Mirac proteins

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 on March 9, 2010. In this international patent application, the applicant in all designated countries except the United States is in the name of BioAtla, LLC, a United States limited liability company, and in the designated country of the 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 a method for conditionally generating biologically active proteins from wild-type proteins of certain therapeutic proteins, which are reversibly or irreversibly inactive in their wild-type form under normal physiological conditions. For example, the developed protein is substantially inactive at body temperature, but active at low temperatures. [Background technology]

[0003] There is a considerable body of literature describing the possibilities for developing proteins in various characteristics, for example, particularly enzymes, to stabilize them for operation under different conditions. For example, enzymes have been developed 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 presumed in the case of enzymes where turnover doubles for every 10 degrees Celsius increase. In addition, there are also examples of natural mutations that destabilize proteins at normal operating conditions, such as the wild-type activity temperature of the molecule. In temperature mutants, these mutations may be active at low temperatures, but usually at a lower level compared to the wild molecule (and usually described by a decrease in activity as guided by Q10 or similar rules).

[0004] It is desirable to generate useful molecules that are conditionally active, e.g., substantially inactive under wild-type conditions, but active at levels equal to or better than wild-type conditions, or active or inactive in a particular microenvironment, or active or inactive over time. In addition to temperature, other conditions in which proteins may be developed or optimized include pH, osmolality, osmolality, oxidation, and electrolyte concentration. Other desirable properties that may be optimized during development include chemical 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) in their wild-type operating conditions requires coexistence of destabilizing mutations and increasing activity of mutations that do not counteract the destabilizing effect, while maintaining equal or better activity than in the wild-type conditions in the new conditions. It is speculated that destabilization can reduce the activity of a protein to a greater extent than the effect predicted by standard rules such as Q10, and thus the ability to develop proteins that work 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 after the date of such disclosure as described and claimed herein. Summary of the Invention [Means for solving the problem]

[0007] The present disclosure provides a method for preparing a conditionally active biological protein, the method comprising: selecting a wild-type biological protein; developing a DNA encoding the wild-type biological protein using one or more of 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, the conditionally active biological protein, that exhibit both (a) decreased activity in the normal physiological condition assay compared to the wild-type protein; and (b) increased activity in the abnormal condition assay compared to the wild-type protein. In various embodiments, the normal physiological condition is selected from one or more of temperature, pH, osmolality, osmolality, oxidation, and electrolyte concentration. In certain embodiments, the normal physiological condition is temperature, where the conditionally active biological protein is substantially inactive at the normal physiological temperature, but active at an abnormal temperature lower than the normal physiological temperature. In other embodiments, the conditionally active biological protein is reversibly or irreversibly inactive in the wild-type normal physiological condition. In one particular embodiment, the protein is reversibly inactive under the wild-type normal physiological conditions, whereas the conditionally active biological protein is selected from those proteins that exhibit a change in activity, either reversible or irreversible, under two or more distinct physiological conditions.

[0008] In one embodiment, the wild-type biological protein is an enzyme. In certain aspects, 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 of preparing a conditionally active biological response modifier, the method comprising selecting an inflammatory response mediator, identifying a wild-type antibody to the mediator, developing the wild-type antibody, specifically screening for variants that exhibit reduced 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 reduced 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 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 pharma- ceutical acceptable carrier. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[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 a person 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 used. Unless otherwise specified, "about" relates to a variation of + / - 10% 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 a biological material such as a bacterial, plant, fungal, or animal (especially mammalian) cell or tissue. 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 is not a specific base (such as, 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 express 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; N=A or C or G or T.

[0017] The term "amino acid" as used herein refers to any organic compound containing an amino group (-NH2) 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 non-homologous to a first reference molecule, and further includes, but does not exclude, the possibility that the molecule may be 2) partially homologous and partially non-homologous to a second reference molecule, and 3) partially homologous and simultaneously non-homologous to one or more additional reference molecules. In non-limiting embodiments, chimeric molecules may be prepared by assembling a reassembly of partial molecular sequences. In non-limiting aspects, chimeric polynucleotide molecules may be prepared by synthesizing said polynucleotides using multiple molecular templates, thereby resulting in a chimeric polynucleotide having the properties of multiple templates.

[0020] The term "homologous" as used herein refers to gene sequences that are developmentally and functionally related between species. For example, but not limited to, in humans, the human CD4 gene is a homologous gene to the mouse 3d4 gene, the sequences and structures of these two genes show high homology, 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 additions or deletions (i.e., gaps) of 20% percent or less compared to the reference sequence (which has no additions or deletions) for optimal alignment of the two sequences.The optimal alignment of sequences to place 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 searches (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 may be derived by inspection and the best alignment (i.e., resulting in the highest percentage of homology over the comparison window) generated 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 also exhibits activity in selected regions of the body or exhibits increased or decreased activity under physiological conditions that are permissive to abnormal or infection. Normal physiological conditions are temperature, pH, osmolality, osmolality, oxidation, and electrolyte concentrations that are considered within the normal ranges in 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 ranges. In one embodiment, the conditionally active biological protein is substantially inactive in wild-type conditions but active in other wild-type conditions at a level equal to or better than the wild-type conditions. For example, in a variety of sites, the developed conditionally active biological protein is substantially inactive at body temperature but active at low temperature. In other embodiments, the conditionally active biological protein is reversibly or irreversibly inactive in wild-type conditions. In further embodiments, the wild-type protein is a therapeutic protein. In other embodiments, the conditionally active biological proteins are used as drugs or therapeutic agents. In yet another embodiment, the proteins exhibit more or less activity in the highly oxygenated blood, such as after passing through the lungs, or at low pH, such as found in the kidneys.

[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. Suitable 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 refers 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 set of defined sequences selected from non-random bases, typically 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 is (20) 10 and (2) a pseudorandom 10 base long sequence of 20 conventional amino acids can be any of the sequences. 10 (3) A defined sequence kernel is a subset of sequences where each residue site is any of the 20 conventional amino acids that are acceptable. A defined sequence kernel generally has either mutated or inmutated residue sites, and / or has mutated residue sites that may have residues selected from a defined subset of amino acid residues and the like throughout or segmentally throughout the length of each selected library member sequence. A defined sequence kernel may relate to amino acid sequences or polynucleotide sequences. 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] "Digestion" of DNA refers to the catalytic cleavage of DNA by a restriction enzyme that acts only on specific sequences within the DNA. The various restriction enzymes used herein are commercially available and their reaction conditions, cofactors and other requirements were used as 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 purposes 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 solution and amount of substrate for a particular restriction enzyme are specified by the manufacturer. Incubation at 37°C for about 1 hour is usually 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 that originally has two blunt ends typically does not have a preferred ligation direction when ligated into a cloning vector that is digested to generate blunt ends at 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 digested with EcoRI and BamHI.

[0029] The term "DNA shuffling" is used herein to refer to recombination between substantially homologous, but non-identical, sequences, and in some embodiments DNA shuffling may include 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 that includes 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 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 bodily fluids that carry electrical charge. For example, in one embodiment, the normal physiological conditions and abnormal conditions may be conditions of "electrolyte concentration." In one embodiment, the electrolyte concentrations tested are 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 8.5-10.2 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 96-106 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 1.7-2.2 mg / dL. In this embodiment, the abnormal serum magnesium concentration may be selected from above or below the normal range. In another embodiment, in one aspect, the normal range for serum phosphate is 2.4-4.1 mg / dL. In this embodiment, the abnormal serum phosphate concentration may be selected from above or below the normal range. In another embodiment, in one aspect, the normal range for serum or blood sodium is 135-145 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, in one aspect, the normal range for serum or blood potassium is 3.7-5.2 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 20-29 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 a particular electrolyte concentration 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 concentrations 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 a chemical surface active group of molecules, such as amino acids or side chains, and may also have specific three-dimensional structural properties and a specific charge. As used herein, "antigenic determinant" refers to that portion of an antigen or other macromolecule that can form a binding interaction that interacts 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 that has certain 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, in wild-type enzymes, Q10 (temperature coefficient) describes the increase in reaction rate for every 10°C increase in temperature. In wild-type enzymes, Q10=2-3, in other words, 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 term "development" or "developing" refers to the use of mutagenesis methods to generate novel polynucleotides encoding one or more novel polypeptides, which 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 Publication No. 2009 / 0130718, which is 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 have enhanced activity compared to the wild-type enzyme under one or more abnormal conditions.

