MIRAC protein

Abstract

To provide a method of generating conditionally active biological proteins from wild-type proteins in specific therapeutic proteins.SOLUTION: The method includes: selecting a wild-type biological protein; evolving a DNA which encodes the wild-type biological protein using one or more evolutionary techniques to create mutant DNAs; expressing the mutant DNAs to obtain a mutant protein; subjecting the mutant protein and the wild-type protein to an assay under a normal physiological condition and to an assay under an aberrant condition; and selecting the conditionally active biological protein from the at least one mutant protein which exhibits both (a) a decrease in activity in the assay at the normal physiological condition compared with the wild-type protein, and (b) an increase in activity in the assay under the aberrant condition compared with the wild-type protein.SELECTED DRAWING: None

Description

[Technical Field]

[0001] This application was filed as a PCT application on March 9, 2010. In all designated countries except the United States, the applicant in this international patent application is in the name of BioAtla, LLC, a United States limited liability company, and in the designated United States only, the applicants are Jay M Short, Hwai Wen Chang, both U.S. nationals, and Gerhard Frey, a German national, and claims priority to U.S. Provisional Patent Application No. 61 / 209,489, filed March 9, 2009, the entire contents of which are incorporated herein by reference.

[0002] The present disclosure relates to the field of protein development and activity. Specifically, the present disclosure relates to methods for conditionally generating biologically active proteins from wild-type proteins of certain therapeutic proteins, where the proteins are reversibly or irreversibly inactive in their wild-type form under normal physiological conditions. For example, the developed proteins are substantially inactive at body temperature but active at low temperatures. [Background technology]

[0003] There is a considerable body of literature describing the possibilities for evolving proteins in various aspects, particularly enzymes, to stabilize them for operation under different conditions. For example, enzymes have been evolved to be stable at higher temperatures by altering their activity. In the context of improved activity at high temperatures, a substantial portion of the improvement can be attributed to higher kinetic activity, commonly described by the Q10 rule, which is assumed in the case of enzymes where turnover doubles for every 10-degree Celsius increase. In addition, there are examples of natural mutations that destabilize proteins at normal operating conditions, such as the molecule's wild-type activity temperature. In temperature mutants, these mutations may be active at low temperatures, but typically result in lower levels of activity compared to the wild-type molecule (and typically described by a decrease in activity as determined by Q10 or a similar rule).

[0004] It is desirable to generate useful molecules that are conditionally activated, e.g., substantially inactive under wild-type conditions but active at levels equal to or better than wild-type conditions, or activated or inactivated in a particular microenvironment, or activated or inactivated over time. In addition to temperature, other conditions under which proteins can be developed or optimized include pH, osmolality, osmolality, oxidation, and electrolyte concentration. Other desirable properties that can be optimized during development include chemo-resistance and resistance to proteolysis.

[0005] Many strategies for developing or engineering molecules have already been published. However, engineering or developing proteins to be inactive or essentially inactive (below 10% activity, and especially below 1% activity) under their wild-type operating conditions requires the coexistence of destabilizing mutations and increasing activity of mutations that do not counteract the destabilizing effect, while maintaining equal or better activity than under wild-type conditions under the new conditions. It is speculated that destabilization can reduce protein activity to a greater extent than the effect predicted by standard rules such as Q10, and thus the ability to develop proteins that function effectively at low temperatures, for example, while inactive under their normal operating conditions, creates unexpected novel proteins that we call Mirac proteins.

[0006] Throughout this application, various publications are referenced by author and date. The disclosures of these publications in their entireties are hereby incorporated by reference into this application in order to more fully describe the state of the art as known to those skilled in the art from the date of the disclosure as described and claimed herein. The prior art documents relevant to the invention of this application are as follows (including documents cited in the international phase after the international filing date and documents cited when the application entered the national phase in other countries). (Prior art document) (Patent document) (Patent Document 1) International Publication No. 2003 / 105757 (Patent Document 2) International Publication No. 2009 / 125825 (Non-patent literature) (Non-Patent Document 1) Examination report for corresponding Australian application no. 2023203304; 2024-11-06 (4 pages) (Non-Patent Document 2) Examination report for corresponding Australian application no. 2023203304; 2025-03-11 (4 pages) Summary of the Invention [Means for solving the problem]

[0007] The present disclosure provides methods for preparing a conditionally active biological protein, the method comprising selecting a wild-type biological protein, developing DNA encoding the wild-type biological protein using one or more developmental techniques to create mutant DNA, expressing the mutant DNA to obtain a mutant protein, analyzing the mutant protein and the wild-type protein under normal physiological conditions and abnormal conditions, and selecting those mutant proteins that exhibit both (a) decreased activity in the assay under normal physiological conditions compared to the wild-type protein, and (b) increased activity in the assay under abnormal conditions compared to the wild-type protein. In various embodiments, the normal physiological conditions are selected from one or more of temperature, pH, osmolality, osmolality, oxidation, and electrolyte concentration. In certain embodiments, the normal physiological conditions are temperature, wherein the conditionally active biological protein is substantially inactive at the normal physiological temperature but active at abnormal temperatures below the normal physiological temperature. In other embodiments, the conditionally active biological protein is reversibly or irreversibly inactive in the wild-type normal physiological conditions. In one particular embodiment, the protein is reversibly inactive under the wild-type normal physiological conditions, while the conditionally active biological protein is selected from those proteins that exhibit a change in activity, either reversible or irreversible, under two or more different physiological conditions.

[0008] In one embodiment, the wild-type biological protein is an enzyme. In a particular aspect, the wild-type biological protein is selected from the group consisting of tissue plasminogen activator, streptokinase, urokinase, renin, and hyaluronidase.

[0009] In other embodiments, the wild-type protein is selected from calcitonin gene-related peptide (CGRP), substance P (SP), neuropeptide Y (NPY), vasoactive intestinal peptide (VIP), vasopressin, and angiostatin.

[0010] In other embodiments, the biological protein is an antibody.

[0011] In another embodiment, the disclosure provides a method for preparing a conditionally active biological response modifier, the method comprising selecting an inflammatory response mediator, identifying a wild-type antibody to the mediator, cultivating the wild-type antibody, specifically screening for variants that exhibit decreased binding to the mediator compared to the wild-type antibody under a first condition and increased binding affinity to the mediator under a second condition to identify an up-mutant, recombining the heavy and light chains of the up-mutant to create a recombinant up-mutant, and screening the recombinant up-mutant for variants that exhibit decreased binding to the mediator compared to the wild-type antibody under the first condition and increased binding affinity to the mediator under the second condition to identify the conditionally active biological response modifier. In one embodiment, the inflammatory response mediator is selected from IL-6, IL-6 receptor, TNF-alpha, IL-23, and IL-12. In another embodiment, the first and second conditions are selected from conditions of pH, osmolality, osmolality, oxidation, and electrolyte concentration.

[0012] In other embodiments, the disclosure provides a pharmaceutical composition comprising a conditionally active biological protein and a pharmaceutically acceptable carrier. DETAILED DESCRIPTION OF THE INVENTION

[0013] To facilitate understanding of the examples provided herein, certain frequently occurring methods and / or terms are described below.

[0014] The term "about," as used herein in connection with a measured quantity, relates to the normal variation in the measured quantity that would be expected by one of ordinary skill in the art making the measurement and exercising a level of care appropriate to the purpose of the measurement and the precision of the measuring equipment being used. Unless otherwise specified, "about" relates to a + / - 10% variation of the given value.

[0015] The term "agent" is used to refer to a chemical compound, a mixture of chemical compounds, a spatially localized array of compounds (e.g., VLSIPS peptide arrays, polynucleotide arrays, and / or combinatorial small molecule arrays), a biopolymer, a bacteriophage peptide display library, a bacteriophage antibody (e.g., scFV) display library, a polysome peptide display library, or an extract made from biological material such as bacterial, plant, fungal, or animal (e.g., mammalian) cells or tissues. Agents are evaluated for their potential enzymatic activity as conditionally active biological therapeutic enzymes by inclusion in the screening assays described herein below. Agents are evaluated for their potential activity as conditionally active biological therapeutic enzymes by inclusion in the screening assays described herein below.

[0016] An "ambiguous base requirement" in a restriction site refers to a nucleotide base requirement that is not maximally specific. For example, it may not be a specific base (e.g., but is not limited to, a specific base selected from A, C, G, and T), but may be any one of at least two or more bases. Commonly accepted abbreviations used in the prior art as well as herein to represent base ambiguity include: R=G or A; Y=C or T; M=A or C; K=G or T; S=G or C; W=A or T; H=A or C or T; B=G or T or C; V=G or C or A; D=G or A or T; and N=A or C or G or T.

[0017] As used herein, the term "amino acid" refers to any organic compound containing an amino group (-NH) and a carboxyl group (-COOH), preferably either as a free group or after condensation as part of a peptide. The "20 naturally encoded polypeptide forming alpha-amino acids" are known in the art and relate to alanine (ala or A), arginine (arg or R), asparagine (asn or N), aspartic acid (asp or D), cysteine ​​(cys or C), glutamic acid (glu or G), histidine (his or H), isoleucine (ile or I), leucine (leu or L), lysine (lys or K), methionine (met or M), phenylalanine (phe or F), proline (Pro or P), serine (ser or S), threonine (thr or T), tryptophan (tip or W), tyrosine (tyr or Y), and valine (val or V).

[0018] The term "amplification" means increasing the number of copies of a polynucleotide.

[0019] A molecule with "chimeric properties" is 1) partially homologous and partially heterologous to a first reference molecule, and further includes, but does not exclude, the possibility that the molecule may be 2) partially homologous and partially heterologous to a second reference molecule, or 3) partially homologous and partially heterologous to one or more additional reference molecules. In a non-limiting embodiment, chimeric molecules may be prepared by assembling a reassembly of partial molecular sequences. In a non-limiting aspect, chimeric polynucleotide molecules may be prepared by synthesizing the polynucleotide using multiple molecular templates, thereby resulting in a chimeric polynucleotide with the properties of multiple templates.

[0020] As used herein, the term "homologous" refers to gene sequences that are developmentally and functionally similar between species. For example, but not limited to, in humans, the human CD4 gene is homologous to the mouse 3d4 gene; the sequences and structures of these two genes are highly homologous, and both genes encode proteins that function in signaling T cell activation through MHC class II-restricted antigen recognition.

[0021] As used herein, a "comparison window" refers to a portion of at least 20 contiguous nucleotides in which a polynucleotide sequence is compared to a reference sequence of at least 20 contiguous nucleotides, and the portion of the polynucleotide sequence in the comparison window may have 20% or less additions or deletions (i.e., gaps) compared to the reference sequence (which has no additions or deletions) for optimal alignment of the two sequences.The optimal alignment of sequences for placing the comparison window was determined by Smith's local homology algorithm (Smith and Waterman, 1981, "Comparison of biosequences", Adv Appl Math, 2:482-489; Smith and Waterman, 1981, "Overlapping genes and information theory", J Theor Biol, 91:379-380; Smith and Waterman, J MoI Biol, "Identification of common molecular subsequences", 1981, 147:195-197; Smith et al., 1981, "Comparative biosequence metrics", J MoI Evol, 18:38-46) or by Needleman's homology algorithm (Needleman and Wunsch, 1970, "A general method applicable to the search for similarities in the amino acid sequence of two proteins", J MoI Biol, 48(3):443-453), by Pearson similarity search (Pearson and Lipman, 1988, "Improved tools for biological sequence comparison", Proc Nat Acad Sci USA, 85:2444-2448), by computer implementations of these algorithms (GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics Software Package Release 7.0, Genetics Computer Group, 575 Science Dr., Madison, Wis.), or by inspection of the best alignment (i.e., resulting in the highest percentage of homology over the comparison window) produced by the various methods selected.

[0022] The term "conditionally active biological protein" refers to a mutant or variant of a wild-type protein that is more or less active than the parent wild-type protein under one or more normal physiological conditions. The conditionally active protein may also be active in selected regions of the body or may exhibit increased or decreased activity under physiological conditions permissive to abnormalities or infection. Normal physiological conditions are those temperature, pH, osmolality, osmolality, oxidation, and electrolyte concentrations considered within the normal ranges for a tissue or organ at the site of administration or at the site of administration or action in a subject. Abnormal conditions refer to conditions that deviate from the normally accepted range. In one embodiment, a conditionally active biological protein is substantially inactive under wild-type conditions but active under other wild-type conditions at levels equal to or better than those under wild-type conditions. For example, in a variety of regions, a conditionally active biological protein developed is substantially inactive at body temperature but active at low temperatures. In other embodiments, the conditionally active biological protein is reversibly or irreversibly inactive under wild-type conditions. In further embodiments, the wild-type protein is a therapeutic protein. In other embodiments, the conditionally active biological protein is used as a drug or therapeutic agent. In yet another embodiment, the protein exhibits more or less activity in the highly oxygenated blood, such as after passage through the lungs, or at low pH, such as found in the kidney.

[0023] "Conservative amino acid substitution" refers to the interchangeability of residues with similar side chains. For example, the group of amino acids with aliphatic side chains is glycine, alanine, valine, leucine, and isoleucine; the group of amino acids with carboxylic acid side chains is serine and threonine; the group of amino acids with amide-containing side chains is asparagine and glutamine; the group of amino acids with aromatic side chains is phenylalanine, tyrosine, and tryptophan; the group of amino acids with basic side chains is arginine and histidine; and the group of amino acids with sulfur-containing side chains is cysteine ​​and methionine. Preferred conservative amino acid substitution groups are valine-leucine-isoleucine, phenylalanine-tyrosine, lysine-arginine, alanine-valine, and asparagine-glutamine.

[0024] The term "corresponding," as used herein, means that a polynucleotide sequence is homologous to all or a portion of a reference polynucleotide sequence (i.e., identical, even if not strictly developmentally related) or that a polynucleotide sequence is identical to a reference polynucleotide sequence. In contrast, the term "complementary," as used herein, means that a complementary sequence is homologous to all or a portion of a reference polynucleotide sequence. For example, a nucleotide sequence "TATAC" corresponds to the reference "TATAC" and is complementary to the reference sequence "GTATA."

[0025] The term "effective degradation" amount relates to the amount of enzyme required to process at least 50% of the substrate compared to the substrate not contacted with the enzyme.

[0026] As used herein, a "defined sequence framework" refers to a defined set of sequences selected from non-random bases, typically bases from experimental or structural data. For example, a defined sequence framework may consist of a set of amino acid sequences predicted to form a beta-sheet structure, or may consist of a leucine zipper heptad repeat motif, a zinc finger region, among other variations. A "defined sequence kernel" is a set of sequences encompassing a limited range of variability. (1) A completely random 10-base-long sequence of the 20 conventional amino acids would be (20) 10 (2) a pseudorandom 10-base long sequence of 20 conventional amino acids can be either 10 (3) A defined sequence kernel is a subset of sequences where each residue position is one of the 20 conventional amino acids that are acceptable. A defined sequence kernel generally has either mutated or invariant residue positions, and / or has mutated residue positions that may have residues selected from a defined subset of amino acid residues and the like across the entire length or segments of each selected library member sequence. A defined sequence kernel may relate to an amino acid sequence or a polynucleotide sequence. By way of example and not limitation, the sequence (NNK) 10 and (NNM) 10 wherein N represents A, T, G, or C, K represents G or T, and M represents A or C, and the sequence (NNK) 10 and (NNM) 10 is the defined array kernel.

[0027] DNA "digestion" refers to the catalytic cleavage of DNA by a restriction enzyme that acts only at specific sequences within the DNA. The various restriction enzymes used herein are commercially available, and their reaction conditions, cofactors, and other requirements are known to those skilled in the art. For analytical purposes, typically 1 microgram of plasmid or DNA fragment is used with about 2 units of enzyme in about 20 microliters of buffer. For the purpose of isolating DNA fragments for plasmid construction, typically 5 to 50 micrograms of DNA are digested with a larger volume of enzyme, 20 to 250 units. The appropriate buffer and substrate amounts for a particular restriction enzyme are specified by the manufacturer. An incubation time of about 1 hour at 37°C is typically used but may vary according to the supplier's instructions. After digestion, the reaction is directly subjected to electrophoresis to separate and obtain the desired fragments.

[0028] "Directional ligation" means that the ligations at the 5' and 3' ends of a polynucleotide are sufficiently different to specify a preferred ligation direction. For example, an untreated and undigested PCR product, which originally has two blunt ends, typically does not have a preferred ligation direction when ligated into a cloning vector that has been digested to generate blunt ends in the multiple cloning site. Thus, directional ligation is typically not demonstrated in these situations. In contrast, directional ligation is typically demonstrated when a digested PCR product with a 5' EcoRI treated end and a 3' BamHI is ligated into a cloning vector with a multiple cloning site that has been digested with EcoRI and BamHI.

[0029] The term "DNA shuffling" is used herein to refer to recombination between substantially homologous, but non-identical, sequences; in some embodiments, DNA shuffling may involve crossing over via non-homologous recombination, such as via the cer / lox and / or flp / frt systems.

[0030] The term "drug" or "drug molecule" refers to a therapeutic agent comprising a substance that has a beneficial effect on the human or animal body when administered to the human or animal body. Preferably, the drug is capable of treating, curing, or alleviating one or more symptoms, diseases, or abnormal conditions in the human or animal body, or of promoting the health of the human or animal body.

[0031] An "effective amount" is an amount of a conditionally active biological protein or fragment that is effective to treat or prevent a condition in a living organism of a person to whom it is administered over some period of time, e.g., to provide a therapeutic effect during a desired dosage period.

[0032] As used herein, the term "electrolyte" is used to define minerals in blood or other body fluids that carry electrical charge. For example, in one embodiment, normal physiological conditions and abnormal conditions may be conditions of "electrolyte concentration." In one embodiment, the electrolyte concentration tested is selected from one or more of ionized calcium, sodium, potassium, magnesium, chloride, bicarbonate, and phosphate concentrations. For example, in one embodiment, the normal range for serum calcium is 8.5 to 10.2 mg / dL. In this embodiment, the abnormal serum calcium concentration may be selected from above or below the normal range. In another embodiment, in one embodiment, the normal range for serum chloride is 96 to 106 milliequivalents per liter (mEq / L). In this embodiment, the abnormal serum chloride concentration may be selected from above or below the normal range. In another embodiment, in one embodiment, the normal range for serum magnesium concentration is 1.7 to 2.2 mg / dL. In this embodiment, the abnormal serum magnesium concentration may be selected from above or below the normal range. In another embodiment, the normal range for serum phosphate is 2.4 to 4.1 mg / dL. In this embodiment, the abnormal serum phosphate concentration may be selected from above or below the normal range. In another embodiment, the normal range for serum or blood sodium is 135 to 145 mEq / L. In this embodiment, the abnormal serum or blood sodium concentration may be selected from above or below the normal range. In another embodiment, the normal range for serum or blood potassium is 3.7 to 5.2 mEq / L. In this embodiment, the abnormal serum or blood potassium concentration may be selected from above or below the normal range. In a further embodiment, the normal range for serum bicarbonate is 20 to 29 mEq / L. In this embodiment, the abnormal serum or blood bicarbonate concentration may be selected from above or below the normal range. In a different embodiment, the bicarbonate level may be used to indicate the normal level of acidity (pH) in the blood. The term "electrolyte concentration" may also be used to define the concentration of a particular electrolyte in a tissue or a body fluid other than blood or plasma.In this case, normal physiological conditions are considered to be the clinically normal range for that tissue or body fluid. In this embodiment, the abnormal tissue or body fluid electrolyte concentration may be selected from above or below the normal range.

[0033] As used in this disclosure, the term "antigenic determinant" refers to an antigenic determinant on an antigen, such as an enzyme polypeptide, to which the antigen-binding site of an antibody, such as an enzyme-specific antibody, binds. An antigenic determinant usually consists of chemical surface-active groups of molecules, such as amino acids or side chains, and may also have specific three-dimensional structural properties and specific charge characteristics. As used herein, "antigenic determinant" refers to that portion of an antigen or other macromolecule that can form binding interactions with the variable region that binds to the body of an antibody. Typically, such binding interactions are manifested as intermolecular interactions with amino acid residues of one or more CDRs.

