated from cells or by extraction from cell-based expression systems.

[0165] Specific start signals may also be used to achieve more efficient translation of the sequence encoding the polypeptide of interest. Such signals include the ATG start codon and its adjacent sequences. If the polypeptide, its start codon, and the sequence encoding the upstream sequence are inserted into a suitable expression vector, additional transcriptional or translational regulatory signals may not be required. However, if only the coding sequence, or a portion thereof, is inserted, an exogenous translational regulatory signal, including the ATG start codon, should be provided. Furthermore, the start codon should be within the correct reading frame to ensure translation of the entire insertion. Exogenous translational elements and start codons can be of various origins, both natural and synthetic. Expression efficiency may be enhanced by the inclusion of enhancers appropriate for the specific cell line being used, such as those described in the literature (Scharf et al., Results Probl. Cell Differ. 20:125-162 (1994)).

[0166] In addition, host cell lines may be selected for their ability to modify the expression of the inserted sequence or to process the expressed protein in a desired manner. Such modifications of polypeptides include, but are not limited to, post-translational modifications such as acetylation, carboxylation, glycosylation, phosphorylation, lipidation, and acylation. Post-translational processing that cleaves the "prepro" form of the protein may also be used to facilitate correct insertion, folding, and / or function. In addition to bacterial cells, different host cells such as yeast, CHO, HeLa, MDCK, HEK293, and W138, which may or may not have specific cellular and characteristic mechanisms for such translational activity, may be selected to ensure the correct modification and processing of foreign proteins.

[0167] For long-term, high-yield recombinant protein production, stable expression is generally preferred. For example, cell lines that stably express a polynucleotide of interest can be transformed using expression vectors that may contain a viral replication origin and / or endogenous expression elements, as well as a selectable marker gene, on the same or separate vector. After vector introduction, cells may be allowed to grow in concentrated medium for about 1-2 days before being switched to selective medium. The purpose of the selectable marker is to confer resistance to selection, and its presence allows for the growth and harvesting of cells that successfully express the introduced sequence. Resistant clones of stably transformed cells can be propagated using tissue culture techniques appropriate to the cell type. Transient production, such as transient transfection or infection, can also be used. Exemplary mammalian expression systems suitable for transient production include HEK293 and CHO-based systems.

[0168] Any number of selection systems can be used to recover transformed or transduced cell lines. These include, but are not limited to, the herpes simplex virus thymidine kinase (Wigler et al., Cell 11:223-232 (1977)) and adenine phosphoribosyltransferase (Lowy et al., Cell 22:817-823 (1990)) genes, which can be adopted into tk- or aprt- cells, respectively. Furthermore, resistance to antimetabolites, antibiotics, or herbicides can be used as a basis for selection; for example, dhfr (Wigler et al., Proc. Natl. Acad. Sci. USA 77:3567-70 (1980)) conferring resistance to methotrexate, npt (Colbere-Garapin et al., J. Mol. Biol. 150:1-14 (1981)) conferring resistance to aminoglycosides, neomycin, and G-418, and als or pat (Murry, above) conferring resistance to chlorsulfuron and phosphinotricin acetyltransferase, respectively. Additional selectable genes, such as trpB, which allows cells to utilize indole instead of tryptophan, or hisD, which allows cells to utilize histidine instead of histinol, have been described (Hartman & Mulligan, Proc. Natl. Acad. Sci. USA 85:8047-51 (1988)). The use of visible markers, such as green fluorescent protein (GFP) and other fluorescent proteins (e.g., RFP, YFP), anthocyanins, β-glucuronidase and its substrate GUS, and luciferase and its substrate luciferin, is widely used not only to identify transformants but also to quantify the amount of transient or stable protein expression resulting from specific vector systems (e.g., Rhodes et al., Methods Mol. Biol. 55:121-131 (1995)).