[0036] The terms "fragment," "derivative," and "analog" when referring to a reference polypeptide include 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 a template polypeptide 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 polypeptide forming 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 regions (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 loss of repeated sequences. Deletions can include loss of one copy of a repeat and all of the space between the repeats.

[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 the sequence that cannot hybridize to another nucleic acid or polynucleotide. Such a region or portion is, for example, a mutation portion.

[0041] The term "homologous" or "homology" means 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 the 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), the term being 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 the fields of 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 occurs in nature). 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 polynucleotides may be part of a vector and / or such polynucleotides or enzymes may be part of a composition and are still isolated in that such vectors or compositions are not part of their 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 adjacent to the 5' and 3' flanking sequences that are normally immediately adjacent when present in a naturally occurring gene of the organism from which it is derived. Thus, the term refers to a nucleic acid incorporated into a vector, e.g., a plasmid or viral vector, a nucleic acid 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 a nucleic acid that exists as an isolated molecule, e.g., a DNA fragment produced by PCR amplification or restriction enzyme digestion, or an RNA molecule produced by in vitro transcription. The term also refers to a recombinant nucleic acid that forms part of a hybrid gene encoding an additional protein that can be used, for example, in the production of a fusion protein.

[0046] As used herein, "ligand" refers to a molecule that is recognized by a specific receptor, such as a random peptide or a variable segment sequence. As those skilled in the art will appreciate, a molecule (or a macromolecular complex) can be either a receptor or a ligand. In general, 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 having 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 that binds a random peptide, and to position the two molecules in a suitable configuration, such that the random peptide can bind to a receptor with minimal steric hindrance from the DNA that binds the 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 that consist of polynucleotide sequences, molecules that consist of polypeptide sequences, and molecules that consist 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, osmolarity, pH, oxidation, and concentrations of glycerol, DMSO, detergents, and / or any other molecular types that are 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 that exists 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. In general, the term "naturally occurring" refers to an object that exists in a non-pathological (non-diseased) individual, as is typical for a 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 ranges at the site of administration or site of action in a subject.

[0055] As used herein, a "nucleic acid molecule" is comprised of at least one base or one base pair, depending on whether it is single-stranded or double-stranded, respectively. Furthermore, a nucleic acid molecule can belong solely or chimerically to any group of nucleotides including molecules such as, but not limited to, groups of nucleic acid molecules such as RNA, DNA, genetic nucleic acids, non-genic nucleic acids, naturally occurring and non-naturally occurring nucleic acids, and synthetic nucleic acids. This includes, as non-limiting examples, 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 in part nucleotide based and in part protein based, is considered a "nucleic acid molecule."

[0057] Additionally, by way of non-limiting example, 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" or "nucleotide sequence encoding" refer in particular 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 is capable of binding RNA polymerase in a cell and initiating transcription in the downstream direction (3' direction) of the coding sequence. A promoter is a part of a DNA sequence. This sequence region has a start 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 bound the sequence, transcription is initiated at the start codon (the 3' end with the promoter) and then transcription proceeds downstream in the 3' direction. In the promoter, sequences are found at the transcription initiation site (conveniently defined by mapping with nuclease SI) as well as protein binding regions (consensus sequences) responsible for the binding of RNA polymerase.

[0059] The terms "enzyme (protein)-encoding nucleic acid" or "enzyme (protein)-encoding DNA" or "enzyme (protein)-encoding polynucleotide" 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 a function of the nucleic acid species, or by a 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 attributable to it, including an activity or property attributable 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 a PCR primer to facilitate amplification by PCR.

[0062] Also, in a non-limiting embodiment, a "nucleic acid library" comprises one or more vector based collections of nucleic acid molecules. In other preferred embodiments, a "nucleic acid library" comprises a non-vector based collection of nucleic acid molecules. In yet other preferred embodiments, 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 enzyme polynucleotide) that may optionally be chemically linked to one or more additional molecular moieties, such as a vector or a portion of a vector. In certain embodiments, and not meant to be limiting of the embodiment, 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 a phosphate in the presence of a kinase. Synthetic oligonucleotides are ligated to fragments that are not dephosphorylated. To obtain 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 polynucleotides that are 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 do not have to 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 one or more distinct polynucleotide species. In general, the term is preferably used to refer to a progeny polynucleotide set obtained by mutagenesis of a parental set, in which case the terms "parental," "startup," and "template" are interchangeable.

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

[0069] As used herein, the term "physiological conditions" refers to biochemical limiting factors that are compatible with living organisms and / or that are normally present intracellularly in viable cultured yeast or mammalian cells, such as temperature, pH, osmolality, ionic strength, viscosity, etc. For example, the intracellular conditions are physiological conditions in yeast cells grown under typical laboratory culture conditions. Suitable in vitro reaction conditions for the in vitro transcription cocktail are normal physiological conditions. In general, 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% non-specific protein (e.g., BSA). A non-ionic detergent (Tween, NP-40, Triton X-100) is often present, usually at about 0.001-2%, typically 0.05-0.2% (v / v). The particular aqueous solution conditions are selected by the practitioner according to the prior art. As a general guide, the following buffered aqueous solution conditions are applicable: 10-250 mM sodium chloride, 5-50 mM Tris-HCl, pH 5-8, with the addition of any divalent cations and / or metal chelators and / or non-ionic detergents and / or membrane fractions and / or antifoams and / or scintillants. Normal physiological conditions refers to the temperature, pH, osmolality, osmolality, acidity and electrolyte concentrations in the subject at the site of administration or at the site of action, in the subject's body or at the site of administration, that are considered to be within the normal range in the subject.

[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 acid or protein, but differ in their sequence (i.e., not identical) and therefore differ in their physiological intellectual activity.

[0072] A molecule having a "surrogate form" 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-induced or pH-induced conformational changes, association with cofactors, etc.) en route to a more mature molecular form with distinct properties (e.g., increased activity) in comparison to a reference surrogate form 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 a mature molecule, the reference precursor molecule is referred to as a "precursor surrogate form" molecule.

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

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

[0075] As used herein, a "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" refers to an amino acid sequence that is composed 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 unaltered state or as aggregates with other species. Receptors may be covalently or non-covalently bound to a binding member, either directly or via a specific binding agent. Examples of receptors include, but are not limited to, monoclonal antibodies and antiserum reagents with specific antigenic determinants (e.g., 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," "percentage of sequence identity," and "substantially identical" are used to describe sequence relationships 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, for example, a segment of a full-length cDNA or a segment of a gene sequence given in a sequence listing, or a 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., a portion 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] "Repetition 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 expression of the action of a restriction enzyme, and includes the site of contact cleavage. It is recognized that the site of cleavage may or may not be contained in the portion of the restriction site that consists of a low ambiguity sequence (i.e., a sequence that contains the major determinant of the frequency of occurrence of the restriction site). Thus, in many cases, the relevant restriction site contains only a low ambiguity sequence 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, the relevant restriction enzyme (e.g., an Eco57I site or CTGAAG(16 / 14)) contains a low ambiguity sequence (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 the 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). Thus, it is part 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). Thus, it is part or all of the 3' half of a polynucleotide. As used in this non-limiting illustration, there may 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 "percentage of 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 percentage of 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, where 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 to nonspecific proteins. (Similarly, a nucleic acid probe can be described as specifically binding to a target nucleic acid if 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 difference from the first polynucleotide but substantially the same sequence) 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 with different ends, are composed of two different specific polynucleotides.

[0090] "Stringent hybridization conditions" means that hybridization will occur only if 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 disclosure also includes polypeptides having sequences that are "substantially identical" to those of the enzyme polypeptides. A "substantially identical" amino acid sequence is one that differs from a reference sequence only by conservative amino acid substitutions, such as 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] In addition, a "substantially identical" amino acid sequence is a sequence that differs from a reference sequence, or by one or more non-conservative substitutions, deletions, or insertions, when such substitutions occur at sites other than the active site of the molecule, provided that the polypeptide essentially retains its behavioral properties. 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 active enzyme polypeptides.