[0034] As used herein, an "enzyme" is a protein with specific catalytic properties. For example, factors such as substrate concentration, pH, temperature, and the presence or absence of inhibitors can affect the rate of catalysis. Typically, for wild-type enzymes, the Q10 (temperature coefficient) describes the increase in reaction rate for every 10°C increase in temperature. For wild-type enzymes, Q10 = 2-3, meaning that the reaction rate doubles or triples for every 10°C increase in temperature. At high temperatures, proteins denature. At pH values ​​slightly different from the enzyme optimum, small changes occur in the charge of the enzyme and possibly the substrate molecules. Changes in ionization can affect the binding of substrate molecules. At extreme pH levels, enzymes denature, where the active site is distorted and the substrate site no longer fits.

[0035] As used herein, the terms "development" or "developing" refer to the use of mutagenesis methods to generate novel polynucleotides encoding one or more novel polypeptides, which novel polypeptides are improved biological molecules themselves and / or contribute to the generation of other improved biological molecules. In certain non-limiting embodiments, the present disclosure relates to the development of conditionally active biological proteins from parent wild-type proteins. In one embodiment, for example, development refers to methods that perform both non-stochastic polynucleotide chimerization and non-stochastic site-directed point mutagenesis, as disclosed in U.S. Patent Application No. 2009 / 0130718, incorporated herein by reference. More specifically, the present disclosure provides methods for the development of conditionally active biological enzymes that exhibit reduced activity compared to the wild-type parent enzyme under normal physiological conditions, but enhance activity compared to the wild-type enzyme under one or more abnormal conditions.

[0036] The terms "fragment," "derivative," and "analog," when used in reference to a reference polypeptide, refer to a polypeptide that retains at least one, at least essentially the same physiological function or activity as the reference polypeptide. Further, the terms "fragment," "derivative," and "analog" are exemplified by "preform" molecules, such as low-activity precursor proteins, that can be modified by cleavage to produce mature enzymes with significantly higher activity.

[0037] Provided herein are methods for generating progeny polypeptides from template polypeptides in which a "full range of single amino acid substitutions" is indicated at each amino acid position. As used herein, "full range of single amino acid substitutions" refers to the 20 naturally encoded polypeptides that form the alpha-amino acids as described herein.

[0038] The term "gene" refers to a DNA segment involved in the production of a polypeptide chain, including the regions preceding and following the coding region (leader and leader) as well as intervening sequences (nitrones) between individual coding segments (exons).

[0039] As used herein, "genetic instability" refers to the natural tendency of highly repetitive sequences to be lost by a process of reduction events that usually involves sequence simplification through the loss of repeated sequences. Deletions can include the loss of one copy of a repeat and all inter-repeat sequences.

[0040] The term "heterologous" means that a single-stranded nucleic acid sequence cannot hybridize to another single-stranded nucleic acid sequence or its complementary sequence. Thus, a non-homologous portion means that a polynucleotide or polynucleotide has a portion or region in its sequence that cannot hybridize to another nucleic acid or polynucleotide. Such a region or portion is, for example, a mutation portion.

[0041] The terms "homologous" or "homologous" mean that a single-stranded nucleic acid sequence is capable of hybridizing to a complementary single-stranded nucleic acid sequence. The degree of hybridization can depend on a number of factors, including the amount of identity between the sequences and hybridization conditions such as temperature and salt concentration, as described below. Preferably, the region of identity is greater than about 5 bp, and more preferably, the region of identity is greater than 10 bp.

[0042] The benefit of this disclosure extends to "industrial applications" (or industrial processes), which term is used to include appropriate commercial industrial (or simply industrial) applications as well as non-commercial industrial applications (e.g., positive medical research at non-profit institutions). Relevant applications include those in diagnostics, medicine, agriculture, manufacturing, and academia.

[0043] "Identical" or "identical" means that two nucleic acids have the same or complementary sequences. Thus, "identical portion" means that a region or portion of a polynucleotide, or an entire polynucleotide, is identical to or complementary to a portion of another polynucleotide.

[0044] The term "isolated" means that the material is removed from its original environment (e.g., the natural environment, if it is naturally occurring). For example, a naturally occurring polynucleotide or enzyme present in a living animal is not isolated, but the same polynucleotide or enzyme separated from some or all of the coexisting materials in the natural system is isolated. Such a polynucleotide may be part of a vector and / or such a polynucleotide or enzyme may be part of a composition and still be isolated in that such a vector or composition is not part of its natural environment.

[0045] The term "isolated nucleic acid" is used to define a nucleic acid, e.g., a DNA or RNA molecule. A nucleic acid, such as a DNA or RNA molecule, is not immediately flanked by 5' and 3' flanking sequences that normally flank it when present in a naturally occurring gene of the organism from which it is derived. Thus, the term refers to nucleic acids incorporated into a vector, e.g., a plasmid or viral vector, nucleic acids incorporated into a gene of a heterologous cell (or into a gene of a heterologous cell, but at a site different from that in which it naturally occurs), and nucleic acids that exist as isolated molecules, e.g., DNA fragments produced by PCR amplification or restriction enzyme digestion, or RNA molecules produced by in vitro transcription. The term also refers to recombinant nucleic acids that form part of a hybrid gene encoding an additional protein that can be used, for example, in the production of fusion proteins.

[0046] As used herein, "ligand" refers to a molecule that is recognized by a specific receptor, such as a random peptide or variable segment sequence. As one skilled in the art will recognize, a molecule (or macromolecular complex) can be either a receptor or a ligand. Generally, the binding partner with the smaller molecular weight is called the ligand, and the binding partner with the larger molecular weight is called the receptor.

[0047] "Ligation" refers to the process of forming a phosphodiester bond between two double-stranded nucleic acid fragments (Sambrook et al., (1982). Molecular Cloning: A Laboratory Manual. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY., p. 146; Sambrook et al., Molecular Cloning: a laboratory manual, 2 nd Ed., Cold Spring Harbor Laboratory Press, 1989). Unless otherwise provided, ligation may be accomplished using known buffers and conditions with 10 units of T4 DNA ligase ("ligase") per 0.5 micrograms of approximately equimolar amounts of the DNA fragments to be ligated.

[0048] As used herein, a "linker" or "spacer" serves to join two molecules, such as a protein and a DNA binding random peptide, and to position the two molecules in a suitable configuration, e.g., so that the random peptide can bind to a receptor with minimal steric hindrance from the DNA binding protein.

[0049] As used herein, "microenvironment" means any part or region of a tissue or body that has a physical or chemical difference, either regularly or temporally, from other regions of the tissue or body.

[0050] As used herein, "molecular properties developed" includes reference to molecules consisting of polynucleotide sequences, molecules consisting of polypeptide sequences, and molecules consisting of portions of polynucleotide sequences and portions of polypeptide sequences. Particularly relevant, but not meant to be limiting, examples of molecular properties developed include protein activity under specific conditions such as temperature, salinity, osmolality, pH, oxidation, and the concentration of glycerol, DMSO, detergent, and / or any other molecular species contacted in the reaction environment. Additionally, particularly relevant, but not meant to be limiting, examples of molecular properties developed include stability, e.g., the amount of molecular property remaining after a specified exposure time to a specified environment.

[0051] The term "mutation" refers to a change in the sequence of a wild-type nucleic acid sequence or a change in the sequence of a peptide. Such a mutation may be a point mutation, such as a transition or transversion. A mutation may be a deletion, insertion, or duplication.

[0052] As used herein, a degenerate "N,N,G / T" nucleotide sequence represents 32 possible triplets, where "N" can be A, C, G, or T.

[0053] The term "naturally occurring," as used herein, refers to the fact that an object is found in nature. For example, a polypeptide or polynucleotide sequence present in an organism (including a virus) that can be isolated from a source in nature and has not been intentionally modified by humans in a laboratory is naturally occurring. Generally, the term "naturally occurring" refers to an object as it exists in a non-pathological (non-diseased) individual, as is typical for the species.

[0054] As used herein, "normal physiological conditions" or "wild-type operating conditions" are conditions of temperature, pH, osmolality, osmolality, acidity, and electrolyte concentrations that are considered within the normal range at the site of administration or site of action in a subject.

[0055] As used herein, a "nucleic acid molecule" refers to a molecule that is single-stranded or double-stranded and consists of at least one base or one base pair, respectively. Furthermore, a nucleic acid molecule can belong solely or chimerically to any group of nucleic acid molecules, including, but not limited to, RNA, DNA, genetic nucleic acids, non-genic nucleic acids, naturally occurring and non-naturally occurring nucleic acids, and groups of nucleic acid molecules such as synthetic nucleic acids. This includes, by way of non-limiting example, nucleic acids associated with any organelle, such as mitochondria, ribosomal RNA, and nucleic acid molecules that are chimeric from one or more naturally occurring and non-naturally occurring components.

[0056] In addition, a "nucleic acid molecule" may contain, in part, non-nucleotide-based components such as, but not limited to, one or more amino acids and sugars. Thus, by way of example and not limitation, a ribozyme, which is partially nucleotide-based and partially protein-based, is considered a "nucleic acid molecule."

[0057] Additionally, by way of example and not limitation, a nucleic acid molecule that is labeled with a detectable moiety, such as a radioactive or non-radioactive label, is also considered a "nucleic acid molecule."

[0058] The terms "nucleic acid sequence encoding" or "DNA encoding a sequence of" or "nucleotide sequence encoding" refer specifically to a nucleotide sequence encoding an enzyme, as well as other synonymous terms, a DNA sequence that is transcribed and converted into an enzyme when placed under the control of appropriate regulatory sequences. A "promoter sequence" is a DNA regulatory region that allows RNA polymerase to bind in a cell and initiate downstream (3') transcription of the coding sequence. A promoter is a portion of a DNA sequence. This sequence region has an initiation codon at its 3' end. A promoter sequence contains the minimum number of bases necessary to initiate transcription at a detectable level above background. However, once RNA polymerase has identified the sequence, transcription begins at the initiation codon (the 3' end containing the promoter), and then proceeds downstream in the 3' direction. In a promoter, sequences are found at the transcription initiation site (conveniently defined by mapping with nuclease S1), as well as protein binding regions (consensus sequences) responsible for RNA polymerase binding.

[0059] The terms "nucleic acid encoding an enzyme (protein)" or "DNA encoding an enzyme (protein)" or "polynucleotide encoding an enzyme (protein)" and other synonymous terms are intended to encompass polynucleotides that contain only the coding sequence for an enzyme as well as polynucleotides that contain additional coding and / or non-coding sequences.

[0060] In one preferred embodiment, a "specific nucleic acid species" is defined by its chemical structure, as exemplified, but not limited to, by its primary sequence. In other preferred embodiments, a "specific nucleic acid species" is defined by the function of the nucleic acid species or by the function of a product derived from the nucleic acid species. Thus, as a non-limiting example, a "specific nucleic acid species" is defined by one or more activities or properties attributed to it, including an activity or property attributed to its expressed product.

[0061] The instant definition of "constructing a working nucleic acid sample into a nucleic acid library" includes incorporating a nucleic acid sample into a collection-based vector, such as by ligation into a vector and transformation of a host. A description of relevant vectors, hosts, and other reagents is provided below, as well as specific, non-limiting examples thereof. The instant definition of "constructing a working nucleic acid sample into a nucleic acid library" also includes incorporating a nucleic acid sample into a non-vector-based collection, such as by ligation to an adhesin. Preferably, the adhesin is capable of annealing to PCR primers to facilitate amplification by PCR.

[0062] Also, in a non-limiting embodiment, a "nucleic acid library" comprises a vector-based collection of one or more nucleic acid molecules. In another preferred embodiment, a "nucleic acid library" comprises a non-vector-based collection of nucleic acid molecules. In yet another preferred embodiment, a "nucleic acid library" comprises a combination of partially vector-based and partially non-vector-based collections of nucleic acid molecules. Preferably, the collection of molecules comprising the library is searchable and separable according to the type of individual nucleic acid molecule.

[0063] The present disclosure provides a "nucleic acid construct" or "nucleotide construct" or "DNA construct." The term "construct" is used herein to describe a molecule such as a polynucleotide (e.g., an enzymatic polynucleotide) that may optionally be chemically linked to one or more additional molecular moieties, such as a vector or vector portion. In certain embodiments, and not meant to be limiting, the nucleotide construct is exemplified by a DNA expression construct suitable for transformation of a host cell.

[0064] "Oligonucleotide" (or synonymous with "oligo") refers to a single-stranded polydeoxynucleotide or a chemically synthesized complementary polydeoxynucleotide strand. Such synthetic oligonucleotides may or may not have a 5' phosphate. They will not ligate to other oligonucleotides without adding an ATP and phosphate in the presence of a kinase. Synthetic oligonucleotides are ligated to fragments that are not dephosphorylated. For purposes of polymerase-based amplification (e.g., by PCR), reference is made to "a 32-fold degenerate oligonucleotide consisting, in sequence, of at least a first homologous sequence, a degenerate N, N, G / T sequence, and a second homologous sequence." As used in this context, "homologous" refers to the homology between the oligo and the parent polynucleotide that is subjected to polymerase-based amplification.

[0065] As used herein, the term "operably linked" refers to the linkage of polynucleotide elements in a functional relationship. A nucleic acid is "operably linked" when it is placed into a functional relationship with another nucleic acid sequence. For example, a promoter or enhancer is operably linked to a coding sequence if it affects the transcription of the coding sequence. Operatively linked means that the DNA sequences being linked are typically contiguous, and, where necessary to join two protein-coding regions, contiguous and in reading frame.

[0066] A coding sequence becomes "operably linked" to another coding sequence when RNA polymerase transcribes the two coding sequences into a single mRNA that is translated into a single polypeptide having amino acids from both coding sequences. The coding sequences need not be contiguous to each other, so long as the expressed sequences are ultimately processed to produce the desired protein.

[0067] As used herein, the term "parental polynucleotide set" refers to a set of one or more distinct polynucleotide species. Typically, the term is used to refer to a progeny polynucleotide set, preferably obtained by mutagenesis of a parental set, in which case the terms "parental," "starting," and "template" are interchangeable.

[0068] The term "patient" or "subject" means an animal, e.g., a mammal, such as a human, who is the object of treatment. Subjects or patients can be male or female.

[0069] As used herein, the term "physiological conditions" refers to biochemical constraints, such as temperature, pH, osmolality, ionic strength, and viscosity, that are compatible with living and / or viable cultured yeast or mammalian cells and that are normally present intracellularly. For example, the intracellular conditions are physiological conditions in yeast cells grown under typical laboratory culture conditions. Suitable in vitro reaction conditions for in vitro transcription cocktails are normal physiological conditions. Generally, in vitro physiological conditions consist of 50-200 mM sodium chloride or potassium chloride, pH 6.5-8.5, 20-45°C, and 0.001-10 mM divalent cations (e.g., Mg++, Ca++), preferably about 150 mM sodium chloride or potassium chloride, pH 7.2-7.6, 5 mM divalent cations, and often 0.01-1.0% nonspecific protein (e.g., BSA). Non-ionic detergents (Tween, NP-40, Triton X-100) are often present, usually at about 0.001-2%, typically 0.05-0.2% (v / v). Specific aqueous solution conditions are selected by the practitioner according to prior art. As a general guide, the following aqueous buffer conditions are applicable: 10-250 mM sodium chloride, 5-50 mM Tris-HCl, pH 5-8, with the addition of optional divalent cations and / or metal chelators and / or non-ionic detergents and / or membrane fractions and / or antifoaming agents and / or scintillants. Normal physiological conditions refer to the temperature, pH, osmolality, osmolality, acidity, and electrolyte concentrations that are considered normal in a patient, in vivo, or at the site of administration or site of action.

[0070] The standard convention (5' to 3') is used herein to describe the sequence of a double-stranded polynucleotide.

[0071] The term "population" refers to a collection of compositions such as polynucleotides, portions of polynucleotides, or proteins. A "mixed population" is a collection of compositions that are of the same genus (i.e., related) of nucleic acids or proteins, but that differ in their sequence (i.e., are not identical) and therefore differ in their physiological and intellectual activity.

[0072] A molecule having a "surrogate" refers to a molecule that has undergone any combination of one or more covalent and non-covalent chemical modifications (e.g., glycosylation, proteolytic cleavage, dimerization or oligomerization, temperature- or pH-induced conformational changes, association with cofactors, etc.) en route to a more mature molecular form with distinct properties (e.g., increased activity) compared to the reference surrogate molecule. When two or more chemical modifications (e.g., two proteolytic cleavages, or a proteolytic cleavage and no glycosylation) can be distinguished en route to producing the mature molecule, the reference precursor molecule is referred to as a "precursor surrogate" molecule.

[0073] As used herein, the term "pseudorandom" refers to a set of sequences with limited variability, e.g., the degree of residue variability at other positions is limited, while allowing for a degree of residue variation outside any pseudorandom position.

[0074] As used herein, "quasi-repeat units" refer to reassorted repeats that are not, by definition, identical. Indeed, this method proposes not only the reassortment of virtually identical encoding units produced by mutagenesis of the same starting sequence, but also of similar or related sequences that may diverge significantly in some regions. Nevertheless, if sequences contain sufficient homology to be reassorted by this method, they can be referred to as "quasi-repeat" units.

[0075] As used herein, "random peptide library" refers to a set of polynucleotide sequences that encode a set of random peptides, and the set of random peptides encoded by these polypeptide sequences, as well as fusion proteins that contain these random peptides.

[0076] As used herein, "random peptide sequence" means an amino acid sequence that consists of two or more amino acid monomers and that is constructed by a stochastic or random process. Random peptides may include a framework or scaffold and may have an invariant sequence.

[0077] As used herein, "receptor" refers to a molecule that has affinity for a given ligand. Receptors may be naturally occurring or synthetic molecules. Receptors may be used in their native state or in association with other species. Receptors may be covalently or non-covalently bound to a binding member directly or via a specific binding substance. Examples of receptors include, but are not limited to, monoclonal antibodies and antiserum reagents with specific antigenic determinants (e.g., on viruses, cells, or other materials), cell membrane receptors, sugar and glycoprotein complexes, enzymes, and hormone receptors.

[0078] A "recombinant" enzyme is an enzyme produced by recombinant DNA techniques, i.e., produced from cells transformed with an exogenous DNA construct encoding the desired enzyme. A "synthetic" enzyme is prepared by chemical synthesis.

[0079] The term "related polynucleotides" refers to regions or portions of polynucleotides that are identical, as well as regions or portions of polynucleotides that are non-homologous.

[0080] As used herein, "reductive reassortment" refers to an increase in molecular diversity that occurs through deletions (and / or insertions) mediated by repeated sequences.

[0081] The following terms "reference sequence," "comparison window," "sequence identity," "percent sequence identity," and "substantially identical" are used to describe the sequence relationship between two or more polynucleotides.

[0082] A "reference sequence" is a defined sequence used as a basis for sequence comparison. A reference sequence may consist of a subset of a larger sequence, e.g., a segment of a full-length cDNA or gene sequence given in a sequence listing, or the complete cDNA or gene sequence. Generally, a reference sequence is at least 20 nucleotides in length, often at least 25 nucleotides in length, and often at least 50 nucleotides in length. Because two polynucleotides each consist of (1) similar sequences between the two polynucleotides (i.e., portions of the complete polynucleotide sequence), and (2) sequences that further diverge between the two polynucleotides, sequence comparison between two (or more) polynucleotides is typically performed by comparing the sequences of the two polynucleotides over a "comparison window" to identify and compare local regions of sequence similarity.

[0083] "Repetitive index (RI)," as used herein, is the average number of copies of a quasi-repeated unit contained within a cloning vector.