[0169] This also includes high-throughput protein production systems or micro-production systems. Specific embodiments may utilize, for example, hexahistidine fusion tags for protein expression and purification on metal-chelated sliding surfaces or MagneHis Ni-Particles (see, e.g., Kwon et al., BMC Biotechnol. 9:72, 2009, and Lin et al., Methods Mol Biol. 498:129-41, 2009). High-throughput cell-free protein expression systems are also included (see, e.g., Sitaraman et al., Methods Mol Biol. 498:229-44, 2009).

[0170] Various protocols for detecting and measuring the expression of polynucleotide-encoded products using product-specific binders or antibodies such as polyclonal or monoclonal antibodies are known in the art. Examples include enzyme-linked immunosorbent assay (ELISA), Western immunoblotting, radioimmunoassay (RIA), and fluorescence-activated cell sorting (FACS). These and other assays are described elsewhere, among others, in Hampton et al., Serological Methods, a Laboratory Manual (1990), and Maddox et al., J. Exp. Med. 158:1211-1216 (1983).

[0171] A wide range of labeling and conjugation techniques are known to those skilled in the art and can be used in various nucleic acid and amino acid assays. Means for generating labeled hybridization or PCR probes for detecting sequences related to polynucleotides include oligo-labeling, nick translation, end-labeling, or PCR amplification using labeled nucleotides. Alternatively, a sequence, or any portion thereof, can be cloned into a vector for the production of mRNA probes. Such vectors are known and commercially available in the art and can be used to synthesize RNA probes in vitro by adding a suitable RNA polymerase such as T7, T3, or SP6 and labeled nucleotides. These procedures can be carried out using various commercially available kits. Suitable reporter molecules or labels that can be used include radionuclides, enzymes, fluorescent agents, chemiluminescent agents, or colorants, and substrates, cofactors, inhibitors, magnetic particles, etc.

[0172] Host cells transformed with one or more polynucleotide sequences of interest can be cultured under conditions suitable for protein expression and recovery from cell cultures. Certain embodiments utilize serum-free cell expression systems. Examples include HEK293 cells and CHO cells that can be grown in serum-free medium (see, for example, Rosser et al., Protein Expr. Purif. 40:237-43, 2005, and U.S. Patent No. 6,210,922).

[0173] Modified PPE proteins or proproteins produced by recombinant cells may be secreted or contained within the cell, depending on the sequence and / or vector used. As will be understood by those skilled in the art, polynucleotide-containing expression vectors may be designed to contain a signal sequence that directs the secretion of the encoded polypeptide across the prokaryotic or eukaryotic cell membrane. Other recombinant constructs may be used to conjugate a sequence encoding the polypeptide of interest to a nucleotide sequence encoding a polypeptide domain, which will facilitate the purification and / or detection of the soluble protein. Examples of such domains include cleavable and non-cleavable affinity purifiers, as well as epitope tags such as avidin, FLAG tags, polyhistidine tags (e.g., 6xHis), cMyc tags, V5- tags, glutathione S-transferase (GST) tags, and others.

[0174] Proteins produced by recombinant cells can be purified and characterized according to various techniques known in the art. Exemplary systems for performing protein purification and analyzing protein purity include high-performance protein liquid chromatography (FPLC) (e.g., AKTA and Bio-Rad FPLC systems) and high-performance liquid chromatography (HPLC) (e.g., Beckman and Waters HPLC). Exemplary chemicals for purification, among those known in the art, include ion exchange chromatography (e.g., Q, S), size exclusion chromatography, salt gradients, affinity purification (e.g., Ni, Co, FLAG, maltose, glutathione, protein A / G), gel filtration, reversed phase, ceramic HYPERD® ion exchange chromatography, and hydrophobic interaction columns (HIC). Also typically included are analytical methods such as SDS-PAGE (e.g., Coomassie, silver staining), immunoblotting, Bradford, and ELISA, which can be used during any step of the production or purification process to measure the purity of the protein composition.