[0093] The present disclosure provides a "substantially pure enzyme." The term "substantially pure enzyme" is used herein to describe a molecule, such as a polypeptide (e.g., an enzyme polypeptide, or a fragment thereof), that is substantially free of other proteins, lipids, sugars, nucleic acids, and other physiological materials with which it is naturally associated. 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., on a molar basis, more abundant than any other individual molecule in the composition), and preferably, a substantially purified fragment is a composition in which the species of interest consists of at least about 50% (on a molar basis) of all macromolecular species present. Generally, a substantially pure composition will consist of 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 (no contaminant species can 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 basic ionic species are not considered macromolecular species.

[0095] The term "treating" 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, reversal thereof or at least one clinical or subclinical symptom); and / or (3) relieving the condition (i.e., causing a 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 is comprised of random, pseudorandom, or defined nucleolar sequences. A "variable segment" refers to a portion of a nascent peptide that is comprised of random, pseudorandom, or defined nucleolar sequences. A variable segment may be comprised of both variant and invariant residue positions, and the degree of variant residues at variant residue positions may be limited, both options being selected at the discretion of the practitioner. Typically, a variable segment is about 5-20 amino acid residues in length (e.g., 8-10), however, a variable segment may be longer and may be comprised of an antibody protein or a receptor protein, such as an antibody fragment, a protein-binding nucleic acid, a receptor protein, and the like.

[0097] The term "mutant" 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. Mutants are 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. Disclosed herein are techniques for producing mutant proteins that have reduced activity under normal physiological conditions, such as one or more of temperature, pH, osmolality, osmolality, oxidation, and electrolyte concentration, and enhanced activity under abnormal conditions, as compared to the wild-type protein. Mutants are additionally selected for properties that enhance chemical and proteolytic resistance, as 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 is active at a level of activity found in nature and that consists of an amino acid sequence found in nature, as may be isolated from nature. The terms "parent molecule" and "target protein" refer to wild-type proteins.

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

[0100] The present disclosure also directs methods of engineering or developing proteins to generate novel molecules that are reversibly or irreversibly inactive in wild-type conditions, but active in non-normal conditions at the same or equal levels as 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 in a short or limited period of time within the host. This is particularly valuable in the extended engineering of administered proteins that are detrimental 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 combination of administration and the rate of inactivation of the protein. This condition-based inactivation is particularly important in enzyme therapy, when catalytic activity causes 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 over time, reversibly or irreversibly, 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. Therapeutic protein enzymes currently in use 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 a currently used therapeutic protein to avoid or minimize adverse side effects associated with the wild-type protein or enzyme. Alternatively, an enzyme not currently in use 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 diseases, 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 in parenteral, topical, or oral dosage forms, 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 that include interactions between blood clotting factors, platelets, red blood cells, and the vessel wall. Platelets are intravascular aggregates of platelets, fibrin, and trapped blood cells that can cause vasculopathy. By impeding or blocking blood flow, thrombi deprive tissues of oxygen supply. Pieces of thrombus (emboli) can detach and block smaller blood vessels. Arterial thrombus formation can be triggered by any of a variety of factors including subclinical stenosis-atherosclerosis, low-flow cardiac function, hypercoagulability or clotting factor deficiency 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 major cause of morbidity and mortality is the formation of arterial thrombi (coronary and cerebral arterial thrombi) and pulmonary thrombi. Venous thrombosis may result from trauma, stasis due to quiescence, or endothelial injury such as hypercoagulability, but atherosclerosis is not a contributing factor. Treatment options include mechanical thrombectomy, pharmacodynamic thrombectomy, and thrombolysis. Treatments for thrombosis are used to minimize the formation of thrombi and aid in their 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 substances 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 in acute myocardial infarction is well established. The use of thrombolytic agents has become the standard emergency treatment. Although effective, these products achieve complete reperfusion in only about 50% of patients, and side effects include the risk of bleeding (especially intracranial bleeding) 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 by plasminogen activators that activate the protein plasminogen, thereby forming plasmin. Proteolytic plasmin breaks down fibrin strands to dissolve the clot. Fibrin-specific plasminogen activators include tissue plasminogen activator or variants. Non-specific 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 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 dissolution of acute pulmonary emboli, and for dissolution of pulmonary emboli associated with unstable hemodynamics, 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 streptococci that can bind and activate human plasminogen. The complex of human plasminogen and streptokinase can hydrolytically activate otherwise unbound plasminogen by cleavage of the bond to generate plasmin. Normal activation of plasminogen occurs by 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 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 (blood clot in the lungs) and deep vein thrombosis (blood clot in the legs). Streptokinase belongs to a group of drugs called fibrinolytics. Streptokinase is given as soon as possible after the onset of a heart attack to dissolve blood 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 establish immunity to the protein. Therefore, it is recommended that this product not be given after 4 days from the first dose, as it may not be effective and may cause an allergic reaction. For this reason, it is usually given only after the first heart attack, and further thrombosis is typically treated with tissue plasminogen activator (TPA). Streptokinase is also sometimes used to prevent post-operative 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, or by bolus intravenous administration, or by mechanical infusion systems. Side effects can be severe and include 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 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 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 such that blood clots are cleared and rapid inactivation of the streptokinase mutant avoids 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 the endothelium-bound angiotensin-converting enzyme (ACE) into angiotensin II. Angiotensin II constricts blood vessels, resulting in an increase in blood pressure. However, angiotensin π 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 increases blood pressure. An overactive renin-angiotensin system leads to vasoconstriction and retention of sodium and water. These effects lead to hypertension. There are many drugs that interrupt different steps in this system to lower blood pressure. These drugs are one of the primary 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 loss of large amounts of blood and / or fluids prevents the heart from adequately perfusing oxygenated blood to the body's cells. 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 will reveal signs of shock including low blood pressure, low body temperature, 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 fluid volume and blood pressure in the body.

[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 loss. When exposed to cold, the extremities lose heat. Blood flow to the fingers and toes is normally slowed down to maintain the body's core temperature. Blood flow is reduced by narrowing of the small arteries under the skin of the extremities. Stress causes a reaction similar to that of the body becoming cold. In Raynaud's phenomenon, the normal reaction is exaggerated. The condition can cause pain, discoloration, and a feeling of cold and numbness. The phenomenon is a vasospasm that results in a reduction in blood supply to the respective area. In Raynaud's disease (primary Raynaud's phenomenon), the condition is idiopathic. In Raynaud's syndrome (secondary Raynaud's phenomenon), the phenomenon is caused by other instigating factors. Measurement of the temperature gradient in the hand is one way of distinguishing between the primary and secondary forms. The primary form may progress to a 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 essentially caused by microvascular spasm. Overactivation of the sympathetic nervous system causes excessive vasoconstriction of peripheral blood vessels, leading to hypoxia. In chronic, recurrent cases, atrophy of the skin, subcutaneous tissue, and muscle may result. Rarely, ulceration and ischemic gangrene may occur.

[0120] Conventional treatment options for Raynaud's phenomenon include prescription drug treatments that dilate blood vessels and improve circulation. These include calcium channel blockers such as nifepidine or diltiazem, alpha blockers such as noradrenaline, prazosin or doxazosin, which counteract the effects of hormones that constrict blood vessels, and vasodilators to relax blood vessels, such as nitroglycerin cream or the angiotensin II inhibitors losartan, sildenafil or prostaglandins. Fluoxetine, selective serotonin reuptake inhibitors and other antidepressants can reduce the frequency and severity of episodes due to physiological stressors. These medications can cause side effects such as headaches, flushing and ankle edema. The medications may also lose their effectiveness over time.

