Conditionally activated biological proteins

Abstract

Methods are provided for producing conditionally active biological proteins, particularly therapeutic or diagnostic proteins, that have greater activity under abnormal conditions compared to normal physiological conditions. The method includes a discovery method using a protein library and an assay utilizing physiological concentrations of body fluid components. The conditionally active biological proteins may be further developed to reduce activity by conjugating with other molecules, masking, or adding cleavable moieties. Selection criteria for the starting protein for the discovery method, as well as protein formats, are also disclosed.

Description

Technical Field

[0001] The present disclosure relates to the field of protein development and activity. Specifically, the present disclosure relates to a method for generating conditionally active biological proteins that are reversibly or irreversibly inactivated under wild-type normal physiological conditions from wild-type proteins, particularly therapeutic proteins. For example, the developed protein is substantially inactive at body temperature but active at lower temperatures.

Background Art

[0002] There are a number of documents describing the possibility of developing proteins in various characteristics, for example, particularly stabilizing enzymes for operation under different conditions. For example, enzymes have been developed to be stable at higher temperatures by changing their activity. In relation to improved activity at high temperatures, a substantial part of the improvement may be due to the high kinetic activity commonly described by the Q10 rule, which is estimated for enzymes where the metabolic turnover doubles for every 10-degree Celsius increase. In addition, there are examples of natural mutations that destabilize proteins under their normal operating conditions, such as the wild-type temperature activity of the molecule. For temperature mutants, these mutants can be active at low temperatures but typically have lower activity levels compared to the wild-type molecule. (Typically, it can also be explained by a decrease in activity derived from Ql0 or a similar law).

[0003] For example, it is desirable to generate useful molecules that are conditionally active and can be substantially inactive under wild-type conditions, but are active at levels equal to or better than wild-type conditions under conditions other than wild-type molecules, or are activated or inactivated in a specific microenvironment, or are activated or inactivated over time. In addition to temperature, other conditions under which proteins can be developed or optimized include pH, osmotic pressure, weight osmolarity, oxidative stress, and electrolyte concentration. Other desirable properties that can be optimized during development include chemical resistance and proteolytic resistance.

[0004] Many strategies for developing or manipulating molecules have already been published. However, manipulating or developing proteins to be inactive or substantially inactive (less than 10% activity, and especially less than 1% activity) under their wild-type operating conditions requires coexistence with activity that increases destabilizing mutations and mutations that do not counteract destabilizing effects, while maintaining activity equal to or better than that under wild-type conditions under new conditions. It is hypothesized that destabilization can reduce protein activity more significantly than the effect predicted by standard rules such as Q10, and therefore the ability to develop proteins that act effectively at low temperatures creates, for example, novel proteins that are inactive under their normal operating conditions, which we call conditionally active biological proteins.

[0005] Throughout this application, various publications are referenced by author and date. The disclosures of these publications in their entirety are incorporated by reference within this application to provide a more detailed description of the prior art known to those skilled in the art since the date of this disclosure described herein and claimed herein. [Overview of the project] [Means for solving the problem]

[0006] This disclosure provides a method for preparing a conditionally active biological protein, the method comprising the steps of: selecting a wild-type biological protein; developing the DNA encoding the wild-type biological protein using one or more developmental techniques to create mutant DNA; expressing the mutant DNA to obtain a mutant protein; subjecting the mutant protein and the wild-type protein to analysis under normal physiological conditions and under abnormal conditions; and selecting a conditionally active biological protein from the mutant protein that exhibits both (a) decreased activity compared to the wild-type protein in analysis under normal physiological conditions and (b) increased activity compared to the wild-type protein in analysis under abnormal conditions. In various embodiments, the normal physiological conditions are selected from one or more of temperature, pH, osmotic pressure, osmolality, oxidative stress, and electrolyte concentration. In a particular embodiment, the normal physiological condition is temperature, where the conditionally active biological protein is substantially inactive at normal physiological temperatures but active at abnormal temperatures lower than normal physiological temperatures. In other embodiments, the conditionally active biological protein is reversibly or irreversibly inactive under wild-type normal physiological conditions. In one particular embodiment, a protein is reversibly inactive under wild-type normal physiological conditions. On the other hand, conditionally active biological proteins are selected from these proteins that exhibit reversible or irreversible changes in activity under two or more different physiological conditions.

[0007] In one embodiment, the wild-type biological protein is an enzyme. In a particular embodiment, the wild-type biological protein is selected from the group consisting of tissue plasminogen activator, streptokinase, urokinase, renin, and hyaluronidase.

[0008] 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.

[0009] In another embodiment, the biological protein is an antibody.

[0010] In another embodiment, the disclosure provides a method for preparing a conditionally active biological reaction modifier, the method comprising: selecting an inflammatory response mediator; identifying a wild-type antibody against the mediator; developing the wild-type antibody; differentially screening for mutants that exhibit decreased binding to the mediator compared to the wild-type antibody under a first condition and increased binding affinity to the mediator under a second condition to identify an elevated mutant; recombining the heavy and light chains of the elevated mutant to create a recombinant elevated mutant; and screening the recombinant elevated mutant for mutants that exhibit decreased binding to the mediator compared to the wild-type antibody under a first condition and increased binding affinity to the mediator under a second condition to identify a conditionally active biological reaction modifier. In one embodiment, the inflammatory response mediator is selected from IL-6, IL-6 receptor, TNF-α, IL-23, and IL-12. In another embodiment, the first and second conditions are selected from conditions of pH, osmotic pressure, gravimetric osmolality, oxidative stress, and electrolyte concentration.

[0011] In another embodiment, the disclosure provides a pharmaceutical composition comprising a conditionally active biological protein and a pharmaceutically acceptable carrier. [Brief explanation of the drawing]

[0012] [Figure 1] This plot shows the conditionally activated antibodies selected in Example 9, and their selectivity at pH 6.0 compared to pH 7.4. [Modes for carrying out the invention]

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

[0014] As used herein in relation to a measured quantity, the term "approximately" refers to the normal variation in the measured quantity expected by a person skilled in the art who performs the measurement and exercises a level of care and precision appropriate to the purpose of the measurement. Unless otherwise specified, "approximately" refers to a variation of + / - 10% of the given value.

[0015] The term "activity," as used herein, refers to any function that a protein may perform, including catalysis of a reaction and binding to a partner. For enzymes, activity may refer to the enzymatic activity of the enzyme. For antibodies, activity may refer to the binding activity (i.e., binding affinity) between the antibody and its antigen. For receptors or ligands, activity may refer to the binding affinity between the receptor and its ligand.

[0016] The term “drug” is used to refer to chemical compounds, mixtures of chemical compounds, spatially localized sequences of compounds (e.g., VLSIPS peptide sequences, polynucleotide sequences, and / or combinatorial small molecule sequences), biomacromolecules, bacteriophage peptide display libraries, bacteriophage antibody (e.g., scFV) display libraries, polysomal peptide display libraries, or extracts made from biomaterials such as cells or tissues of bacteria, plants, fungi, or animals (especially mammals). Drugs are evaluated for their potential enzymatic activity as conditionally active biological therapeutic enzymes by being included in the screening analyses described below in this specification.

[0017] The “ambiguous base requirement” at the restriction site relates to a nucleotide base requirement that is not specified to the maximum extent. For example, it may be any one of at least two bases, but not a specific base (for example, a specific base selected from A, C, G, and T, but not limited to this example). Commonly accepted abbreviations used in the prior art as in this specification 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.

[0018] As used herein, the term “amino acid” refers to any organic compound containing an amino group (-NH2) and a carboxyl group (-COOH), preferably attached either as a free group or as a peptide portion after condensation. The “20 naturally encoded polypeptides that form alpha-amino acids” are known in the prior 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).

[0019] The term "amplification" refers to an increase in the copy number of polynucleotides.

[0020] A molecule possessing "chimeric properties" does not exclude the possibility of 1) being partially homologous and partially heterohomologous to a first reference molecule, 2) being partially homologous and partially heterohomologous to a second reference molecule, and 3) being partially homologous and partially heterohomologous to one or more additional reference molecules. In non-limiting embodiments, chimeric molecules may also be prepared by assembling a rearrangement of partial molecular sequences. In non-limiting embodiments, chimeric polynucleotide molecules may also be prepared by synthesizing chimeric polynucleotides using multiple molecular templates. As a result, the chimeric polynucleotides possess the properties of multiple templates.

[0021] As used herein, the term "homologous" refers to gene sequences that are developmentally and functionally related to each species. For example, but not limited to, in human genes, the human CD4 gene is homologous to the mouse 3d4 gene, the sequences and structures of these two genes exhibit high homology, and both genes encode proteins that function in signaling T cell activation through restricted antigen recognition of MHC class II.

[0022] As used herein, “comparison window” means a partial concept of at least 20 adjacent nucleotide sites, where the polynucleotide sequence is compared to at least 20 adjacent nucleotide reference sequences, 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 (without additions or deletions) for the optimal arrangement of the two sequences.The optimal placement of sequences for arranging the comparison window is 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 Mol Biol, “Identification of common molecular subsequences”, 1981, 147:195-197; Smith et al., 1981, “Comparative biosequence metrics”, J Mol Evol, 18:38-46) and 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 Mol The best arrangement may be derived from, or from, the best arrangement produced by, various selected methods (i.e., the arrangement that results in a high proportion of homology on the comparison window), by, or by, Biol, 48(3):443-453, by, Pearson's similarity search (Pearson and Lipman, 1988, “Improved tools for biological sequence comparison”, Proc Nat Acad Sci USA, 85:2444-2448), by, or by, computer implementation of, these algorithms (GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics Software Package Release 7.0, Genetics Computer Group, 575 Science Dr., Madison, Wis.), or by, or by, or by, or by, a best arrangement produced by, various selected methods (i.e., the arrangement that results in a high proportion of homology on the comparison window).

[0023] The term "conditionally active biological protein" means a variant or mutation of a wild-type protein that has more or less activity than one or more parent wild-type proteins under one or more normal physiological conditions. This conditionally active protein exhibits activity in selected regions of the body or shows an increase or decrease in activity under physiological conditions that are permissive to abnormalities or infections. Normal physiological conditions are the temperature, pH, osmotic pressure, osmolality, oxidative stress, and electrolyte concentrations that are considered within the normal range in tissues or organs at the site of administration or at the site of administration or site of action to the subject. Abnormal conditions refer to conditions that deviate from the normally acceptable range. In one aspect, the conditionally active biological protein is substantially inactive under wild-type conditions but is active under other wild-type conditions at a level equal to or better than wild-type conditions. For example, in diverse sites, the developed conditionally active biological protein is substantially inactive at body temperature but is active at low temperatures. In another aspect, the conditionally active biological protein is reversibly or irreversibly inactive under wild-type conditions. In a further aspect, the wild-type protein is a therapeutic protein. In another aspect, the conditionally active biological protein is used as a drug or therapeutic agent. In yet another aspect, the protein exhibits more or less activity in blood with a high oxygen concentration, such as after passing through the lungs, or at a low pH found in the kidneys.

[0024] "Conservative amino acid substitution" means the interchangeability of residues having similar side chains. For example, the group of amino acids having aliphatic side chains includes glycine, alanine, valine, leucine, and isoleucine; the group of amino acids having carboxylic acid side chains includes serine and threonine; the group of amino acids having side chains containing amide includes asparagine and glutamine; the group of amino acids having aromatic side chains includes phenylalanine, tyrosine, and tryptophan; the group of amino acids having basic side chains includes arginine and histidine; and the group of amino acids having sulfur-containing side chains includes cysteine and methionine. Preferred groups of conservative amino acid substitutions are valine-leucine-isoleucine, phenylalanine-tyrosine, lysine-arginine, alanine-valine, and asparagine-glutamine.

[0025] The term "corresponding" as used herein means that a polynucleotide sequence is identical (i.e., not necessarily strictly evolutionarily related) to all or part of a reference polynucleotide sequence or that the polynucleotide sequence is identical to the reference polynucleotide sequence. In contrast, the term "complementary" as used herein means that a complementary sequence is identical to all or part of a reference polynucleotide sequence. For example, the nucleotide sequence "TATAC" corresponds to the reference "TATAC" and is complementary to the reference sequence "GTATA".

[0026] The term "effective breakdown" amount relates to the amount of enzyme required to process at least 50% of a substrate as compared to the substrate not in contact with the enzyme.

[0027] As used herein, “defined sequence framework” refers to a defined set of sequences selected from non-random bases, generally 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, and in other variations, it may consist of a leucine zipper seven repeat motif, a zinc finger region. A “defined sequence kernel” is a set of sequences that encompass a limited range of variability. (1) A completely random 10-base sequence of 20 conventional amino acids may be any of the (20)10 sequences, and (2) a pseudo-random 10-base sequence of 20 conventional amino acids may be any of the (20)10 sequences, but will show a bias for certain residues at certain sites and / or as a whole, whereas (3) a defined sequence kernel is a subset of sequences such that each residue site is any of the 20 acceptable conventional amino acids. A defined sequence kernel generally has mutant or non-mutant residue sites, and / or mutant residue sites that may have residues selected from a defined subset of amino acid residues and their kind, either over the entire length or segmentally, of individual selected library member sequences. A defined sequence kernel may be associated with an amino acid sequence or a polynucleotide sequence. For example, but not limited to, sequences (NNK)10 and (NNM)10 are given, where N represents A, T, G, or C, K represents G or T, and M represents A or C, and sequences (NNK)10 and (NNM)10 are defined sequence kernels.

[0028] DNA “digestion” refers to the catalytic cleavage of DNA by restriction enzymes that act only on specific sequences within the DNA. The various restriction enzymes used herein are commercially available, and their reaction conditions, cofactors, and other requirements are known to those skilled in the art. For analytical purposes, typically 1 microgram of plasmid or DNA fragment is used with about 2 units of enzyme in about 20 microliters of buffer. For the purpose of separating DNA fragments for plasmid preparation, typically 5 to 50 micrograms of DNA are digested with a larger volume of enzyme, 20 to 250 units. The appropriate amounts of buffer and substrate for a particular restriction enzyme are specified by the manufacturer. Incubation at 37°C for about 1 hour is commonly used, but may vary according to the supplier's instructions. After digestion, the reaction is subjected to direct electrophoresis to separate and obtain the desired fragment.

[0029] "Directed ligation" means that ligation at the 5' and 3' ends of a polynucleotide is sufficiently different to identify a preferred ligation direction. For example, untreated and undigested PCR products, which originally have two blunt ends, typically do not have a preferred ligation direction when ligated in a cloning vector that is digested to generate blunt ends at multiple cloning sites. Therefore, directed ligation is not typically shown in these cases. In contrast, directed ligation is typically shown when digested PCR products having a 5'EcoRI-treated end and a 3'BamHI end are ligated in a cloning vector having multiple cloning sites digested with EcoRI and BamHI.