[0084] The term "restriction site" refers to a recognition sequence necessary for the action of a restriction enzyme and includes the site of catalytic cleavage. It is recognized that the site of cleavage may or may not be contained in the portion of the restriction site consisting of low-ambiguity sequences (i.e., sequences containing the major determinants of the frequency of occurrence of the restriction site). Thus, in many cases, relevant restriction sites contain only low-ambiguity sequences with an internal cleavage site (e.g., G / AATTC in an EcoRI site) or an immediately adjacent cleavage site (e.g., / CCWGG in an EcoRII site). In other cases, relevant restriction enzymes (e.g., Eco57I site or CTGAAG(16 / 14)) contain low-ambiguity sequences (e.g., the CTGAAG sequence in an Eco57I site) with an external cleavage site (e.g., in the N.Sub.16 portion of an Eco57I site). It is understood that an enzyme (e.g., a restriction enzyme) "cleaves" a polynucleotide to mean that the restriction enzyme catalyzes or facilitates cleavage of the polynucleotide.

[0085] In a non-limiting embodiment, a "selectable polynucleotide" consists of a 5'-terminal region (or termination region), an intermediate region (e.g., an internal or central region), and a 3'-terminal region (or termination region). As used in this embodiment, a 5'-terminal region is a region located toward the 5' polynucleotide end (or 5' polynucleotide end). It is thus part of or all of the 5' half of a polynucleotide. Similarly, a 3'-terminal region is a region located toward the 3' polynucleotide end (or 3' polynucleotide end). It is thus part of or all of the 3' half of a polynucleotide. As used in this non-limiting illustration, there can be sequence overlap between any two regions or between all three regions.

[0086] The term "sequence identity" means that two polynucleotide sequences are identical (i.e., nucleotide-by-nucleotide) over a comparison window. The term "percent sequence identity" is calculated by comparing two optimally aligned sequences over a comparison window, determining the number of positions at which the same nucleobase (e.g., A, T, C, G, U, or I) occurs in both sequences to obtain the number of matching positions, dividing the number of matching positions by the total number of positions in the comparison window (i.e., the window size), and multiplying the result by 100 to obtain the percent sequence identity. As used herein, "substantially identical" refers to a characteristic of a polynucleotide sequence, wherein the polynucleotide has at least 80% sequence identity, preferably at least 85% identity, often 90-95% sequence identity, and most commonly at least 99% sequence identity, compared to a reference sequence over a comparison window of at least 25-50 nucleotides, wherein the percent sequence identity is calculated by comparing the polynucleotide sequence to the reference sequence, which may include deletions or additions of 20% or less of the total of the reference sequence over the comparison window.

[0087] As known to those skilled in the art, "similarity" between two enzymes is determined by comparing the amino acid sequence and conserved amino acid subunits of that enzyme to the sequence of a second enzyme. Similarity is determined by the procedure of the BLAST program (Basic Local Alignment Search Tool at the National Center for Biotechnology Information), as is well known to those skilled in the art.

[0088] Members of a molecular pair (e.g., an antibody-antigen pair or a nucleic acid pair) are said to "specifically bind" to one another when they bind to one another with greater affinity than to other nonspecific molecules. For example, an antibody can be described as specifically binding to an antigen because it binds to the antigen more efficiently than nonspecific proteins. (Similarly, a nucleic acid probe can be described as specifically binding to a target nucleic acid when it specifically forms a duplex with the target through base-pairing interactions (see above).) "Specific hybridization" is defined herein as a form of hybridization between a first polynucleotide and a second polynucleotide (e.g., a polynucleotide having a sequence that differs from, but is substantially identical to, the first polynucleotide), where substantially unrelated polynucleotide sequences in the mixture do not form hybrids.

[0089] The term "specific polynucleotide" refers to a polynucleotide that has a specific end point and a specific nucleic acid sequence. Two polynucleotides, one of which has the same sequence as a portion of a second polynucleotide, but whose different ends are composed of two different specific polynucleotides.

[0090] "Stringent hybridization conditions" means that hybridization will occur only when there is at least 90% identity between the sequences, preferably at least 95% identity, and most preferably at least 97% identity. Sambrook et al., Molecular Cloning: a laboratory manual, 2 nd Ed., Cold Spring Harbor Laboratory Press, 1989, which is incorporated herein by reference in its entirety.

[0091] The present disclosure also includes polypeptides having a sequence "substantially identical" to that of an enzyme polypeptide. A "substantially identical" amino acid sequence is one that differs from a reference sequence only by conservative amino acid substitutions, e.g., the substitution of one amino acid of the same type for another (e.g., a hydrophobic amino acid such as isoleucine, valine, leucine, or methionine for another hydrophobic amino acid, or a polar amino acid for another polar amino acid, such as arginine for lysine, glutamic acid for aspartic acid, or glutamine for asparagine).

[0092] Additionally, a "substantially identical" amino acid sequence is one that differs from a reference sequence, or by one or more non-conservative substitutions, deletions, or insertions, provided that the polypeptide essentially retains its behavioral properties when the substitutions occur at sites other than the active site of the molecule. For example, one or more amino acids may be removed from an enzyme polypeptide, resulting in a modification of the polypeptide's structure without significantly altering its biological activity. For example, amino- or carboxyl-terminal amino acids that are not required for the enzyme's physiological activity may be removed. Such modifications may lead to the development of smaller, more active enzyme polypeptides.

[0093] The present disclosure provides "substantially pure enzymes." The term "substantially pure enzymes" is used herein to describe molecules such as polypeptides (e.g., enzyme polypeptides, or fragments thereof) that are substantially free from other proteins, lipids, sugars, nucleic acids, and other physiological materials with which they are naturally associated. For example, a substantially pure molecule, such as a polypeptide, can be at least 60% by dry weight of the molecule of interest. Purity of a polypeptide can be determined using standard methods, including, for example, polyacrylamide gel electrophoresis (e.g., SDS-PAGE), column chromatography (e.g., high-performance liquid chromatography (HPLC)), and amino-terminal amino acid sequence analysis.

[0094] As used herein, "substantially pure" means that the species of interest is the predominant species (i.e., more abundant than any other individual molecule in the composition, on a molar basis), and preferably, a substantially purified fragment is one in which the species of interest comprises at least about 50% (on a molar basis) of all macromolecular species present. Generally, a substantially pure composition will comprise about 80-90 percent or more of the macromolecular species present in the composition. Most preferably, the species of interest is purified to essential homogeneity (contaminant species cannot be detected in the composition by conventional detection methods), wherein the composition consists essentially of a single macromolecular species. Dissolved species, small molecules (<500 Daltons), and elementary ionic species are not considered macromolecular species.

[0095] The term "treat" means (1) preventing or delaying the appearance of clinical symptoms of a condition, disease, or condition that progresses in an animal afflicted with or susceptible to a clinical or subclinical symptom of the condition, disease, or condition, but that has not yet experienced or exhibited a clinical or subclinical symptom of the condition, disease, or condition; (2) inhibiting the condition, disease, or condition (i.e., suppressing, reducing, or delaying the progression of the disease or, in the case of maintenance treatment, its reversal or at least one clinical or subclinical symptom); and / or (3) ameliorating the condition (i.e., causing regression of the condition, disease, or condition or at least one clinical or subclinical symptom), where the benefit to the patient treated is statistically significant or at least perceptible to the patient or physician.

[0096] As used herein, the term "variable segment" refers to a portion of a nascent peptide that consists of random, pseudorandom, or defined nucleolar sequences. A "variable segment" refers to a portion of a nascent peptide that consists of random, pseudorandom, or defined nucleolar sequences. A variable segment may consist of both variant and invariant residue positions, and the degree of variant residues at variant residue positions may be limited; both options are selected at the discretion of the practitioner. Typically, a variable segment is about 5-20 amino acid residues (e.g., 8-10) in length, however, variable segments may be longer and may consist of antibody proteins or receptor proteins, such as antibody fragments, protein-binding nucleic acids, receptor proteins, etc.

[0097] The term "variant" refers to a disclosed polynucleotide or polypeptide modified in one or more base pairs, codons, introns, exons, or amino acid residues (respectively) of a wild-type protein parent molecule. Variants may be produced by any number of means, including, for example, methods such as error-prone PCR, shuffling, oligonucleotide-directed mutagenesis, assembly PCR, sexual PCR mutagenesis, in vivo mutagenesis, cassette mutagenesis, recursive ensemble mutagenesis, exponential ensemble mutagenesis, site-specific mutagenesis, gene remodeling, saturation mutagenesis, and any combination thereof. Techniques for producing mutant proteins that have reduced activity under normal physiological conditions, e.g., one or more of temperature, pH, osmolality, oxidation, and electrolyte concentration, and enhanced activity under abnormal conditions, compared to the wild-type protein, are disclosed herein. Variants may additionally be selected for properties that enhance chemical and proteolytic resistance, compared to the wild-type protein.

[0098] As used herein, the term "wild-type" refers to a polynucleotide that does not have any mutations. "Wild-type protein," "wild-type protein," "wild-type biological protein," or "wild-type biological protein" refers to a protein that can be isolated from nature that is active at the level of activity found in nature and consists of an amino acid sequence found in nature. The terms "parent molecule" and "target protein" refer to wild-type proteins.

[0099] The term "action" in "action sample" refers to, for example, the sample on which one acts. Similarly, "action molecule" refers to, for example, the molecule on which one acts.

[0100] The present disclosure also teaches methods for engineering or developing proteins to generate novel molecules that are reversibly or irreversibly inactive under wild-type conditions but active under non-normal conditions at levels similar to or equal to those of wild-type conditions. These novel proteins are also referred to herein as "Mirac" proteins. Mirac proteins are particularly valuable in the development of novel therapeutics that are active within a host for a short or limited period of time. This is particularly valuable in the extended engineering of administered proteins that are harmful to the host but where limited activity is necessary to effect the desired treatment. Examples of beneficial applications include local or systemic treatment at high doses, as well as localized treatment at high concentrations. Inactivation under physiological conditions may be determined by the administration combination and the rate of inactivation of the protein. This condition-based inactivation is particularly important in enzyme therapy, when catalytic activity can cause substantial negative effects in a relatively short period of time.

[0101] The present disclosure also directs methods for engineering or developing proteins to generate novel molecules that differ from wild-type molecules, that are active or inactive reversibly or irreversibly over time, or that are active or inactive only in specific microenvironments within the body, including specific organs within the body (e.g., the bladder or kidney).

[0102] Target wild-type protein Any therapeutic protein can serve as a target protein or wild-type protein in the production of a conditionally active biological protein. In one embodiment, the target protein is a wild-type enzyme. Currently used therapeutic protein enzymes include urokinase and streptokinase, which are used in the treatment of blood clots, and hyaluronidase, which is used as an adjuvant to increase the absorption and distribution of other drugs. In one embodiment, the wild-type protein selected for the production of a conditionally active biological protein is one currently used therapeutic protein to avoid or minimize adverse side effects associated with the wild-type protein or enzyme. Alternatively, an enzyme not currently used as a therapeutic may be selected for the production of a conditionally active biological protein. Specific non-limiting examples are discussed in detail below.

[0103] Therapeutic proteins may be used alone or in combination with other therapies to treat various diseases or medical conditions. The conditionally active biological proteins of the present disclosure may be adapted for use in one or more indications, including circulatory disorders, arthritis, multiple sclerosis, autoimmune diseases, cancer, dermatological conditions, and may be used in a variety of diagnostic formats. Depending on the protein and indication, the conditionally active biological enzyme protein may be administered parenterally, topically, or orally, as discussed below.

[0104] Circulatory disorders - thrombosis and thrombolytic therapy A thrombus (blood clot) is defined as a solid mass derived from blood components that forms in the circulatory system. Thrombi form through a series of events involving interactions between blood clotting factors, platelets, red blood cells, and the blood vessel wall. Platelet aggregation is an intravascular aggregation of platelets, fibrin, and trapped blood cells that can cause vasculopathy. By impeding or blocking blood flow, thrombi deprive tissues of oxygen. Thrombus fragments (emboli) can detach and obstruct smaller blood vessels. Arterial thrombus formation can be triggered by any of a variety of factors, including occult stenosis—atherosclerosis, low-flow cardiac function, hypercoagulability or clotting factor deficiencies in cancer, or foreign bodies such as stents or catheters. Thrombi leading to arterial ischemia can result in limb or tissue damage, acute myocardial infarction (AMI), stroke, amputation, or intestinal infarction. A significant cause of morbidity and mortality is the formation of arterial thrombi (coronary and cerebral artery thrombi) and pulmonary thrombi. Venous thrombus formation can occur due to trauma, e.g., stasis due to quiescence, or endothelial damage such as hypercoagulability, but atherosclerosis is not a contributing factor. Treatments include mechanical thrombectomy, pharmacodynamic thrombectomy, and thrombolysis. Treatments for thrombosis are used to minimize thrombus formation and aid in its removal.

[0105] Treatment of thrombosis includes the use of antiplatelet drugs to inhibit platelet activation, anticoagulant therapy, and / or thrombolytic therapy to break down blood clots. Examples of antiplatelet agents include aspirin, dipyridamole, and ticlopidine. Examples of anticoagulants include heparin, warfarin, hirudin, and activated human protein C. Examples of thrombolytic agents include tissue plasminogen activator (tPA) / tPA variants, urokinase, and streptokinase. Thrombolytic agents exhibit a catalytic type of action.

[0106] Thrombolytic therapy for acute myocardial infarction has been established. The use of thrombolytic agents has become the standard emergency treatment. Despite their effectiveness, these products achieve complete reperfusion in only approximately 50% of patients, and side effects include the risk of bleeding (particularly intracranial hemorrhage) as well as hypertension. The breakdown of clots from injured or diseased blood vessels is referred to as "fibrinolysis" or the "fibrinolytic process." Fibrinolysis is a proteolytic process in which plasminogen activators activate the protein plasminogen, thereby forming plasmin. Proteolytic plasmin breaks down fibrin strands to dissolve the clot. Fibrin-specific plasminogen activators include tissue plasminogen activators or variants. Nonspecific plasminogen activators may include streptokinase and urokinase.

[0107] Certain commonly used thrombolytic therapies utilize one of several available tissue plasminogen activator (tPA) variants. For example, tPA variants based on products previously approved for use are Alteplase (rt-PA), Reteplase (r-PA), and Tenecteplase (TNK). Approved uses of tPA variants include, for example, acute myocardial infarction (AMI) in improving ventricular function following AMI, reducing the incidence of congestive heart failure, and reducing mortality associated with AMI, management of ischemic stroke in adults to improve neurological recovery and reduce the incidence of disability, for the lysis of acute pulmonary emboli, and for the lysis of pulmonary emboli associated with unstable hemodynamics, and management of acute massive pulmonary emboli in adults.

[0108] Another commonly used thrombolytic therapy utilizes urokinase, a standard lytic agent used in the treatment of peripheral vascular disease.

[0109] Streptokinase is a protein secreted by some species of streptococcus that can bind and activate human plasminogen. The complex of human plasminogen and streptokinase is activated by bond cleavage to generate plasmin, which can then hydrolytically activate otherwise unbound plasminogen. Normal activation of plasminogen occurs via proteolysis of the Arg561-Val562 bond. The amino group of Val562 forms a salt bridge with Asp740, causing a conformational change to generate the active protease plasmin. Plasmin is produced in the blood to break down fibrin, the main component of blood clots.

[0110] Streptokinase is used as an effective clot-dissolving agent in some cases of myocardial infarction (heart attack), pulmonary embolism (lung clot), and deep vein thrombosis (leg clot). Streptokinase belongs to a group of drugs called fibrinolytic agents. Streptokinase is given as soon as possible after the onset of a heart attack to dissolve clots in the arteries of the heart wall and reduce damage to the heart muscle. Because streptokinase is a bacterial product, the body has the ability to build up an immune response to the protein. Therefore, it is recommended that this product not be given more than four days after the initial administration, as it may not be effective and may cause an allergic reaction. For this reason, it is usually given only after the initial heart attack, and further thrombosis is typically treated with tissue plasminogen activator (TPA). Streptokinase is also sometimes used to prevent postoperative adhesions.

[0111] Side effects of streptokinase include bleeding (major and minor), hypotension, and respiratory depression and allergic reactions. In addition, anticoagulants and drugs that alter platelet function (e.g., aspirin, other NSAIDs, dipyridamole) may increase the risk of bleeding.

[0112] Thrombolytic agents are usually administered by injection, intravenous bolus, or mechanical infusion system. Side effects can be severe, including intracranial, gastrointestinal, retroperitoneal, or pericardial bleeding. If bleeding occurs, administration must be discontinued immediately.

[0113] In certain embodiments of the present disclosure, tPA, streptokinase, or urokinase are selected as the target or wild-type protein.

[0114] In one embodiment, the method of the present disclosure is used to select a conditionally active recombinant or synthetic streptokinase mutant that has high activity at abnormal temperature conditions, lower than normal physiological conditions, and is substantially inactive or inactive at normal physiological conditions (e.g., 37°C). In one aspect, the abnormal temperature conditions are room temperature, e.g., 20-25°C. In another aspect, the present disclosure provides a method of treating stroke or heart attack, comprising administering a high dose of a conditionally active streptokinase mutant to a stroke or heart attack patient to eliminate blood clots and allow rapid inactivation of the streptokinase mutant to avoid excessive bleeding.

[0115] Circulatory disorders - Renin / Angiotensin The renin-angiotensin system is a hormonal system that regulates blood pressure and water (fluid) balance. The kidneys secrete renin when blood volume is low. Renin is an enzyme that hydrolyzes angiotensinogen, secreted by the liver, into the peptide angiotensin I. Angiotensin I is further cleaved in the lungs by endothelium-bound angiotensin-converting enzyme (ACE) to angiotensin II. Angiotensin II constricts blood vessels, resulting in increased blood pressure. However, angiotensin II also stimulates the secretion of the hormone aldosterone from the adrenal cortex. Aldosterone increases the reabsorption of sodium and water in the renal tubules. This increase increases body fluids and blood pressure. An overactive renin-angiotensin system leads to vasoconstriction and sodium and water retention. These effects contribute to hypertension. Many drugs are available that disrupt different steps in this system to lower blood pressure. These drugs are one of the main methods for controlling the harmful effects of high blood pressure (hypertension), heart failure, kidney failure and diabetes.

[0116] Hypovolemic shock is an emergency condition in which the heart cannot adequately perfuse body cells with oxygenated blood due to the loss of a large amount of blood and / or fluid. Blood loss can occur due to trauma, injury, and internal bleeding. Circulating blood volume can decrease due to excessive fluid loss from burns, diarrhea, excessive sweating, or vomiting. Signs of hypovolemic shock include anxiety, cold, clammy skin, confusion, rapid breathing, or loss of consciousness. Examination reveals signs of shock, including low blood pressure, hypothermia, and a rapid pulse that may be weak or feeble. Treatment includes fluid infusion, blood or blood products, treatment for shock, and medications such as dopamine, dobutamine, epinephrine, and norepinephrine to increase blood pressure and cardiac output.

[0117] In one embodiment, the present disclosure provides a method for selecting conditional recombinant renin mutants that are reversibly inactivated at normal physiological temperatures but are reactivated when a patient is hypothermic due to hypovolemic shock. The conditionally active proteins may be used to treat hypovolemic shock to promote an increase in body fluid volume and blood pressure.

[0118] Circulatory disorders - Raynaud's phenomenon Raynaud's phenomenon (RP) is a vasospastic disorder that causes discoloration of the fingers, toes, and sometimes other extremities. Emotional stress and exposure to cold are typical triggers of this decline. Exposure to cold causes the extremities to lose heat. Blood flow to the fingers and toes is normally slowed to maintain the body's core temperature. Blood flow is reduced due to narrowing of small arteries under the skin in the extremities. Stress triggers a response similar to that of exposure to cold. In Raynaud's phenomenon, this normal response is exaggerated. The condition can cause pain, discoloration, and a feeling of coldness and numbness. This phenomenon results from vasospasm, which reduces blood supply to the respective area. In Raynaud's disease (primary Raynaud's phenomenon), the condition is sudden. In Raynaud's syndrome (secondary Raynaud's phenomenon), the phenomenon is caused by other inciting factors. Measurement of the temperature gradient in the hand is one way to distinguish between the primary and secondary forms. The primary form may progress to the secondary form, which in extreme cases may progress to necrosis or gangrene of the fingertip.