[0121] The regulation of cutaneous vasoconstriction and vasodilation involves altered sympathetic activity and many neuromodulations, including adrenergic and non-adrenergic, as well as other signaling such as REDOX signaling and the RhoA / ROCK pathway. Vasoconstriction of cutaneous vascular smooth muscle cells (vSMC) 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 vSMC in response to stimuli, and the 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 the mitochondria of vSMC. 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 vSMC. 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 concentrations of CGRP. CGRP dilates blood vessels in the brain and transmits vascular pain sensations. CGRP receptor antagonists have been experimented with as a treatment for migraine. Arulmani et al., 2004, "Calcitonin gene-related peptide and it 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 that vary in function to modulate the action of the peptide 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 and its role are understood. Indian J. Pharmacol. 36(5): 277-283. One study of the effect of intravenous administration of three vasodilators, the endothelium-dependent vasodilator adenosine triphosphate (ATP), the non-endothelium-dependent vasodilator prostacyclin (epoprostenol, PGI2) and CGRP in patients with Raynaud's phenomenon, and a similar number of age and sex matched patients with Raynaud's phenomenon and controls using CGRP and laser Doppler flowmetry (LDF), showed that in Raynaud's patients, redness of the face and hands was induced by CGRP through increased cutaneous blood flow, whereas in the control group, CGRP caused redness of the face only. PG12 caused similar blood flow to the hands and face in both groups.ATP did not cause any significant changes in the blood flow in the hands or face of patients, but increased the 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 a protein associated with Raynaud's syndrome that are reversibly inactive at normal physiological temperatures but are reactivated when the digits are subjected to abnormally low temperatures. The conditionally active proteins may be used to treat Raynaud's phenomenon, to prevent or reduce loss of digit 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 the most important roles of vasopressin is to regulate water retention in the body. At high concentrations, it increases blood pressure by inducing moderate vasoconstriction. Vasopressin has three effects that result in an increase in urine osmolality (high concentration) and a decrease in water excretion. First, vasopressin causes an increase in the permeability of collecting duct cells to water in the kidney, which allows for the reabsorption of water 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 causes an increase in the permeability of the inner medullary portion of the collecting duct to urea, allowing for increased reabsorption of urine in the medullary interstitium. Third, vasopressin increases the activity of the Na+, K+, 2Cl- cotransporter, stimulating sodium and chloride reabsorption in the thickened upper limb of the loop of Henle. Sodium chloride reabsorption is by a process of increased reflux, providing an osmotic gradient across aquaporins that results in water reabsorption in the collecting duct medulla.

[0125] The hypertonic interstitial fluid around the collecting ducts of the kidney provides a high osmotic pressure for the removal of water. 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 a receptor (called the V2 receptor) on the basolateral surface of the collecting duct cells. Binding of the hormone triggers an increase in the level of cAMP in the cells. This "second messenger" initiates a chain of events that results in insertion of aquaporin 2 channels in the apical membrane of the collecting duct cells. Aquaporins increase water reabsorption by pumping water out of the renal unit and returning urine to the bloodstream.

[0126] The primary stimulus for the release of vasopressin from the pituitary gland is an increase in plasma osmolality. Any dehydration of the body, such as heavy sweating, increases the osmolality of the blood, activating vasopressin to the V2 receptor in the aquaporin 2 pathway. As a result, as little as 0.5 L / day of urine may remain, leaving 180 L / day of the original renal filtrate. The salt concentration in urine may be four times higher than in blood. When the blood becomes too dilute, for example by drinking large amounts of water, vasopressin secretion is inhibited and the aquaporin 2 channels are endocytosed back into the cells. As a result, a large amount of dilute urine is produced with a small salt concentration, about one-quarter that of blood.

[0127] Decreased vasopressin release or decreased sensitivity of the kidney to AVP can result in diabetes insipidus, hypernatremic states (increased sodium concentration 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. This can cause hyponatremia for several days.

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

[0130] In one embodiment, the disclosure provides a method of selection for conditional biological recombinant or synthetic proteins of proteins involved in 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 vasopressin response are activated under sodium depletion conditions but inactivated under normal serum sodium concentrations. In one aspect, the sodium depletion 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 metastasis. Angiostatin is a 38 kD fragment of plasmin (itself a fragment of plasminogen). Angiostatin consists of 1-3 kringles of plasminogen. Angiostatin is produced by autolytic cleavage of plasminogen, including extracellular disulfide bond reduction by, for example, 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 a number of 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 has shown 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 and inhibits ATP synthesis on the endothelial cell surface. 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 (pHe). 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. It has been hypothesized that the mechanism of tumor cytotoxicity is through 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 embodiment, low pH is about pH 7.2 or less. In a particular embodiment, low pH is about pH 6.7.

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

[0135] Increased tissue permeability – Hyaluronidase Hyaluronidase is a family of enzymes that degrade hyaluronic acid. By causing the degradation of hyaluronic acid, the main component of the interstitial barrier, hyaluronidase reduces the viscosity of hyaluronic acid, thereby increasing tissue permeability. It is used in medicine in conjunction with drugs to speed up drug dispersion and delivery. The most common application is in eye surgery, where it is used in conjunction with local anesthesia. Animal-derived hyaluronidases include Hydase™ (PrimaPharm Inc.; Akorn me), Vitrase (ISTA Pharmaceuticals), and Amphadase (Amphastar Pharmaceuticals). Human recombinant hyaluronidase is currently approved as an adjunct to increase the absorption of other drugs, such as Hypodermocyclis (subcutaneous injection of fluids), an adjunct in subcutaneous urography to improve absorption of radiopaque drugs (Hylenex; Halozyme Therapeutics, Inc.; Baxter Healthcare Corp). In one embodiment, hyaluronidase is used as a wild-type protein (parent molecule) for the preparation of conditionally active biological proteins. Hyaluronidase can play a role in cancer metastasis and angiogenesis. Thus, 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 in 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 mechanism that leads to inflammation of the joints and swelling due to the progressive destruction of the joints. RA can also affect the skin, connective tissues, 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 are ideal, especially in long-term use.

[0137] Biological response modifiers that target inflammatory mediators provide 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-a), a proinflammatory cytokine involved in the pathogenesis of RA. Several anti-TNF-alpha drugs are currently on the market for the treatment of RA. For example, Enbrel® (etanercept, Amgen) is a TNF-alpha blocker. Etanercept is a dimeric fusion protein consisting of the extracellular tether binding portion of the human 75 kilodalton (p75) tumor necrosis factor receptor (TNFR) bound to the Fc portion of human IgGI. The Fc component of etanercept contains the CH2 domain, the CH3 domain, and the hinge region, but not the Ch1 domain of IgGI. 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 IgG KI 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 to treat 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 reduces the signs and symptoms of RA, both as a monotherapy and in combination with MTX or other DMARDs. 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 that lead to synovial thickening 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 IgGIk monoclonal antibody against the p40 subunit of the IL-12 and IL-23 cytokines. STELARA has been shown to provide relief from certain symptoms related to psoriasis plaques, such as plaque thickening, skin peeling, and redness. The formulation for STELARA 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 infections, including tuberculosis and infections caused by bacteria, fungi, or viruses, as well as increasing the risk of certain types of cancer.

[0143] The side effects of biological response modifiers can be severe and injecting high levels into patients can make them more susceptible to serious infections or death. This is the major side effect associated with this important class of drug. One challenge is to avoid the high initial levels of activity from the administration of antibodies that 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, which avoids the high levels of activity from administration of antibodies required to provide a long therapeutic effect after injection. The methods of the present disclosure may be used to design antibodies against inflammatory mediators such as IL-6, IL-6 receptor, TNF-alpha, IL-23, and IL-12, which 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 hours to 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 disclosure provides a method for the preparation of conditionally active antibodies or fragments thereof to inflammatory mediators that are inactive at administration conditions such as room temperature, but slowly refold the protein (reversibly or irreversibly) at body temperature. The method comprises the steps of: selecting an inflammatory mediator; screening to identify antibodies to inflammatory mediators via fusion cells; humanizing an anti-inflammatory mediator antibody; developing an anti-inflammatory mediator antibody and differentially screening for binding in two or more conditions, e.g., two or more temperature conditions, e.g., room temperature and 37°C or higher; selecting for mutations that are inactive in a first condition compared to wild type, but show increased activity (e.g., binding) in a second condition compared to wild type antibody activity (binding). The identified up-mutants in the heavy and light chains are recombined in the heavy and light chains as well as through combinatorial binding of the heavy and light chains. The screening of these recombined heavy and light chains is repeated in 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, hi 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 the preparation of conditionally active antibodies or fragments thereof to IL-6 that are inactive at administration conditions such as room temperature, but slowly refold the protein (reversibly or irreversibly) at body temperature. The method includes the steps of: 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 show increased activity (e.g., binding) compared to the wild type antibody activity (binding). The identified up-mutants in the heavy and light chains are recombined in the heavy and light chains as well as through combinatorial combination of the heavy and light chains. The screening of these recombined heavy and light chains is repeated at room temperature and higher temperatures. In addition, the recombined antibodies or fragments are tested for activity and stability under storage and physiological conditions.