[0030] The term "DNA shuffling" is used herein to describe recombination between substantially homologous but non-identical sequences, and in some embodiments, DNA shuffling may include crossover via non-homologous recombination, such as via the cer / lox and / or flp / frt system.

[0031] The term "drug" or "drug molecule" means a therapeutic agent containing a substance that, when administered to the human or animal body, has a beneficial effect on the human or animal body. Preferably, a drug is one that can treat, cure or alleviate one or more symptoms, diseases, or abnormal conditions in the human or animal body, or that can promote the health of the human or animal body.

[0032] "Effective dose" refers to the amount of a conditionally active biological protein or fragment that is effective in treating or preventing a condition in a living organism of a person administered it over a certain period of time, for example, in providing a therapeutic effect during a desired period of administration.

[0033] As used herein, the term “electrolyte” is used to define minerals in blood or other body fluids that carry electric charge. For example, in one embodiment, normal physiological conditions and abnormal conditions may be conditions of “electrolyte concentration.” In one embodiment, the electrolyte concentration to be tested is selected from one or more of the concentrations of ionized calcium, sodium, potassium, magnesium, chloride, bicarbonate, and phosphate. For example, in one embodiment, the normal range for serum calcium is 8.5 to 10.2 mg / dL. In this embodiment, the abnormal serum calcium concentration may be selected from above or below the normal range. In another embodiment, serum chloride is 96 to 106 milligram equivalents (mEq / L) per liter. In this embodiment, the abnormal serum chloride concentration may be selected from above or below the normal range. In another embodiment, serum magnesium concentration is 1.7 to 2.2 mg / dL. In this embodiment, the abnormal serum magnesium concentration may be selected from above or below the normal range. In other embodiments, in one aspect, the normal range for serum phosphate is 2.4–4.1 mg / dL. In this embodiment, abnormal serum phosphate concentrations may be selected from above or below the normal range. In other embodiments, in one aspect, the normal range for serum or blood sodium is 135–145 mEq / L. In this embodiment, abnormal serum or blood sodium concentrations may be selected from above or below the normal range. In other embodiments, in one aspect, the normal range for serum or blood potassium is 3.7–5.2 mEq / L. In this embodiment, abnormal serum or blood potassium concentrations 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, abnormal serum or blood bicarbonate concentrations may be selected from above or below the normal range. In different embodiments, bicarbonate levels may be used to indicate the normal level of acidity (pH) in the blood. The term “electrolyte concentration” may also be used to define specific electrolyte concentrations in tissues or body fluids other than blood or plasma. In this case, normal physiological conditions are considered to be within the clinically normal range for the tissue or body fluids in question.In this embodiment, abnormal tissue or fluid electrolyte concentrations may be selected from above or below the normal range.

[0034] As used in this disclosure, the term “antigenic determinant” refers to antigenicity-determining elements on an antigen, such as enzyme polypeptides, or to the antigen-binding site of an antibody, such as an enzyme-specific antibody. Antigenic determinants typically consist of a chemical surface activity group of molecules, such as amino acids or their side chains, and their specific three-dimensional structural properties may also have specific charge properties. As used herein, “antigenic determinant” means other macromolecules that can form binding interactions with the variable region that binds to that portion of an antigen or the body of an antibody. Typically, such binding interactions manifest as intermolecular interactions with amino acid residues of one or more CDRs.

[0035] As used herein, “enzyme” refers to a protein possessing specific catalytic properties. 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) indicates an increase in the reaction rate for every 10°C increase in temperature. In wild-type enzymes, Q10 = 2-3, meaning 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's optimal value, small changes occur in the charge of the enzyme and possibly the substrate molecule. Changes in ionization can affect the binding of the substrate molecule. At extreme pH levels, the enzyme denatures, where the active site is distorted and the substrate site is no longer compatible.

[0036] As used herein, the terms “develop” or “to develop” mean using a mutagenesis method to produce a novel polynucleotide encoding one or more novel polypeptides, the novel polypeptide being the improved biomolecule itself and / or contributing to the production of other improved biomolecules. In certain non-limiting embodiments, this disclosure relates to the development of a conditionally active biological protein from a parent wild-type protein. In one embodiment, for example, development means a method that performs both non-stochastic polynucleotide chimeration and non-stochastic mutation at a directed site, as disclosed in U.S. Patent Application Publication No. 2009 / 0130718. More specifically, this disclosure provides a method for developing a conditionally active biological enzyme that exhibits reduced activity compared to the wild-type parent enzyme under normal physiological conditions but enhances activity compared to the wild-type enzyme under one or more abnormal conditions.

[0037] The terms “fragment,” “derivative,” and “similar organ” refer to a reference polypeptide that has at least one polypeptide that possesses at least essentially the same physiological function or activity as the reference polypeptide. Furthermore, the terms “fragment,” “derivative,” and “similar organ” are exemplified by “pre-morphological” molecules such as low-activity precursor proteins that can be modified by cleavage to produce mature enzymes with significantly higher activity.

[0038] A method for generating progeny polypeptides from a template polypeptide is provided herein, wherein the "entire range of single amino acid substitutions" is indicated for each amino acid site. As used herein, the "entire range of single amino acid substitutions" refers to 20 naturally encoded polypeptides that form alpha-amino acids as described herein.

[0039] The term "gene" refers to a DNA segment involved in the formation of a polypeptide chain, including the regions preceding and following the coding region (leader and trader), as well as intervening sequences (nitrons) between individual coding segments (exons).

[0040] As used herein, “genetic instability” means the natural tendency of highly repetitive sequences to be lost through processes of reduction events, which typically involve sequence simplification by the loss of repetitive sequences. Deletions may include the loss of one copy of a repeat and all copies between repeats.

[0041] The term "non-homologous" means that a single-stranded nucleic acid sequence cannot hybridize with another single-stranded nucleic acid sequence or its complementary sequence. Therefore, a non-homologous region means that a polynucleotide or polynucleotide has a portion or region in its sequence that cannot hybridize with another nucleic acid or polynucleotide. Such a region or portion is, for example, a mutated portion.

[0042] The term "homologous" means that a single-stranded nucleic acid sequence can hybridize to a complementary single-stranded nucleic acid sequence. The degree of hybridization may depend on the amount of identity between the sequences and a number of factors, including hybridization conditions such as temperature and salt concentration, as described later. Preferably, the identical region is larger than about 5 bp, and more preferably, the identical region is larger than 10 bp.

[0043] The benefits of this disclosure extend to “industrial applications” (or industrial processes), and the term is used to include appropriate commercial industrial (or simply industrial) applications as well as non-commercial industrial applications (e.g., orthodontic research in non-profit organizations). Relevant applications include the fields of diagnostics, medicine, agriculture, manufacturing, and the academic world.

[0044] "Identical" or "identical" means that two nucleic acids have the same or complementary sequence. Therefore, "identical portion" means that a region or portion of a polynucleotide, or an entire polynucleotide, is identical or complementary to a portion of another polynucleotide.

[0045] The term "isolated" means that the material is removed from its original environment (for example, the natural environment if it occurs naturally). For example, naturally occurring polynucleotides or enzymes present in living animals are not isolated, but the same polynucleotide or enzyme is isolated if it has been separated from some or all of the materials that coexist in the natural system. Such polynucleotides may be part of a vector, and / or such polynucleotides or enzymes may be part of a composition, and such vectors or compositions are still isolated in that they are not part of their natural environment.

[0046] The term “isolated nucleic acid” is used to define nucleic acids, such as DNA or RNA molecules. Nucleic acids, such as DNA or RNA molecules, do not directly adjoin the 5' and 3' facies that are normally directly adjacent when they exist in the spontaneously occurring genes of the organism from which they are induced. Therefore, the term refers to nucleic acids incorporated into vectors, such as plasmids or viral vectors, nucleic acids integrated into the genes of heterologous cells (or in heterologous cells, but at a site different from that of spontaneous generation), and nucleic acids that exist as isolated molecules, such as DNA fragments produced by PCR amplification or restriction enzyme digestion, or RNA molecules produced by in vitro transcription. The term also refers to recombinant nucleic acids that form part of a hybrid gene encoding additional proteins, which can be used, for example, in the production of fused proteins.

[0047] As used herein, “ligand” means a molecule recognized by a specific receptor, such as a random peptide or a variable segment sequence. As those skilled in the art will recognize, a molecule (or polymeric complex) can be both a receptor and a ligand. Generally, binding pairs with smaller molecular weights are called ligands, and binding pairs with larger molecular weights are called receptors.

[0048] "Ligation" refers to a method of forming a phosphate diester bond between two double-stranded nucleic acid fragments (Sambrook et al., (1982). Molecular Cloning: A Laboratory Manual. Cold Spring Harbour Laboratory, Cold Spring Harbor, NY., p.146; Sambrook et al., Molecular Cloning: a laboratory manual, 2nd Ed., Cold Spring Harbor Laboratory Press, 1989). If not by the method provided, ligation may also be achieved using known buffers and conditions with 10 units of T4 DNA ligase ("ligase") per 0.5 micrograms of approximately equimolar amounts of the DNA fragment to be ligated.

[0049] When used herein, a “linker” or “spacer” helps to link two molecules, such as a protein and DNA that binds a random peptide, and to position the two molecules into a preferred configuration, so that the random peptide can bind to a receptor with minimal steric hindrance from the DNA that binds the protein.

[0050] As used herein, “microenvironment” means any part or area of ​​tissue or body that has physical or chemical differences, at any given time or in any given period, from other areas of tissue or body.

[0051] As used herein, “developing molecular properties” includes referring to molecules consisting of polynucleotide sequences, molecules consisting of polypeptide sequences, and molecules consisting of a portion of a polynucleotide sequence and a portion of a polypeptide sequence. In particular, but not limited to, examples of developing molecular properties include protein activity under specific conditions such as temperature, salinity, osmotic pressure, pH, oxidative stress, and concentrations of glycerol, DMSO, detergents, and / or any other types of molecules brought into contact in the reaction environment. In addition, but not limited to, examples of developing molecular properties include stability, for example, the amount of residual molecular properties after a specified exposure time in a specified environment.

[0052] 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 mutations can be point mutations, such as transpositions or base changes. Mutations can also be deletions, insertions, or replications.

[0053] As used herein, the degenerate "N,N,G / T" nucleotide sequence represents 32 possible triplets, where "N" may be A, C, G, or T.

[0054] The term "spontaneous generation," as used herein, applies in relation to the fact that the subject is found in nature. For example, a polypeptide or polynucleotide sequence present in an organism (including a virus) that can be isolated from raw materials in nature and has not been intentionally modified by a human in a laboratory is spontaneous generation. In general, the term "spontaneous generation" relates to subjects that are present in non-pathological (not diseased) individuals, as is typical in species.

[0055] As used herein, “normal physiological conditions” or “wild-type operating conditions” refer to the temperature, pH, osmotic pressure, osmolality, oxidative stress, and electrolyte concentration conditions that are considered to be within the normal range at the site of administration or action to the subject.

[0056] As used herein, “nucleic acid molecule” consists of at least one nucleotide or one nucleotide pair, depending on whether it is single-stranded or double-stranded. Furthermore, nucleic acid molecules may belong only to any group of nucleotides, or chimerically, including, but not limited to, a group of nucleic acid molecules such as RNA, DNA, genetic nucleic acids, non-genetic nucleic acids, spontaneously occurring and non-spontaneously occurring nucleic acids, and synthetic nucleic acids. This includes, as non-limited examples, nucleic acids associated with any organelle, such as mitochondria, ribosomal RNA, and nucleic acid molecules that are chimeric from one or more spontaneously occurring and non-spontaneously occurring components.

[0057] In addition, "nucleic acid molecules" may, in a non-limiting sense, include components that are not nucleotide-based, such as one or more amino acids and sugars. Therefore, as an example and not limiting, ribozymes that are partially nucleotide-based and partially protein-based are considered "nucleic acid molecules."

[0058] In addition, but not limited to, examples include nucleic acid molecules labeled by a detectable portion, such as a radioactive or non-radioactive label, which are also considered “nucleic acid molecules.”

[0059] The term "nucleotide sequence encoding ~," or "DNA encoding ~," or "nucleotide sequence encoding ~," particularly nucleotide sequences encoding enzymes—as with other synonymous terms—refers to DNA sequences that are transcribed and converted into enzymes under the control of appropriate regulatory sequences. A "promoter sequence" is a DNA regulatory region within a cell that allows RNA polymerase to bind and initiate downstream (3' direction) transcription that encodes the sequence. The promoter is a portion of a DNA sequence. This sequence region has a start codon at its 3' end. The promoter sequence contains a minimal number of bases necessary to initiate transcription at a detectable level higher than the background. However, once RNA polymerase binds the sequence and transcription begins at the start codon (the 3' end containing the promoter), transcription proceeds downstream in the 3' direction. Within the promoter, the sequence is found at the transcription start site (defined for convenience by mapping with nuclease S1), as well as at the protein that binds the region (common sequence) that bears the binding of RNA polymerase.

[0060] The terms “enzyme-encoding nucleic acid,” “enzyme-encoding DNA,” or “enzyme-encoding polynucleotide,” and other synonyms encompass both polynucleotides containing only the coding sequence for the enzyme and polynucleotides containing additional coding sequences and / or non-coding sequences.

[0061] In one preferred embodiment, the “specific nucleic acid species” is defined by its chemical structure, as exemplified by its primary sequence, but is not limited thereto. In another preferred embodiment, the “specific nucleic acid species” is defined by the function of the nucleic acid species or by the function of a product derived from the nucleic acid species. Thus, in non-limiting examples, the “specific nucleic acid species” is defined by one or more activities or properties resulting therefrom, including activities or properties resulting from the products expressed therefrom.

[0062] The immediate definition of "constructing a working nucleic acid sample in a nucleic acid library" includes the step of incorporating the nucleic acid sample into a vector based on collection, such as by ligation within the vector and host conversion. Descriptions of relevant vectors, hosts, and other reagents, as well as their specific and non-limiting examples, are provided below. The immediate definition of "constructing a working nucleic acid sample in a nucleic acid library" also includes the step of incorporating the nucleic acid sample into a non-vector based on collection, such as by ligation to an adherend. Preferably, the adherend can be annealed to PCR primers to facilitate amplification by PCR.