[0175] The invention also includes methods for concentrating modified PPE protein or proprotein, and compositions comprising concentrated soluble modified PPE protein or proprotein. In some embodiments, such concentrated solutions of modified PPE protein or proprotein contain protein at concentrations of about or at least about 5 mg / mL, 8 mg / mL, 10 mg / mL, 15 mg / mL, 20 mg / mL or higher.

[0176] In some embodiments, such compositions may be substantially monodisperse, meaning that the modified PPE protein or proprotein is predominantly (i.e., at least about 90%) present in one apparent molecular weight form when evaluated, for example, by size exclusion chromatography, dynamic light scattering, or analytical ultracentrifugation.

[0177] In some embodiments, such compositions have a purity of at least about 90%, or in some embodiments, at least about 95%, or in some embodiments, at least 98%. Purity can be determined by any conventional analytical method known in the art.

[0178] In some embodiments, such compositions have a high molecular weight aggregate content of less than about 10% relative to the total amount of protein present, or in some embodiments, such compositions have a high molecular weight aggregate content of less than about 5%, or in some embodiments, such compositions have a high molecular weight aggregate content of less than about 3%, or in some embodiments, such compositions have a high molecular weight aggregate content of less than about 1%. The high molecular weight aggregate content can be determined by various analytical techniques, including, for example, size exclusion chromatography, dynamic light scattering, or analytical ultracentrifugation.

[0179] Examples of concentration approaches intended herein include lyophilization, which is typically used when the solution contains little to no soluble components other than the protein of interest. Lyophilization is often performed after HPLC and can remove most or all volatile components from the mixture. Ultrafiltration techniques are also included, which typically use one or more selectively permeable membranes to concentrate the protein solution. The membranes allow water and small molecules to pass through while retaining the protein, and the solution can be pressed against the membrane by mechanical pumps, gas pressure, or centrifugation, among other techniques.

[0180] In certain embodiments, the modified PPE protein or proprotein in the composition has a purity of at least about 90% when measured according to the conventional art. In certain embodiments, such as diagnostic compositions or certain pharmaceutical or therapeutic compositions, the modified PPE protein or proprotein in the composition has a purity of at least about 95%, or at least about 97%, 98%, or 99%. In some embodiments, such as when used as a reference or research reagent, the modified PPE protein or proprotein may be of lower purity, having a purity of at least about 50%, 60%, 70%, or 80%. Purity may be measured as a whole or against selected components, such as other proteins, and may be, for example, protein-based purity.

[0181] Purified proteins can also be characterized according to their biological characteristics. Binding affinity and binding kinetics can be measured according to various techniques known in the art, such as Biacore® and related technologies, which utilize surface plasmon resonance (SPR), an optical phenomenon that enables real-time detection of unlabeled interacting substances. SPR-based biosensors can be used to determine activity concentrations, screening, and characterization in terms of both affinity and kinetics. The presence or level of one or more biological activities can be measured according to in vitro or cell-based assays as described herein, and they are functionally and optionally bound to readouts or indicators, such as fluorescence or luminescence indicators of biological activity.

[0182] In certain embodiments, as described above, the composition is substantially endotoxin-free, for example, containing about 95% endotoxin-free, preferably about 99% endotoxin-free, and more preferably about 99.99% endotoxin-free. The presence of endotoxins can be detected according to the conventional techniques of the art described herein. In certain embodiments, the modified PPE protein or proprotein is prepared from eukaryotic cells, such as mammalian or human cells, in substantially serum-free medium. In certain embodiments, as described herein, the composition has an endotoxin content of less than about 10 EU / mg of protein, or less than about 5 EU / mg of protein, less than about 3 EU / mg of protein, or less than about 1 EU / mg of protein.

[0183] In certain embodiments, the composition comprises high molecular weight aggregates of less than about 10% by weight, or less than about 5% by weight, or less than about 2% by weight, or less than 1% by weight.