[0149] The thus identified and produced conditionally active anti-IL-6 antibodies are 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 that it allows for smoothing and leveling of drug dosage over the treatment period, compared to the current high levels of biological response modifiers that have half-life clearances lasting 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 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 under non-wild-type conditions that remain the same or greater than the activity under wild-type conditions and have reduced activity under wild-type conditions.

[0152] The disclosure provides a method for generating a nucleic acid variant that encodes a polypeptide having enzymatic activity, wherein the variant has an abnormal physiological activity from that which occurs naturally, the method comprising: (a) (i) substituting one or more nucleotides with a different nucleotide, wherein the nucleotide comprises a natural or unnatural nucleotide; (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 comprises inosine. In another embodiment, the method comprises analyzing the polypeptides encoded by the modified nucleic acid for abnormal enzymatic activity, thereby identifying the modified nucleic acid that encodes a polypeptide having 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 the modification step (a).

[0153] The 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, where at least one of the nucleic acids comprises a nucleic acid of the 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 the situation where 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 is, for example, a specifically unplanned product whose production is not intended. Rather, it is indeterminate and therefore random with respect to the exact nature of the mutations obtained and thus with respect to the product produced. Stochastic mutagenesis is illustrated in methods where mutations are random or unplanned, such as error-prone PCR and stochastic shuffling. Mutant formation is by error-prone transcription, such as error-prone PCR, or the use of a polymerase lacking proofreading activity (see Liao (1990) Gene 88: 107-111), or by a first form of replication in a mutant strain (mutant host cells are discussed in more detail below, which is generally well known). Mutagen strains can include any mutants that are defective in the function of mismatch repair. These include mutant gene products such as mutS, mutT, mutH, mutL, ovrD, dcm, vsr, umuC, umuD, sbcB, recJ, etc. Deletions can be obtained by gene mutation, allelic exchange, small chemical compounds or expressed antisense RNA or other techniques. Deletions can be in the described gene or in a homologous gene in any organism.

[0155] Currently, the mutagenesis methods widely used to create selective proteins from starting molecules are oligonucleotide-directed mutagenesis techniques, 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 are generated 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 may 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. Generally, these agents are added to the PCR reaction in place of the nucleotide precursors that displace the sequence. Insertion agents such as proflavine, acriflavine, quinacrine, and others may also be used. Random mutagenesis of polynucleotide sequences may be obtained by X-ray or ultraviolet irradiation. Generally, mutagenized plasmid polynucleotides are introduced into E. coli and propagated as a pool or library of hybridizing plasmids.

[0157] Error-prone PCR uses low-fidelity polymerization conditions to introduce low levels 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 interruptions 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 the situation where a parent molecule is mutated (modified or changed) to obtain a molecule with one or more pre-determined mutations. It is well understood that the presence of background products in some amount is real in many reactions where molecular processing occurs, and the presence of background products does not detract from the non-stochastic nature of the mutagenesis process with unplanned products. Site-saturation mutagenesis and synthetic ligation reassembly are examples of gene mutagenesis techniques where the exact chemical structure of the intended product is pre-determined.

[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 that correspond to the codons of a template polynucleotide and performing a polymerase extension to produce progeny polynucleotides that contain sequences that correspond 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: (a) providing copies of a template polypeptide, each copy of the template polynucleotide comprising a plurality of codons encoding the template polypeptide sequence; and (b) for each codon of the template polynucleotide, (1) providing a series of degenerate primers, each primer comprising a degenerate codon corresponding to the codon in the template polynucleotide and at least one flanking sequence that is homologous to a sequence flanking the codon in the template polynucleotide, (2) providing conditions that permit the primers to anneal to the copy of the template polynucleotide, and (3) performing a polymerase extension reaction from the primers along the template, thereby producing a series of progeny polynucleotides, each of which comprises a sequence that corresponds to the degenerate codon of the annealed primer.

[0161] Site-saturation mutagenesis involves the directed evolution of a nucleic acid and screening of clones containing the evolved nucleic acid for a resulting activity of interest, such as a nucleic acid activity and / or a specific protein, particularly an enzyme, activity 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), which is mutagenesis to obtain at least one point mutation, addition, deletion and / or chimerization from one or more ancestral or parent generation templates; 2) screening the progeny generation molecules, preferably using high throughput methods, for at least one property of interest (e.g., improvements in enzymatic activity, or increased stability, or novel chemotherapeutic effects); 3) optionally obtaining and / or cataloging structural and / or functional information on the parent 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 from the new codon), such that every codon (or the entire family of degenerate codons encoding the same amino acid) is represented at each codon position. Corresponding to and encoded by the progeny generations of this polynucleotide are a series of progeny polypeptides generated, each having 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, more specifically, methods for preparing polynucleotides encoding polypeptides by methods of 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 the production of a polypeptide having useful properties.

[0165] In other embodiments, mutagenesis techniques take advantage of the natural propensity of cells to recombine molecules and / or 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 having enhanced activity. In achieving these and other objects, 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 the 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 separate ancestral (parent) molecule. Another example is the mutagenesis of a single codon position (i.e., to obtain a codon substitution, addition, 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 gene crosses 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 expansion 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 that functionally belong to a specific structural grouping (e.g., a specific single amino acid position or a sequence of two or more amino acid positions) and to classify and compare specific groupings of mutations.

[0170] These, or any other developed methods, in this disclosure, generate 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 a cloning vector, and transfected into appropriate host cells.

[0172] Expression of developmental molecules Once a library of mutant molecules has been generated, the DNA can be expressed using conventional molecular biology techniques, and thus protein expression can 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 the individual mutants 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 polynucleotide thus selected and isolated is introduced into a suitable host cell. A suitable host cell is any cell capable of promoting genetic recombination and / or reductive reassortment. The selected polynucleotide is preferably already in a vector containing appropriate control 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 the host cell may be 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 expression vectors that can be used 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 vectors specific to a particular target host (e.g., Bacillus, Aspergillus, and yeast). Thus, for example, the 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 given as examples: Bacterial: pQE vectors (Qiagen), pBluescript plasmids, pNH vectors, lambda ZAP vectors (Stratagene); ptrc99a, ρKK223-3, pDR540, pRIT2T (Pharmacia); Eukaryotic: pXT1, ρSG5 (Stratagene) pSVK3, pBPV, pMSG, pSVLSV40 (Pharmacia). However, any other plasmids or other vectors can be used as long as they are replicable and viable in the host. Low copy number vectors 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. Particular 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. The promoter region can be selected from any desired gene using a chloramphenicol transferase (CAT) vector 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 a method of reductive reassortment. The method involves the generation of constructs containing consecutive sequences (original coding sequence 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 constructs 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, the reassortment method can involve the natural properties of homologous recombination or quasi-repeated sequences to guide their own development.

[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 E. 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 a suitable host is considered to be within the scope of a person skilled in the art from the teachings herein.

[0179] With particular reference to the various mammalian cell culture systems that may 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 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 necessary. In vivo reassortment is directed to "intermolecular" processes collectively referred to as "genetic recombination", which in bacteria is usually observed as a "RecA-dependent" phenomenon. The 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 method 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 transformed cells, 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 of skill in the art.

[0182] Protein expression can be induced by a variety of well-known methods, and many genetic systems have been published for induction of protein expression. For example, for a suitable system, protein expression is induced by the addition of an inducer product. The cells are then pelleted by centrifugation and the supernatant is removed. Periplasmic proteins can be enriched by culturing the cells with DNAse, RNAse and lysozyme. After centrifugation, the supernatant containing the novel protein is transferred to a new multi-well 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 cycles, sonication, mechanical separation, or the use of cell lysing agents. Such methods are well known to those skilled in the art. The expressed polypeptide or fragment thereof can be recovered and purified from the 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 in completing the structure of the polypeptide, if necessary. High performance liquid chromatography (HPLC) can be used for the final purification process, if necessary.