[0063] In a non-limiting embodiment, the "nucleic acid library" consists of a vector based on a collection of one or more nucleic acid molecules. In another preferred embodiment, the "nucleic acid library" consists of a non-vector based on a collection of nucleic acid molecules. In yet another preferred embodiment, the "nucleic acid library" consists of a combination of a collection of nucleic acid molecules that is partially vector-based and partially non-vector-based. Preferably, the collection of molecules comprising the library is searchable and separable according to the type of individual nucleic acid molecule.

[0064] This disclosure provides “nucleic acid constructs,” “nucleotide constructs,” or “DNA constructs.” The term “construct” is used herein to describe molecules such as polynucleotides (e.g., enzymatic polynucleotides) that may optionally be chemically bonded to one or more additional molecular portions, such as vectors or portions of vectors. In particular embodiments, without presumably limiting the embodiments, nucleotide constructs are exemplified by DNA expression constructs suitable for transformation of host cells.

[0065] "Oligonilocytes" (or synonymous with "oligo") refer to single-stranded polydeoxynucleotides or chemically synthesized complementary polydeoxynucleotide chains. Such synthetic oligonucleotides may or may not have a 5' phosphate. They do not ligate to other oligonucleotides without the addition of ATP and 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), "32-fold denatured oligonucleotides consisting of at least a first homologous sequence, a denatured N, N, G / T sequence, and a second homologous sequence in sequence" are mentioned. As used in this context, "homologous" refers to homology between the oligo and the parent polynucleotides subjected to polymerase-based amplification.

[0066] As used herein, the term “operable binding” means the binding of polynucleotide elements in a functional relationship. Nucleic acids are “operable binding” when they are placed in a functional relationship with other nucleic acid sequences. For example, a promoter or enhancer is operablely bound to a coding sequence if it affects the transcription of that coding sequence. Operable binding means that the bound DNA sequences are typically adjacent and, if necessary to join two protein coding regions, adjacent and within a reading frame.

[0067] When RNA polymerase transcribes two coding sequences in a single mRNA molecule, the coding sequences "bind in an operable state" to the other coding sequence and are translated into a single polypeptide containing amino acids derived from both coding sequences. The coding sequences do not need to be adjacent to each other, as long as the expressed sequences are ultimately processed to produce the desired protein.

[0068] As used herein, the term “parental polynucleotide set” consists of one or more different polynucleotide species. Typically, this term is used to refer to a descendant polynucleotide set obtained by mutagenesis of a parental set, in which case the terms “parental,” “startup,” and “template” are interchangeable.

[0069] The terms "patient" or "subject" refer to an animal, such as a human or mammal, that is the target of treatment. A subject or patient can be male or female.

[0070] As used herein, the term “physiological conditions” means biochemical parameters normally present intracellularly in viable cultured yeast or mammalian cells, such as temperature, pH, osmotic pressure, ionization strength, and viscosity, that are suitable for viable cells. For example, intracellular conditions in yeast cells grown under typical experimental culture conditions are physiological conditions. Appropriate in vitro reaction conditions in an in vitro transcription cocktail are normal physiological conditions. Generally, in vitro physiological conditions include 50-200 mM NaCl or KCl, pH 6.5-8.5, 20-45°C, and 0.001-10 mM divalent cations (e.g., Mg++, Ca++); preferably, about 150 mM NaCl or KCl, pH 7.2-7.6, and 5 mM divalent cations, often containing 0.01-1.0% nonspecific proteins (e.g., BSA). Nonionic surfactants (Tween, NP-40, Triton X-100) are often present, usually around 0.001-2%, typically 0.05-0.2% (v / v). Detailed aqueous solution conditions can be selected by the physician using conventional methods. As a general guideline, the following buffered aqueous solution conditions can be applied: 10-250 mM NaCl, 5-50 mM Tris-HCl, pH 5-8, and optionally the addition of divalent cations and / or metal chelating agents and / or nonionic surfactants and / or membrane fractions and / or antifoaming agents and / or scintillants. Normal physiological conditions refer to temperature, pH, osmotic pressure, osmolality, oxidative stress, and electrolyte concentrations at the administration or action site in the patient or subject's body that can be considered within the normal range for the patient.

[0071] The standard (5'-3') is used herein to describe the sequence of double-stranded polynucleotides.

[0072] The term "population" refers to a collection of compositions such as polynucleotides, polynucleotide segments, or proteins. A "mixed population" is a collection of compositions that belong to the same genus (i.e., related) of nucleic acids or proteins, but differ in their sequence (i.e., are not identical), and therefore differ in their physiological and intellectual activity.

[0073] A molecule having a "substitute form" is 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 higher-order structural changes, association with cofactors, etc.) in order to obtain a more mature molecular form with different properties (e.g., increased activity) compared to a reference substitute form molecule. A reference precursor molecule is called a "precursor substitute form" molecule when two or more chemical modifications (e.g., two proteolytic cleavages, or proteolytic cleavage and non-glycosylation) can be distinguished in the process of producing a mature molecule.

[0074] As used herein, the term “protein” refers to a polymer in which monomers are amino acids, linked together by peptide bonds or disulfide bonds. “Protein” refers to a full-length, naturally occurring amino acid chain or fragment thereof, for example, a specific region of a polypeptide that is beneficial in binding interactions, or a synthetic amino acid chain, or a combination thereof. Thus, the fragment refers to an amino acid sequence that is part of a full-length protein, with a length of about 8 to about 500 amino acids, preferably about 8 to about 300 amino acids, more preferably about 8 to about 200 amino acids, and even more preferably about 10 to about 50 or 100 amino acids. In addition, amino acids other than naturally occurring amino acids, such as β-alanine, phenylglycine, and homoarginine, may be included in proteins. Commonly found non-gene-coding amino acids may also be used in the present invention. All amino acids used in the present invention may be either D-type or L-type optical isomers. D-type isomers are preferred for use in certain contexts, which are described in detail below. In addition, other peptidomimetics are also useful, for example, in the linker sequences of the polypeptides of the present invention (see Spatola, 1983, Chemistry and Biochemistry of Amino Acids. Peptides and Proteins, Weinstein, ed., Marcel Dekker, New York, p. 267). In general, the term "protein" is not intended to convey any significant difference from the term "polypeptide" other than that it encompasses structures containing two or more polypeptide chains held together by covalent or noncovalent bonds.

[0075] As used herein, the term “pseudorandom” means a set of sequences with limited variability. For example, the degree of residue variability at other positions is limited, although it gives a certain degree of residue variation other than at any pseudorandom position.

[0076] As used herein, a “quasi-repeat unit” refers to a reassembled repeat that is not identical by definition. In practice, this method proposes not only the reassemblies of actually identical encoding units produced by mutagenesis of the same starting sequence, but also the reassemblies of similar or related sequences that may diverge significantly in some regions. Nevertheless, if a sequence contains enough homology to be reassembled by this method, it can be called a “quasi-repeat” unit.

[0077] As used herein, “random peptide library” means a set of polynucleotide sequences encoding a set of random peptides, and a set of random peptides encoded by these polypeptide sequences, as well as a fused protein containing these random peptides.

[0078] As used herein, "random peptide sequence" means an amino acid sequence consisting of two or more amino acid monomers and constructed by a probabilistic or random process. A random peptide may include a framework or scaffolding material and may have an invariant sequence.

[0079] As used herein, “receptor” means a molecule that has affinity for a given ligand. Receptors may be spontaneously occurring or synthetic molecules. Receptors may be used in an immutable state or as aggregates with other species. Receptors may be covalently or noncovalently bound to binding members directly or via specific binding agents. Examples of receptors include, but are not limited to, antiserum reagents with monoclonal antibodies and specific antigenic determinants (e.g., viruses, cells, or other materials), cell membrane receptors, sugar and glycoprotein complexes, enzymes, and hormone receptors.

[0080] "Recombinant" enzymes are enzymes produced by recombinant DNA technology, i.e., from cells transformed with an exogenous DNA configuration that encodes the desired enzyme. "Synthetic" enzymes are prepared by chemical synthesis.

[0081] The term "related polynucleotide" means that the regions or parts of a polynucleotide are identical, or that the regions or parts of a polynucleotide are non-homologous.

[0082] As used herein, “decreasing reassembly” means an increase in molecular diversity resulting from deletions (and / or insertions) mediated by repeating sequences.

[0083] The following terms, “reference sequence,” “comparison window,” “sequence identity,” “sequence identity ratio,” and “substantially identical,” are used to describe the sequence relationship between two or more polynucleotides.

[0084] A “reference sequence” is a defined sequence used as the 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 list, or a complete cDNA or gene sequence. Generally, a reference sequence is at least 20 nucleotides in length, often at least 25 nucleotides, and often at least 50 nucleotides. Since two polynucleotides may each consist of (1) sequences similar between the two polynucleotides (i.e., a portion of a complete polynucleotide sequence) and (2) sequences that further branch between the two polynucleotides, sequence comparison between two (or more) polynucleotides is performed by comparing the sequences of the two polynucleotides on a “comparison window” to identify and compare local regions of sequence similarity.

[0085] The "Repetition Index (RI)," as used herein, is the average number of copies of a quasi-repeating unit contained within a cloning vector.

[0086] The term "restriction site" refers to the recognition sequence necessary for the action of a restriction enzyme, and includes the site of contact cleavage. It is recognized that the cleavage site may or may not be present in the portion of the restriction site consisting of low-ambiguity sequences (i.e., sequences containing the main determinants of the frequency of restriction site occurrence). Therefore, in many cases, the relevant restriction site consists only of low-ambiguity sequences having an internal cleavage site (e.g., G / AATTC in the EcoRI site) or a directly adjacent cleavage site (e.g., / CCWGG in the EcoRII site). In other cases, the relevant restriction enzyme (e.g., the Eco57I site or CTGAAG(16 / 14)) contains a low-ambiguity sequence (e.g., the CTGAAG sequence in the Eco57I site) having an external cleavage site (e.g., in the N.Sub.16 portion of the Eco57I site). When an enzyme (e.g., a restriction enzyme) "cleaves" a polynucleotide, it is understood that the restriction enzyme catalyzes or facilitates the cleavage of the polynucleotide.

[0087] 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, the 5' terminal region is the region located toward the 5' polynucleotide terminal (or 5' polynucleotide termination). Thus, it is a portion or the whole of the 5' half of the polynucleotide. Similarly, the 3' terminal region is the region located toward the 3' polynucleotide terminal (or 3' polynucleotide termination). Thus, it is a portion or the whole of the 3' half of the polynucleotide. As used in this non-limiting example, there may be sequence overlaps between any two regions or between all three regions.

[0088] The term "sequence identity" means that two polynucleotide sequences are identical across a comparison window (i.e., for each nucleotide). The term "sequence identity percentage" is calculated by comparing two optimally aligned sequences across a comparison window, determining the number of positions in both sequences where the same nucleic acid base (e.g., A, T, C, G, U, or I) appears to determine the number of match positions, dividing that number of match 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 sequence identity percentage. When used herein, “substantial identity” refers to a characteristic of a polynucleotide sequence, where the polynucleotide includes sequences having at least 80 percent sequence identity, preferably at least 85 percent, often 90 to 95 percent, and most commonly at least 99 percent, when compared to a reference sequence over a comparison window of at least 25 to 50 nucleotides, where the sequence identity percentage is calculated by comparing the reference sequence with a polynucleotide sequence that may contain no more than 20 percent of the total deletions or additions of the reference sequence over the comparison window.

[0089] As is well known in the art, the "similarity" between two enzymes is determined by comparing the amino acid sequence and its conserved amino acid substitutions of one enzyme with the sequence of the second enzyme. Similarity can be determined by procedures well known in the art, such as the BLAST program (Basic Local Alignment Search Tool of the National Center for Biological Information).

[0090] Members of a molecular pair (e.g., an antibody-antigen pair or a nucleic acid pair) are said to be "specifically bound" to each other when they bind to one another with a stronger affinity than to other nonspecific molecules. For example, an antibody can be described as specifically binding to an antigen because it binds to the antigen more efficiently than nonspecific proteins. (Similarly, a nucleic acid probe can be described as specifically binding to a target nucleic acid if it forms a specific double helix with the target through base-pair interactions (see above)).

[0091] "Specific hybridization" is defined herein as a form of cross between a first polynucleotide and a second polynucleotide (for example, a polynucleotide having a different but substantially identical sequence from the first polynucleotide), where substantially unrelated polynucleotide sequences do not form a cross in the mixture.

[0092] The term "specific polynucleotide" refers to a polynucleotide that has a specific endpoint and a specific nucleic acid sequence. In two polynucleotides, one polynucleotide has the same sequence as the second polynucleotide, but the different ends consist of two different specific polynucleotides.

[0093] "Strict hybridization conditions" mean that hybridization occurs only when the sequences are at least 90% identical, preferably at least 95% identical, and most preferably at least 97% identical. See Sambrook et al., Molecular Cloning: A Laboratory Manual, 2nd Ed., Cold Spring Harbor Laboratory Press, 1989.

[0094] The disclosure also includes polypeptides having sequences that are "substantially identical" to those of enzyme polypeptides. A "substantially identical" amino acid sequence is one that differs from the reference sequence only by a conserved amino acid substitution, for example, a substitution of one other amino acid of the same type (e.g., a substitution from one hydrophobic amino acid to another, such as isoleucine, valine, leucine, or methionine, or a substitution from one polar amino acid to another, such as arginine to lysine, glutamic acid to aspartic acid, or glutamine to asparagine).

[0095] In addition, a “substantially identical” amino acid sequence is a sequence that differs from the reference sequence, or a sequence that differs due to one or more non-conservative substitutions, deletions, or insertions, such as non-conservative substitutions, deletions, or insertions, when such substitutions occur at a site other than the active site of the molecule, and is defined as a sequence that essentially retains the behavioral properties of the polypeptide. For example, one or more amino acids may be removed from an enzyme polypeptide, resulting in a modification of the polypeptide structure without significantly altering its biological activity. For instance, amino- or carboxyl-terminal amino acids that are not required for the physiological activity of the enzyme may be removed. Such modifications can lead to the development of smaller active enzyme polypeptides.

[0096] This disclosure provides “substantially pure enzymes.” The term “substantially pure enzyme” is used herein to describe molecules such as polypeptides (e.g., enzyme polypeptides, or fragments thereof) that are substantially free of other naturally associated proteins, lipids, sugars, nucleic acids, and other physiological materials. For example, a substantially pure molecule, such as a polypeptide, may be at least 60% by dry weight in the molecule in question. The purity of a polypeptide is 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.