[0119] Raynaud's phenomenon is an exaggerated response to cold or emotional stress. Early RP is primarily caused by microvascular spasms. Overactivation of the sympathetic nervous system causes excessive vasoconstriction of peripheral blood vessels, leading to hypoxia. Chronic, recurrent cases can result in atrophy of the skin, subcutaneous tissue, and muscle. Rarely, ulceration and ischemic gangrene can occur.

[0120] Conventional treatment options for Raynaud's phenomenon include prescription medications that dilate blood vessels and improve circulation. These include calcium channel blockers like nifepidine or diltiazem, alpha-blockers like prazosin or doxazosin, which counteract the effects of norepinephrine, a hormone that constricts blood vessels, and vasodilators like nitroglycerin cream or the angiotensin II inhibitor losartan, sildenafil, or prostaglandins, which relax blood vessels. Fluoxetine, selective serotonin reuptake inhibitors, and other antidepressants can reduce the frequency and severity of episodes caused by physiological stressors. These medications can cause side effects like headache, flushing, and ankle edema. Medications may also lose their effectiveness over time.

[0121] Regulation of cutaneous vasoconstriction and vasodilation involves altered sympathetic activity and numerous neuromodulators, including adrenergic and non-adrenergic, as well as other signaling pathways such as REDOX signaling and the RhoA / ROCK pathway. Vasoconstriction of cutaneous vascular smooth muscle cells (vSMCs) is thought to be activated by norepinephrine, mediated by alpha1 and alpha2 adrenergic receptors. Alpha2C-ARs translocate from the trans-Golgi to the cell surface of vSMCs in response to stimuli, and signaling of these responses involves the RhoA / Rhokinase (ROCK) signaling pathway. Cold stimulation of cutaneous arteries leads to the immediate generation of reactive oxygen species (ROS) in vSMC mitochondria. ROS are involved in REDOX signaling via the RhoA / ROCK pathway. RhoA is a GTP-binding protein that plays a role in regulating actin-myosin-dependent processes such as migration and cell contraction in vSMCs. Non-adrenergic neuropeptides with well-known functions in the vasculature, with possible involvement in RP, include calcitonin gene-related peptide (CGRP), substance P (SP), neuropeptide Y (NPY), and vasoactive intestinal peptide (VIP). Fonseca et al., 2009, "Neuronal regulators and vascular dysfunction in Raynaud's phenomenon and systemic sclerosis", Curr. Vascul. Pharmacol. 7:34-39. Newer treatments for RP include alpha-2C adrenoceptor blockers, protein tyrosine kinase inhibitors, Rho-kinase inhibitors and calcitonin gene-related peptide.

[0122] Calcitonin gene-related peptide (CGRP) is a member of the calcitonin family of peptides and exists in two forms: alpha-CGRP and beta-CGRP. Alpha-CGRP is a 37-amino acid peptide formed by alternative splicing of the calcitonin / CGRP gene. CGRP is one of the most common peptides produced in the peripheral and central nervous systems. It is a potent peptide vasodilator and can affect pain conduction. Migraine is a common neurological disorder associated with elevated levels of CGRP. CGRP dilates blood vessels in the brain and transmits vascular pain sensations. CGRP receptor antagonists have been experimentally tested as a treatment for migraine. Arulmani et al., 2004, "Calcitonin gene-related peptide and its role in migraine pathophysiology," Eur. J. Pharmacol. 500(1-3): 315-330. At least three receptor subtypes have been identified, and CGRP acts through G protein-coupled receptors, varying in function to regulate the peptide's actions in various tissues. CGRP signaling through the receptor depends on two auxiliary proteins, receptor activity modifier protein 1 (RAMP1) and receptor component protein (RCP). Ghatta 2004, Calcitonin gene-related peptide, its role is now understood. Indian J. Pharmacol. 36(5): 277-283. One study of the effects of intravenous administration of three vasodilators, the endothelium-dependent vasodilator adenosine triphosphate (ATP), the endothelium-independent vasodilator prostacyclin (epoprostenol, PGI2), and CGRP, in patients with Raynaud's phenomenon, in an age- and sex-matched population of patients with Raynaud's phenomenon and controls using CGRP and laser Doppler flowmetry (LDF), showed that in Raynaud's patients, CGRP induced flushing of the face and hands by increasing cutaneous blood flow, whereas in the control group, CGRP caused flushing of only the face. PG12 induced similar changes in blood flow in the hands and face in both groups.ATP did not cause any significant changes in blood flow in the hands or face of patients, but increased blood flow in the face of the control group. Shawket et al., 1989, "Selective suprasensitivity to calcitonin-gene-related peptide in the hands in Reynaud's phenomenon". The Lancet, 334(8676):1354-1357. In one embodiment, the wild-type protein target molecule is CGRP.

[0123] In one embodiment, the present disclosure provides a method for selecting conditionally active recombinant protein variants of proteins associated with Raynaud's syndrome that are reversibly inactive at normal physiological temperatures but are reactivated when the fingers are exposed to abnormally low temperatures. The conditionally active proteins may be used to treat Raynaud's phenomenon, preventing or reducing loss of finger function due to hypocirculation.

[0124] Circulatory disorders - vasopressin Arginine vasopressin (AVP, vasopressin, antidiuretic hormone (ADH)) is a peptide hormone found in many mammals that controls the reabsorption of molecules in the renal tubules related to tissue permeability. One of vasopressin's most important roles is to regulate water retention in the body. At high concentrations, it elevates blood pressure by inducing moderate vasoconstriction. Vasopressin has three effects that result in increased urine osmolality (high concentration) and decreased water excretion. First, vasopressin increases the water permeability of collecting duct cells in the kidney, allowing for water reabsorption and the excretion of smaller amounts of concentrated urine (antidiuretic). This occurs via the insertion of aquaporin 2 water channels in the apical membrane of collecting duct cells. Second, vasopressin increases the permeability of the inner medullary portion of the collecting duct to urea, allowing for increased urine reabsorption in the medullary interstitium. Third, vasopressin increases the activity of Na+, K+, 2Cl- cotransporters, stimulating sodium and chloride reabsorption in the thickened superior limb of the loop of Henle. Sodium chloride reabsorption is by a process of increased backflow, providing an osmotic gradient across aquaporins that results in water reabsorption in the collecting duct medulla.

[0125] The hypertonic interstitial fluid surrounding the kidney's collecting ducts provides a high osmotic pressure for water removal. Transmembrane channels created by proteins called aquaporins are inserted into the plasma membrane, making it highly permeable to water. When open, aquaporin channels can pass 3 billion molecules of water per second. Insertion of aquaporin 2 channels requires signaling by vasopressin. Vasopressin binds to receptors (called V2 receptors) on the basolateral surface of collecting duct cells. Hormone binding triggers an increase in intracellular cAMP levels. This "second messenger" initiates a cascade that results in insertion of aquaporin 2 channels in the apical membrane of collecting duct cells. Aquaporins increase water reabsorption by pumping water out of the nephron and returning urine to the bloodstream.

[0126] The primary stimulus for vasopressin release from the pituitary gland is an increase in plasma osmolality. Any dehydration, such as intense sweating, increases blood osmolality and activates vasopressin at the V2 receptor via the aquaporin 2 pathway. As a result, as little as 0.5 L / day of urine may remain from the original 180 L / day of renal filtrate. The salt concentration in urine may be four times higher than that in blood. When the blood becomes too diluted, such as by drinking large amounts of water, vasopressin secretion is inhibited, and aquaporin 2 channels are transported back into the cell by endocytosis. This results in the production of large volumes of dilute urine with only a quarter of the salt concentration found in blood.

[0127] Decreased vasopressin release or decreased renal sensitivity to AVP can lead to diabetes insipidus, hypernatremic conditions (increased sodium levels in the blood), polyuria (excessive urine production), and polydipsia (dry mouth).

[0128] High levels of AVP secretion (syndrome of inappropriate antidiuretic hormone (SIADH)) and resulting hyponatremia (low blood sodium concentration) occur in brain and pulmonary (small cell lung cancer) conditions. In the perioperative period, surgical stress and the effects of several commonly used drugs (e.g., opiates, syntocinon, antiemetics) lead to a similar state of excessive vasopressin secretion, which can cause hyponatremia for several days.

[0129] Vasopressin agonists are used therapeutically in a variety of conditions, and their long-acting synthetic analog, desmopressin, is used in conditions characterized by low vasopressin secretion, as well as for the control of bleeding (in various forms of von Willebrand disease) and extreme cases of bedwetting in children. Terlipressin and related analogs are used as vasoconstrictors in certain conditions. Vasopressin infusions are used as a second line of management in patients with septic shock who do not respond to high doses of inotropes (e.g., dopamine or norepinephrine). Vasopressin receptor antagonists are drugs that block the action of vasopressin at the receptor. They may also be used to treat hyponatremia.

[0130] In one embodiment, the present disclosure provides a method of selection for conditional biological recombinant or synthetic proteins of proteins involved in the vasopressin response that are reversibly inactive under normal physiological osmolality but are reactivated under abnormal osmolality in blood. In another embodiment, mutants of proteins involved in the vasopressin response are activated under sodium-deficient conditions but inactivated under normal serum sodium concentrations. In one aspect, the sodium-deficient condition is serum sodium <135 mEq / L.

[0131] Cancer-Angiostatin Angiostatin is a naturally occurring protein in several animal species. It acts as an endogenous angiogenesis inhibitor (i.e., inhibits the growth of new blood vessels). Angiostatin suppresses tumor cell growth and metastasis by inhibiting endothelial cell proliferation and migration. Angiostatin is a 38 kD fragment of plasmin (which is itself a fragment of plasminogen). Angiostatin consists of kringles 1 to 3 of plasminogen. Angiostatin is produced by autolytic cleavage of plasminogen, involving extracellular disulfide bond reduction, for example, by phosphoglycerate kinase. Angiostatin can be cleaved from plasminogen by different matrix metalloproteinases, including MMP2, MMP12, and MMP9, and serine proteases (neutral elastase, prostate-specific antigen (PSA)). In vivo, angiostatin inhibits tumor growth and maintains experimental metastases in a dormant state. Angiostatin is elevated in animals with early tumors and other inflammatory and degenerative diseases.

[0132] Angiostatin is known to bind to many proteins, including angiomotin and endothelial cell surface ATO synthase, integrins, annexin π, C-met receptor, NG2-proteoglycan, tissue plasminogen activator, chondroitin surface glycoprotein, and CD26. One study showed that IL-12, a TH1 cytokine with potent antiangiogenic activity, is a mediator of angiostatin activity. Albin", J. Translational Medicine. Jan. 4, 2009, 7:5. Angiostatin binds to the endothelial cell surface and inhibits ATP synthesis. ATP synthesis also occurs on the surface of various cancer cells. Tumor cell surface ATP synthesis is more active at low extracellular pH, a characteristic of the tumor microenvironment. Angiostatin appears to act on tumor cell surface ATP synthesis activity at acidic extracellular pH (pH). At low extracellular pH, angiostatin is directly antitumorigenic. At low pH, angiostatin and anti-beta subunit antibodies induce intracellular acidification of A549 cancer cells, as well as direct toxicity, which is lacking in tumor cells with low levels of extracellular ATP synthesis. The mechanism of tumor cytotoxicity is hypothesized to be intracellular pH deregulation due to inhibition of cell surface ATP synthesis. Chi and Pizzo, "Angiostatin is directly cytotoxic to tumor cells at low extracellular pH: a mechanism dependent on cell surface-associated ATP synthase", Cancer Res. 2006, 66(2): 875-82.

[0133] In one embodiment, the present disclosure provides a method for identifying conditionally active angiostatin mutants that are less active than wild-type angiostatin at normal physiological blood pH, but exhibit enhanced activity at low pH. Low pH is defined as lower than normal physiological pH. In one aspect, low pH is about pH 7.2. less than In certain embodiments, the low pH is about pH 6.7.

[0134] In one embodiment, the conditionally active angiostatin variants may be formulated and utilized as anti-cancer drugs.

[0135] Increased tissue permeability - hyaluronidase Hyaluronidase is a family of enzymes that degrades hyaluronic acid. By causing the degradation of hyaluronic acid, a major component of the interstitial barrier, hyaluronidase reduces hyaluronic acid viscosity, thereby increasing tissue permeability. It is used in medicine in conjunction with drugs to speed drug dispersion and delivery. Its most common application is in ophthalmic surgery, where it is used in conjunction with local anesthesia. Animal-derived hyaluronidases include Hydase™ (PrimaPharm Inc.; Akorn), Vitrase (ISTA Pharmaceuticals), and Amphadase (Amphastar Pharmaceuticals). Human recombinant hyaluronidase is currently approved as an adjuvant to increase the absorption of other drugs, such as Hypodermocyclis (subcutaneous injection of fluid), an adjuvant in subcutaneous urography that improves the absorption of radiopaque agents (Hylenex; Halozyme Therapeutics, Inc.; Baxter Healthcare Corp). In one embodiment, hyaluronidase is used as a wild-type protein (parent molecule) to prepare a conditionally active biological protein. Hyaluronidase can play a role in cancer metastasis and angiogenesis. Therefore, overexposure to these enzymes can be harmful. In one aspect, the conditionally active biological hyaluronidase protein is irreversibly or reversibly inactive at normal physiological temperatures, but is active at levels equal to or greater than those of wild-type hyaluronidase within a specific temperature range below normal physiological temperatures.

[0136] Autoimmune diseases - Conditionally active biological response modifiers Rheumatoid arthritis is an autoimmune disease characterized by an abnormal immune system that leads to inflammation and swelling due to the progressive destruction of joints. RA (rheumatoid arthritis) can also affect the skin, connective tissue, and internal organs. Conventional treatments include nonsteroidal anti-inflammatory drugs (NSAIDS), COX-2 inhibitors, and disease-modifying antirheumatic drugs (DMARDS) such as methotrexate. None of the conventional treatment modalities is ideal, especially when used long-term.

[0137] Biological response modifiers that target inflammatory mediators offer a relatively novel approach to the treatment of rheumatoid arthritis and other autoimmune diseases. Such biological response modifiers include antibodies or active portions thereof against various inflammatory mediators, such as IL-6, IL-6 receptor, TNF-alpha, IL-23, and IL-12.

[0138] Some of the first biological response modifiers are drugs that target tumor necrosis factor alpha (TNF-α), a pro-inflammatory cytokine involved in the pathogenesis of RA. Several anti-TNF-α drugs are currently marketed for the treatment of RA. For example, Enbrel® (etanercept, Amgen) is a TNF-α blocker. Etanercept is a dimeric fusion protein consisting of the extracellular tethered binding portion of the human 75-kilodalton (p75) tumor necrosis factor receptor (TNFR) bound to the Fc portion of human IgG1. The Fc component of etanercept contains the CH2 domain, CH3 domain, and hinge region, but not the Ch1 domain of IgG1. Etanercept is produced in a Chinese hamster ovary (CHO) mammalian cell expression system. It consists of 934 amino acids and has an apparent molecular weight of approximately 150 kilodaltons. Enbrel® is used to treat rheumatoid arthritis, psoriatic arthritis, ankylosing spondylitis, and plaque psoriasis. Serious side effects of Enbrel® include infections, including tuberculosis, fungal infections, and viral infections caused by opportunistic pathogens. Sepsis may occur. Lymphoma or other malignancies have also been reported.

[0139] Remicade® (infliximab) is a chimeric anti-TNF-alpha IgG1 monoclonal antibody consisting of human constant regions and murine variable regions. Remicade is administered intravenously and is used to treat rheumatoid arthritis, psoriasis, Crohn's disease, ulcerative colitis, and ankylosing spondylitis. Side effects of Remicade include serious infections or sepsis and, rarely, certain T-cell lymphomas. Other side effects include hepatotoxicity, certain serious hematologic events, hypersensitivity reactions, and certain neurologic events.

[0140] Other biological response modifiers include humanized anti-interleukin-6 (IL-6) receptor antibodies. IL-6 is a cytokine that contributes to inflammation, swelling, and joint damage in RA. One humanized anti-IL-6 receptor antibody, Actemra (tocilizumab, Roche), has been approved by the FDA and the European Commission for the treatment of adult patients with rheumatoid arthritis. Actemra is also approved in Japan for the treatment of RA and juvenile rheumatoid arthritis (sJIA). Phase III studies have shown that, compared with other therapeutic agents, treatment with Actemra, both as monotherapy and in combination with MTX or other DMARDs, reduces the signs and symptoms of RA. Actemra is a humanized IL-6 receptor monoclonal antibody that competitively blocks the binding of IL-6 to its receptor. Thus, it inhibits the proliferative effects of IL-6, which lead to synovial hyperplasia and pannus formation in RA. Serious side effects of Actemra include serious infections and hypersensitivity reactions, including some cases of anaphylaxis. Other side effects include upper respiratory tract infections, headache, nasopharyngitis, hypertension, and increased ALT.

[0141] Another common autoimmune disease is psoriasis. Hyperactivity of the immune system can lead to high concentrations of IL-12 and IL-23, two cytokine proteins found in psoriatic skin plaques. IL-12 and IL-23 are involved in inflammatory and immune responses, such as natural killer cell activation and CD4+ T cell differentiation and activation.

[0142] One treatment for moderate or severe psoriasis involves subcutaneous injection of STELARA™ (ustekinumab, Centocor Ortho Biotech, Inc.), a humanized IgG1k monoclonal antibody against the p40 subunit of the IL-12 and IL-23 cytokines. STELARA has been shown to provide relief from certain symptoms associated with psoriasis plaques, such as plaque thickening, peeling, and redness. The STELARA formulation contains L-histidine and L-histidine hydrochloride hydrate, polysorbate 80, and sucrose in an aqueous solution. Use of STELARA™ affects the immune system and increases the chance of infection, including tuberculosis and infections caused by bacteria, fungi, or viruses, as well as the risk of certain types of cancer.

[0143] The side effects of biological response modifiers can be severe, and infusion of high levels into patients can make them more susceptible to serious infection or death. This is a major side effect associated with this important class of drug. One challenge is avoiding the high initial levels of activity from antibody administration, which are necessary to provide a long therapeutic effect after infusion.

[0144] In one embodiment, the present disclosure provides a method for preparing conditionally active biological response mediators or fragments thereof, avoiding the high levels of activity required from antibody administration to provide a prolonged therapeutic effect after injection. The disclosed method may be used to design antibodies against inflammatory mediators, such as IL-6, IL-6 receptor, TNF-alpha, IL-23, and IL-12, that are inactive at administration conditions, such as room temperature, but slowly refold (reversibly or irreversibly) at body temperature. These antibodies or fragments thereof are inactive upon initial injection, but refold or reactivate over a period of several hours to several days upon exposure to blood infusion. This allows for higher dosages and longer half-lives (or administration periods) with reduced side effects.

[0145] In one aspect, the present disclosure provides a method for preparing conditionally active antibodies or fragments thereof to inflammatory mediators that are inactive under administration conditions, such as room temperature, but slowly refold (reversibly or irreversibly) at body temperature. This method comprises the following steps: selecting for the inflammatory mediator; screening to identify antibodies to the inflammatory mediator via hybridoma cells; humanizing the anti-inflammatory mediator antibody; developing the anti-inflammatory mediator antibody and differentially screening for binding under two or more temperature conditions, e.g., room temperature and 37°C or higher; selecting for mutations that are inactive under a first condition relative to the wild-type but that exhibit increased activity (e.g., binding) compared to the wild-type antibody activity (binding) under a second condition. The identified upregulation mutants in the heavy and light chains are recombined in the heavy and light chains as well as through combinatorial heavy and light chain binding. Screening of these recombined heavy and light chains is repeated under two conditions, e.g., room temperature and 37°C or higher. In addition, the recombinant antibodies or fragments are screened for activity and stability under storage and physiological conditions.

[0146] Alternatively, wild-type antibodies to inflammatory mediators are known antibodies or variants or active fragments thereof.