[0184] For example, this includes protein-based analytical assays and methods that can be used to evaluate, among other properties, protein purity, size, solubility, and degree of aggregation. Protein purity can be evaluated in several ways. For example, purity can be evaluated based on primary structure, higher-order structure, size, charge, hydrophobicity, and glycosylation. Examples of methods for evaluating primary structure include N- and C-terminal sequencing, as well as peptide mapping (see, e.g., Allen et al., Biologicals. 24:255-275, 1996). Examples of methods for evaluating higher-order structure include circular dichroism (see, e.g., Kelly et al., Biochim Biophys Acta. 1751:119-139, 2005), fluorescence spectroscopy (see, e.g., Meagher et al., J. Biol. Chem. 273:23283-89, 1998), FT-IR, amide hydrogen-deuterium exchange dynamics, differential scanning calorimetry, NMR spectroscopy, and immunoassay using structure-sensitive antibodies. Higher-order structure can also be evaluated as a function of various parameters such as pH, temperature, or added salts. Examples of methods for evaluating protein properties such as size include analytical ultracentrifugation and size exclusion HPLC (SEC-HPLC), and exemplary methods for measuring charge include ion exchange chromatography and isoelectric focusing. Hydrophobicity can be evaluated, for example, by reversed-phase HPLC and hydrophobic interaction chromatography-HPLC. Glycosylation can affect pharmacokinetics (e.g., clearance), conformation or stability, receptor binding, and protein function, and can be evaluated, for example, by mass spectrometry and nuclear magnetic resonance (NMR) spectroscopy.

[0185] As described above, certain embodiments include the use of SEC-HPLC for evaluating protein properties such as purity, size (e.g., size homogeneity), or degree of aggregation, and / or for purifying proteins, among other applications. SEC, including gel filtration chromatography (GFC) and gel permeation chromatography (GPC), refers to a chromatographic method in which molecules in solution are separated by a porous material based on their size, or more specifically, on their hydrodynamic volume, diffusion coefficient, and / or surface properties. The process is commonly used to separate biomolecules and determine the molecular weight and molecular weight distribution of polymers. Typically, a biological or protein sample (such as a protein extract produced according to a protein expression method provided herein and known in the art) is loaded into a selected size exclusion column having a defined stationary phase (porous material), preferably a phase that does not interact with the proteins in the sample. In certain embodiments, the stationary phase consists of inert particles packed in a high-density three-dimensional matrix within a glass or steel column. The mobile phase may be pure water, aqueous buffer, organic solvent, or a mixture thereof. Stationary phase particles typically have small pores and / or channels that allow only molecules below a certain size to enter. Therefore, larger particles are excluded from these pores and channels, and their limited interaction with the stationary phase causes them to elute as "completely excluded" peaks at the start of the experiment. Smaller molecules that can fit within the pores are removed from the flowing mobile phase, and the time they spend immobilizing in the stationary phase pores depends, in part, on how far they penetrate into the pores. Their removal from the mobile phase flow results in separation between particles based on their size differences, causing them to take longer to elute from the column. A given size exclusion column has a range of molecular weights that can be separated. Overall, molecules larger than the upper limit will not be captured by the stationary phase, molecules smaller than the lower limit will enter the solid phase completely and elute as a single band, and molecules within the range will elute at different rates defined by their properties, such as hydrodynamic volume.For practical examples of these methods using pharmaceutical proteins, see Bruner et al., Journal of Pharmaceutical and Biomedical Analysis. 15:1929-1935, 1997.

[0186] Protein purity for clinical application has also been studied, for example, by Anicetti et al. (Trends in Biotechnology. 7:342-349, 1989). More recent techniques for analyzing protein purity include, without limitation, the LabChip GXII, an automated platform for rapid analysis of proteins and nucleic acids, which provides high-throughput analysis of protein titer, sizing, and purity. In certain non-limiting embodiments, clinical-grade proteins can be obtained by utilizing a combination of chromatographic materials in at least two orthogonal steps, among other methods (see, for example, Therapeutic Proteins: Methods and Protocols. Vol. 308, Eds., Smales and James, Humana Press Inc., 2005). Typically, protein agents are substantially endotoxin-free when measured according to techniques known in the art and techniques described herein.