[0184] Clones identified as having the desired activity can then be sequenced to identify the polynucleotide sequence that encodes 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 mouse systems. 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 yeast systems. 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 invention can be synthetic or recombinantly produced polypeptides. The peptides and proteins can be expressed by recombinant techniques in vitro or in vivo. The peptides and polypeptides of the invention can be made and isolated using any method known in the art. The polypeptides and peptides of the 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 a variety of 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) following instructions provided by the manufacturer.

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

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

[0191] The polypeptide mimetic compositions of the invention can include any combination of non-natural structural components. In other embodiments, the mimetic compositions of the 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 bonds 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 invention can be characterized as a mimetic when all or some of its residues are linked by chemical means other than natural peptide bonds. The residues of the individual peptidomimetics can be linked by peptide bonds, other chemical bonds, or coupling means (e.g., glutaraldehyde, N-hydroxysuccinimide esters, bifunctional maleimides, N,N'-dicyclohexylcarbodiimide (DCC), or N,N'-diisopropylcarbodiimide (DIC), etc.). Linking groups which may 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 invention can also be characterized as mimetics by containing all or some non-natural residues in place of naturally occurring amino acid residues. Non-natural residues are well described in the scientific and patent literature. The following provides a few examples and guidelines of non-natural 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 ... Dp-phenylalanine; 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 non-acidic amino acids. Aromatic rings of non-natural 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, for example, 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 substitution with 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 converted to asparaginyl and glutaminyl residues by reaction with ammonium ions. It can be unaminated to the corresponding aspartyl or glutamyl residue. Arginine residue mimetics can be generated by reacting arginyl, for example, with one or more conventional reagents, including, for example, phenylglyoxal, 2,3-butanedione, 1,2-cyclohexanedione, or ninhydrin, preferably under alkaline conditions. Tyrosine residue mimetics can be generated by reacting tyrosyl, for example, with aromatic diazonium compounds or tetranitromethane. N-acetylimidazole and tetranitromethane can be used to form O-acetyltyrosyl species and 3-nitro derivatives, respectively. Cysteine ​​residue mimetics can be generated by reacting cysteinyl residues, for example, with α-haloacetates, such as 2-chloroacetic acid or acetamide chloride, and the corresponding amines to give carboxymethyl or carboxyamidomethyl derivatives.Cysteine ​​residue mimetics can be generated by reacting cysteinyl residues with, for example, bromo-trifluoroacetone, α-bromo-β-(5-imidazoyl)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 altered) 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 salts, pyridoxal phosphate, pyridoxal, chloroborohydride, trinitro-benzenesulfonic acid, O-methylisourea, 2,4,pentanedione, and transamidase-catalyzed reaction 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, for example, with diethylprocarboxylate or para-bromophenacyl bromide. Other mimetics include those generated, for example, by hydroxylation of proline and lysine; phosphorylation of the hydroxyl groups of seryl or threonyl residues; methylation of the α-amino groups of lysine, arginine, and histidine; acetylation of the N-terminal amine; 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 D-amino acid, also called R- or S-form.

[0195] The invention also provides methods for modifying the polypeptides of the 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. Also, 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, cross-linking cyclization, 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 HM 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 a number of "rods" or "pins," all of which are connected to a single plate. When such a system is utilized, the plate of rods or pins is inverted and inserted into a second plate of corresponding wells or reservoirs, which contain solutions for attaching or immobilizing the appropriate amino acids to the tips of the pins or rods. By repeating these process steps, 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. In addition, 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 which can be coupled using other well known techniques.

[0197] The synthetic polypeptide or fragment 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 steps can be used, if necessary, in completing the structure of the polypeptide. If necessary, high performance liquid chromatography (HPLC) can be used for a final purification step.

[0198] The present invention provides a conditionally active protein variant formulation or dosage form having at least one protein variant, the formulation being liquid or dry. The protein formulation optionally includes a buffer, a cofactor, a second or additional protein, or one or more excipients. In one embodiment, the formulation is utilized as a therapeutic conditionally active biological protein that is active under abnormal or non-physiological conditions, e.g., with respect to temperature, pH, or osmolarity, oxidation, or osmolality, and is 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 quite directly by measuring protein activity in permissive and wild-type conditions. The mutants exhibiting the highest activity ratios (permissive / wild-type) are then selected and point mutation permutations can be generated by combining the individual mutations using standard methods. This combinatorial permutation protein library is then screened for proteins that exhibit the greatest difference between permissive and wild-type activity.

[0201] The activity of the supernatants can be screened using a variety of methods, for example high-throughput activity assays such as fluorescent assays, to identify protein variants that are sensitive to a desired property (temperature, pH, etc.). For example, to screen for time-sensitive variants, enzyme or antibody activity measurements are performed 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 performed initially in a multi-well assay format, such as a 96-well assay, and confirmed using a different format, such as a 14 ml tube format.

[0202] The present disclosure further provides a screening assay for identifying enzymes, 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 the candidate polypeptides that exhibit higher enzymatic activity than the wild-type enzyme protein under abnormal or non-physiological conditions, e.g., conditions of temperature, pH, oxidation, osmolarity, electrolyte concentration, or osmolarity, 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 a host cell or host organism. In a further embodiment, the testing step (c) further comprises testing for enzymatic activity in a pH range of about pH 3 to about pH 12. In a further embodiment, the testing step (c) further comprises testing for enzymatic activity in a pH range of about pH 5 to about pH 10. In a further embodiment, the testing step (c) further comprises testing for enzymatic activity in a pH range of about pH 6 to about pH 8. In a further embodiment, the testing step (c) further comprises testing for enzymatic activity in a pH range of about pH 6.7 to about pH 7.5. In another embodiment, the testing step (c) further comprises testing for enzymatic activity in a temperature range of about 4° C. to about 55° C. In another embodiment, the testing step (c) further comprises a step of testing the enzyme activity in a temperature range of about 15°C to about 47°C. In another embodiment, the testing step (c) further comprises a step of testing the enzyme activity in a temperature range of about 20°C to about 40°C. In another embodiment, the testing step (c) further comprises a step of testing the enzyme activity in a temperature range of about 25°C to about 37°C. In another embodiment, the testing step (c) further comprises a step of testing the enzyme activity under normal osmotic pressure and under abnormal osmotic pressure (positive or negative). In another embodiment, the testing step (c) further comprises a step of testing the enzyme activity under normal electrolyte concentration and under abnormal electrolyte concentration (positive or negative). The electrolyte concentration tested is selected from the concentration 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 disclosure provides purified antibodies that specifically bind to a polypeptide of the disclosure or a fragment thereof that has enzymatic activity. In one aspect, the disclosure provides fragments of said 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. Thus, the present disclosure provides methods of inhibiting enzymes using the antibodies of the present disclosure.

[0206] The antibodies can be used in immunoprecipitation, staining, immunoaffinity columns, and the like. If necessary, 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 to the arrays of the present disclosure. Alternatively, the methods of the present disclosure can be used to modify the structure of the antibodies produced by the cells, modifying the antibodies, for example, to increase or decrease the affinity of the antibodies. Furthermore, the ability to make or modify antibodies can be a phenotype engineered into the cells by the methods of the present disclosure. Methods for immunization, generation and isolation of antibodies (polyclonal and monoclonal) are known to those of skill 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 (2d 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 animal methods.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, such as an enzyme, of the 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 disclosure.

[0208] In immunoaffinity techniques, the antibody is attached 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 disclosure. After washing to remove non-specifically 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 of skill in the art. For example, binding may be determined by labeling the antibody with a detectable label, such as a fluorescent substance, an enzyme label, a radioisotope, etc. Alternatively, the ability of the antibody to bind to the sample may be determined by detection using a secondary antibody that has such a detectable label on its surface. Particular 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 direct injection of the polypeptides into an animal or by administration of the polypeptides to a non-human animal. 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 which may bind to the native full-length polypeptide. Such antibodies can then be used to isolate the polypeptides from cells expressing the polypeptides.

[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 this disclosure. Alternatively, transgenic mice can be used to express humanized antibodies to the polypeptides or fragments thereof. Antibodies generated against the polypeptides of this disclosure can be used to screen for similar polypeptides (e.g., enzymes) from other organisms and samples. In such techniques, a polypeptide from an organism is contacted with the antibody and polypeptides that specifically bind to the antibody are detected. Any of the techniques described above may be used to detect antibody binding.

[0213] Screening method and "on-line" monitoring device 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 that are 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, for screening cells that express the polypeptides of the present disclosure, and the like.