[0147] In one embodiment, the first and second conditions are selected from pH, osmolality, osmolality, oxidation, and charge concentration. In another embodiment, the inflammatory mediator is selected from IL-6, IL-6 receptor, TNF-alpha, IL-23, and IL-12.

[0148] In another aspect, the present disclosure provides a method for preparing conditionally active antibodies to IL-6 or fragments thereof that are inactive at administration conditions such as room temperature but slowly refold (reversibly or irreversibly) at body temperature. This method includes the following steps: screening a fully human library for antibodies to IL-6; developing IL-6 antibodies and differentially screening the molecules at room temperature and at 37°C or higher; selecting mutations that are inactive at room temperature compared to the wild-type but exhibit increased activity (e.g., binding) compared to the wild-type antibody activity (binding). The identified upregulation mutants in the heavy and light chains are recombined in the heavy and light chains as well as through combinatorial heavy and light chain binding. Screening of these recombined heavy and light chains is repeated at room temperature and higher temperatures. Additionally, the recombined antibodies or fragments are tested for activity and stability under storage and physiological conditions.

[0149] The conditionally active anti-IL-6 antibodies thus identified and produced can be used in methods for treating autoimmune diseases such as rheumatoid arthritis or psoriasis by administering an effective amount to a patient in need thereof, with reduced side effects compared to the administration of conventional biological response modifier anti-IL-6 antibodies. One advantage of this method is the smoothing and leveling of drug dosage over the treatment period, compared to the current high levels of biological response modifiers whose clearance half-lives last for weeks or months.

[0150] One or more mutagenesis techniques are used to develop DNA encoding the wild-type protein to create a library of mutant DNA. The mutant DNA is expressed to create a library of mutant proteins, and this library is then subjected to screening assays under normal physiological conditions and one or more abnormal conditions. Conditionally active biological proteins are selected from those proteins that exhibit both (a) decreased activity when assayed under normal physiological conditions compared to the wild-type protein and (b) increased activity when assayed under abnormal conditions compared to the wild-type protein. Alternatively, conditionally active biological proteins are selected from those proteins that exhibit altered activity, either reversibly or irreversibly, under two or more different physiological conditions.

[0151] Generation of evolved molecules from parent molecules Mirac proteins are generated through a process of mutagenesis and screening for individual mutations that have activity in non-wild-type conditions that remains the same or greater than the activity in wild-type conditions and have reduced activity in wild-type conditions.

[0152] The present disclosure provides a method for generating nucleic acid variants encoding polypeptides with enzymatic activity, wherein the variants have abnormal physiological activity from naturally occurring variants, the method comprising: (a) (i) substituting one or more nucleotides with different nucleotides, where the nucleotides consist of natural or unnatural nucleotides; (ii) deleting one or more nucleotides; (iii) adding one or more nucleotides; or (iv) modifying the nucleic acid by any combination thereof. In one embodiment, the unnatural nucleotide consists of inosine. In another embodiment, the method comprises analyzing polypeptides encoded by the modified nucleic acids for abnormal enzymatic activity, thereby identifying modified nucleic acids that encode polypeptides with abnormal enzymatic activity. In one embodiment, the modification of step (a) is accomplished by PCR, error-prone PCR, shuffling, oligonucleotide-directed mutagenesis, assembly PCR, sexual PCR mutagenesis, in vivo mutagenesis, cassette mutagenesis, recursive ensemble mutagenesis, exponential ensemble mutagenesis, site-specific mutagenesis, gene reassembly, gene site saturation mutagenesis, ligase chain reaction, in vitro mutagenesis, ligase chain reaction, oligonucleotide synthesis, any gene production technique, and combinations thereof. In another embodiment, the method further comprises at least one repetition of modifying step (a).

[0153] This method further provides a method for producing a polynucleotide from two or more nucleic acids, the method comprising: (a) identifying regions of identity and variance between two or more nucleic acids, at least one of which consists of a nucleic acid of the present disclosure; (b) providing a set of oligonucleotides corresponding to at least two sequences of the two or more nucleic acids; and (c) extending the oligonucleotides with a polymerase, thereby producing a polynucleotide.

[0154] Any mutagenesis technique may be used in various embodiments of the present disclosure. Stochastic or random mutagenesis is exemplified by a situation in which a parent molecule is mutated (modified or altered) to obtain a series of progeny molecules with unplanned mutations. Thus, an in vitro stochastic mutagenesis reaction, for example, is an unplanned product, the production of which is not specifically intended. Rather, the exact nature of the resulting mutations, and therefore the resulting product, is uncertain and therefore random. Stochastic mutagenesis is also demonstrated in methods in which mutations are random or unplanned, such as error-prone PCR and stochastic shuffling. Mutant formation can be achieved by error-prone transcription, such as error-prone PCR, or by the use of polymerases lacking proofreading activity (see Liao (1990) Gene 88: 107-111), or by primary replication in a mutant strain (mutant host cells are discussed in more detail below and are generally well known). Mutagen strains can include any mutants defective in mismatch repair function. These include mutant gene products such as mutS, mutT, mutH, mutL, ovrD, dcm, vsr, umuC, umuD, sbcB, and recJ. Deletions can be obtained by gene mutation, allelic exchange, small chemical compounds, or expressed antisense RNA, or other techniques. The deletion can be in the described gene or a homologous gene in any organism.

[0155] Currently, widely used mutagenesis methods for generating selective proteins from starting molecules are oligonucleotide-directed mutagenesis, error-prone polymerase chain reaction (error-prone PCR), and cassette mutagenesis, in which a specific region to be optimized is replaced with a synthetically mutagenized oligonucleotide. In these cases, multiple mutation sites occur around a specific site in the original sequence.

[0156] In oligonucleotide-directed mutagenesis, short sequences are replaced with synthetically mutagenized oligonucleotides. In oligonucleotide-directed mutagenesis, short sequences of polynucleotides are removed from synthetic polynucleotides using restriction enzyme digestion and replaced with synthetic polynucleotides in which various bases are altered from the original sequence. Polynucleotide sequences can also be altered by chemical mutagenesis. Chemical mutagens include, for example, sodium bisulfite, nitrous acid, hydroxylamine, hydrazine, or formic acid. Other agents similar to nucleotide precursors include nitrosoguanidine, 5-bromouracil, 2-aminopurine, or acridine. These agents are typically added to PCR reactions in place of the nucleotide precursors that alter the sequence. Insertion agents such as proflavine, acriflavine, and quinacrine may also be used. Random mutagenesis of polynucleotide sequences can be achieved by X-ray or ultraviolet irradiation. Mutagenized plasmid polynucleotides are typically introduced into E. coli and propagated as pools or libraries of hybridizing plasmids.

[0157] Error-prone PCR uses low-fidelity polymerization conditions to introduce a low level of point mutations randomly over a long sequence. Given a mixture of fragments of unknown sequence, error-prone PCR can be used to mutagenize the mixture.

[0158] In cassette mutagenesis, sequence blocks of a single template are typically (partially) replaced by random sequences. Reidhaar-Olson JF and Sauer RT: Combinatorial cassette mutagenesis as a probe of the information content of protein sequences. Science 241(4861):53-57, 1988.

[0159] Alternatively, any technique of non-stochastic or non-random gene mutagenesis can be used in various embodiments of the present disclosure. Non-stochastic mutagenesis is exemplified by a situation in which a parent molecule is mutated (modified or changed) to obtain a molecule with one or more predetermined mutations. It is well understood that the presence of background products in some amount is inherent in many reactions in which molecular processing occurs, and the presence of background products does not detract from the non-stochastic nature of the mutagenesis process, which involves unplanned products. Site-saturation mutagenesis and synthetic ligation reassembly are examples of gene mutagenesis techniques in which the precise chemical structure of the intended product is predetermined.

[0160] One method of site-saturation point mutagenesis is described in U.S. Application Publication No. 2009 / 0130718, incorporated herein by reference. This method involves providing a series of degenerate primers corresponding to codons of a template polynucleotide and performing polymerase extension to produce progeny polynucleotides containing sequences corresponding to the degenerate primers. The progeny polynucleotides are expressed and screened in a directed developmental fashion. Specifically, this is a method for producing a series of progeny polypeptides, comprising the steps of: (a) providing copies of a template polypeptide, each copy consisting of a plurality of codons encoding the template polypeptide sequence; and (b) for each codon in the template polynucleotide, (1) providing a series of degenerate primers, each primer consisting of a degenerate codon corresponding to the codon in the template polynucleotide, and at least one flanking sequence homologous to the sequence flanking the codon in the template polynucleotide; (2) providing conditions in which the primers can anneal to the copy of the template polynucleotide; and (3) performing a polymerase extension reaction from the primers along the template, thereby providing progeny polynucleotides, each containing a sequence corresponding to the degenerate codon of the annealed primer, thereby producing a series of progeny polynucleotides.

[0161] Site-saturation mutagenesis involves the directed evolution of nucleic acids and screening of clones containing the evolved nucleic acids for the resulting activity of interest, such as the activity of a nucleic acid and / or a specific protein, particularly an enzyme, of interest. Mutagenized molecules provided by this technique can include chimeric molecules and molecules with point mutations, including biological molecules comprising sugar, lipid, nucleic acid, and / or protein compositions, and specific, but non-limiting examples of these include antibiotics, antibodies, enzymes, and steroid and non-steroid hormones.

[0162] Site saturation mutagenesis generally refers to a method comprising the steps of: 1) preparing progeny generations of molecules (including molecules consisting of polynucleotide sequences, molecules consisting of polypeptides, and molecules consisting of portions of polynucleotide sequences and portions of polypeptide sequences) by mutagenesis to obtain at least one point mutation, addition, deletion, and / or chimerization from one or more ancestral or parental generation templates; 2) screening the progeny molecules, preferably using high-throughput methods, for at least one property of interest (e.g., an improvement in enzymatic activity, or increased stability, or a novel chemotherapeutic effect); 3) optionally obtaining and / or cataloging structural and / or functional information about the parental and / or progeny generation molecules; and 4) optionally repeating any of steps 1)-3).

[0163] In site-saturation mutagenesis, progeny generations of polynucleotides generated (e.g., from a parent polynucleotide template), referred to as "codon site saturation mutagenesis," each have at least one set of three or more adjacent point mutations (i.e., different bases comprising the new codon) such that all codons (or the entire family of degenerate codons encoding the same amino acid) are represented at each codon position. Corresponding to and encoded by these progeny generations of polynucleotides are a series of progeny polypeptides generated, each with at least one single amino acid point mutation. In a preferred embodiment, an example of the progeny generated, referred to as "amino acid site saturation mutagenesis," is a mutant polypeptide in each of the 19 naturally encoded polypeptides, forming alpha amino acid substitutions at each and every amino acid position along the polypeptide. This yields a total of 20 different progeny polypeptides containing the original amino acid—at each and every amino acid position along the parent polypeptide—or potentially 21 or more different progeny polypeptides if additional amino acids are used either in place of or in addition to the 20 naturally encoded amino acids.

[0164] Other mutagenesis techniques may be used, including recombination, and more specifically, methods for preparing polynucleotides encoding polypeptides by in vivo reassortment of polynucleotide sequences containing regions of partial homology, methods for assembling polynucleotides to form at least one polynucleotide, and methods for screening polynucleotides for production of polypeptides with useful properties.

[0165] In other embodiments, mutagenesis techniques take advantage of the natural ability of cells to recombine molecules and / or to deliver reduced methods of reducing the complexity of sequences and stretches of repetitive or contiguous sequences with homologous regions.

[0166] Various mutagenesis techniques may be used alone or in combination to provide a method for generating hybrid polypeptides that encode biologically active hybrid polypeptides with enhanced activity. In achieving these and other objectives, in accordance with one aspect of the present disclosure, a method is provided for introducing a polypeptide into a suitable host cell and growing the host cell under conditions for producing a hybrid polynucleotide.

[0167] Chimeric genes are created by joining two polynucleotide fragments using compatible sticky ends generated by restriction enzymes, where each fragment is derived from a different ancestral (parent) molecule. Another example is the mutagenesis of a single codon position (i.e., to obtain a codon substitution, addition, or deletion) in a parent polynucleotide to generate a single progeny polynucleotide that encodes for a single-site mutagenized polypeptide.

[0168] Additionally, in vivo site-specific recombination systems have been utilized to generate genetic crossovers and recombinations between homologous but truncated genes on a plasmid, as well as random methods of in vivo recombination. Mutagenesis has also been reported by overlap extension and PCR.

[0169] Non-random methods are used to obtain a larger number of point mutations and / or chimerizations, for example, comprehensive or global methods are used to generate all molecular species in a particular mutation grouping in a template molecule to functionally belong to a specific structural grouping (e.g., a specific single amino acid position or sequence of two or more amino acid positions) and to classify and compare specific groupings of mutations.

[0170] These, or any other developed method, in the present disclosure, generates a population (library) of novel molecules from one or more parent molecules.

[0171] Once formed, the constructs may or may not be sized to be fractionated on agarose gels according to published protocols, inserted into cloning vectors, and transfected into suitable host cells.

[0172] Expression of developmental molecules Once the library of mutant molecules is generated, the DNA can be expressed using conventional molecular biology techniques, and protein expression can thus be controlled using a variety of well-known methods.

[0173] For example, wild-type genes can be developed using any of a variety of random or non-random methods, as described herein. The mutant DNA molecules are then digested and ligated into vector DNA, such as plasmid DNA, using standard molecular biology techniques. The vector DNA containing each mutant is transformed into bacteria or other cells using standard protocols. This can be done in individual wells of a multi-well tray, such as a 96-well tray, for high-throughput expression and screening. This process is repeated for each mutant molecule.

[0174] The selected and isolated polynucleotide is then introduced into a suitable host cell. Suitable host cells are any cells capable of promoting genetic recombination and / or reductive reassortment. The selected polynucleotide is preferably already in a vector containing appropriate regulatory sequences. The host cell may be a higher eukaryotic cell, such as a mammalian cell, or a lower eukaryotic cell, such as a yeast cell, or preferably a prokaryotic cell, such as a bacterial cell. Introduction of the construct into the host cell can be accomplished by calcium phosphate transfection, DEAE-dextran-mediated transfection, or electroporation (e.g., Ecker and Davis, 1986, Inhibition of gene expression in plant cells by expression of antisense RNA, Proc Natl Acad Sci USA, 83:5372-5376).

[0175] Representative examples of usable expression vectors include virus particles, baculoviruses, phages, plasmids, phagemids, cosmids, fosmids, bacterial artificial chromosomes, artificial chromosomes based on viral DNA (e.g., vaccinia, adenovirus, foul pox virus, pseudorabies, and SV40 derivatives), yeast plasmids, yeast artificial chromosomes, and any other vector specific to a particular target host (e.g., Bacillus, Aspergillus, and yeast). Thus, for example, DNA can be included in any one of a variety of expression vectors for expressing a polypeptide. Such vectors include chromosomal, non-chromosomal, and synthetic DNA sequences. Many suitable vectors are well known to those skilled in the art and are commercially available. The following vectors are exemplary: Bacterial: pQE vector (Qiagen), pBluescript plasmid, pNH vector, lambda ZAP vector (Stratagene); ptrc99a, ρKK223-3, pDR540, pRIT2T (Pharmacia); eukaryotic: pXT1, ρSG5 (Stratagene), pSVK3, pBPV, pMSG, pSVLSV40 (Pharmacia). However, any other plasmid or vector can be used as long as it is replicable and viable in the host. Low copy number or high copy number vectors can be used in the present invention.

[0176] The DNA sequence in the expression vector is operably linked to an appropriate expression control sequence (promoter) to direct RNA synthesis. Specific named bacterial promoters include lad, lacZ, T3, T7, gpt, lambda PR, PL, and trp. Eukaryotic promoters include immediate-early CMV, HSV thymidine kinase, early and late SV40, LTRs from retrovirus, and mouse metallothionein-1. Selection of an appropriate vector and promoter is well within the level of ordinary skill in the art. The expression vector also contains a ribosome binding site for translation initiation and a transcription terminator. The vector may also contain appropriate sequences for amplifying expression. Promoter regions can be selected from any desired gene using chloramphenicol transferase (CAT) vectors or other vectors with selectable markers. In addition, the expression vector preferably contains one or more selectable marker genes to provide a phenotypic trait for selection of transformed host cells, such as dihydrofolate reductase or neomycin resistance for eukaryotic cell culture, or tetracycline or ampicillin resistance in E. coli.

[0177] Thus, in another embodiment of the present invention, novel polynucleotides can be generated by the method of reductive reassortment. The method involves the generation of constructs containing consecutive sequences (original coding sequences), their insertion into a suitable vector, and their subsequent introduction into a suitable host cell. Reassortment of individual molecular identities occurs by combinatorial methods between consecutive sequences in the construct with homologous regions or between quasi-repeated units. The reassortment method recombines and / or reduces the complexity and extent of repeated sequences, resulting in the generation of new molecular species. Various treatments can be applied to enhance the rate of reassortment. These can include UV treatment or DNA-damaging chemicals, and / or the use of host cell lines that exhibit enhanced levels of "genetic instability." Thus, reassortment methods can utilize the natural properties of homologous recombination or quasi-repeated sequences to guide their own evolution.

[0178] In one embodiment, the host organism or cell comprises a Gram-negative bacterium, a Gram-positive bacterium, or a eukaryote. In another embodiment of the invention, the Gram-negative bacterium comprises Escherichia coli or Pseudomonas fluorescens. In another embodiment of the invention, the Gram-positive bacterium comprises Streptomyces diversa, Lactobacillus gasseri, Lactococcus lactis, Lactococcus cremoris, or Bacillus subtilis. In another embodiment of the invention, the eukaryote comprises Saccharomyces cerevisiae, Schizosaccharomyces pombe, Pichia pastoris, Kluyveromyces lactis, Hansenula plymorpha, or Aspergillus niger. Representative examples of suitable hosts include bacterial cells such as Escherichia coli, Streptomyces, Salmonella typhimurium, etc.; fungal cells such as yeast; insect cells such as Drosophila S2 and Spodoptera Sf9; animal cells such as CHO, COS, or Bowes melanoma; adenovirus; and plant cells. The selection of an appropriate host is considered to be within the scope of those skilled in the art from the teachings herein.

[0179] With particular reference to the various mammalian cell culture systems that can be used to express recombinant proteins, examples of mammalian expression systems include the COS-7 line of monkey kidney fibroblasts, described in "SV40-transformed simian cells support the replication of early SV40 mutants" (Gluzman, 1981), and other cell lines capable of expressing compatible vectors, such as the C127, 3T3, CHO, HeLa, and BHK cell lines. Mammalian expression vectors will also contain an origin of replication, a suitable promoter and enhancer, and any necessary ribosome binding sites, polyadenylation sites, splice donor and acceptor sites, transcription termination sequences, and 5' flanking nontranscribed sequences. DNA sequences derived from SV40 splicing and polyadenylation sites can be used to provide the necessary nontranscribed genetic elements.

[0180] The cells are then propagated, and "reduced reassortment" is achieved. The rate of the reductive reassortment process can be stimulated by the introduction of DNA damage, if desired. In vivo reassortment is directed by "intermolecular" processes collectively referred to as "genetic recombination," which in bacteria is commonly observed as a "RecA-dependent" phenomenon. The present invention can rely on the host cell's ability to regulate the genetic recombination process to recombine and reassort sequences, or the reduction process to reduce the complexity of quasi-repeated sequences in the cell by deletion. This process of "reduced reassortment" occurs by an "intramolecular," RecA-independent process. The end result is the reassortment of molecules into all possible combinations.

[0181] Host cells containing the polynucleotide of interest can be cultured in conventional nutrient media modified as appropriate for activating promoters, selecting transformants, or amplifying genes. The culture conditions (e.g., temperature, pH, etc.) will be those previously used with the host cell selected for expression and will be apparent to those skilled in the art.

[0182] Protein expression can be induced by a variety of well-known methods, and many genetic systems have been published for inducing protein expression. For example, for a suitable system, protein expression is induced by the addition of an inducer. The cells are then pelleted by centrifugation, and the supernatant is removed. Periplasmic proteins can be enriched by incubating the cells with DNAse, RNAse, and lysozyme. After centrifugation, the supernatant containing the novel protein is transferred to a new multiwell tray and stored prior to assay.