[0097] As used herein, “substantially pure” means that the species in question is the dominant species (i.e., abundant in the composition on a molar basis compared to any other distinct molecule), and preferably, a substantially purified fragment is a composition consisting of at least about 50% (on a molar basis) of all polymer species in which the species in question exists. Generally, a substantially pure composition consists of about 80-90 percent or more of the polymer species present in the composition. Most preferably, the species in question is purified to a substantially homogeneous degree (contaminant species cannot be detected in the composition by conventional detection methods), where the composition consists essentially of a single polymer species. Solubilous species, small molecules (<500 Daltons), and basic ionic species are not considered polymer species.

[0098] The term “to treat” means (1) preventing or delaying the appearance of the clinical symptoms of a progressive state, disease, or condition in an animal that suffers from or is susceptible to the clinical or latent symptoms of a state, disease, or condition but has not yet experienced or shown the clinical or latent symptoms of the state, disease, or condition; (2) inhibiting the state, disease, or condition (i.e., inhibiting, reducing, or delaying their relapse or the progression of at least one clinical or latent symptom in the case of disease or maintenance therapy); and / or (3) alleviating the state (i.e., causing a regression of at least one clinical or latent symptom of the state, disease, or condition). The benefit to the treated patient is statistically significant, or at least recognizable to the patient or physician.

[0099] As used herein, the term “variable segment” means a portion of a developing peptide consisting of random, pseudorandom, or defined kernel sequences. A variable segment may consist of both mutant and non-mutant residue positions, and the degree of mutant residues at mutant residue positions may be limited, with both options being chosen at the discretion of the practitioner. Typically, a variable segment is about 5–20 amino acid residues (e.g., 8–10) in length, however, variable segments may be longer and may consist of antibody proteins or receptor proteins, such as antibody fragments, protein-binding nucleic acids, or receptor proteins.

[0100] The term “mutant” means a polynucleotide or polypeptide of the disclosure modified at base pairs, codons, introns, exons, or amino acid residues (each) of one or more wild-type protein parent molecules. Mutants are produced by any number of means, including, for example, error-prone PCR, shuffling, oligonucleotide-designated mutation, assembly PCR, sex-PCR mutation, in vivo mutation, cassette mutation, recurrent ensemble mutation, exponential ensemble mutation, site-directed mutation, gene rearrangement, saturation mutation, and any combination thereof. Techniques for producing mutant proteins in which activity is reduced under one or more normal physiological conditions, such as temperature, pH, osmotic pressure, osmolality, oxidative stress, and electrolyte concentration, and enhanced under abnormal conditions, compared to wild-type proteins, are disclosed herein. Mutants are additionally selected for their properties of enhancing chemical and proteolytic resistance compared to wild-type proteins.

[0101] As used herein, the term “wild-type” means that the polynucleotide is free from any mutations. “Wild-type protein,” “wild-type biological protein,” or “wild-type biological protein” refers to a protein that can be isolated from nature, is active at naturally occurring levels of activity, and contains naturally occurring amino acid sequences. The terms “parent molecule” and “target protein” also refer to wild-type proteins.

[0102] For example, the term "working" in "working sample" simply refers to a sample that a person is working on. Similarly, "working molecule" refers to a molecule that a person is working on.

[0103] The term "conditionally activated antibody" refers to a variant or mutant of a wild-type antibody that exhibits higher or lower activity compared to the parent wild-type antibody under one or more normal physiological conditions. This conditionally activated antibody may exhibit activity in specific areas of the body and / or increased or decreased activity under abnormal or tolerable physiological conditions. In one embodiment, the conditionally activated antibody is virtually inactive under normal physiological conditions but is more active under other conditions than it is under normal physiological conditions. For example, in one embodiment, a developed conditionally activated antibody may be virtually inactive at body temperature but active at lower temperatures. In another embodiment, the conditionally activated antibody may be reversibly or irreversibly inactivated under normal physiological conditions. In yet another embodiment, the wild-type antibody is a therapeutic antibody. In yet another embodiment, the conditionally activated antibody is used as a drug or therapeutic agent. In yet another embodiment, the antibody is more or less active in hyperoxygenated blood, such as after passing through the lungs, or in lower pH environments found in the kidneys.

[0104] The term "antibody-dependent cell-mediated cytotoxicity" or "ADCC" refers to a form of cytotoxicity in which secretory immunoglobulins bind to Fc receptors (FcRs) present on specific cytotoxic cells (e.g., natural killer (NK) cells, neutrophils, and macrophages), thereby enabling these cytotoxic effector cells to specifically bind to antigen-carrying target cells and subsequently kill them with cytotoxicity. Ligand-specific high-affinity IgG antibodies against the surface of target cells stimulate the cytotoxic cells and are necessary for such cell death. Lysis of target cells occurs extracellularly, requires direct cell-to-cell contact, and does not involve complement.

[0105] The ability of any particular antibody to mediate the lysis of target cells by ADCC can be analyzed. To assess ADCC activity, the antibody of interest is added to target cells presenting the target ligand in conjunction with immune effector cells. When the immune effector cells are activated by the antigen-antibody complex, cell lysis of the target cells may occur. Cell lysis is generally detected by the release of labels (e.g., radioactive substrates, fluorescent dyes, or innate intracellular proteins) from the lysed cells. Effector cells useful for such analysis include peripheral blood mononuclear cells (PBMCs) and natural killer (NK) cells. Specific examples of in vitro ADCC analysis are described in Bruggemann et al, 1987, J. Exp. Med., vol. 166, page 1351; Wilkinson et al, 2001, J. Immunol. Methods, vol. 258, page 183; and Patel et al, 1995, J. Immunol. Methods, vol. 184, page 29. Alternatively, or in addition, the ADCC activity of the antibody of interest may be evaluated in vivo, for example, in an animal model, as disclosed in Clynes et al, 1998, PNAS USA, vol. 95, p. 652.

[0106] The terms “cancer” and “malignant” refer to or describe a physiological condition in mammals typically characterized by uncontrolled cell growth / proliferation. “Tumor” includes one or more cancerous cells. Examples of cancer include, but are not limited to, carcinomas, lymphomas, blastomas, sarcomas, and leukemias or lymphoid malignancies. More specific examples of such cancers include squamous cell carcinoma (e.g., epithelial squamous cell carcinoma), lung cancer (including small cell lung cancer, non-small cell lung cancer ("NSCLC"), lung adenocarcinoma and lung squamous cell carcinoma), peritoneal cancer, hepatocellular carcinoma, gastric cancer or stomach cancer (including gastrointestinal cancer), pancreatic cancer, glioblastoma, cervical cancer, ovarian cancer, liver cancer, bladder cancer, hepatoma, breast cancer, colon cancer, rectal cancer, colorectal cancer, endometrial cancer or uterine cancer, salivary gland cancer, kidney cancer or renal cancer, prostate cancer, vulvar cancer, thyroid cancer, liver cancer, anal cancer, penile cancer, and head and neck cancer.

[0107] The term “multispecific antibody,” as used herein, refers to an antibody that has binding specificity to at least two different epitopes. Exemplary multispecific antibodies may bind to both BBB-R and brain antigens. Multispecific antibodies can be prepared as full-length antibodies or antibody fragments (e.g., F(ab')2 bispecific antibodies). Manipulated antibodies containing two, three, or more (e.g., four) functional antigen-binding sites are also conceived (see, for example, U.S. Patent Application Publication 2002 / 0004587 A1). Multispecific antibodies can be prepared as full-length antibodies or antibody fragments.

[0108] The term "full-length antibody" refers to an antibody that includes an antigen-binding variable region (VH or VL) and light chain constant domains (CL) and heavy chain constant domains CH1, CH2, and CH3. The constant domains may be natural sequence constant domains (e.g., human natural sequence constant domains) or amino acid sequence variants thereof.

[0109] Full-length antibodies can be assigned to different "classes" depending on the amino acid sequence of the constant domain of their heavy chain. There are five main classes of full-length antibodies: IgA, IgD, IgE, IgG, and IgM, some of which can be further divided into "subclasses" (isotypes), such as IgG1, IgG2, IgG3, IgG4, IgA, and IgA2. The constant domains of the heavy chain corresponding to different antibody classes are called α, δ, ε, γ, and μ, respectively.

[0110] The term “library,” as used herein, refers to a collection of proteins as a single pool. Libraries of the present invention are preferably prepared using DNA recombination techniques. For example, a protein library may be prepared by inserting a collection of cDNA or any other protein-coding DNA into an expression vector. Expression vectors may be selected from plasmids, cosmids, artificial chromosomes, and viral expression vectors. Alternatively, a collection of cDNA or protein-coding DNA may be inserted into a phage genome to prepare a bacteriophage display library of wild-type proteins. Collections of cDNA may be prepared from specific cell populations or tissue samples, such as by the method disclosed by Sambrook et al. (Molecular Cloning, Cold Spring Harbor Laboratory Press, 1989). Collections of cDNA derived from specific cell types are also commercially available from suppliers such as Stratagene®. A library of wild-type proteins as used herein is not a collection of biological samples.

[0111] The term “recombinant antibody” as used herein refers to an antibody expressed by a recombinant host cell containing a nucleic acid encoding the antibody (e.g., a chimeric, humanized, or human antibody or its antigen-binding fragment). Examples of "host cells" that produce recombinant antibodies include: (1) mammalian cells, e.g., Chinese hamster ovary (CHO), COS, myeloma cells (including Y0 and NS0 cells), baby hamster kidney (BHK), Hela and Vero cells; (2) insect cells, e.g., sf9, sf21 and Tn5; (3) plant cells, e.g., plants belonging to the genus Nicotiana (e.g., Nicotiana tabacum); (4) yeast cells, e.g., those belonging to the genus Saccharomyces (e.g., Saccharomyces cerevisiae) or those belonging to the genus Aspergillus (e.g., Aspergillus niger); (5) bacterial cells, e.g., Escherichia coli cells or Bacillus subtilis cells, etc.

[0112] The “individual” or “subject” is a mammal. Mammals include, but are not limited to, domestic animals (e.g., cows, sheep, cats, dogs, and horses), primates (e.g., humans and non-human primates such as monkeys), rabbits, and rodents (e.g., mice and rats). In certain embodiments, the individual or subject is a human.

[0113] Detailed explanation This disclosure relates to methods for creating novel molecules that are reversibly or irreversibly inactivated under wild-type conditions but are active at the same or equivalent levels under normal conditions as under wild-type conditions, by manipulating or developing proteins. These novel proteins are referred to herein as conditionally active biological proteins. These conditionally active biological proteins and methods for producing them are described in U.S. Patent Application Publication No. 2012 / 0164127. Conditionally active biological proteins are of particular value in the development of novel therapeutic agents that are active only for a short period or limited duration in the host body. This is particularly valuable in the extended manipulation of administered proteins that are harmful to the host but whose limited activity is necessary to perform the desired treatment. Examples of beneficial applications include topical or systemic treatment at high doses, as well as topical treatment at high concentrations. Inactivation under physiological conditions may be determined by the combination of administrations and the rate of protein inactivation. This condition-based inactivation is particularly important in enzyme therapy when catalytic activity causes substantially negative effects for a relatively short period.

[0114] This disclosure also directs methods for manipulating or developing proteins to generate novel molecules different from wild-type molecules, novel molecules that are reversibly or irreversibly active or inactive over time, or that are active or inactive only in specific microenvironments within the body, including specific organs within the body (e.g., the bladder or kidneys). In some embodiments, the conditionally active biological protein is an antibody against one or more target proteins as described herein.

[0115] Target wild-type protein Any therapeutic protein can function 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, used in the treatment of blood clots, and hyaluronidase, used as an adjuvant to increase the absorption and dispersion of other drugs. In one embodiment, a wild-type protein is selected for the production of a conditionally active biological protein, or a therapeutic protein is used to avoid or minimize adverse side effects associated with the wild-type protein or enzyme. Alternatively, an enzyme not currently used therapeutically may be selected for the production of a conditionally active biological protein. Specific non-limiting embodiments are discussed in detail below.

[0116] 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 this disclosure may be applied for use in one or more conditions including circulatory disorders, arthritis, multiple sclerosis, autoimmune diseases, cancer, and dermatological conditions, and may be used in various diagnostic forms. Depending on the protein and condition, conditionally active biological enzyme proteins may be administered in parenteral, topical, or oral dosage forms, as discussed below.

[0117] Circulatory disorders - thrombosis and thrombolytic therapy A thrombus (blood clot) is defined as a solid mass derived from blood components that form in the circulatory system. Thrombi are formed by a series of events involving blood clotting factors, platelets, red blood cells, and interactions with the blood vessel wall. Platelets are intravascular aggregations of captured blood cells, including platelets, fibrin, and other components that can cause vascular damage. By obstructing or blocking blood flow, thrombi deprive tissues of oxygen. Fragments of a thrombus (embolus) can break off and obstruct smaller blood vessels. Arterial thrombus formation is triggered by any of a variety of factors, including latent stenosis—atherosclerosis, low-flow cardiac function, hypercoagulation 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 injury, acute myocardial infarction (AMI), stroke, amputation, or intestinal infarction. The formation of arterial thrombi (coronary and cerebral thrombi) and pulmonary thrombi is a major cause of morbidity and mortality. Venous thrombosis can result from trauma, such as congestion due to rest, or endothelial damage due to hypercoagulation, but atherosclerosis is not a contributing factor. Treatment options include mechanical thrombectomy, pharmacodynamic thrombectomy, and thrombolysis. Treatment for thrombosis is used to minimize thrombus formation and aid in its removal.

[0118] Treatment of thrombosis includes the use of antiplatelet agents that 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 catalytic action.

[0119] Thrombolytic therapy for acute myocardial infarction is well-established. The use of thrombolytic agents has become standard emergency treatment. Despite their effectiveness, these products achieve complete reperfusion in only about 50% of patients, and side effects include the risk of bleeding (particularly intracranial hemorrhage) as well as hypertension. The breakdown of blood clots from damaging or diseased blood vessels is called "fibrinolysis" or "fibrinolytic methods." Fibrinolysis is a proteolytic method by plasminogen activators that activate the protein plasminogen, thereby forming plasmin. Proteolytic plasmin breaks down fibrin strands to dissolve blood clots. Fibrin-specific plasminogen activators include tissue plasminogen activators or their variants. Non-specific plasminogen activators may include streptokinase and urokinase.

[0120] Certain commonly used thrombolytic therapies utilize one of several available tissue plasminogen activator (tPA) variants. For example, tPA variants based on previously approved products include Alteplase (rt-PA), Reteplase (r-PA), and Tenecteplase (TNK). Approved uses of tPA variants include, for example, the management of acute myocardial infarction following AMI, the reduction of the incidence of congestive heart failure and AMI-associated mortality, the management of ischemic stroke and the reduction of disability in adults to improve neurological recovery, the dissolution of acute pulmonary embolism, and the management of acute massive pulmonary embolism in adults, for the dissolution of pulmonary embolism associated with unstable hemodynamics.