[0214] Arrays or "Biochips" The nucleic acids or polypeptides of the disclosure can be immobilized or applied to an array. Arrays can be used to screen or monitor a library 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 disclosure. For example, in one embodiment of the disclosure, the monitored parameter is the transcriptional expression of an enzyme gene. One or more or all of the transcripts of a cell can be measured by hybridizing a sample having the transcripts of the cell or a nucleic acid representative or complementary to the transcripts of the cell 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 having genomic nucleic acids can be used to determine the genotype of a newly engineered strain created by the method of the disclosure. A polypeptide "array" can be used to simultaneously quantify multiple proteins. The present disclosure can be practiced with any known "array," which may also be considered a "microarray" or a "nucleic acid array" or a "polypeptide array" or an "antibody array" or a "biochip" or variations thereof. An array generally comprises a plurality of "spots" or "target elements," each having a defined amount of one or more biomolecules, e.g., oligonucleotides, immobilized in defined areas on a substrate surface that specifically binds 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 modifications 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 No. WO 99 / 51773; International Publication No. WO 99 / 09217; International Publication No. WO 97 / 46313; International Publication No. 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, Calif.) can be used in the methods of the disclosure. The nucleic acids or polypeptides of the disclosure can be immobilized or applied to an array, including a capillary array. The array can be used to screen or monitor a library 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 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 adjacent capillary array, each capillary having at least one wall that forms 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 is adapted to be coupled to a capillary array and may include a first wall defining a lumen for holding a sample and a second wall formed of a filter material for filtering excitation energy provided to the lumen for exciting 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 of the capillary array. Each capillary of 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 sample of interest is introduced into one of the capillaries in the capillary array as a first liquid labeled with detectable particles, each capillary in the capillary array having 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 that defines a lumen. The outer wall may be one or more walls fused together. Similarly, the walls may define a lumen that is cylindrical, rectangular, hexagonal, or of any other geometric shape, so long as the walls form a lumen that holds a liquid or sample. The capillaries in the capillary array may be in close proximity to each other and support each other to form a planar structure. The capillaries may be attached to each other by fusing (e.g., here the capillaries are made of glass), gluing, bonding, or fixing adjacent ones. The capillary array may be formed from any number of individual capillaries, for example, from 100 to 4,000,000. The capillary array may form a microtiter plate with about 100,000 or more individual capillaries attached to each other.

[0217] Pharmaceutical Compositions 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 as described herein, and (b) a suitable carrier or diluent. The carrier or diluent can optionally be pharma- ceutically 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 pharma- ceutically acceptable salt, which includes those salts commonly used in the pharmaceutical industry for therapeutic proteins, such as sodium, potassium, calcium, and other salts, as well as amine salts, such as procaine, dibenzylamine, ethylenediamine, ethanolamine, methylglucamine, taurine, and other salts, and acid addition salts, such as hydrochlorides and basic amino acids.

[0219] The present disclosure further provides methods or compositions of at least one conditionally active biological protein for administration in a therapeutically effective amount 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 of 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. The method may optionally further comprise administering to the cell, tissue, organ, or animal an effective amount of 0.001-50 mg / kg of a conditionally active biological protein of the present disclosure. The method may optionally further comprise using a contacting or administering step by at least one mode selected from parenteral, subcutaneous, intramuscular, intravenous, intraarticular, 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 the step of administering, prior to, concurrently with, or after the contacting or administration of the conditionally active biological protein, at least one composition having 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 drug, 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 drug, 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 drug, a beta agonist, an inhaled steroid, epinephrine or its analogs, a cytotoxic drug or other anticancer drug, an antimetabolite such as methotrexate, a proliferation 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 to be 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, etc. Such solutions are sterile and generally free of undesirable substances. Such solutions may be sterilized by conventional, known sterilization methods. The compositions may contain pharma-ceutically acceptable auxiliary substances, such as pH adjusting and buffering agents, isotonicity 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., according to 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 PEG400; (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. The pharmaceutical compositions and formulations for oral administration of the present invention can be formulated using pharma- ceutically acceptable carriers known in the art in appropriate dosages. Such carriers allow the pharmaceutical agent to be formulated into a unit dosage form suitable for patient intake, such as tablets, pills, powders, dragees, capsules, liquids, lozenges, gels, syrups, slurries, suspensions, etc. Pharmaceutical preparations for oral use are formulated with solid excipients, but in some cases, the resulting mixture is ground to obtain a core of a tablet or dragee, and the mixture of granules is processed after adding suitable additional compounds. 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 arabic and tragacanth; and proteins such as gelatin and collagen. Disintegrants or solubilizers may be added, such as cross-linked polyvinylpyrrolidone, agar, alginic acid, or salts thereof, such as sodium alginate. Tablet forms may include 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, hydrating agents, preservatives, flavorings, dyes, disintegrants, and pharma- ceutically acceptable carriers.

[0223] The present invention provides an aqueous suspension having a conditionally active biological protein in admixture with excipients suitable for the manufacture of an aqueous suspension, which may include suspending agents such as sodium carboxymethylcellulose, methylcellulose, hydroxypropylmethylcellulose, sodium alginate, polyvinylpyrrolidone, gum tragacanth, and gum arabic, and nucleic acid 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 fatty acids and partial esters derived from hexitols (e.g., polyethylene sorbitol monooleate), or condensation products of ethylene oxide with fatty acids and partial esters derived from 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 preparations may also be adjusted for osmolality.

[0224] Lozenge dosage forms have the active ingredient in a flavoring, usually sucrose and acacia or tragacanth, and pastilles have the active ingredient in an inert base, such as gelatin and glycerin or sucrose and acacia emulsion, gel, etc., which may have 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 accomplished 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 may be encapsulated, for example, in a liposome or in a formulation that allows 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., capable of being inhaled by nebulization) and administered by inhalation. The aerosol formulation can be placed into a compressed acceptable propellant, such as dichlorofluoromethane, propane, nitrogen, and the like. Suitable formulations 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, and 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 pharma- ceutically acceptable carrier in the form of a cream, ointment, solution or hydrogel formulation, and may contain other compounds, so long as the added ingredients do not adversely affect the delivery of the therapeutic protein. Conventional pharma- ceutically acceptable emulsifiers, surfactants, suspending agents, antioxidants, osmolality enhancing 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 blood of the intended recipient, and aqueous and non-aqueous sterile suspension solutions, which may contain suspending agents, solubilizers, thickeners, stabilizers, and preservatives. In the practice of the invention, compositions can be administered, for example, by intravenous infusion, orally, topically, intraperitoneally, or intrathecally. In one embodiment, parenteral forms of administration are a suitable method for administering compositions having conditionally active biological proteins. The compositions may be conveniently administered in unit dosage form and may be prepared by any method known in the pharmaceutical art, for example, as described in Remington's Pharmaceutical Sciences, Mack Publishing Co. Easton Pa., 18th Ed., 1990. Preparations for intravenous administration may contain pharma- ceutically 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 for further details.

[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. Injection 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 methods of delivery 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 pharma- ceutically acceptable carrier or diluent, which may 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 drug, an antimetabolite such as methotrexate, a proliferation inhibitor, a cytokine or cytokine antagonist, a TNF antagonist, an antirheumatic drug, 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 drug, an antipsoriatic drug, 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 drug, a beta agonist, an inhaled steroid, epinephrine, or the like.

[0231] Also provided is a medical device having at least one conditionally active biological protein of the present disclosure, wherein the device is suitable for contacting or administering 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, intracelial, 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 at least one preservative selected from the group consisting of sterile water, sterile buffered water, or 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 isotonicity agent. In another embodiment, the second container further comprises a physiologically acceptable buffer. In one embodiment, the 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 a packaging material and a solution or lyophilized form of at least one conditionally active biological protein of the present disclosure, which may optionally comprise a container that comprises 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 of the 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 novel type lower reaction temperature (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) with a fluorescent plate reader. Determine relative fluorescence units (RFU). Supernatants from wild type molecules and cells transformed with plasmids / vectors are used as positive and negative controls. Perform duplicate reactions for each sample, reaction temperature, positive and negative controls.