[0183] Cells are typically harvested by centrifugation, separated by physical or chemical means, and the resulting crude extract is retained for further purification. Microbial cells used for protein expression can be separated by any convenient method, including freeze-thaw cycling, sonication, mechanical separation, or the use of cell lysing agents. Such methods are well known to those of skill in the art. The expressed polypeptide or fragment thereof can be recovered and purified from recombinant cell culture by methods including ammonium sulfate or ethanol precipitation, acid extraction, anion or cation exchange chromatography, phosphocellulose chromatography, hydrophobic interaction chromatography, affinity chromatography, hydroxylapatite chromatography, and lectin chromatography. Protein refolding processes can be used, if necessary, to refine the structure of the polypeptide. Optionally, high-performance liquid chromatography (HPLC) can be used for final purification.

[0184] Clones identified as having the desired activity can then be sequenced to identify the polynucleotide sequence encoding the enzyme with the enhanced activity.

[0185] Polypeptides identified from such libraries can be used for therapeutic, diagnostic, research, and related purposes, and / or can be subjected to one or more additional cycles of shuffling and / or selection. The present invention provides fragments of conditionally active biological proteins that are at least 10 amino acids in length, wherein the fragments have activity.

[0186] The present invention provides codon-optimized polypeptides or fragments thereof having enzymatic activity, wherein the codon usage is optimized for a particular organism or cell. Narum et al., "Codon optimization of gene fragments encoding Plasmodium falciparum merzoite proteins enhances DNA vaccine protein expression and immunogenicity in mice", Infect.Immun. 2001 December; 69(12): 7250-3, describes codon optimization in a mouse system. Outchkourov et al., "Optimization of the expression of Equistatin in Pichia pastoris, protein expression and purification", Protein Expr. Purif. 2002 February; 24(1): 18-24, describes codon optimization in a yeast system. Feng et al., "High-level expression and mutagenesis of recombinant human phosphatidylcholine transfer protein using a synthetic gene: evidence for a C-terminal membrane binding domain," Biochemistry 2000 Dec. 19, 39(50): 15399-409, describes codon optimization in E. coli.Humphreys et al., "High-level periplasmic expression in Escherichia coli using a eukaryotic signal peptide: importance of codon usage at the 5' end of the coding sequence," Protein Expr. Purif. 2000 Nov. 20(2): 252-64, describes how codon usage affects secretion in E. coli.

[0187] The development of conditionally active biological proteins can be aided by the use of convenient high-throughput screening or selection processes.

[0188] Once identified, the polypeptides and peptides of the present invention can be synthetic or recombinantly produced polypeptides. Peptides and proteins can be expressed in vitro or in vivo by recombinant techniques. The peptides and polypeptides of the present invention can be made and isolated using any method known in the art. The polypeptides and peptides of the present invention can also be synthesized, in whole or in part, using chemical methods known in the art. For example, Caruthers (1980) "New chemical methods for synthesizing polynucleotides", Nucleic Acids Res. Symp. Ser. 215-223; Horn (1980), "Synthesis of oligonucleotides on cellulose. Part II: design and synthetic strategy to the synthesis of 22 oligodeoxynucleotides coding for Gastric Inhibitory Polypeptide (GIP)1", Nucleic Acids Res. Symp. Ser. 225-232; see Banga, AK, Therapeutic Peptides and Proteins, Formulation, Processing and Delivery Systems (1995) Technomic Publishing Co., Lancaster, Pa.For example, peptide synthesis can be carried out using various solid-phase methods (e.g., Roberge (1995) "A strategy for a convergent synthesis of N-linked glycopeptides on a solid support," Science 269:202; Merrifield (1997) "Concept and early development of solid-phase peptide synthesis," Methods Enzymol. 289:3-13), and automated synthesis can be accomplished, for example, by using an ABI 43 IA Peptide Synthesizer (Perkin Elmer) according to instructions provided by the manufacturer.

[0189] The peptides and polypeptides of the invention can also be glycosylated. Glycosylation can be added chemically or post-translationally by cellular biosynthetic machinery, the latter incorporating the use of known glycosylation motifs, which can be inherent in the sequence or can be added as a peptide or added to the nucleic acid coding sequence. Glycosylation can be O-linked or N-linked.

[0190] The peptides and polypeptides of the present invention, as described above, include all "mimetic" and "peptidomimetic" forms. The terms "mimetic" and "peptidomimetic" refer to synthetic chemical compounds having substantially the same structural and / or functional characteristics as the polypeptides of the present invention. Mimetics can be entirely composed of synthetic, non-natural amino acid analogs, or can be chimeric molecules of partly natural peptide amino acids and partly non-natural amino acid analogs. Mimetics can also incorporate any number of conservative substitutions of natural amino acids, as long as the mimetic's structure and / or activity are not substantially altered. As with polypeptides of the present invention that are conservative variations, routine testing will determine whether a mimetic is within the scope of the present invention, i.e., whether its structure and / or function are not substantially altered.

[0191] The polypeptide mimetic compositions of the present invention can include any combination of non-natural structural components. In other embodiments, the mimetic compositions of the present invention include one or all of the following three structural groups: a) residue linkage groups other than the natural amide bond ("peptide bond"); b) non-natural linkages in place of naturally occurring amino acid residues; or c) residues that induce secondary structure mimicry, i.e., induce or stabilize secondary structure (e.g., β-turn, γ-turn, β-sheet, α-helical conformation, etc.). For example, a polypeptide of the present invention can be characterized as a mimetic when all or some of its residues are joined by chemical means other than natural peptide bonds. Individual peptidomimetic residues can be joined by peptide bonds, other chemical bonds, or coupling means (e.g., glutaraldehyde, N-hydroxysuccinimide ester, bifunctional maleimide, N,N'-dicyclohexylcarbodiimide (DCC), N,N'-diisopropylcarbodiimide (DIC), etc.). Linking groups that can be alternatives to the traditional amide bond ("peptide bond") linkage include, for example, ketomethylene (e.g., --C(.dbd.O)--CH.sub.2--for--C(.dbd.O)--NH--), aminomethylene (CH.sub.2--NH), ethylene, olefin (CH.dbd.CH), ether (CH.sub.2--O), thioether (CH.sub.2--S), tetrazole (CN.sub.4--), thiazole, retroamide, thioamide, or ester (see, e.g., Spatola (1983) in Chemistry and Biochemistry of Amino Acids, Peptides and Proteins, Vol. 7, pp 267-357, "Peptide Backbone Modifications," Marcell Dekker, NY).

[0192] The polypeptides of the present invention can also be characterized as mimetics by containing all or some non-naturally occurring residues in place of naturally occurring amino acid residues. Non-naturally occurring residues are well described in the scientific and patent literature. The following provides a few examples and guidelines of non-naturally occurring compositions useful as mimetics of natural amino acid residues. Mimetics of aromatic amino acids include, for example, D- or L-naphthylalanine; D- or L-phenylglycine; DL-2 thienylalanine; D- or L-1, -2, 3-, or 4-pyrenylalanine; D- or L-3 thienylalanine; D- or L-(2-pyridinyl)-alanine; D- or L-(3-pyridinyl)-alanine; D- or L-(2-pyrazinyl)-alanine; D- or L-(4-isopropyl)-phenylglycine; D-(trifluoromethyl)-phenylglycine; D-(trifluoromethyl)-alan ... D- or L-2-indole(alkyl)alanine; Dp-fluoro-phenylalanine; D- or Lp-biphenylphenylalanine; D- or Lp-methoxy-biphenylphenylalanine; D- or L-2-indole(alkyl)alanine; and D- or L-alkylamines, where the alkyl can be unsubstituted methyl, ethyl, propyl, hexyl, butyl, pentyl, isopropyl, isobutyl, sec-isotyl, isopentyl, or a non-acidic amino acid. Aromatic rings of unnatural amino acids include, for example, thiazolyl, thiophenyl, pyrazolyl, benzimidazolyl, naphthyl, furanyl, pyrrolyl, and pyridyl aromatic rings.

[0193] Mimetics of acidic amino acids can be generated by substituting, for example, non-carboxylate amino acids while maintaining the negative charge; (phosphono)alanine; sulfated threonine. Carboxyl side groups (e.g., aspartyl or glutamyl) can also be selectively modified by reaction with carbodiimides (R'--N--C--N--R'), such as 1-cyclohexyl-3(2-morpholinyl-(4-ethyl)carbodiimide or 1-ethyl-3(4-azonia-4,4-dimethylpentyl)carbodiimide. Aspartyl or glutamyl can also be converted to asparaginyl and glutaminyl residues by reaction with ammonium ions. Mimetics of basic amino acids can be generated, for example, by substituting the amino acids ornithine, citrulline, or (guanidino)-acetic acid (in addition to lysine and arginine) or (guanidino)alkyl-acetic acid, where alkyl is as defined above. Nitrile derivatives (e.g., containing a CN moiety instead of COOH) can be substituted for asparagine or glutamine. Asparaginyl and glutaminyl residues can be It can be deaminated to the corresponding aspartyl or glutamyl residue. Arginine residue mimics can be generated by reacting arginyl with, for example, one or more conventional reagents, including, for example, phenylglyoxal, 2,3-butanedione, 1,2-cyclohexanedione, or ninhydrin, preferably under alkaline conditions. Tyrosine residue mimics can be generated by reacting tyrosyl with, for example, aromatic diazonium compounds or tetranitromethane. N-acetylimidazole and tetranitromethane can be used to form O-acetyltyrosyl species and 3-nitro derivatives, respectively. Cysteine ​​residue mimics can be generated by reacting cysteinyl residues with, for example, α-haloacetates, such as 2-chloroacetic acid or acetamide chloride, and the corresponding amine to give carboxymethyl or carboxyamidomethyl derivatives.Cysteine ​​residue mimetics can be generated by reacting cysteinyl residues with, for example, bromotrifluoroacetone, α-bromo-β-(5-imidazolyl)propionic acid; acetyl phosphate chloride, N-alkylmaleimide, 3-nitro-2-pyridyl disulfide; methyl 2-pyridyl disulfide; p-chloromercuric benzoate; 2-chloromercuri-4-nitrophenol; or chloro-7-nitrobenzo-oxa-1,3-diazole. Lysine mimetics can be generated (and the amino-terminal residue can be modified) by reacting lysinyl with, for example, succinic acid or other carboxylic acid anhydrides. Lysine and other α-amino-containing residue mimetics can be generated by reaction with imidoesters such as methylpicolinamide, pyridoxal phosphate, pyridoxal, chloroborohydride, trinitrobenzenesulfonic acid, O-methylisourea, 2,4-pentanedione, and transamidase-catalyzed reactions with glyoxylic acid. Methionine mimetics can be generated, for example, by reaction with methionine sulfoxide. Proline mimetics include, for example, pipecolic acid, thiazolidine carboxylic acid, 3- or 4-hydroxyproline, dehydroproline, 3- or 4-methylproline, or 3,3-dimethylproline. Histidine residue mimetics can be generated by reacting histidyl with, for example, diethylprocarbonate or para-bromophenacyl bromide. Other mimetics include those generated, for example, by hydroxylation of proline and lysine; phosphorylation of the hydroxyl group of seryl or threonyl residues; methylation of the α-amino group of lysine, arginine, and histidine; acetylation of N-terminal amines; methylation of backbone amide residues or substitution with N-methyl amino acids; or amidation of the C-terminal carboxyl group.

[0194] Residues of the polypeptides (e.g., amino acids) of the invention can also be replaced by amino acids (or peptidomimetic residues) of the opposite chirality. Thus, any amino acid that naturally occurs in the L-configuration (also called R or S, depending on the chemical structure) can be replaced by an amino acid of the same chemical structure type or peptidomimetic, but of the opposite chirality, also called a D-amino acid, also called the R- or S-form.

[0195] The present invention also provides methods for modifying the polypeptides of the present invention by natural processes such as post-translational processing (e.g., phosphorylation, acylation, etc.) or by chemical modification techniques. Modifications can occur anywhere in the polypeptide, including the peptide backbone, the amino acid side-chains, and the amino or carboxyl termini. It will be appreciated that the same type of modification can be present in the same or varying degrees at several sites in a given polypeptide. A given polypeptide can contain many different types of modifications. Modifications include acetylation, acylation, pegylation, ADP-ribosylation, amidation, covalent attachment of flavin, covalent attachment of a heme moiety, covalent attachment of a nucleotide or nucleotide derivative, covalent attachment of a lipid or lipid derivative, covalent attachment of phosphatidylinositol, cyclization cross-linking, disulfide bond formation, demethylation, formation of covalent cross-links, formation of cysteine, formation of pyroglutamate, formylation, gamma-carboxylation, glycosylation, GPI anchor formation, hydroxylation, iodination, methylation, myristoylation, oxidation, pegylation, proteolytic processing, phosphorylation, prenylation, racemization, selenoylation, sulfation, and tRNA-mediated addition of amino acids to proteins such as arginylation. See Creighton, TE, Proteins-Structure and Molecular Properties 2nd Ed., WH Freeman and Company, New York (1993); Posttranslational Covalent Modification of Proteins, BC Johnson, Ed., Academic Press, New York, pp. 1-12 (1983).

[0196] Solid-phase chemical peptide synthesis methods can also be used to synthesize the polypeptides or fragments of the present invention. Such methods have been well known in the art since the early 1960s (Merrifield, RB, "Solid-phase synthesis. I. The synthesis of a tetrapeptide", J. Am. Chem. Soc, 85:2149-2154, 1963) (see also Stewart, JM and Young, JD, Solid Phase Peptide Synthesis, 2nd Ed., Pierce Chemical Co., Rockford, 111., pp. 11-12) and have recently been used in commercially available laboratory peptide design and synthesis kits (Cambridge Research Biochemicals). Such commercially available laboratory kits typically use the teachings of H.M. Geysen et al., "Use of peptide synthesis to probe viral antigens for epitopes to a resolution of a single amino acid," Proc. Natl. Acad. Sci., USA, 81:3998 (1984), and provide for the synthesis of peptides on the tips of numerous "rods" or "pins," all of which are connected to a single plate. When such a system is used, the plate of rods or pins is inverted and inserted into corresponding wells or reservoirs in a second plate, which contains a solution for attaching or immobilizing the appropriate amino acids to the tips of the pins or rods. By repeating this process step, i.e., by inverting and inserting the tips of the rods and pins into the appropriate solutions, amino acids are incorporated into the desired peptide. Additionally, many available FMOC peptide synthesis systems are available. For example, assembly of polypeptides or fragments can be performed on a solid support using an Applied Biosystems, Inc. 431A® Automated Peptide Synthesizer.Such equipment provides ready access to the peptides of the invention, either by direct synthesis or by synthesis of a series of fragments that can be coupled using other well-known techniques.

[0197] Synthetic polypeptides or fragments thereof can be recovered and purified by well-known methods, including ammonium sulfate or ethanol precipitation, acid extraction, anion or cation exchange chromatography, phosphocellulose chromatography, hydrophobic interaction chromatography, affinity chromatography, hydroxylapatite chromatography, and lectin chromatography. Protein refolding procedures can be used, if necessary, to complete the structure of the polypeptide. If necessary, high-performance liquid chromatography (HPLC) can be used for final purification.

[0198] The present invention provides conditionally active protein variant formulations or dosage forms comprising at least one protein variant, the formulations being liquid or dry. Protein formulations optionally include a buffer, cofactor, secondary or additional protein, or one or more excipients. In one embodiment, the formulations are utilized as therapeutic conditionally active biological proteins that are active under abnormal or non-physiological conditions, e.g., with respect to temperature, pH, or osmolality, oxidation, or osmolality, and are less active or inactive under normal or physiological conditions.

[0199] Standard purification techniques can be used for any recombinant or synthetic conditionally active biological protein.

[0200] Mutation screening to identify reversible or irreversible mutations Identification of desirable molecules is accomplished most directly by measuring protein activity under permissive and wild-type conditions. Mutants exhibiting the highest activity ratio (permissive / wild-type) are then selected, and point mutation permutations can be generated by combining individual mutations using standard methods. This combinatorial permutation protein library is then screened for proteins exhibiting the greatest difference between permissive and wild-type activity.

[0201] The activity of the supernatant can be screened using various methods, e.g., high-throughput activity assays such as fluorescent assays, to identify protein variants sensitive to desired properties (e.g., temperature, pH, etc.). For example, to screen for time-sensitive variants, enzyme or antibody activity is measured for each variant using commercially available substrates at a low temperature (e.g., 25°C) and at a temperature where the original protein is functional (e.g., 37°C). Reactions may be initially performed in a multiwell assay format, such as a 96-well assay, and then confirmed using a different format, such as a 14 ml tube format.

[0202] The present disclosure further provides a screening assay for identifying enzymes, the assay comprising the steps of: (a) providing a plurality of nucleic acids or polypeptides; (b) obtaining candidate polypeptides from the plurality of nucleic acids or polypeptides to test for enzymatic activity; (c) testing the candidate polypeptides for enzymatic activity; and (d) identifying candidate polypeptides that exhibit higher enzymatic activity than the wild-type enzyme protein under abnormal or non-physiological conditions, e.g., temperature, pH, oxidation, osmolarity, electrolyte concentration, or osmolality, and exhibit lower enzymatic activity under normal physiological conditions.

[0203] In one embodiment, the method further comprises modifying at least one of the nucleic acids or polypeptides prior to testing the candidate conditional bioactivity. In another embodiment, the testing step (c) further comprises testing for increased expression of the polypeptide in the host cell or host organism. In a further embodiment, the testing step (c) further comprises testing for enzyme activity within a pH range of about pH 3 to about pH 12. In a further embodiment, the testing step (c) further comprises testing for enzyme activity within a pH range of about pH 5 to about pH 10. In a further embodiment, the testing step (c) further comprises testing for enzyme activity within a pH range of about pH 6 to about pH 8. In a further embodiment, the testing step (c) further comprises testing for enzyme activity within a pH range of about pH 6.7 to about pH 7.5. In another embodiment, the testing step (c) further comprises testing for enzyme activity within a temperature range of about 4°C to about 55°C. In another embodiment, the testing step (c) further comprises testing the enzyme activity within a temperature range of about 15°C to about 47°C. In another embodiment, the testing step (c) further comprises testing the enzyme activity within a temperature range of about 20°C to about 40°C. In another embodiment, the testing step (c) further comprises testing the enzyme activity within a temperature range of about 25°C to about 37°C. In another embodiment, the testing step (c) further comprises testing the enzyme activity under normal osmotic pressure and under abnormal (positive or negative) osmotic pressure. In another embodiment, the testing step (c) further comprises testing the enzyme activity under normal electrolyte concentrations and under abnormal (positive or negative) electrolyte concentrations. The electrolyte concentrations tested are selected from the group consisting of calcium, sodium, potassium, magnesium, chloride, bicarbonate, and phosphate.In another embodiment, the testing step (c) further comprises testing for an enzymatic activity that results in a stable reaction product.

[0204] In another aspect, the present disclosure provides purified antibodies that specifically bind to a polypeptide of the present disclosure or a fragment thereof that has enzymatic activity. In one aspect, the present disclosure provides fragments of the antibodies that specifically bind to a polypeptide with enzymatic activity.

[0205] Antibodies and antibody-based screening methods The present disclosure provides isolated or recombinant antibodies that specifically bind to the enzymes of the present disclosure. These antibodies can be used to isolate, identify, or quantify the enzymes of the present disclosure, or related polypeptides. These antibodies can be used to isolate other polypeptides within the scope of the present disclosure, or other related enzymes. The antibodies can be designed to bind to the active site of the enzyme. Accordingly, the present disclosure provides methods for inhibiting enzymes using the antibodies of the present disclosure.