[0121] Another commonly used thrombolytic therapy utilizes urokinase. Urokinase is a standard dissolving agent used in the treatment of peripheral vascular diseases.

[0122] Streptokinase is a protein secreted by several types of streptococci that can bind to and activate human plasminogen. The complex of human plasminogen and streptokinase can hydrolytically activate other unbound plasminogens by being activated by binding cleavage to produce plasmin. The normal activity of plasminogen occurs by proteolysis of the Arg561-Val562 bond. The amino group of Val562 forms a salt bridge with Asp740, causing a higher-order structural change to create the active protease plasmin. Plasmin is produced in the blood to break down fibrin, the main component of blood coagulation.

[0123] Streptokinase is used as an effective clot-dissolving agent in some cases of myocardial infarction (heart attack), pulmonary embolism (pulmonary clots), and deep vein thrombosis (leg clots). Streptokinase belongs to a group of drugs called fibrinolytics. Streptokinase is administered 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 myocardium. Because streptokinase is a bacterial product, it has the ability to establish immunity against proteins. Therefore, it is recommended not to administer this product more than four days after the initial dose, as it may not be effective and may cause allergic reactions. For this reason, it is usually administered only after the first heart attack, and further thrombosis is typically treated with tissue plasminogen activators (TPAs). Streptokinase may also be used to prevent postoperative adhesions.

[0124] Side effects of streptokinase include bleeding (both heavy and minor), hypotension, 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.

[0125] Thrombolytic agents are usually administered by injection, rapid intravenous injection, or mechanical infusion system. Serious side effects may include intracranial, gastrointestinal, retroperitoneal, or pericardial bleeding. If bleeding occurs, administration must be immediately discontinued.

[0126] In certain embodiments of this disclosure, tPA, streptokinase, or urokinase is selected as the target or wild-type protein.

[0127] In one embodiment, the method of the present disclosure is used to select a conditionally active recombinant or synthetic streptokinase variant that exhibits high activity under abnormal temperature conditions lower than normal physiological conditions and is substantially inactive or non-active under normal physiological conditions (e.g., 37°C). In one embodiment, the abnormal temperature conditions are room temperature, e.g., 20-25°C. In another embodiment, the present disclosure provides a method for treating stroke or heart attack, the method comprising administering a high dose of a conditionally active streptokinase variant to a stroke or heart attack patient so as to clear the blood clot and so as to prevent excessive bleeding by rapidly inactivating the streptokinase variant.

[0128] 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 from the liver, into the peptide angiotensin I. Angiotensin I is further cleaved in the lungs by endothelial-bound angiotensin-converting enzyme (ACE) into angiotensin II. Angiotensin II constricts blood vessels, resulting in increased blood pressure. However, angiotensin π also promotes 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 the body's fluids and raises blood pressure. An overly active renin-angiotensin system leads to vasoconstriction and retention of sodium and water. These effects lead to hypertension. There are many drugs that disrupt different processes in this system to lower blood pressure. These medications are one of the primary methods for controlling the adverse effects of hypertension, heart failure, renal failure, and diabetes.

[0129] Hypovolemic shock is an emergency condition in which the heart is unable to adequately perfuse oxygenated blood to body cells due to the loss of a large amount of blood and / or fluids. Blood loss can occur due to trauma, injury, and internal bleeding. The volume of circulating blood may 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. Tests may show signs of shock, including hypotension, hypothermia, and a rapid pulse, which may be weak or frail. Treatment includes intravenous fluids, blood or blood products, treatment of shock, and medications such as dopamine, dobutamine, epinephrine, and norepinephrine to increase blood pressure and cardiac output.

[0130] In one embodiment, the disclosure provides a method for selecting a conditional recombinant renin variant that is reversibly inactivated at normal physiological temperatures but reactivated when a patient is abnormally cold due to hypovolemic shock. Conditionally activated proteins may be used to treat hypovolemic shock by promoting an increase in bodily fluid volume and blood pressure.

[0131] Circulatory disorders - Raynaud's phenomenon Raynaud's phenomenon (RP) is a vasospasmodic disorder that causes discoloration of the fingers, toes, and sometimes other extremities. Emotional stress and cold are typical triggers for this reduction. When exposed to cold, the extremities lose heat. Blood supply to the fingers and toes normally slows down to maintain the body's core temperature. Blood flow is reduced by narrowing of the small subcutaneous arteries in the extremities. Stress triggers a similar reaction to the body getting cold. In Raynaud's phenomenon, the normal reaction is amplified. The condition may cause pain, discoloration, and sensations of cold and numbness. The phenomenon is vasospasm, which results in reduced blood supply to the respective areas. In Raynaud's disease (primary Raynaud's phenomenon), the disease is sudden. In Raynaud's syndrome (secondary Raynaud's phenomenon), the phenomenon is triggered by other instability factors. Measuring the temperature gradient of the hand is one way to distinguish between the primary and secondary forms. The initial stage can progress to the second stage, and in extreme cases, the second stage can progress to necrosis or gangrene of the fingertips.

[0132] Raynaud's phenomenon is an exaggerated response to cold or emotional stress. Early RP is primarily caused by microvasoconstriction. Overactivation of the sympathetic nervous system leads to excessive vasoconstriction of peripheral blood vessels, resulting in hypoxia. In chronic, recurrent cases, atrophy of the skin, subcutaneous tissue, and muscles may occur. Rarely, ulcer formation and ischemic gangrene may also occur.

[0133] Conventional treatment options for Raynaud's phenomenon include prescription drug therapy that dilates blood vessels and promotes circulation. These include calcium channel blockers such as nifepidin or diltiazem, alpha-blockers such as prazosin or doxazosin that counteract the effects of norepinephrine and vasoconstricting hormones, and vasodilators to relax blood vessels such as nitroglycerin cream or the angiotensin II inhibitor losartan, sildenafil, or prostaglandins. Fluoxetine, selective serotonin reuptake inhibitors, and other antidepressants can reduce the frequency and severity of onset caused by physiological stressors. These drugs may cause side effects such as headache, flushing, and ankle edema. The effects of these drugs may also diminish over time.

[0134] The regulation of cutaneous vasoconstriction and vasodilation involves altered sympathetic nerve activity and numerous neuromodulations, including both adrenaline and non-adrenergic signals, as well as other signaling pathways such as REDOX signaling and the RhoA / ROCK pathway. Vasoconstriction in cutaneous vascular smooth muscle cells (vSMCs) is thought to be activated by norepinephrine transmitted by alpha-1 and alpha-2 adrenergic receptors. Alpha-2C-ARs are translocated from the trans-Golgi to the cell surface of vSMCs 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 production of reactive oxygen species (ROS) in the mitochondria of vSMCs. ROS are involved in REDOX signaling via the RhoA / ROCK pathway. RhoA is a GTP-binding protein that plays a role in regulating actin-myosin-dependent processes such as migration and cell contraction in vSMCs. Known functional non-adrenergic neuropeptides of vascular structures with possible inclusion of 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.

[0135] Novel therapies for RP include alpha-2C adrenergic receptor blockers, protein tyrosine kinase inhibitors, Rho kinase inhibitors, and calcitonin gene-related peptides.

[0136] 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 an effective peptide vasodilator and can act on pain conduction. Migraine is a common neurological disorder associated with elevated CGRP levels. CGRP dilates blood vessels in the brain and conducts vascular pain. CGRP receptor antagonists have been experimented with as a treatment for migraine. Arulmani et al., 2004, “Calcitonin gene-related peptide and its role in migraine pathophysiology”, Eur.J.Pharmacol.500(1-3):315-330. At least three receptor subtypes have been identified, and CGRP acts via G protein-coupled receptors and varies in its function of regulating peptide action in various tissues. Receptor-mediated CGRP signaling depends on two co-proteins, receptor activity modifier protein 1 (RAMP1) and receptor constituent protein (RCP). Ghatta 2004, the role of calcitonin gene-related peptide is understood. Indian J. Pharmacol. 36(5):277-283. One study of the effects of intravenous administration of three vasodilators—endothelium-dependent vasodilator adenosine triphosphate (ATP), endothelium-independent vasodilator prostacyclin (epoprosterol, PGI2), and CGRP in patients with Raynaud's phenomenon, and a study of CGRP, showed that in Raynaud's patients, increased skin blood flow induced facial and hand redness with CGRP and laser Doppler flow measurement (LDF) in a similar number of patients of age and sex with Raynaud's phenomenon and in a control group of similar age and sex. In Raynaud's patients, CGRP induced facial redness only in the face, while in the control group, CGRP caused redness only in the face. PG12 caused similar effects on hand and facial blood flow in both groups. ATP did not cause any significant changes in hand or facial blood flow in patients, but increased facial blood flow in 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.

[0137] In one embodiment, the disclosure provides a method for selecting a conditionally active recombinant protein variant of a protein associated with Raynaud's syndrome that is reversibly inactive at normal physiological temperatures but reactivated when the fingers are abnormally cold. Conditionally active proteins may be used to treat Raynaud's phenomenon to prevent or reduce loss of finger function due to hypocirculation.

[0138] Circulatory disorders - vasopressin Arginine vasopressin (AVP, vasopressin, antidiuretic hormone (ADH)) is a peptide hormone found in many mammals that controls the reabsorption of molecules in the renal tubules related to tissue permeability. One of vasopressin's most important roles is to regulate water retention in the body. At high concentrations, it raises blood pressure by inducing moderate vasoconstriction. Vasopressin has three effects that result in increased urine osmolality (high concentration) and decreased water excretion. Firstly, vasopressin causes increased water permeability of collecting duct cells in the kidney, which allows for water reabsorption and the excretion of smaller amounts of concentrated urine (antidiuretic). This occurs via the insertion of aquaporin 2 water channels in the apical membrane of collecting duct cells. Secondly, vasopressin causes increased permeability of the inner medullary portion of the collecting duct to urea, allowing for increased urine reabsorption in the medullary interstitium. Thirdly, vasopressin increases the activity of the Na+, K+, 2Cl- cotransporter, leading to sodium stimulation and chloride reabsorption in the thickened upper limb of the loop of Henle. Sodium chloride reabsorption is due to an increased reflux process, providing an osmotic gradient in aquaporins that results in water reabsorption in the collecting duct medulla.

[0139] The hypertonic interstitial fluid surrounding the collecting ducts of the kidneys provides high osmotic pressure for water removal. Transmembrane channels made of proteins called aquaporins are inserted into the plasma membrane, which greatly increases water permeability. When open, aquaporin channels can permeate 3 billion molecules of water per second. Insertion of aquaporin 2 channels requires signaling by vasopressin. Vasopressin binds to receptors (called V2 receptors) on the basal surface of collecting duct cells. Hormone binding triggers an increase in intracellular cAMP levels. This "second messenger" initiates a chain reaction at the apical membrane of collecting duct cells that results in the insertion of aquaporin 2 channels. Aquaporins increase water reabsorption by draining water out of the renal unit and returning urine to the bloodstream.

[0140] The primary stimulus for vasopressin release from the pituitary gland is an increase in plasma osmolality. Any dehydration, such as heavy sweating, increases blood osmolality and activates vasopressin to V2 receptors in the aquaporin 2 pathway. As a result, a small amount, as small as 0.5 liters / day of urine, may remain compared to 180 liters / day of original renal filtrate. The salt concentration in urine can be four times that of blood. If the blood becomes too dilute, such as by drinking large amounts of water, vasopressin secretion is inhibited, and aquaporin 2 channels are returned to cells by endocytosis. As a result, a large amount of dilute urine is produced with a salt concentration about one-quarter that of blood.

[0141] Reduced vasopressin release or reduced renal sensitivity to AVP can lead to diabetes insipidus, hypernatremia (increased blood sodium levels), polyuria (excessive urine production), and polydipsia (thirst).

[0142] High levels of AVP secretion (inappropriate antidiuretic hormone (SIADH) syndrome) and resulting hyponatremia (low blood sodium concentration) occur in brain diseases and lung conditions (small cell lung cancer). During the perioperative period, surgical stress and the effects of some commonly used drugs (e.g., opium, syntocinone, antiemetics) can lead to a similar condition of excessive vasopressin secretion. This can cause hyponatremia for several days.

[0143] Vasopressin agonists are used therapeutically in a variety of conditions, and their long-acting synthetic analog, desmopressin, is used in conditions characterized by low vasopressin secretion, as well as in the control of bleeding (in various forms of von Willebrandt disease) and in extreme cases of bedwetting in children. Terlipressin and related analogs are used as vasoconstrictors in certain conditions. Vasopressin infusion is used as a second line of treatment in infectious shock patients who do not respond to high doses of inotropes (e.g., dopamine or norepinephrine). Vasopressin receptor antagonists are drugs that interfere with the action of vasopressin receptors. They may also be used in the treatment of hyponatremia.

[0144] In one embodiment, the disclosure provides a method for selecting a conditional biologically recombinant or synthetic protein of a protein involved in the vasopressin response that is reversibly inactive under normal physiological osmotic pressure but reactivated under abnormal osmotic pressure in the blood. In another embodiment, a variant of the protein involved in the vasopressin response is activated under sodium-deficient conditions but inactivated at normal serum sodium concentrations. In one embodiment, the sodium-deficient condition is serum sodium <135 mEq / L.

[0145] Cancer-Angiostatin Angiostatin is a naturally occurring protein in several animal species. It acts as an intrinsic 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 38kD fragment of plasmin (which is itself a fragment of plasminogen). Angiostatin consists of 1 to 3 kringles of plasminogen. Angiostatin is produced by autolysis cleavage of plasminogen, for example, by reduction of extracellular disulfide bonds by phosphoglycerate kinase. Angiostatin can be cleaved from plasminogen by different substrate metalloproteinases, including MMP2, MMP12, and MMP9, and serine proteases (neutropenic elastase, prostate-specific antigen (PSA)). In vivo, angiostatin inhibits tumor growth and maintains experimental metastases in a dormant state. Angiostatin levels are elevated in animals with early-stage tumors and other inflammatory and degenerative diseases.