[0187] Protein solubility assays are also included. Such assays may be used, for example, to determine optimal growth and purification conditions for recombinant production, to optimize the selection of buffers, and to optimize the selection of modified PPE proteins or proproteins and their variants. Solubility or aggregation may be evaluated according to a variety of parameters, including temperature, pH, salt, and the presence or absence of other additives. Examples of solubility screening assays include, in particular, microplate-based methods that measure protein solubility using turbidity or other measurements as endpoints, high-throughput assays for the analysis of the solubility of purified recombinant proteins (see, e.g., Stenvall et al., Biochim Biophys Acta. 1752:6-10, 2005), assays that use structural complementation of genetic marker proteins to monitor and measure protein folding and solubility in vivo (see, e.g., Wigley et al., Nature Biotechnology. 19:131-136, 2001), and electrochemical screening of recombinant protein solubility in Escherichia coli using scanning electrochemical microscopy (SECM) (see, e.g., Nagamine et al., Biotechnology and Bioengineering. 96:1008-1013, 2006). Modified PPE proteins or proproteins having increased solubility (or reduced aggregation) may be identified or selected according to conventional techniques in the art, including simple in vivo assays for protein solubility (see, for example, Maxwell et al., Protein Sci. 8:1908-11, 1999).

[0188] Protein solubility and aggregation can also be measured by dynamic light scattering techniques. Aggregation is a general term encompassing several types of interactions or features, including soluble / insoluble, covalent / non-covalent, reversible / irreversible, and intrinsic / denatured interactions and features. For protein therapeutics, the presence of aggregates is typically considered undesirable due to concerns that the aggregates may cause immunogenic reactions (e.g., small aggregates) or adverse events at administration (e.g., microparticles). Dynamic light scattering refers to a technique that can be used to determine the size distribution profile of small particles in suspension or polymers such as proteins in solution. This technique, also known as photon correlation spectroscopy (PCS) or quasi-elastic light scattering (QELS), uses scattered light to measure the diffusion rate of protein particles. Variations in scattering intensity can be observed due to Brownian motion of molecules and particles in solution. This motion data can conventionally be processed to derive the size distribution for a sample, where size is given by the Stokes radius or hydrodynamic radius of the protein particles. Hydrodynamic size depends on both mass and shape (morphology). Dynamic scattering can detect the presence of very small amounts of aggregated protein (<0.01% by weight) even in samples containing a wide range of masses. It can also be used to compare the stability of different formulations, for example, in applications that rely on real-time monitoring of changes at high temperatures. Accordingly, certain embodiments include the use of dynamic light scattering to analyze the solubility and / or presence of aggregates in samples containing modified PPE proteins or proproteins of this disclosure.

[0189] While the embodiments described above have been explained in some detail by examples and embodiments for the purpose of clarity of understanding, it will be readily apparent to those skilled in the art that certain changes and modifications can be made in light of the teachings of this disclosure without departing from the spirit or scope of the appended claims. The following embodiments are provided by illustration only and not by limitation. Those skilled in the art will readily recognize various non-essential parameters that can be changed or modified to produce essentially similar results. [Examples]

[0190] Example 1 Activity of PPE mutants Ten mutants of porcine pancreatic elastase (PPE) protein were prepared and tested. The mutants were prepared as enzymatically inactive PPE proproteins containing an N-terminal modified signal peptide (SEQ ID NO: 2), a trypsin-cleavable activating peptide (SEQ ID NO: 3), a mutant PPE peptidase domain, and a C-terminal 6xHis tag. The mutant names are provided in Table E1 below, and residue numbering is defined with respect to SEQ ID NO: 1. [Table 3]