[0206] The antibodies can be used in immunoprecipitation, staining, immunoaffinity columns, and the like. If desired, nucleic acid sequences encoding specific antigens can be generated by immunization, followed by isolation, amplification, or cloning of the polypeptide or nucleic acid, and immobilization of the polypeptide on the arrays disclosed herein. Alternatively, the methods disclosed herein can be used to modify the structure of antibodies produced by cells, altering the antibody to, for example, increase or decrease the affinity of the antibody. Furthermore, the ability to generate or modify antibodies can be a phenotype engineered into cells using the methods disclosed herein. Methods for immunization, antibody generation, and isolation (both polyclonal and monoclonal) are known to those skilled in the art and are described in the scientific and patent literature. See, e.g., Coligan, CURRENT PROTOCOLS IN IMMUNOLOGY, Wiley / Greene, NY (1991); Stites (eds.), BASIC AND CLINICAL IMMUNOLOGY (7th ed.), Lange Medical Publications, Los Altos, Calif. ("Stites"); Goding, MONOCLONAL ANTIBODIES: PRINCIPLES AND PRACTICE (2nd ed.), Academic Press, New York, NY (1986); Kohler (1975), "Continuous cultures of fused cells secreting antibody of predefined specificity," Nature 256:495; Harlow (1988), ANTIBODIES, A LABORATORY MANUAL, Cold Spring Harbor Publications, New York. Antibodies can also be produced in vitro, for example, by using phage display libraries expressing recombinant antibody binding sites, in addition to traditional in vivo methods using animals.For example, Hoogenboom (1997) "Designing and optimizing library selection strategies for generating high-affinity antibodies", Trends Biotechnol. 15:62-70; and Katz (1997) "Structural and mechanistic determinants of affinity and specificity of ligands discovered or engineered by phage" See "Display", Annu.Rev.Biophys.Biomol.Struct.26:27-45.

[0207] Polypeptides or peptides can be used to generate antibodies that specifically bind to a polypeptide, e.g., an enzyme, of the present disclosure. The resulting antibodies can be used in immunoaffinity chromatography procedures to isolate or purify the polypeptide or to determine whether the polypeptide is present in a biological sample. In such procedures, a protein preparation, such as an extract, or a biological sample is contacted with an antibody that can specifically bind to one of the polypeptides of the present disclosure.

[0208] In immunoaffinity techniques, the antibody is bound to a solid support, such as beads or other column substrate. The protein preparation is placed in contact with the antibody under conditions in which the antibody specifically binds to one of the polypeptides of the present disclosure. After washing to remove nonspecifically bound proteins, the specifically bound polypeptide is eluted.

[0209] The ability of proteins in a biological sample to bind to an antibody can be determined using a variety of techniques known to those skilled in the art. For example, binding ability may be determined by labeling the antibody with a detectable label, such as a fluorescent substance, an enzyme label, or a radioisotope. Alternatively, the ability of the antibody to bind to the sample may be determined by detection using a secondary antibody bearing such a detectable label on its surface. Specific assays include ELISA assays, sandwich assays, radioimmunoassays, and Western blots.

[0210] Polyclonal antibodies raised against the polypeptides of the present disclosure can be obtained by directly injecting the polypeptides into animals or by administering the polypeptides to non-human animals. The antibodies thus obtained are then allowed to bind to the polypeptide itself. In this manner, even sequences encoding only fragments of the polypeptides can be used to generate antibodies that may bind to the native, full-length polypeptide. Such antibodies can then be used to isolate the polypeptides from cells expressing them.

[0211] To prepare monoclonal antibodies, any technique that provides antibodies produced by continuous cell lines can be used, including, for example, the hybridoma technique, the trioma technique, the human B cell hybridoma technique, and the EBV hybridoma technique (see, e.g., Cole (1985) in Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, Inc., pp. 77-96).

[0212] Techniques described for producing single chain antibodies (see, e.g., U.S. Pat. No. 4,946,778) can be adapted to produce single chain antibodies to the polypeptides of the present disclosure. Alternatively, transgenic mice can be used to express humanized antibodies to the polypeptides or fragments thereof. Antibodies generated against the polypeptides of the present disclosure can be used to screen for similar polypeptides (e.g., enzymes) from other organisms and samples. In such techniques, a polypeptide from the organism is contacted with the antibody and polypeptides that specifically bind to the antibody are detected. Any of the techniques described above can be used to detect antibody binding.

[0213] Screening methods and "online" monitoring devices In practicing the methods of the present disclosure, various devices and methods can be used in conjunction with the polypeptides and nucleic acids of the present disclosure, including, for example, devices and methods for screening peptides for enzymatic activity, for screening compounds for potential modulators, such as activators or inhibitors, of enzymatic activity, for antibodies that bind to the polypeptides of the present disclosure, for nucleic acids that hybridize to the nucleic acids of the present disclosure, and for screening cells that express the polypeptides of the present disclosure.

[0214] Arrays or "biochips" The nucleic acids or polypeptides of the present disclosure can be immobilized or applied to an array. Arrays can be used to screen or monitor libraries of compositions (e.g., small molecules, antibodies, nucleic acids, etc.) for their ability to bind to or modulate the activity of the nucleic acids or polypeptides of the present disclosure. For example, in one embodiment of the present disclosure, the monitored parameter is the transcriptional expression of an enzyme gene. One, several, or all of a cell's transcripts can be measured by hybridizing a sample containing the cell's transcripts or representative or complementary nucleic acids to nucleic acids immobilized on an array or "biochip." An "array" of nucleic acids on a microchip can be used to simultaneously quantify some or all of the transcripts. Alternatively, an array containing genomic nucleic acids can be used to genotype newly engineered strains generated by the methods of the present disclosure. Polypeptide "arrays" can also be used to simultaneously quantify multiple proteins. The present disclosure can be practiced with any known "array," which may also be considered a "microarray," "nucleic acid array," "polypeptide array," "antibody array," or "biochip," or variations thereof. Arrays generally comprise a plurality of "spots" or "target elements," each having a defined amount of one or more biomolecules, e.g., oligonucleotides, immobilized in defined regions on a substrate surface that specifically bind to sample molecules, e.g., mRNA transcripts.

[0215] In practicing the methods of the present disclosure, any known arrays and / or methods of making and using arrays may be used, in whole or in part, or in variations thereof, such as those described in the following documents:See, for example, U.S. Patent Nos. 6,277,628; 6,277,489; 6,261,776; 6,258,606; 6,054,270; 6,048,695; 6,045,996; 6,022,963; 6,013,440; 5,965,452; 5,959,098; 5,856,174; 5,830,645; 5,770,456; 5,632,957; 5,556 752; 5143854; 5807522; 5800992; 5744305; 5700637; 5556752; 5434049; see also, e.g., International Publication Nos. WO 99 / 51773; WO 99 / 09217; WO 97 / 46313; WO 96 / 17958; see, e.g., Johnston (1998) "Gene chips: Array of hope for understanding gene regulation",Curr.Biol.8:R171-R174;Schummer(1997)"Inexpensive Handheld Device for the Construction of High-Density Nucleic Acid Arrays",Biotechniques 23:1087-1092;Kern(1997)"Direct hybridization of large-insert genomic clones on high-density gridded cDNA filter arrays",Biotechniques 23:120-124;Solinas-Toldo(1997)"Matrix-Based Comparative Genomic Hybridization: Biochips to Screen for Genomic Imbalances",Genes,Chromosomes & Cancer 20:399-407;Bowtell(1999)"Options Available-From Start to Finish￣for Obtaining Expression Data by Microarray",Nature Genetics See also Supp. 21:25-32.See also U.S. Patent Application Publication No. 20010018642; U.S. Patent Application Publication No. 20010019827; U.S. Patent Application Publication No. 20010016322; U.S. Patent Application Publication No. 20010014449; U.S. Patent Application Publication No. 20010014448; U.S. Patent Application Publication No. 20010012537; U.S. Patent Application Publication No. 20010008765.

[0216] Capillary Array Capillary arrays, such as GIGAMATRIX™ (Diversa Corporation, San Diego, CA), can be used in the methods of the present disclosure. The nucleic acids or polypeptides of the present disclosure can be immobilized or applied to arrays, including capillary arrays. Arrays can be used to screen or monitor libraries of compositions (e.g., small molecules, antibodies, nucleic acids, etc.) for their ability to bind to or modulate the activity of the nucleic acids or polypeptides of the present disclosure. Capillary arrays provide another system for holding and screening samples. For example, a sample screening device can include a plurality of capillaries formed as an array of adjacent capillaries, each capillary having at least one wall forming a lumen for holding a sample. The device can further include an interstitial material disposed between adjacent capillaries in the array, with one or more reference indicia formed within the interstitial material. A capillary for screening a sample may be adapted to be coupled to a capillary array and may include a first wall defining a lumen for holding the sample and a second wall formed of a filter material for filtering excitation energy applied to the lumen to excite the sample. A polypeptide or nucleic acid, e.g., a ligand, may be introduced into a first component of at least some of the capillaries in the capillary array. Each capillary in the capillary array may have at least one wall defining a lumen for holding the first component. An air bubble may be introduced into the capillary behind the first component. A second component may be introduced into the capillary, where the second component is separated from the first component by the air bubble. A target sample is introduced into one capillary in the capillary array as a first liquid labeled with detectable particles, and each capillary in the capillary array has at least one wall defining a lumen for holding the first liquid and the detectable particles, the at least one wall being coated with a binding material for binding the detectable particles.The method further includes removing the first liquid from the capillary tube holding the bound detectable particles and introducing a second liquid into the capillary. The capillary array includes a plurality of individual capillaries having at least one outer wall forming a lumen. The outer wall may be one or more walls fused together. Similarly, the walls may form a cylindrical, rectangular, hexagonal, or other geometric shape lumen, so long as the wall forms a lumen that holds a liquid or sample. The capillaries in the capillary array can be adjacent to each other and support each other to form a planar structure. The capillaries can be connected to each other by fusing (e.g., here the capillaries are made of glass), gluing, bonding, or fixing adjacent capillaries. The capillary array can be formed from any number of individual capillaries, e.g., from 100 to 4,000,000. The capillary array can form a microtiter plate with approximately 100,000 or more individual capillaries connected to each other.

[0217] Pharmaceutical Composition The present disclosure provides at least one composition comprising (a) a conditionally active biological protein and (b) a suitable carrier or diluent. The present disclosure also provides at least one composition comprising (a) a conditionally active biological protein encoding a nucleic acid described herein and (b) a suitable carrier or diluent. The carrier or diluent can optionally be pharmaceutically acceptable, in accordance with known carriers or diluents. The composition can further optionally comprise at least one additional compound, protein, or composition.

[0218] The conditionally active biological protein may be in the form of a pharmaceutically acceptable salt, such as those commonly used in the pharmaceutical industry as salts of therapeutic proteins, including, for example, sodium, potassium, and calcium salts, amine salts such as procaine, dibenzylamine, ethylenediamine, ethanolamine, methylglucamine, and taurine, and acid addition salts such as hydrochlorides and basic amino acids.

[0219] The present disclosure further provides methods or compositions for administering a therapeutically effective amount of at least one conditionally active biological protein to modulate or treat a condition associated with at least one parent molecule in a cell, tissue, organ, animal, or patient, and / or before, after, or during the associated condition, as known in the art and / or as described herein. Accordingly, the present disclosure provides a method for diagnosing or treating a condition associated with a wild-type protein in a cell, tissue, organ, or animal, comprising contacting or administering to the cell, tissue, organ, or animal a composition having an effective amount of at least one conditionally active biological protein of the present disclosure. Optionally, the method may further comprise administering to the cell, tissue, organ, or animal an effective amount of 0.001 to 50 mg / kg of a conditionally active biological protein of the present disclosure. The method may optionally further comprise using at least one form of contacting or administering selected from parenteral, subcutaneous, intramuscular, intravenous, intra-articular, intrabronchial, intraperitoneal, intracapsular, intrachondral, intracavitary, intracelial, intracerebellar, intraventricular, intracolonic, intracervical, intragastric, intrahepatic, intramyocardial, intraosseous, intrapelvic, intrapericardial, intraperitoneal, intrapleural, intraprostatic, intrapulmonary, intrarectal, intrarenal, intraretinal, intraspinal, intrasynovial, intrathoracic, intrauterine, intravesical, bolus, vaginal, rectal, buccal, sublingual, intranasal, or transdermal.The method may optionally further comprise administering, prior to, concurrently with, or after the contacting or administration of the conditionally active biological protein, at least one composition comprising an effective amount of at least one compound or protein selected from at least one of a detectable label or reporter, a TNF antagonist, an antirheumatic agent, a muscle relaxant, an anesthetic, a nonsteroidal anti-inflammatory drug (NSAID), an analgesic, an anesthetic, a sedative, a local anesthetic, a neuromuscular blocker, an antibacterial agent, an antipsoriatic agent, a corticosteroid, an anabolic steroid, erythropoietin, an immunizing agent, an immunoglobulin, an immunosuppressant, a growth hormone, a hormone replacement drug, a radiopharmaceutical, an antidepressant, an antipsychotic, a stimulant, an asthma medication, a beta agonist, an inhaled steroid, epinephrine or its analogs, a cytotoxic agent or other anticancer agent, an antimetabolite such as methotrexate, a growth inhibitor, a cytokine, or a cytokine antagonist.

[0220] The present disclosure further provides methods of at least one conditionally active biological protein for diagnosing a condition associated with at least one wild-type protein in a cell, tissue, organ, animal, or patient, and / or before, after, or during the associated condition, as known in the art and / or as described herein.

[0221] Pharmaceutically acceptable carriers are determined in part by the particular composition being administered and by the particular method used to administer the composition. Accordingly, suitable formulations for the pharmaceutical compositions of the present invention are widely varied. A variety of liquid carriers may be used, such as buffered saline solutions. Such solutions are sterile and generally free of undesirable substances. Such solutions may be sterilized by conventional, known sterilization methods. The compositions may also contain pharmaceutically acceptable auxiliary substances, such as pH adjusting and buffering agents, tonicity adjusting agents, etc., required to approximate physiological conditions, e.g., sodium acetate, sodium chloride, potassium chloride, calcium chloride, sodium lactate, etc. The concentration of the conditionally active biological protein in such formulations may vary widely and will be selected primarily based on fluid volume, viscosity, body weight, etc., in accordance with the particular mode of administration chosen.

[0222] Formulations suitable for oral administration can consist of (a) liquid solutions, such as an effective amount of the administered nucleic acid, suspended in a diluent such as water, saline, or PEG 400; (b) capsules, sachets, or tablets, each containing a predetermined amount of the active ingredient as a liquid, solid, granule, or gelatin; (c) suspensions in a suitable liquid; and (d) suitable emulsions. Pharmaceutical compositions and formulations for oral administration of the present invention can be formulated using pharmaceutically acceptable carriers known in the art in appropriate dosage amounts. Such carriers allow the pharmaceutical to be formulated into a unit dosage form suitable for patient administration, such as tablets, pills, powders, dragees, capsules, liquids, lozenges, gels, syrups, slurries, suspensions, etc. Pharmaceutical formulations for oral administration are formulated with solid excipients, or, in some cases, to obtain tablet or dragee cores, by grinding the resulting mixture and adding suitable additional compounds, followed by processing the granule mixture. Suitable solid excipients are carbohydrate or protein fillers, including, for example, sugars such as lactose, sucrose, mannitol, or sorbitol; starches derived from corn, wheat, rice, potato, or other plants; celluloses such as methylcellulose, hydroxymethylcellulose, hydroxypropylmethylcellulose, or sodium carboxymethylcellulose; and gums including acacia and tragacanth; and proteins such as gelatin and collagen. Disintegrants or solubilizers may be added, such as cross-linked polyvinylpyrrolidone, agar, alginic acid, or a salt thereof, such as sodium alginate. Tablet forms may contain one or more of lactose, sucrose, mannitol, sorbitol, calcium phosphate, corn starch, potato starch, microcrystalline cellulose, gelatin, colloidal silicon dioxide, talc, magnesium stearate, stearic acid, other excipients, colorants, fillers, binders, diluents, buffers, wetting agents, preservatives, flavorings, dyes, disintegrants, and pharmaceutically acceptable carriers.

[0223] The present invention provides aqueous suspensions having a conditionally active biological protein in admixture with excipients suitable for the manufacture of an aqueous suspension. Such excipients can include suspending agents such as sodium carboxymethylcellulose, methylcellulose, hydroxypropylmethylcellulose, sodium alginate, polyvinylpyrrolidone, gum tragacanth, and gum arabic, and nucleating agents or wetting agents such as naturally occurring phosphatides (e.g., lecithin), condensation products of alkylene oxides with fatty acids (e.g., polyoxyethylene stearate), condensation products of ethylene oxide with long-chain aliphatic alcohols (e.g., heptadecaethyleneoxycetanol), condensation products of ethylene oxide with partial esters derived from fatty acids and hexitols (e.g., polyethylene sorbitol monooleate), or condensation products of ethylene oxide with partial esters derived from fatty acids and hexitol anhydrides (e.g., polyoxyethylene sorbitan monooleate). The said aqueous suspensions may also contain one or more preservatives, such as ethyl or n-propyl-p-hydroxybenzoate, one or more coloring agents, one or more flavoring agents, and one or more sweetening agents, such as sucrose, aspartame, or saccharin. The preparation may also be adjusted for osmotic pressure.

[0224] Lozenge dosage forms typically contain the active ingredient in a flavoring such as sucrose and acacia or tragacanth, while pastilles contain the active ingredient in an inert base, such as gelatin and glycerin or sucrose and acacia emulsion, gel, or the like, which may contain carriers known in the art in addition to the active ingredient. It is understood that the conditionally active biological protein must be protected from digestion when administered orally. This is generally achieved by complexing the conditionally active biological protein with a composition that confers resistance to acidic and enzymatic hydrolysis, or by packaging the conditionally active biological protein in a suitable resistant carrier, such as a liposome. Methods for protecting proteins from digestion are known in the art. The pharmaceutical composition can be encapsulated, for example, in liposomes or in formulations that allow for sustained release of the active ingredient.

[0225] The packaged conditionally active biological protein, alone or in combination with other suitable components, can be formulated into an aerosol formulation (e.g., one that can be inhaled as a nebulizer) and administered by inhalation. The aerosol formulation can be placed in a compressed acceptable propellant, such as dichlorofluoromethane, propane, or nitrogen. Formulations suitable for rectal administration include, for example, suppositories consisting of the packaged nucleic acid and a suppository base. Suitable suppositories include natural or synthetic triglycerides or paraffin hydrocarbons. In addition, gelatin rectal capsules consisting of a combination of the packaged nucleic acid and a base, such as liquid triglycerides, polyethylene glycols, and paraffin hydrocarbons, can also be used.

[0226] In addition to the conditionally active biological protein, the transdermal or topical delivery compositions of the present invention may contain a pharmaceutically acceptable carrier in the form of a cream, ointment, solution, or hydrogel, and may contain other compounds, so long as the added ingredients do not adversely affect delivery of the therapeutic protein. Conventional pharmaceutically acceptable emulsifiers, surfactants, suspending agents, antioxidants, osmolality-increasing agents, bulking agents, diluents, and preservatives may also be added. Water-soluble polymers may also be used as carriers.

[0227] Suitable formulations for parenteral administration, such as intra-articular (into a joint), intravenous, intramuscular, intradermal, intraperitoneal, and subcutaneous routes, include aqueous and non-aqueous isotonic sterile injection solutions, which may contain antioxidants, buffers, bacteriostats, and solutes that render the formulation isotonic with the intended recipient's blood, and aqueous and non-aqueous sterile suspension solutions, which may contain suspending agents, solubilizers, thickeners, stabilizers, and preservatives. In the practice of the present invention, compositions can be administered, for example, by intravenous infusion, orally, topically, intraperitoneally, or intrathecally. In one embodiment, parenteral dosage forms are a preferred method for administering compositions having conditionally active biological proteins. The compositions may conveniently be administered in unit dosage form and may be prepared by any method known in the pharmaceutical arts, for example, as described in Remington's Pharmaceutical Sciences, Mack Publishing Co., Easton Pa., 18th Ed., 1990. Preparations for intravenous administration may contain pharmaceutically acceptable carriers such as sterile water or saline, polyalkylene glycols such as polyethylene glycol, vegetable oils, hydrogenated naphthalenes, etc. See also U.S. Patent No. 4,318,905.