[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., activity ratios greater than or equal to 1.1 or greater (e.g., activity ratios at 25° C. or 37° C. (25° C. / 37° C.) greater than or equal to 1.1 or greater), can be considered as putative temperature-sensitive primary hits. The 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 wild-type temperature (e.g., 37° C.) and at lower temperatures (e.g., 25° C.). Proteins are expressed and purified for use in multiwall format as described above, but also expressed in a different format (14 ml tubes) that is not multiwall (96-well plates).

[0238] Transfer the supernatant of each mutant to a multi-wall plate, e.g. a 96-well microplate. Add a fluorescent substrate to each tube and place at the specified temperature (wild type temperature, lower temperature) for the appropriate time. Use the wild type molecule as a positive control and supernatant from cells transformed with the vector only as a negative control. Detect the fluorescence by measuring the fluorescence in the appropriate emission spectrum (e.g. excitation spectrum of 320 nm, emission spectrum of 405 nm) with a fluorescent plate reader. Determine the relative fluorescence units (RFU). Perform duplicate reactions 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 2 or greater, and if the mutant exhibits reduced overall activity when compared to the wild-type molecule at the wild-type temperature (37° C.), the phenotype of the mutant is confirmed to be 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 screened again 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 the enzyme activity at low temperature (e.g., 25°C) is reversible or irreversible by exposing the mutant to a higher temperature and then returning it to a lower temperature (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 exposed again to the required lower temperature (e.g., 25°C). Mutants that are active at low temperature and show reduced activity when raised to a higher or wild-type temperature (i.e., the ratio of activity at low temperature to the higher temperature is equal to or greater than 1, 1.5, 2, or higher) and show baseline activity when lowered back to low temperature are determined to be "reversible hits." Mutants that are active at low temperatures, that show reduced activity when raised to higher or wild-type temperatures (i.e., the ratio of activity at low temperature to higher temperature is equal to or greater than 1, 1.5, 2, or more), and that show at least as much activity when lowered back to low temperature are determined to be "irreversible hits."

[0243] Example 5: Materials and methods for screening for conditionally active angiostatin mutants. Materials and methods for screening for conditionally active angiostatin mutants 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 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 Fl-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 (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 (HUVEC) can be isolated from human umbilical veins as described (Grant et al., "Matrigel induces thymosin h 4 gene in differentiating endothelial cells", J Cell Sci 1995;108:3685-94.). HUVEC cells are a cell line that expresses ATP synthase on the cell surface and can be used as a positive control. Cells can be cultured in DMEM (Life Technologies, Carlsbad, CA) with 1% penicillin-streptomycin and 10% serum replacement medium 3 (Sigma, St. Louis, MO) to minimize the presence of plasminogen. Low pH (6.7) media 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 media in 17% CO2. The method of reducing 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, N2 balance) versus normoxia (21% O2, 5% CO2) media of different pH (10, 22, and 44 mmol / L bicarbonate DMEM) for 0, 12, 24, 48, and 72 hours. Live cells can be blocked, incubated with anti-β subunit antibody, washed, blocked, incubated with secondary antibody goat anti-rabbit FITC (Southern Biotech, Birmingham, Alabama), 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 removing 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 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 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 in 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 by 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium, inner salt (MTS) proliferation assay in serum-free medium. After 20 hours of incubation at 37°C and 5% CO2 in the presence or absence of angiostatin, the relative cell numbers 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 per well) treated with angiostatin, angiostatin mutants, anti-β subunit antibody, rabbit IgG, cariporide, and Triton X (a detergent that permeabilizes cells and is used as a positive control) can be incubated at 37°C for 15 hours in 5% CO2 or 17% CO2 (neutral and low pH conditions, respectively). An index of cytotoxicity can be calculated by dividing the mean absorbance of quadruplicate treated samples by the mean absorbance of quadruplicate untreated samples in corresponding media of the same pH. Assessment of cell necrosis and apoptosis. To determine the effect of angiostatin in causing cell death, a histone-DNA ELISA can be performed. The effects of angiostatin, angiostatin mutants, anti-β subunit antibody, rabbit IgG, and cariporide on A549 cells (5,000 per well) can be determined using the 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 the reagents, apoptosis or necrosis can be determined from cell lysates or supernatants of quadruplicate samples, respectively. Apoptosis or necrosis can be 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 can be adjusted by incubation in 5% CO2 or 17% CO2.

[0250] Measurement of intracellular pH (pHi). pHi can be measured by fluorescence of cells plated on 35 mm microwell dishes with cover slips (MatTek, Ashland, MA). Cells can be plated on growth factor-reduced, phenol red-free Matrigel (BD Biosciences). After overnight incubation, medium is changed and cells are incubated with the pH-sensitive fluorescent dye cSNARF (Molecular Probes, Eugene, OR) for 15 min followed by recovery in fresh medium for 20 min. Cells are then mounted on a microscope platform and emission spectra are collected for 1 h at 37°C and 5% CO2, and pHi 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, and sodium-proton exchange inhibitor. The pH of the medium can be adjusted by bicarbonate concentration as described above in a fixed % CO2 atmosphere.

Claims

Claim 1 An in vitro method for identifying and selecting a modified antibody or an antigen-binding fragment thereof prepared by performing at least one of amino acid substitution, insertion or deletion on an unmodified antibody or an antigen-binding fragment thereof, wherein the modified antibody or an antigen-binding fragment thereof has conditional activity, and the binding activity of the antibody or an antigen-binding fragment thereof to a target in a tumor microenvironment with a pH less than 7.2 is greater than the binding activity to the same target in a normal physiological environment with a pH of 7.2 to 7.

6. The method comprises: 1) a) a step of testing the binding activity of a plurality of modified antibodies or antigen-binding fragments thereof under conditions with a pH less than 7.2; and b) a step of testing the binding activity of the same plurality of modified antibodies or antigen-binding fragments thereof under conditions with a pH of 7.2 to 7.6, wherein the binding activities of the plurality of modified antibodies or antigen-binding fragments thereof in a) and b) are tested in the presence of human serum, and all the test conditions of a) and b) are the same, and each modified antibody or antigen-binding fragment thereof is tested in each of a) and b), the step of testing; 2) a step of comparing the binding activity of a) with the binding activity of b); and 3) a step of selecting and identifying a modified antibody or an antigen-binding fragment thereof having a greater binding activity in a) compared to b), thereby identifying the antibody or an antigen-binding fragment thereof having conditional activity with a binding activity to a target in a tumor microenvironment with a pH less than 7.2 being greater than the binding activity to the same target in a normal physiological environment with a pH of 7.2 to 7.

6. A method comprising the above steps. Claim 2 The method according to claim 1, further comprising repeating steps 1) to 3) a plurality of times, and in each repetition, further modified antibodies or antigen-binding fragments thereof of the selected modified antibody or antigen-binding fragment thereof are prepared and tested, whereby the antibody or antigen-binding fragment thereof is developed to show an increase in activity at a pH lower than neutral pH. Claim 3 The method according to 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. Claim 4 In the method according to claim 1, the plurality of modified antibodies or antigen-binding fragments thereof are modified as compared with 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 contains a single amino acid substitution as compared with the unmodified antibody or antigen-binding fragment thereof, in the collection, the amino acid at each modification position is substituted with up to 1 to 19 other amino acids other than the original amino acid at that position, whereby each modified antibody or antigen-binding fragment thereof contains a different amino acid substitution, in the collection, all amino acids along the length of the antibody or antigen-binding fragment thereof, or a selected portion thereof, are substituted, Method.

5. In the method according to claim 1, the modified antibody or antigen-binding fragment thereof contains an amino acid substitution, and, the amino acid substitution is a substitution with an amino acid selected from Arg, His, and Lys. Method.

6. In the method according to any one of claims 1 to 5, the binding activity is evaluated by an immunoassay. Method.

7. In the method according to claim 6, the immunoassay includes ELISA. Method.

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

9. In the method according to claim 8, the binding of the modified antibody or antigen-binding fragment thereof to the target is detectably labeled or is detectable. Method.

10. In the method according to claim 9, the target is fluorescently labeled or is detected by a secondary reagent that is fluorescently labeled. Method.

11. In the method according to claim 9, the detection or measurement of the binding activity is performed by fluorescence-activated cell sorting (FACS). Method.

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

13. The method according to 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 according to 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 2.