[0191] To test the activation of modified PPE proproteins, natural PPE (wild-type active PPE peptidase domain), wild-type PPE proprotein (also called pro-PPE), and modified PPE proproteins (mutants A-J) were incubated with trypsin (trypsin:PPE in a 1:20 w / w ratio) for variable time points (0-24 hours). Cleavage was monitored by SDS-PAGE and Coomassie blue staining. As shown in Figures 1A-1D, trypsin cleaved both wild-type and modified PPE proproteins, producing bands of the same size as natural PPE, and the cleavage reaction was completed in a 2-hour incubation. The results further demonstrate that prolonged incubation with trypsin does not result in further cleavage, suggesting that trypsin does not further cleave the pro-PPE form after the initial conversion to active PPE. In fact, incubating natural PPE (active PPE peptidase domain) with trypsin did not result in the appearance of low molecular weight bands (see Figure 1A).

[0192] To test the enzymatic activity of trypsin-activated proprotein, wild-type PPE protein and modified PPE proproteins (mutants A-J) were incubated with a vehicle (Veh) or trypsin (trypsin:PPE in a 1:20 w / w ratio) at 37°C for 6 hours (n=4 / condition). Catalytic activity was monitored using a colorimetric substrate activity assay (N-methoxysuccinyl-Ala-Ala-Pro-Val p-nitroanilide, Sigma). Figure 2A shows the increased enzymatic activity of modified PPE after trypsin cleavage.

[0193] To test the enzymatic activity of trypsin-activated proproteins in the presence of serine protease inhibitors, wild-type human neutrophil elastase (ELANE), (activated) wild-type PPE protein (also known as natural PPE), wild-type PPE proprotein (also known as WT rPPE), and (activated) modified PPE protein were incubated with various doses of human alpha-1-antitrypsin (A1AT, 0-20 nM) for 30 minutes at room temperature, and catalytic activity was quantified. Sensitivity to A1AT was evaluated by linear regression analysis of the decrease in enzymatic activity with respect to A1AT concentration (see Table E2 below; P-value = significance of linear regression fit). [Table 4]

[0194] Figure 2B shows complete inhibition curves comparing natural ELANE, WT PPE protein (natural PPE), wild-type PPE proprotein (wt rPPE), and mutant H PPE. *, p<0.05, Student's t-test, and Table E2 below.

[0195] To test cancer cell killing activity, MDA-MB-231 human cancer cells (triple-negative breast cancer) were incubated with wild-type and modified PPE proteins in serum-free medium for 7 hours, either in the presence or absence of A1AT (0-40 nM). Cancer cell killing activity was evaluated by calcein-AM (n=6 / condition). Figure 3A shows the activity at increasing doses of the test protein, and Figure 3B shows the activity of the test protein in the absence or presence of increasing amounts of A1AT (cancer cell killing efficacy in the absence of A1AT was set to 100% *, p<0.05, Student's t-test, compared to wild-type PPE). For example, mutant F (Q211F) showed significantly increased cancer cell killing activity compared to wild-type PPE at equivalent doses (3A), and mutant H (T55A) showed significantly increased cancer cell killing activity compared to wild-type in the presence of increasing amounts of A1AT (3B).

Claims

1. A modified porcine pancreatic elastase (PPE) protein comprising at least one amino acid change from the wild-type PPE protein (SEQ ID NO: 4), wherein the at least one change is in a residue selected from one or more of Q211, T55, D74, R75, S214, R237, and N241, and the residue numbering is defined by SEQ ID NO: 1 (wild-type PPE proprotein).

2. The modified PPE protein according to claim 1, wherein the at least one amino acid change is selected from one or more of Q211F, T55A, D74A, R75A, R75E, Q211A, S214A, R237A, N241A, and N241Y, and the residue numbering is defined by Sequence ID No.

1.