[0146] Angiostatins are known to bind to many proteins, including angiomotin, endothelial cell surface ATO synthase, integrins, annexin II, C-met receptor, NG2-proteoglycan, tissue plasminogen activator, chondroitin surface glycoprotein, and CD26. Some studies have shown that IL-12 and TH1 cytokines, which have potent anti-angiogenic properties, are mediators of angiostatin activity. "Albin"., J. Translational Medicine. Jan. 4, 2009, 7:5. Angiostatin binds to the surface of endothelial cells and inhibits ATP synthesis. ATP synthesis also occurs on the surface of various cancer cells. Tumor cell surface ATP synthesis is more active in 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 antitumor-toxic. At low pH, angiostatin and anti-beta subunit antibodies lead to intracellular acidification of A549 cancer cells, as well as direct toxicity lacking in tumor cells with low levels of extracellular ATP synthesis. The mechanism of tumor cytotoxicity is hypothesized to be due to the disruption of intracellular pH regulation resulting from the 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.

[0147] In one embodiment, the disclosure provides a method for identifying conditionally active angiostatin mutants that exhibit lower activity than wild-type angiostatin at normal physiological blood pH but enhanced activity at low pH. Low pH is defined as a pH lower than normal physiological pH. In one embodiment, low pH is approximately 7.2 or lower. In a particular embodiment, low pH is approximately 6.7.

[0148] In one embodiment, conditionally activated angiostatin variants may be prescribed and used as anticancer agents.

[0149] Enhanced tissue permeability - hyaluronidase Hyaluronidase is a class of enzymes that break down hyaluronic acid. By causing the breakdown of hyaluronic acid, a major component of the interstitial barrier, hyaluronidase reduces the viscosity of hyaluronic acid, thereby increasing its tissue permeability. It is used in medicine in conjunction with drugs to accelerate the dispersion and delivery of those drugs. The most common application is in eye surgery, where it is used in combination with local anesthesia. Animal-derived hyaluronidases include Hydase® (PrimaPharm Inc.; Akorn me), Vitorase (ISTA Pharmaceuticals), and Amphadase (Amphastar Pharmaceuticals). Human recombinant hyaluronidase is currently approved as an adjuvant to increase the absorption of other drugs. Hypodermocyclis (subcutaneous fluid injection), an adjuvant in subcutaneous urography that improves the absorption of radiopaque drugs (Hylenex; Halozyme Therapeutics, Inc.; Baxter Healthcare Corp). In one embodiment, hyaluronidase is used as the wild-type protein (parent molecule) for the preparation of a conditionally active biological protein. Hyaluronidase can play a role in cancer metastasis and angiogenesis. Therefore, excessive exposure to these enzymes can be harmful. In one embodiment, the conditionally active biological hyaluronidase protein is irreversibly or reversibly inactive at normal physiological temperatures, but is active at levels equal to or higher than wild-type hyaluronidase in a specific temperature range lower than normal physiological temperatures.

[0150] Autoimmune diseases - Conditional activated biological response modifiers Rheumatoid arthritis is an autoimmune disease characterized by abnormal immune mechanisms that lead to swelling due to arthritis and progressive joint destruction. RA (rheumatoid arthritis) can also affect the skin, connective tissue, and internal organs. Conventional treatments include nonsteroidal anti-inflammatory drugs (NSAIDs), COX-2 inhibitors, and disease-modifying antirheumatic drugs (DMARDs) such as methotrexate. None of these conventional treatment approaches are ideal, especially with long-term use.

[0151] Biological response modifiers that target inflammatory mediators offer a relatively novel approach to treating rheumatoid arthritis and other autoimmune diseases. Such biological response modifiers include antibodies against various inflammatory mediators such as IL-6, IL-6 receptor, TNF-alpha, IL-23, and IL-12, or their active moieties.

[0152] Some of the first biological response modifiers are drugs that target tumor necrosis factor alpha (TNF-α), a pro-inflammatory cytokine involved in the pathogenesis of rheumatoid arthritis (RA). Several anti-TNF-alpha drugs are currently marketed for the treatment of RA. For example, Enbrel® (etanercept, Amgen) is a TNF-alpha blocker. Etanercept is a dimer-soluble protein consisting of the extracellular linkage 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 includes the CH2 domain, CH3 domain, and hinge region, but does not include 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 psoriasis vulgaris. Serious side effects of Enbrel® include infections, including tuberculosis, fungal infections, and viral infections caused by opportunistic pathogens. Sepsis may also occur. Lymphoma or other malignancies have also been reported.

[0153] Remicade® (infliximab) is a chimeric anti-TNF-alpha-IgGKI monoclonal antibody consisting of a human homeostatic region and a mouse variable region. 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 hematological events, hypersensitivity reactions, and certain neurological events.

[0154] 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), is approved by the FDA and the European Commission for the treatment of adult patients with rheumatoid arthritis. Actemra is also approved in Japan for the treatment of RA and juvenile rheumatoid arthritis (sJIA). A Phase III study has shown that treatment with Actemra, both as monotherapy and in combination with MTX or other DMARDs, reduces the signs and symptoms of RA compared to other therapies. Actemra is a humanized IL-6 receptor monoclonal antibody that competitively blocks the binding of IL-6 to its receptor. Therefore, it inhibits the proliferative activity of IL-6 that leads to synovial thickening and pannus formation in RA. Serious side effects of Actemra include hypersensitivity reactions, including in some cases severe infections and anaphylaxis. Other side effects include upper respiratory tract infections, headache, nasopharyngitis, hypertension, and elevated ALT levels.

[0155] Another common autoimmune disease is psoriasis. Overactivity of the immune system can lead to high concentrations of IL-12 and IL-23, two cytokine proteins found in psoriatic 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.

[0156] One treatment for moderate or severe psoriasis involves subcutaneous injection of Stelara® (ustequimanub, Centocor Ortho Biotech, Inc.), a humanized IgG1k monoclonal antibody against the p40 subunits of the IL-12 and IL-23 cytokines. Stelara has been shown to provide relief from certain symptoms associated with psoriatic plaques, such as plaque thickening, peeling, and redness. The Stelara formulation contains L-histidine and L-histidine hydrochloride hydrate, polysorbate 80, and sucrose in aqueous solution. Use of Stelara® acts on the immune system and increases the chances of infection, including tuberculosis and infections caused by bacteria, fungi, or viruses, as well as increasing the risk of certain types of cancer.

[0157] The side effects of biological response modifiers are serious, and high levels of infusion into a patient can make them more susceptible to serious infections or death. This is a major side effect associated with this important type of drug. One challenge is to avoid high initial levels of activity from the administration of antibodies necessary to provide a long-lasting therapeutic effect after infusion.

[0158] In one embodiment, the Disclosure provides a method for preparing conditionally active biological response mediators or fragments thereof, avoiding high levels of activity from antibody administration required to provide a long-lasting therapeutic effect after infusion. The method of the 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 under administration conditions such as room temperature but slowly refold (reversibly or irreversibly) at body temperature. These antibodies or fragments thereof are inactive upon initial infusion but, upon exposure to blood infusion, refold or reactivate over a period of several hours to several days. This allows for higher doses and longer half-lives (or durations of administration) with reduced side effects.

[0159] In one embodiment, the disclosure provides a method for preparing a conditionally activated antibody against an inflammatory mediator, or a fragment thereof, which is inactive under administration conditions such as room temperature but slowly refolds (reversibly or irreversibly) at body temperature. The method includes the following steps: selecting an inflammatory mediator; screening for the identification of an antibody against the inflammatory mediator using a hybridoma; humanizing the anti-inflammatory mediator antibody; developing the anti-inflammatory mediator antibody; differentially screening for binding under two or more conditions, e.g., room temperature and above 37°C; and selecting for mutations that are inactive under the first condition compared to the wild type but show increased activity (e.g., binding) under the second condition compared to the wild type antibody activity (binding). Next, the identified elevated mutants in the heavy and light chains are recombined within the heavy and light chains and via combinatorial binding between the heavy and light chains. Screening of these recombinant heavy and light chains is repeated under two conditions, e.g., room temperature and above 37°C. In addition, recombinant antibodies or fragments can be screened for their activity and stability under storage and physiological conditions.

[0160] Alternatively, wild-type antibodies against inflammatory mediators are known as antibodies, mutants, or their active fragments.

[0161] In one embodiment, the first and second conditions are selected from pH, osmotic pressure, osmolality, oxidative stress, and charge concentration. In another embodiment, the inflammatory mediator is selected from IL-6, IL-6 receptor, TNF-alpha, IL-23, and IL-12.

[0162] In another embodiment, the present disclosure provides a method for preparing a conditionally active antibody against IL-6, or a fragment thereof, which is inactive under administration conditions such as room temperature but slowly refolds (reversibly or irreversibly) at body temperature. The method comprises the following steps: screening a complete human library for antibodies against IL-6; developing IL-6 antibodies and differentially screening molecules at room temperature and above 37°C; selecting for mutations that are inactive compared to the wild type at room temperature but show increased activity (e.g., binding) compared to wild-type antibody activity (binding); then recombining the identified elevated mutants in the heavy and light chains within the heavy and light chains and via combinatorial binding between the heavy and light chains; repeating the screening of these recombinant heavy and light chains at room temperature and higher temperatures; and in addition, testing the recombinant antibody or fragment for activity and stability under storage and physiological conditions.

[0163] Accordingly, the identified and manufactured conditionally activated anti-IL-6 antibodies are used in methods to treat autoimmune diseases such as rheumatoid arthritis or psoriasis by administering effective doses to patients who need them, reducing 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 doses over the course of treatment, compared to the current high levels of biological response modifiers, which have half-life clearances that last for weeks or months.

[0164] Selection of wild-type proteins from a library Wild-type proteins can be selected from a wild-type protein library, such as a bacteriophage display library. In such embodiments, a number of wild-type protein candidates are expressed in the bacteriophage library, particularly by surface display technology. Candidates from the library are screened for a suitable wild-type protein. A typical bacteriophage library may contain bacteriophages that express the candidates in a bacterial host. In one embodiment, the bacteriophage library may contain multiple bacteriophages.

[0165] To construct a bacteriophage library, a filamentous bacteriophage, typically such as the E. coli phage M13, is genetically modified by inserting a candidate-encoding oligonucleotide into the coding sequence of one of the bacteriophage coat proteins. The bacteriophage particle's coat protein is subsequently expressed along with the candidate, thus presenting the candidate on the surface of the bacteriophage particle. The presented candidate can then be screened against a preferred wild-type protein.

[0166] One common technique for screening for suitable wild-type proteins involves immobilizing bacteriophage particles containing the desired candidate onto a support. The support may be a plastic plate coated with a "bait" capable of binding to the desired candidate. Unbound bacteriophage particles can be washed away from the plate. The bacteriophage particles (containing the desired candidate) bound to the plate are eluted by washing, and the eluted bacteriophage particles are amplified in bacteria. The sequences encoding the candidate in the selected bacteriophage particles can then be determined by sequencing. The relationship between the candidate and the bait may be, for example, a ligand-receptor or antigen-antibody relationship.

[0167] Another common technique for screening for suitable wild-type proteins involves using enzyme assays of individual bacteriophage clones for the desired enzyme activity exhibited by the candidates. Depending on the specific enzyme activity, those skilled in the art can design appropriate assays to screen for candidates with the desired level of enzyme activity.

[0168] In some embodiments, the bacteriophage library is provided as an array such that each bacteriophage clone occupies a specific position on the array. Such an array may be provided on a solid support, such as a membrane, an agar plate, or a microtiter plate, in which case each bacteriophage clone of the library is attached to it at a specific predetermined position on the solid support. In the case of an agar plate, such plate preferably contains a bacterial growth medium to support bacterial growth. If the array is provided on a membrane, for example, a nitrocellulose or nylon membrane, a bacterial culture is added to the membrane and the membrane is immersed in a vegetative growth medium. In addition, bacteriophage clones may also be provided on beads, in which case a single bacteriophage clone can be attached to a single bead. Alternatively, each bacteriophage clone may be provided at the end of an optical fiber, in which case ultraviolet radiation from a light source is optically transmitted using the fiber.

[0169] A typical bacteriophage library may contain 10⁶ to 10¹¹ recombinant bacteriophages, each distinguished by a coat protein carrying a different candidate (e.g., gp3 or gp8 in the case of phage M13). The bacterial hosts for the bacteriophage library may be selected from bacterial genera including, for example, Salmonella, Staphylococcus, Streptococcus, Shigella, Listeria, Campylobacter, Klebsiella, Yersinia, Pseudomonas, and Escherichia.

[0170] Oligonucleotides encoding candidate proteins can be a collection of cDNAs encoding wild-type proteins. Methods for synthesizing cDNA from biological samples capable of expressing suitable wild-type proteins are known. Any genetic information exhibiting physiological activity via transcripts can be collected as cDNA. When preparing cDNA, it is essential to synthesize full-length cDNA. Several methods can be used for synthesizing full-length cDNA. For example, preferred methods include using a cap-binding protein from yeast or HeLa cells to label the 5' cap region (I. Edery et al., “An Efficient Strategy To Isolate Full-length cDNAs Based on a mRNA Cap Retention Procedure (CAPture)”, Mol. Cell. Biol., vol. 15, pages 3363-3371, 1995); and removing the phosphate from incomplete cDNA lacking a 5' cap using alkaline phosphatase, and then treating the entire cDNA with a decapping enzyme from tobacco mosaic virus so that only full-length cDNA retains phosphate (K. Maruyama et al., “Oligo-capping: a simple method to replace the cap structure of eukaryotic mRNAs with oligoribonucleotides”, Gene, vol. 138, pages 171-174, 1995 and S. Kato et al., “Construction of a human full-length cDNA bank”, Gene, vol. 150, pages 3363-3371, 1995). pp. 243-250, 1995) are examples.

[0171] A library of wild-type protein candidates can also be constructed using recombinant antibodies derived from the complete antibody repertoire of an organism. The genetic information representing the repertoire is assembled as a large collection of complete antibodies, which can then be screened for suitable wild-type antibodies having the desired antigen-binding activity and / or one or more other functional properties. In some embodiments, B cells from an animal immunized with an antigen, such as human, mouse, or rabbit B cells, are isolated. mRNA is recovered from the isolated B cells (converted to cDNA) and sequenced. The most frequent cDNA fragments encoding the light chain and the most frequent cDNA fragments encoding the heavy chain are assembled to produce an antibody. In one embodiment, 100 most frequent cDNA fragments encoding the light chain and 100 most frequent cDNA fragments encoding the heavy chain are assembled to produce an antibody. In another embodiment, the most frequent cDNA fragments encode only the variable regions of the heavy chain and the variable regions of the light chain, and therefore the assembled antibody contains only the variable regions and not the constant regions.

[0172] In some embodiments, a cDNA fragment encoding the variable region of the IgG heavy chain, including one derived from mRNA isolated from B cells, is assembled with the most frequent IgK or IgK variable region of the light chain. The assembled antibody contains the IgG-derived heavy chain variable region and the IgK-derived light chain variable region or IgK.