[0228] The formulation of a packaged composition having a conditionally active biological protein can be in unit-dose or multi-dose sealed containers, such as ampoules and vials. Injectable solutions and suspensions can be prepared from sterile powders, granules, and tablets of the kind previously described.

[0229] The present disclosure also provides compositions, devices and / or delivery methods of at least one conditionally active biological protein for diagnosing a condition associated with at least one wild-type protein in accordance with the present disclosure.

[0230] Also provided are compositions comprising at least one conditionally active biological protein and at least one pharmaceutically acceptable carrier or diluent, which can optionally further comprise an effective amount of at least one compound or protein selected from at least one of a detectable label or reporter, a cytotoxic or other anti-cancer agent, an antimetabolite such as methotrexate, an antiproliferative agent, a cytokine or cytokine antagonist, a TNF antagonist, an antirheumatic agent, a muscle relaxant, an anesthetic, a nonsteroidal anti-inflammatory drug (NSAID), an analgesic, an anesthetic, a sedative, a local anesthetic, a neuromuscular blocker, an antibacterial agent, an antipsoriatic agent, a corticosteroid, an anabolic steroid, erythropoietin, an immunizing agent, an immunoglobulin, an immunosuppressant, a growth hormone, a hormone replacement agent, a radiopharmaceutical, an antidepressant, an antipsychotic, a stimulant, an asthma medication, a beta agonist, an inhaled steroid, epinephrine, or the like.

[0231] Also provided is a medical device comprising at least one conditionally active biological protein of the present disclosure, wherein the device is suitable for contacting or administering the at least one conditionally active biological protein by at least one mode selected from parenteral, subcutaneous, intramuscular, intravenous, intra-articular, intrabronchial, intraperitoneal, intracapsular, intrachondral, intracavitary, intracavity, intracerebellar, intraventricular, intracolonic, intracervical, intragastric, intrahepatic, intramyocardial, intraosseous, intrapelvic, intrapericardial, intraperitoneal, intrapleural, intraprostatic, intrapulmonary, intrarectal, intrarenal, intraretinal, intraspinal, intrasynovial, intrathoracic, intrauterine, intracapsular, bolus, vaginal, rectal, buccal, sublingual, intranasal, or transdermal.

[0232] In a further aspect, the disclosure provides a kit comprising a first container having at least one conditionally active biological protein or fragment of the disclosure in lyophilized form, and optionally a second container having sterile water, sterile buffered water, or at least one preservative selected from the group consisting of phenol, m-cresol, p-cresol, o-cresol, chlorocresol, benzyl alcohol, phenylmercuric nitrate, phenoxyethanol, formaldehyde, chlorobutanol, magnesium chloride (e.g., hexahydrate), alkyl parabens (methyl, ethyl, propyl, butyl, etc.), benzalkonium chloride, benzethonium chloride, sodium dehydroacetate, and thimerosal, or mixtures thereof, in an aqueous diluent. In one aspect, the concentration of the conditionally active biological protein or specified portion or variant in the first container of the kit is reconstituted with the contents of the second container to a concentration of about 0.1 mg / ml to about 500 mg / ml. In another embodiment, the second container further comprises an isotonic agent. In another embodiment, the second container further comprises a physiologically acceptable buffer. In one embodiment, the present disclosure provides a method of treating a condition mediated by at least one wild-type protein, comprising administering to a patient in need thereof a formulation provided in the kit and reconstituting prior to administration.

[0233] Also provided is an article of manufacture for human pharmaceutical or diagnostic use, the article of manufacture comprising packaging material and a solution or lyophilized form of at least one conditionally active biological protein of the present disclosure. The article of manufacture can optionally comprise a container comprising a parenteral, subcutaneous, intramuscular, intravenous, intra-articular, intrabronchial, intraperitoneal, intracapsular, intrachondral, intracavitary, intracelial, intracerebellar, intraventricular, intracolonic, intracervical, intragastric, intrahepatic, intramyocardial, intraosseous, intrapelvic, intrapericardial, intraperitoneal, intrapleural, intraprostatic, intrapulmonary, intrarectal, intrarenal, intraretinal, intraspinal, intrasynovial, intrathoracic, intrauterine, intravesical, bolus, vaginal, rectal, buccal, sublingual, intranasal, or transdermal delivery device or system.

[0234] This disclosure further provides all disclosures set forth herein.

[0235] Example 1: Overview of a multi-wall assay (e.g., a 96-well assay) for temperature mutants Add fluorescent substrate to each well of the multiwall plate and place at both the wild-type temperature and the lower reaction temperature for the novel type (e.g., either 37°C or 25°C, as described above) for the appropriate time. Detect fluorescence by measuring fluorescence in the appropriate excitation and emission spectra (e.g., excitation spectrum of 320 nm, emission spectrum of 405 nm) using a fluorescent plate reader. Determine relative fluorescence units (RFU). Supernatant from the wild-type molecule and cells transformed with the plasmid / vector are used as positive and negative controls. Perform duplicate reactions for each sample, reaction temperature, positive control, and negative control.

[0236] Mutants that are active at lower temperatures (e.g., mutants active at 25°C) and have reduced activity at wild-type temperatures (e.g., 10%, 20%, 30%, 40% or more reduced activity at 37°C), i.e., an activity ratio of 1.1 or greater (e.g., an activity ratio (25°C / 37°C) at 25°C or 37°C of 1.1 or greater), can be considered putative temperature-sensitive primary hits. These temperature-sensitive primary hits can then be screened using the same assay to confirm all primary hits.

[0237] Example 2: Overview of different assay formats (e.g., 14 mL assay) for confirmatory testing of temperature mutants Mutants identified as temperature-sensitive primary hits are expressed in 14 mL culture tubes and their enzymatic activity is measured at the wild-type temperature (e.g., 37° C.) and at a lower temperature (e.g., 25° C.). Proteins are expressed and purified for use in a multiwall format as described above, although expression in a different format (14 ml tubes) that is not multiwall (96-well plates) is also performed separately.

[0238] The supernatant of each mutant is transferred to a multi-wall plate, such as a 96-well microplate. A fluorescent substrate is added to each tube and incubated at the designated temperature (wild-type temperature, lower temperature) for the appropriate time. The wild-type molecule is used as a positive control, and supernatant from cells transformed with the vector alone is used as a negative control. Fluorescence is detected by measuring fluorescence in the appropriate emission spectrum (e.g., excitation spectrum of 320 nm, emission spectrum of 405 nm) using a fluorescent plate reader. Relative fluorescence units (RFU) are determined. Duplicate reactions are performed for each sample, reaction temperature, positive control, and negative control.

[0239] Mutants that are active at low temperatures (e.g., 25°C) but show at least a 30% or greater reduction in activity at the wild-type temperature (e.g., 37°C), i.e., the ratio of activity at low temperatures (e.g., 25°C) to activity at wild-type temperatures (e.g., 37°C) is 1.1 or greater, are identified as temperature-sensitive hits.

[0240] The activity of the mutant at a lower temperature (e.g., 25°C) is compared to the activity of the wild-type molecule at the wild-type temperature (e.g., 37°C). If the mutant is more active than the wild-type molecule at a lower temperature (e.g., 25°C), as indicated by a residual activity of greater than 1, preferably greater than 2, and if the mutant exhibits reduced overall activity compared to the wild-type molecule at the wild-type temperature (37°C), the phenotype of the mutant can be confirmed as a temperature-sensitive mutant.

[0241] Example 3: Overview of further development of discovered hits If desired, new combinatorial mutant libraries can be generated from all or a selection of the mutant hits identified above. This new library can be designed to contain all possible amino acid variants for each selected mutant and rescreened as described for the new hits.

[0242] Example 4: Overview of reversibility of enzyme activity after temperature reduction The temperature-sensitive mutants developed can be further assayed to determine whether their enzyme activity at lower temperatures (e.g., 25°C) is reversible or irreversible by exposing the mutants to higher temperatures followed by a return to lower temperatures (e.g., 25°C). The temperature-sensitive mutants are expressed in the desired format, e.g., in 14 mL culture tubes as outlined above. The mutants are tested under several conditions, including the wild-type temperature (e.g., 37°C) and other temperatures, and then re-exposed to the required lower temperature (e.g., 25°C). Mutants active at lower temperatures that exhibit reduced activity when elevated to higher or wild-type temperatures (i.e., the ratio of activity at lower temperature to higher temperature is equal to or greater than 1, 1.5, 2, or higher) and that exhibit baseline activity when lowered back to lower temperatures are considered "reversible hits." A mutant that is active at a low temperature, that exhibits reduced activity when raised to a higher or wild-type temperature (i.e., the ratio of activity at the lower temperature to the higher temperature is equal to or greater than 1, 1.5, 2, or more), and that exhibits at least as much activity when lowered back to the lower temperature is determined to be an "irreversible hit."

[0243] Example 5: Materials and methods for screening for conditionally active angiostatin variants. Materials and methods for screening for conditionally active angiostatin variants can be adapted from Chi and Pizzo, "Angiosatin is directly cytotoxic to tumor cells at low extracellular pH: a mechanism dependent on cell surface-associated ATP synthase," Cancer Res. 2006;66(2):875-882, which is incorporated herein by reference.

[0244] Materials. Wild-type angiostatin kringles 1-3, derived from human plasminogen, are available from Calbiochem (Darmstadt, Germany) and can be reconstituted in sterile PBS. Polyclonal antibodies against the catalytic β subunit of ATP synthase can be generated, and the bovine ATP synthase F1 subunit can be purified, as previously described (Moser et al., "Angiostatin binds ATP synthase on the surface of human endothelial cells," Proc Natl Acad Sci USA 1999;96:2811-6; Moser et al., "Endothelial cell surface F1-FO ATP synthase is active in ATP synthesis and is inhibited by angiostatin," Proc Natl Acad Sci USA 2001;98:6656-61). Cariporide can be solubilized in sterile water and sterile filtered.

[0245] Cell Culture. A549 (a human epithelial cell line derived from lung cancer tissue) or other cancer cell lines (DU145, LNCaP, or PC-3 cells) can be obtained, for example, from ATCC. Human umbilical vein endothelial cells (HUVECs) can be isolated from human umbilical veins as previously described (Grant et al., "Matrigel induces thymosin h4 gene expression in differentiating endothelial cells," J Cell Sci 1995;108:3685-94). HUVEC cells, a cell line that expresses ATP synthase on the cell surface, can be used as a positive control. Cells can be cultured in DMEM (Life Technologies, Carlsbad, CA) supplemented with 1% penicillin-streptomycin and 10% serum replacement medium 3 (Sigma, St. Louis, MO) to minimize the presence of plasminogen. A low pH (6.7) medium can be prepared by reducing bicarbonate to 10 mmol / L in 5% CO2 and adding 34 mmol / L NaCl to maintain osmolality, or by incubating 22 mmol / L bicarbonate medium in 17% CO2. The method for reducing the pH may vary depending on the experimental constraints and assay.

[0246] Flow cytometry. To confirm that ATP synthase is functional on the cell surface of tumor cell lines, flow cytometry experiments can be performed. For example, A549 cell lines can be cultured in hypoxic (0.5% O2, 5% CO2, balanced with N2) versus normoxia (21% O2, 5% CO2) conditions in medium with different pHs (10, 22, and 44 mmol / L bicarbonate DMEM) for 0, 12, 24, 48, and 72 hours. Live cells can be blocked, incubated with an anti-β subunit antibody, washed, blocked, incubated with a secondary antibody, goat anti-rabbit FITC (Southern Biotech, Birmingham, AL), and washed again (all steps performed at 4°C). Propidium iodide (BD Biosciences, San Jose, CA) can be included in all samples to identify cells with compromised cell membranes. The mean fluorescence intensity of FITC in 10,000 cells was quantified using a FACSCalibur flow cytometer (Becton Dickinson, Franklin Lakes, NJ), and detection of mitochondrial ATP synthase was eliminated by excluding cells that had taken up propidium iodide using CELLQuest software (BD Biosciences).

[0247] Cell surface ATP synthesis assay. A549 or 1-LN cells (60,000 cells per well) in 96-well plates can be filled with medium and treated with angiostatin, angiostatin mutants, anti-β subunit antibody, rabbit IgG raised against bovine serum albumin (Organon Teknika, West Chester, PA), piceatannol (a known inhibitor of ATP synthase F1, used as a positive control; Sigma), or medium alone for 30 min at 37°C and 5% CO2. Cells can then be incubated with 0.05 mmol / L ADP for 20 s. Supernatants can be removed, and ATP production can be analyzed by the CellTiterGlo luminescence assay (Promega, Madison, WI) as described (23). Cell lysates can be similarly analyzed to confirm that the intracellular pool of ATP remains unchanged across all conditions. Recordings can be made on a Luminoskan Ascent (Thermo Labsystems, Helsinki, Finland) and data are expressed as moles of ATP per cell based on criteria determined in each independent experiment.

[0248] Cell proliferation assay. The effect of angiostatin on cancer cell lines can be assessed in serum-free medium using the 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium, inner salt (MTS) proliferation assay. After 20 hours of incubation at 37°C and 5% CO2 in the presence or absence of angiostatin, the relative cell number in each well of a 96-well microplate can be determined using the AQueous One cell proliferation assay (Promega) according to the manufacturer's protocol. The pH of the medium can be adjusted by bicarbonate concentration in 5% CO2.

[0249] Assessment of cytotoxicity. To quantify cell death and cell lysis, lactate dehydrogenase (LDH) activity released from the cytosol into the supernatant can be measured using a Cytotoxicity Detection Kit (Roche, Indianapolis, IN). Cancer cells (e.g., A549 cells) (5,000 cells per well) treated with angiostatin, angiostatin mutants, anti-β subunit antibodies, rabbit IgG, cariporide, and Triton X (a detergent used as a positive control to permeabilize cells) can be incubated at 37°C for 15 hours under 5% CO2 or 17% CO2 (neutral and low pH conditions, respectively). Cytotoxicity index can be calculated by dividing the mean absorbance of quadruplicate treated samples by the mean absorbance of quadruplicate untreated samples in medium of the same pH. Assessment of cell necrosis and apoptosis. Histone-DNA ELISA can be performed to determine the effect of angiostatin on cell death. The effects of angiostatin, angiostatin mutants, anti-β subunit antibodies, rabbit IgG, and cariporide on A549 cells (5,000 cells per well) were determined using an ELISA apoptosis and necrosis assay (Roche), based on the detection of extranuclear histone-DNA fragments. After 15 hours of incubation at 37°C in the presence or absence of reagents, apoptosis or necrosis was determined from cell lysates or supernatants of quadruplicate samples, respectively. Apoptosis or necrosis was calculated by dividing the mean absorbance of quadruplicate treated samples by the mean absorbance of quadruplicate untreated samples, corresponding to medium of the same pH. The pH of the medium was adjusted by incubation at 5% CO2 or 17% CO2.

[0250] Measurement of intracellular pH (pHi). pH can be measured by fluorescence of cells plated on 35 mm microwell dishes (MatTek, Ashland, MA) with glass coverslips. Cells can be plated on growth factor-reduced, phenol red-free Matrigel (BD Biosciences). After overnight incubation, the medium is changed, and cells are incubated with the pH-sensitive fluorescent dye cSNARF (Molecular Probes, Eugene, OR) for 15 minutes, followed by a 20-minute recovery period in fresh medium. Cells are then mounted on a microscope stage, and emission spectra are collected for 1 hour at 37°C and 5% CO2. pH can be calculated from regions containing 7–15 cells each, as described (Wahl ML, Grant DS, "Effects of microenvironmental extracellular pH and extracellular matrix proteins on angiostatin's activity and on intracellular pH," Gen Pharmacol 2002;35:277–85). At the start of spectral collection, the medium can be removed from the dish and the cells challenged with 1 mL of fresh medium with or without angiostatin, anti-β subunit antibody, rabbit IgG, cariporide, or sodium-proton exchange inhibitor. The pH of the medium can be adjusted by bicarbonate concentration, as described above, in a fixed % CO atmosphere.

Claims

1. An in vitro method for identifying and selecting a modified antibody or antigen-binding fragment thereof prepared by making at least one of an amino acid substitution, insertion, or deletion from an unmodified antibody or antigen-binding fragment thereof, wherein the modified antibody or antigen-binding fragment thereof has conditional activity and has greater binding activity to a target in a tumor microenvironment at a pH of less than 7.2 compared to the binding activity to the same target in a normal physiological environment at a pH of 7.2 to 7.6; The method comprises: 1) a) testing the binding activity of a plurality of modified antibodies or antigen-binding fragments thereof under conditions of pH below 7.2; and b) testing the binding activity of the same plurality of modified antibodies or antigen-binding fragments thereof under conditions of pH 7.2 to 7.6, the binding activity of the plurality of modified antibodies or antigen-binding fragments thereof in a) and b) is tested in the presence of human serum, and the test conditions in a) and b) are all the same; a testing step, wherein each modified antibody or antigen-binding fragment thereof is tested in each of a) and b); 2) comparing the binding activity of a) with the binding activity of b); and 3) selecting and identifying modified antibodies or antigen-binding fragments thereof that have greater binding activity in a) compared to b), thereby identifying antibodies or antigen-binding fragments thereof that have greater conditional activity for binding to a target in a tumor microenvironment at a pH below 7.2 compared to the binding activity for the same target in a normal physiological environment at a pH of 7.2 to 7.6; A method comprising:

2. 10. The method of claim 1, further comprising repeating steps 1) to 3) multiple times, wherein in each repetition, additional modified antibodies or antigen-binding fragments thereof of a selected modified antibody or antigen-binding fragment thereof are made and tested, whereby the antibody or antigen-binding fragment thereof is developed to exhibit increased activity at a pH below neutral pH.

3. 2. The method of claim 1, wherein each modified antibody or antigen-binding fragment thereof comprises one amino acid substitution or two or more amino acid substitutions compared to the unmodified antibody or antigen-binding fragment thereof.

4. 10. The method of claim 1, wherein the plurality of modified antibodies or antigen-binding fragments thereof are modified relative to the unmodified antibody or antigen-binding fragment thereof to generate a collection of modified antibodies or antigen-binding fragments thereof, whereby each modified antibody or antigen-binding fragment thereof in the collection is tested in each of a) and b); each modified antibody or antigen-binding fragment thereof in the collection comprises a single amino acid substitution compared to an unmodified antibody or antigen-binding fragment thereof; in said collection, the amino acid at each modified position is substituted with up to 1 to 19 other amino acids other than the original amino acid at that position, such that each modified antibody or antigen-binding fragment thereof contains a different amino acid substitution; wherein all amino acids along the length of the antibody or antigen-binding fragment thereof, or a selected portion thereof, are substituted in the collection. method.

5. 10. The method of claim 1, wherein the modified antibody or antigen-binding fragment thereof comprises an amino acid substitution, and The method, wherein the amino acid substitution is with an amino acid selected from Arg, His, and Lys.

6. The method of any one of claims 1 to 5, wherein the binding activity is assessed by immunoassay.

7. The method of claim 6, wherein the immunoassay comprises an ELISA.

8. The method of any one of claims 1 to 7, wherein the modified antibody or antigen-binding fragment thereof is expressed using surface display.

9. 9. The method of claim 8, wherein the binding of the modified antibody or antigen-binding fragment thereof to a target is detectably labeled or detectable.

10. 10. The method of claim 9, wherein the target is fluorescently labeled or is detected by a fluorescently labeled secondary reagent.

11. 10. The method of claim 9, wherein the detection or measurement of binding activity is performed by fluorescence activated cell sorting (FACS).

12. The method of any one of claims 1 to 11, wherein the target of the antibody or antigen-binding fragment thereof is a receptor.

13. The method of any one of claims 1 to 12, wherein the binding activity in a) is greater than the binding activity in b) by a ratio of at least 1.

5.

14. The method of any one of claims 1 to 12, wherein the binding activity in a) is greater than the binding activity in b) by a factor of at least 2.