3. A modified PPE protein according to claim 1 or 2, comprising, consisting of, or essentially comprising an amino acid sequence that is at least 80, 85, 90, 95, 98, or 100% identical to a sequence selected from Table S2, and that retains the at least one amino acid change.

4. A modified PPE protein comprising, consisting of, or essentially comprising an amino acid sequence that is at least 80, 85, 90, 95, 98, or 99% identical to SEQ ID NO: 5 and retains the Q211F amino acid substitution, A modified PPE protein comprising, consisting of, or essentially comprising, an amino acid sequence that is at least 80, 85, 90, 95, 98, or 99% identical to SEQ ID NO: 6 and retains the T55A amino acid substitution, A modified PPE protein comprising, consisting of, or essentially comprising an amino acid sequence that is at least 80, 85, 90, 95, 98, or 99% identical to SEQ ID NO: 7 and retains the N241A amino acid substitution, A modified PPE protein comprising, consisting of, or essentially comprising, an amino acid sequence that is at least 80, 85, 90, 95, 98, or 99% identical to SEQ ID NO: 8 and retains the N241Y amino acid substitution, A modified PPE protein comprising, consisting of, or essentially comprising an amino acid sequence that is at least 80, 85, 90, 95, 98, or 99% identical to SEQ ID NO: 9 and retains the R75A amino acid substitution, A modified PPE protein comprising, consisting of, or essentially comprising an amino acid sequence that is at least 80, 85, 90, 95, 98, or 99% identical to SEQ ID NO: 10 and retains the R75E amino acid substitution, A modified PPE protein comprising, consisting of, or essentially comprising an amino acid sequence that is at least 80, 85, 90, 95, 98, or 99% identical to SEQ ID NO: 11 and retains the Q211A amino acid substitution, A modified PPE protein comprising, consisting of, or essentially comprising, an amino acid sequence that is at least 80, 85, 90, 95, 98, or 99% identical to SEQ ID NO: 12 and retains the R237A amino acid substitution, A modified PPE protein comprising, consisting of, or essentially comprising an amino acid sequence that is at least 80, 85, 90, 95, 98, or 99% identical to SEQ ID NO: 13 and retains the S214A amino acid substitution, A modified PPE protein according to any one of claims 1 to 3, selected from a modified PPE protein comprising, consisting of, or essentially comprising an amino acid sequence that is at least 80, 85, 90, 95, 98, or 99% identical to SEQ ID NO: 14 and retains the D74A amino acid substitution.

5. A modified PPE protein according to any one of claims 1 to 4, having increased cancer cell killing activity compared to the wild-type PPE protein (SEQ ID NO: 4).

6. The modified PPE protein according to claim 5, having cancer cell killing activity that is increased by about or at least about 2 times, 5 times, 10 times, 50 times, 100 times, 500 times, or 1000 times or more compared to the cancer cell killing activity of the wild-type PPE protein (SEQ ID NO: 4).

7. The modified PPE protein according to claim 5 or 6, wherein the increased cancer cell killing activity is observed in vitro or in vivo in the absence of human A1AT protein.

8. The modified PPE protein according to claim 5 or 6, wherein the increased cancer cell killing activity is observed in vitro or in vivo in the presence of human A1AT protein.

9. A modified PPE protein according to any one of claims 1 to 8, having reduced binding to or interaction with human alpha-1 antitrypsin (A1AT) protein compared to the wild-type PPE protein (SEQ ID NO: 4).

10. The modified PPE protein according to claim 9, having reduced binding to the human A1AT protein by about or at least about 2 times, 5 times, 10 times, 50 times, 100 times, 500 times, or 1000 times or more compared to the binding of the wild-type PPE protein to the human A1AT protein.

11. A modified PPE protein according to any one of claims 1 to 10, having serine protease activity of about or at least about 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700, 800, 900, or 1000% or more of that of the wild-type PPE.

12. A modified PPE protein according to any one of claims 1 to 10, having increased serine protease activity compared to the wild-type PPE.