[0173] Next, the cDNA encoding the assembled antibody is cloned and expressed, preferably in a plate-based format. The binding activity of the expressed antibody may be analyzed by bead-based ELISA, and a suitable wild-type antibody can be selected based on the ELISA analysis. The cDNA encoding the assembled antibody may also be expressed in a bacteriophage display library, which can then be screened for one or more desirable wild-type antibodies using any of the techniques disclosed herein.

[0174] In embodiments where the wild-type protein is the antibody, the wild-type antibody preferably has certain properties that facilitate its development into a conditionally active antibody. In certain embodiments, the wild-type antibody may have similar binding activity and / or properties under both normal and abnormal physiological conditions. In such embodiments, the wild-type antibody is selected based on having the most similar binding activity and / or one or more similar properties under both normal and abnormal physiological conditions. For example, if the normal and abnormal physiological conditions are pH 7.4 and pH 6.0, respectively, a wild-type antibody with the most similar binding activity at pH 7.4 and 6.0 may be selected over an antibody with less similar binding activity at pH 7.4 and 6.0.

[0175] After selecting a wild-type protein, the DNA encoding that wild-type protein can be developed using a suitable mutagenesis technique to create mutant DNA, which can then be expressed to produce mutant proteins for screening to identify conditionally active biological proteins. In some embodiments, development may be minimal; for example, introducing only a very small number of mutations into the wild-type protein may produce a mutant protein with the desired conditional activity. For example, it may be sufficient to introduce fewer than 20 changes, or in some cases fewer than 18 changes, into each site by CPE to produce a suitable conditionally active biological protein. For CPS, combinations of fewer than 6, or fewer than 5, or fewer than 4, or fewer than 3, or fewer than 2, uplift mutations in the wild-type protein may be sufficient to produce a desirable conditionally active biological protein.

[0176] In some embodiments, if the wild-type protein library (e.g., bacteriophage library and / or recombinant antibody library) is sufficiently large, the development and expression steps may be unnecessary. Such large libraries may contain wild-type proteins with conditional activity characteristics (having both low activity under normal physiological conditions and high activity under abnormal conditions). In these embodiments, conditional-active biological proteins are discovered by subjecting the wild-type proteins in the library to a selection step, resulting in lower activity under normal physiological conditions compared to the same protein under abnormal conditions. In one embodiment, wild-type proteins in the library are subjected separately to assays under normal physiological conditions and abnormal conditions, along with a reference protein. The conditional-active biological proteins selected from the library exhibit lower activity under normal physiological conditions compared to the activity of the same protein under abnormal conditions. In this embodiment, because the library is sufficiently large, wild-type proteins with conditional activity characteristics are already present in the library. It is not necessary to develop wild-type proteins to discover conditional-active biological proteins.

[0177] In some embodiments, a reference protein may be used in the selection step. The reference protein may not be a conditionally active type in that it has similar or identical activity under both normal and abnormal physiological conditions. The reference protein is a protein of the same type as the wild-type protein in the library, such as a similar enzyme, antibody, or functional peptide. The reference protein may also be a similar tissue plasminogen activator, streptokinase, urokinase, renin, hyaluronidase, calcitonin gene-related peptide (CGRP), substance P (SP), neuropeptide Y (NPY), vasoactive intestinal peptide (VTP), vasopressin, or angiostatin. For example, if the library contains many wild-type antibodies against a certain antigen, the reference protein is an antibody against the same antigen that has the same or similar activity in terms of binding to that antigen under both normal and abnormal physiological conditions.

[0178] Therefore, in one embodiment, wild-type proteins in the library are subjected separately to assays under normal physiological conditions and assays under abnormal conditions, along with a reference protein. From the library, a conditionally active biological protein is selected that exhibits both (a) decreased activity under normal physiological conditions compared to the reference protein, and (b) increased activity under abnormal conditions compared to the reference protein.

[0179] Method for generating conditionally active biological proteins One or more mutagenesis techniques are used to develop DNA encoding wild-type proteins to create a library of mutant DNA. The mutant DNA is expressed to create a library of mutant proteins, and this library is targeted for screening analysis under normal physiological conditions and under one or more abnormal conditions. Conditionally active biological proteins are selected from those proteins that exhibit both (a) decreased activity in analysis under normal physiological conditions compared to wild-type proteins, and (b) increased activity in analysis under abnormal conditions compared to wild-type proteins. Alternatively, conditionally active biological proteins are selected from those proteins that exhibit reversible or irreversible changes in activity under two or more different physiological conditions. In some embodiments, the wild-type protein is an antibody.

[0180] In some embodiments, the protein to be developed may be a fragment of a wild-type protein or a fragment of a wild-type antibody. In some other embodiments, the protein to be developed may be a protein selected by a mutagenesis method, which is selected for having desired properties such as high binding affinity, high expression level, or humanization. The selected protein (in this case, not a wild-type protein) may be used as the protein to be developed in the methods disclosed herein. In such embodiments, the methods of the present invention include the steps of: selecting a protein from a wild-type protein, a fragment of a wild-type protein, and a mutant protein; developing DNA encoding the selected protein to produce mutant DNA; expressing the mutant DNA to produce a mutant protein; and screening the mutant protein to produce a conditionally active biological protein that exhibits both (a) decreased activity compared to the selected protein in an assay under normal physiological conditions, and (b) increased activity compared to the selected protein in an assay under abnormal conditions.

[0181] Generation of developmental molecules from parent molecules Conditionally active biological proteins can be generated by mutagenesis methods and by screening individual mutations for decreased activity under wild-type conditions while maintaining or improving activity under non-wild-type conditions.

[0182] This disclosure provides a method for generating nucleic acid variants encoding a polypeptide having enzymatic activity, wherein the variants range from naturally occurring to having abnormal physiological activity, and the method comprises (a)(i) substituting one or more nucleotides with different nucleotides, where the nucleotides are native or non-native; (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 non-native nucleotide is inosine. In another embodiment, the method comprises analyzing a polypeptide encoded by a nucleic acid modified for abnormal enzymatic activity, thereby identifying the modified nucleic acid encoding the polypeptide having abnormal enzymatic activity. In one embodiment, the modification of step (a) is performed by PCR, error-prone PCR, shuffling, oligonucleotide-directed mutation, assembly PCR, sex-specific PCR mutation, in vivo mutation, cassette mutation, recurrent ensemble mutation, exponential ensemble mutation, site-directed mutation, gene rearrangement, gene site-saturated mutation, ligase chain reaction, in vitro mutation, ligase chain reaction, oligonucleotide synthesis, any gene generation technique, and combinations thereof. In another embodiment, the method further comprises at least one repetition of modification step (a).

[0183] The Disclosure further provides a method for producing polynucleotides from two or more nucleic acids. The method comprises (a) to (c) the following: (a) identifying identical and diverse regions between two or more nucleic acids, wherein at least one of the nucleic acids is 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 to produce a polynucleotide.

[0184] Any mutagenesis technique may be used in various embodiments of this disclosure. Stochastic or random mutagenesis is illustrated by situations in which a parent molecule is mutated (modified or altered) to obtain a set of progeny molecules having unintended mutations. Thus, in vitro stochastic mutagenesis reactions are, for example, unintended products, which are not intended to be produced. Rather, they are uncertain, and therefore random, with respect to the exact nature of the mutations obtained, and therefore to the products produced. Stochastic mutagenesis is demonstrated in methods in which mutations are random or unintended, such as error-prone PCR and stochastic shuffling. Mutagenesis is by error-prone transcription, such as error-prone PCR, or by the use of polymerases lacking proofreading activity (see Liao (1990) Gene 88:107-111), or by a first form of replication in mutant strains (mutant host cells are discussed in more detail below and are generally well known). Mutagenesis strains may include any mutants that are deficient in the function of inconsistent repair. These include mutant gene products such as mutS, mutT, mutH, mutL, ovrD, dcm, vsr, umuC, umuD, sbcB, and recJ. Deletions can be obtained by gene mutation, allele exchange, microcompounds, expressed antisense RNA, or other techniques. Deletions may be the described gene or homologous genes in any organism.

[0185] Currently, widely used mutagenesis techniques for selectively producing proteins from starter molecules include oligonucleotide-targeted mutagenesis, error-prone polymerase chain reaction (error-prone PCR), and cassette mutagenesis, where a specific region to be optimized is replaced with a synthetically mutated oligonucleotide. In these cases, many mutation sites occur around specific locations in the original sequence.

[0186] In oligonucleotide-designated mutagenesis, the short sequence is replaced with a synthetically mutagenerated oligonucleotide. In oligonucleotide-designated mutagenesis, the short sequence of a polynucleotide is removed from a synthetic polynucleotide using restriction enzyme digestion and replaced with a synthetic polynucleotide in which various bases are altered from the original sequence. Polynucleotide sequences can also be altered by chemical mutagenesis. Chemical mutagenesis includes, for example, sodium bisulfite, nitrite, hydroxylamine, hydrazine, or formic acid. Other drugs similar to nucleotide precursors include nitrosoguanidine, 5-bromouracil, 2-aminopurine, or acridine. Generally, these drugs are added to the PCR reaction in place of the nucleotide precursor that thereby displaces the sequence. Insertion of drugs such as proflavin, acriflavin, and quinacrine may also be used. Random mutagenesis of polynucleotide sequences can be obtained by X-ray or ultraviolet irradiation. Generally, mutagenerated plasmid polynucleotides are introduced into Escherichia coli (E. coli) and propagated as a pool or library of hybrid plasmids.

[0187] Error-prone PCR uses low-fidelity polymerization conditions to introduce low levels of random point mutations over long sequences. In mixtures of fragments of unknown sequences, error-prone PCR can be used to mutagenerate the mixture.

[0188] In cassette mutagenesis, single-template sequence blockade is typically (partially) replaced by random sequences. Reidhaar-Olson JF and Sauer RT: Combinatorial cassette mutagenesis as a probe of the informational content of protein sequences. Science 241(4861):53-57, 1988.

[0189] Alternatively, any technique of non-stochastic or non-random gene mutagenesis may also be used in various embodiments of this disclosure. Non-stochastic mutagenesis is illustrated by situations in which a parent molecule is mutated (modified or altered) in order to obtain a molecule having one or more predetermined mutations. It is well understood that the presence of some amount of background product is real in many reactions in which molecular processing occurs, and that the presence of background product does not diminish the non-stochastic nature of a mutagenesis process having an unintended product. Site-saturated mutagenesis and synthetic ligation reassembly are examples of gene mutagenesis techniques in which the precise chemical structure of the intended product is predetermined.

[0190] One method of site-saturation point mutagenesis is described in U.S. Patent Application Publication No. 2009 / 0130718. This method provides a set of denaturing primers corresponding to codons in a template polynucleotide and performs polymerase elongation to produce progeny polynucleotides containing sequences corresponding to the denaturing primers. The progeny polynucleotides are expressed in direct development and screened. Specifically, this is a method for producing a series of progeny polypeptides, comprising the steps of (a) providing a copy of a template polypeptide comprising a plurality of codons encoding a template polypeptide sequence, and (b) for each codon of the template polynucleotide, (1) providing a series of denaturing primers, where each primer comprises a denaturing codon corresponding to a codon of the template polynucleotide, and at least one adjacent sequence homologous to a sequence adjacent to the codon of the template polynucleotide, (2) providing conditions for the primers to be annealable to the copy of the template polynucleotide, and (3) carrying out a polymerase extension reaction from the primers along the template, thereby providing progeny polynucleotides, each comprising a sequence corresponding to the denaturing codon of the annealed primer, thereby producing a series of progeny polynucleotides.

[0191] Site-saturated mutagenesis relates to the induced development of nucleic acids and the screening of clones containing nucleic acids that have developed in the resulting activity of the target, such as nucleic acid activity and / or specific proteins, particularly enzymes.

[0192] The mutagenesized molecules provided by this technology may include chimeric molecules and molecules with point mutations, including biological molecules comprising sugars, lipids, nucleic acids and / or protein compositions, and specific but non-limiting examples of these include antibiotics, antibodies, enzymes, and steroids and non-steroidal hormones.

[0193] Site-saturated mutagenesis generally refers to a method comprising: 1) a step of preparing a progeny generation of a molecule (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 parental generation templates; 2) a step of screening the progeny-generating molecules - preferably a step of using a high-throughput method in at least one property of the target (e.g., improvement in enzyme activity, or increased stability, or novel chemotherapeutic effect); 3) a step of optionally obtaining and / or listing structural and / or functional information relating to parent and / or progeny generation molecules; and 4) an optional repeating of steps 1) to 3).

[0194] In site-saturated mutagenesis, the resulting polynucleotide offspring generation, referred to as “codon-site-saturated mutagenesis” (e.g., from a parent polynucleotide template), each has at least one set of three or more adjacent point mutations (i.e., different bases consisting of new codons) such that all codons (or all lineages of denatured codons encoding the same amino acid) are represented at each codon position. Corresponding to and encoded by this polynucleotide offspring generation is a series of resulting offspring polypeptides, each having at least one single amino acid point mutation. In a preferred embodiment, an example of the resulting “amino acid-site-saturated mutagenesis” is a mutant polypeptide in each of the 19 naturally encoded polypeptides, forming alpha-amino acid substitutions at each amino acid position and all amino acid positions along the polypeptide. The yield is all 20 different offspring polypeptides, including the original amino acids at each amino acid position and all amino acid positions along the parent polypeptide, or potentially 21 or more different offspring polypeptides if the added amino acids are used either in place of or added to the 20 naturally encoded amino acids.

[0195] Other mutagenesis techniques may include recombination, and more specifically, methods for preparing polynucleotides encoding polypeptides by in vivo reassembly of polynucleotide sequences containing partially homologous regions; methods for assembling polynucleotides to form at least one polynucleotide; and methods for screening polynucleotides for the production of polypeptides having useful properties.

[0196] In other embodiments, mutagenesis techniques utilize the inherent properties of cells to recombine molecules and / or to communicate reduced methods that minimize the complexity of ranges of repeating or consecutive sequences having sequences and homologous regions.

[0197] Various mutagenesis techniques may be used alone or in combination to provide methods for producing hybrid polypeptides that encode biologically active hybrid polypeptides with enhanced activity. In achieving these and other objectives, according to one aspect of this disclosure, a method is provided for introducing a polypeptide into a suitable host cell and growing the host cell under conditions for the production of hybrid polynucleotides.