13. The modified PPE protein according to claim 12, wherein the serine protease activity, when measured in the absence of human A1AT protein, is about or at least about 2 times, 5 times, 10 times, 50 times, 100 times, 500 times, or 1000 times or more higher than the serine protease activity of wild-type PPE.

13. The modified PPE protein according to claim 11, wherein the serine protease activity, when measured in the presence of human A1AT protein, is about or at least about 2 times, 5 times, 10 times, 50 times, 100 times, 500 times, or 1000 times or more higher than the serine protease activity of wild-type PPE.

14. A modified PPE proprotein comprising, in an N-terminal to C-terminal orientation, a signal peptide (optionally SEQ ID NO: 2), an activating peptide (optionally SEQ ID NO: 3), and a modified PPE protein as defined in any one of claims 1 to 13, wherein the modified PPE proprotein is activatable via protease cleavage of the activating peptide to produce an enzymatically active modified PPE protein.

15. A recombinant nucleic acid molecule encoding a modified PPE protein or proprotein according to any one of claims 1 to 14, a vector comprising the recombinant nucleic acid molecule, or a host cell comprising the recombinant nucleic acid molecule or the vector.

16. A method for producing a modified PPE protein or proprotein, comprising culturing the host cell described in claim 15 under culture conditions suitable for the expression of the modified PPE protein or proprotein, and isolating the modified PPE protein or proprotein from the culture.

17. A pharmaceutical composition comprising a modified PPE protein or proprotein according to any one of claims 1 to 14, or an expressible polynucleotide encoding the modified PPE protein or proprotein, and a pharmaceutically acceptable carrier.

18. A method for treating cancer, improving its symptoms, and / or reducing its progression in a subject that requires such treatment, comprising administering the pharmaceutical composition according to claim 17 to the subject.

19. The aforementioned cancers are primary or metastatic cancers, and include melanoma (optionally metastatic melanoma), breast cancer (optionally triple-negative breast cancer, TNBC), kidney cancer (optionally renal cell carcinoma), pancreatic cancer, bone cancer, prostate cancer, small cell lung cancer, non-small cell lung cancer (NSCLC), mesothelioma, and leukemia (optionally lymphocytic leukemia, chronic myeloid leukemia, acute myeloid leukemia, or relapsed acute myeloid leukemia). The method according to claim 18, wherein one or more of the following are selected: multiple myeloma, lymphoma, hepatocellular carcinoma, sarcoma, B-cell malignant tumor, ovarian cancer, colorectal cancer, glioma, glioblastoma multiforme, meningioma, pituitary adenoma, vestibular schwannoma, primary CNS lymphoma, primitive neuroectodermal tumor (medulloblastoma), bladder cancer, uterine cancer, esophageal cancer, brain cancer, head and neck cancer, cervical cancer, testicular cancer, thyroid cancer, and gastric cancer.

20. The method according to claim 18 or 19, wherein the pharmaceutical composition comprises the modified PPE proprotein, and the modified PPE proprotein is activated by protease cleavage of the activating peptide in the target cancer tissue or tumor site requiring the pharmaceutical composition to produce an enzymatically active modified PPE protein.

21. The method according to any one of claims 18 to 20, wherein administration of the pharmaceutical composition increases cancer cell death in the subject by about or at least about 2 times, 5 times, 10 times, 50 times, 100 times, 500 times, or 1000 times or more compared to a control or reference.

22. The method according to any one of claims 18 to 21, wherein administration of the pharmaceutical composition results in tumor regression in the subject, such that it is optionally demonstrated by a statistically significant reduction in the amount of surviving tumor or tumor mass, or optionally by a reduction in tumor mass of at least about 10%, 20%, 30%, 40%, or 50%.

23. The method according to any one of claims 18 to 22, comprising administering the pharmaceutical composition to the subject by parenteral administration or intratumor administration.

24. The method according to claim 23, wherein the parenteral administration is intravenous administration.

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