[0198] Chimeric genes are created by joining two polynucleotide fragments that use compatible sticky ends produced by restriction enzymes, where each fragment originates from a separate ancestral (parent) molecule. Another example is single-codon mutagenesis (i.e., to obtain codon substitution, addition, or deletion) in the parent polynucleotide to produce a single offspring polynucleotide to encode a single-site mutagenesis polypeptide.

[0199] Furthermore, in vivo site-specific recombination systems, similar to random methods of in vivo recombination, are used to generate gene hybrids and recombinations between homologous but cleaved genes on plasmids. Mutagenesis has also been reported by duplication expansion and PCR.

[0200] Non-random methods are used to obtain a larger number of point mutations and / or chimeras, while comprehensive or inclusive methods are used to generate all molecular species in a particular mutation group in order for them to functionally belong to specific structural groups (e.g., specific single amino acid positions or sequences consisting of two or more amino acid positions) in a template molecule, and to classify and compare specific groupings of mutations.

[0201] These, or any other development methods, generate a library of novel molecules from one or more parent molecules in this disclosure.

[0202] The constructs may, after formation, be size-fractionated on an agarose gel according to a previously published protocol, inserted into a cloning vector, and transfected into appropriate host cells, or they may not be.

[0203] Expression of developed molecules Once a library of mutant molecules is generated, the DNA can be expressed using standard molecular biological techniques. Therefore, protein expression can be controlled using a variety of well-known methods.

[0204] For example, simply put, wild-type genes can be developed using any variety of random or non-random methods as shown herein. The mutant DNA molecules are then digested and ligated into vector DNA, such as plasmid DNA, using standard molecular biological techniques. The vector DNA containing 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 method is repeated for each mutant molecule.

[0205] The polynucleotides thus selected and isolated are introduced into a suitable host cell. A suitable host cell is any cell capable of promoting genetic recombination and / or decremental reassembly. The selected polynucleotides are preferably already present in a vector containing a suitable regulatory sequence. The host cell may be a higher eukaryotic cell such as a mammalian cell, or a lower eukaryotic cell such as a yeast cell, or preferably a prokaryotic cell such as a bacterial cell. The introduction of the construct into the host cell can be carried out 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).

[0206] Representative examples of usable expression vectors include viral 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., bacilli, aspergillus, and yeast). Thus, for example, DNA can be included in any of the various expression vectors for expressing polypeptides. Such vectors include chromosomal DNA sequences, non-chromosomal DNA sequences, and synthetic DNA sequences. Numerous suitable vectors are well known to those skilled in the art and are commercially available. Examples of vectors include the following: Bacterial: pQE vector (Qiagen), pBluescript plasmid, pNH vector, lambda ZAP vector (Stratagene); ptrc99a, ρKK223-3, pDR540, pRIT2T (Pharmacia); Eukaryotic: pXTl, ρSG5 (Stratagene), pSVK3, pBPV, pMSG, pSVLSV40 (Pharmacia). However, any other plasmid or vector may be used as long as it is replicable and viable in the host. Low copy number vectors or high copy number vectors may be used in this invention.

[0207] The DNA sequence in the expression vector is operably linked to an appropriate expression regulatory sequence (promoter) to guide RNA synthesis. Specific named bacterial promoters include lad, lacZ, T3, T7, gpt, lambda PR, PL, and trp. Eukaryotic promoters include pre-early CMV, HSV thymidine kinase, early and late SV40, retroviral LTR, and mouse metallothionein-1. The selection of appropriate vectors and promoters is well within the scope of those skilled in the art. The expression vector also includes a ribosome binding site and a transcription terminator for translation initiation. This vector may also include appropriate sequences for amplification of expression. The promoter region can be selected from any desired gene using a chloramphenicol transferase (CAT) vector with selectable markers, or other vectors. In addition, the expression vector preferably includes one or more selectable marker genes to provide phenotypic features for selecting transformed host cells, such as resistance to dihydrofolate reductase or neomycin in eukaryotic cell cultures, or resistance to tetracycline or ampicillin in Escherichia coli (E. coli).

[0208] Accordingly, in other embodiments of this disclosure, novel polynucleotides can be generated by decremental recombination methods. These methods involve generating a construct that includes a sequence of sequences (original coding sequences), their insertion into a suitable vector, and their subsequent introduction into a suitable host cell. Recombination of individual molecular identities occurs by combining sequences between sequences or between pseudo-repeating units in a construct having homologous regions. Recombination methods recombine and / or reduce the complexity and range of repetitive sequences, resulting in the generation of new molecular species. Various treatments can be applied to enhance the rate of recombination. These may include ultraviolet treatment or DNA-damaging chemicals, and / or the use of host cell lines exhibiting enhanced levels of "genetic instability." Thus, recombination methods may incorporate the natural properties of homologous recombination or quasi-repetitive sequences to guide their own development.

[0209] In one embodiment, the host organism or cell consists of Gram-negative bacteria, Gram-positive bacteria, or eukaryotes. In another embodiment of the present invention, the Gram-negative bacteria consist of Escherichia coli or Pseudomonas fluorescens. In another embodiment of the present invention, the Gram-positive bacteria consist of Streptomyces diversa, Lactobacillus gasseri, Lactococcus lactis, Lactococcus cremoris, or Bacillus subtilis. In other embodiments of the present invention, the eukaryotes consist of Saccharomyces cerevisiae, Schizosaccharomyces pombe, Pichia pastoris, Kluyveromyces lactis, Hansenula polymorpha, or Aspergillus niger. Representative examples of suitable hosts include bacterial cells such as Escherichia coli, Streptomyces, and Salmonella typhimurium; fungal cells such as yeast; insect cells such as Drosophila S2 and Spodoptera Sf9; animal cells such as CHO, COS, or Bowes melanoma; adenoviruses; and plant cells. The selection of an appropriate host is considered to be within the scope of those skilled in the art based on the teachings herein.

[0210] In particular, with regard to the various mammalian cell culture systems that can be used to express recombinant proteins, examples of mammalian expression systems include the COS-7 strain 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 may also include replication start sites, appropriate promoters and enhancers, and any necessary ribosome binding sites, polyadenylation sites, splice donor and acceptor sites, transcription termination sequences, and 5' adjacent non-transcription sequences. DNA sequences and polyadenylation sites derived from SV40 splicing can be used to provide the necessary non-transcriptional genetic factors.

[0211] The cells then proliferate, and “decreasing reassortment” is achieved. The rate of decreasing reassortment can be stimulated, if necessary, by introducing DNA damage. In vivo reassortment is directed toward “intermolecular” methods, collectively called “recombination,” which is typically observed in bacteria as a “RecA-dependent” phenomenon. The present invention can rely on the cell’s ability to modulate host cell recombination methods for rearranging and reassorting sequences, or reduction methods for reducing the complexity of quasi-repetitive sequences in cells by deletion. This method of “reduction reassortment” is brought about by “intramolecular,” RecA-independent methods. The final result is the reassortment of molecules into all possible combinations.

[0212] Host cells containing the target polynucleotide can be cultured in conventional nutrient media modified to be suitable for activating promoters, selecting transformed cells, or amplifying genes. These culture conditions (e.g., temperature, pH, etc.) have been previously used with host cells selected for expression and are also apparent to those skilled in the art.

[0213] Protein expression can be induced by various well-known methods, and many genetic systems have been published for inducing protein expression. For example, in suitable systems, protein expression is induced by adding an inducer. The cells are then pelleted by centrifugation, and the supernatant is removed. Periplasmic proteins can be enhanced by culturing cells containing DNAse, RNAse, and lysozyme. After centrifugation, the supernatant containing the novel protein is transferred to a new multi-well tray and stored before assay.

[0214] Cells are generally collected 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, sonic treatment, mechanical separation, or the use of cell lysants. Such methods are well known to those skilled in the art. The expressed polypeptide or fragments 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, hydroxyl apatite chromatography, and lectin chromatography. Protein refolding processes can be used, if necessary, to finalize the polypeptide structure. If necessary, high-performance liquid chromatography (HPLC) can be used for the final purification process.

[0215] Clones identified as having the desired activity can then be sequenced to identify the polynucleotide sequence encoding the enzyme with enhanced activity.

[0216] Polypeptides identified from such libraries can be used for therapeutic, diagnostic, research, and related purposes and / or subjected to one or more additional cycles of shuffling and / or selection. The present invention provides a fragment of a conditionally active biological protein having a length of at least 10 amino acids, wherein the fragment is active.

[0217] This disclosure provides codon-optimized polypeptides or fragments thereof having enzymatic activity, where the use of codons is optimized for specific organisms or cells. Narum et al., “Codon optimization of gene fragments encoding Plasmodium falciparum merzoite proteins enhances DNA vaccine protein expression and immunogenicity in mice”. Infect.Immun.2001 December,69(12):7250-3 describes codon optimization in a mouse system. Outchkourov et al., “Optimization of the expression of Equistatin in Pichia pastoris, protein expression and purification”, Protein Expr.Purif.2002 February;24(1):18-24 describes codon optimization in a yeast system. Feng et al., “High level expression and mutagenesis of recombinant human phosphatidylcholine transfer protein using a synthetic gene: evidence for a C-terminal membrane binding domain,” Biochemistry 2000 Dec.19,39(50):15399-409, describes codon optimization in Escherichia 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 use affects secretion in Escherichia coli.

[0218] The development of conditionally active biological proteins can be aided by the use of favorable high-throughput screening or selection processes.

[0219] After identification, the polypeptides and peptides of this disclosure may be synthesized or recombinant polypeptides. The peptides and proteins can be recombinantly expressed in vitro or in vivo. The peptides and polypeptides of this disclosure can be prepared and isolated using any method known in the art. The polypeptides and peptides of this disclosure can also be synthesized in whole or in part using chemical methods well 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 See Res.Symp.Ser.225-232;Banga, AK, Therapeutic Peptides and Proteins, Formulation, Processing and Delivery Systems (1995) Technomic Publishing Co., Lancaster, Pa.For example, peptide synthesis can be carried out using various solid-phase techniques (see, for example, 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 may be achieved using, for example, an ABI 43 IA peptide synthesizer (Perkin Elmer) according to the manufacturer's instructions.

[0220] The peptides and polypeptides of this disclosure can also be glycosylated. Glycosylation can be added chemically or post-translationally by cellular biosynthetic mechanisms, where the cellular biosynthetic mechanism involves the use of known glycosylation motifs (which may be native to the sequence, added as a peptide, or added to the nucleic acid coding sequence). Glycosylation may be O-linked or N-linked.

[0221] As defined above, the peptides and polypeptides of this disclosure include all forms of “mimetics” and “peptide mimetics.” The terms “mimetic” and “peptide mimetic” refer to synthetic chemical compounds having substantially the same structural and / or functional properties as the polypeptides of this disclosure. Mimics may consist entirely of synthetic non-natural amino acid analogs, or they may be chimeric molecules of partially natural peptide amino acids and partially non-natural amino acid analogs. Mimics may also incorporate any amount of conserved natural amino acid substitutions, as long as the substitutions do not substantially alter the structure and / or activity of the mimic. As with the polypeptides of this disclosure, which are conserved variants, whether a mimic is within the scope of this disclosure, i.e., whether its structure and / or function is substantially unaltered, will be determined by routine experimentation.

[0222] The polypeptide mimetic compositions of the present disclosure may contain any combination of non-natural structural components. In an alternative embodiment, the mimetic compositions of the present disclosure may contain one or all of the following three structural groups: a) residue linking groups other than natural amide bonds ("peptide bonds"); b) non-natural residues in place of naturally occurring amino acid residues; or c) residues that induce the mimicry of a secondary structure, i.e., by inducing or stabilizing a secondary structure such as a β-turn, γ-turn, β-sheet, α-helix conformation. For example, the polypeptides of the present disclosure may be characterized as mimetic when all or some of their residues are linked by chemical means other than natural peptide bonds. Individual peptide mimetic residues may be linked by peptide bonds, other chemical bonds or coupling means, such as glutaraldehyde, N-hydroxysuccinimide esters, difunctional maleimides, N,N'-dicyclohexylcarbodiimide (DCC), or N,N'-diisopropylcarbodiimide (DIC). Examples of linking groups that can replace conventional amide bonds ("peptide bonds") include ketomethylene (e.g., ~C(.dbd.O)-CH.sub.2~ or -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, for example, Spatola (1983), Chemistry and Biochemistry of Amino Acids, Peptides and Proteins, Vol.7, pp 267-357, “Peptide Backbone Modifications,” Marcell Dekker, NY).

[0223] The polypeptides of this disclosure can also be characterized as mimics by containing all or some non-natural residues instead of naturally occurring amino acid residues. Non-natural residues are adequately described in the scientific literature and patent documents; some exemplary non-natural compositions and guidelines useful as mimics of natural amino acid residues are described below. Mimics of aromatic amino acids include, for example, D- or L-naphthylalanine; D- or L-phenylglycine; D- or L-2-thienylalanine; D- or L-1,-2, 3-, or 4-pyrenylalanine; D- or L-3-thieneylalanine; D- or L-(2-pyridinyl)-alanine; D- or L-(3-pyridinyl)-alanine; D- or L-(2-pyradinyl)-alanine; D- or L-(4-isopropyl)-phenylglycine; D-(trifluoromethyl)-phenylglycine; D- (trifluoromethyl)-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 [wherein alkyl may be substituted or unsubstituted methyl, ethyl, propyl, hexyl, butyl, pentyl, isopropyl, isobutyl, sec-isobutyl, isopentyl, or a non-acidic amino acid]. Examples of aromatic rings of unnatural amino acids include thiazolyl, thiophenyl, pyrazolyl, benzimidazolyl, naphthyl, furanyl, pyrrolyl, and pyridyl aromatic rings.

[0224] Acidic amino acid mimics can be created, for example, by substitution with non-carboxylated amino acids; (phosphono)alanine; or sulfated threonine, while maintaining a negative charge. Carboxyl side groups (e.g., aspartyl or glutamyl) can also be selectively modified by reaction with carbodiimides (R'~NCN-R'), such as 1-cyclohexyl-3(2-morpholinyl-(4-ethyl)carbodiimide or l-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. Basic amino acid mimics can be created, for example, by substitution with the amino acids ornithine, citrulline, or (guanidino)acetic acid, or (guanidino)alkylacetic acid (where alkyl is defined above), in addition to lysine and arginine. Nitrile derivatives (e.g., containing a CN moiety instead of COOH) can be substituted for asparagine or glutamine. The arginine residue can be deaminated to the corresponding aspartyl or glutamyl residue. Arginine residue mimetic can be prepared by reacting arginyl with, for example, one or more conventional reagents, such as phenylglyoxal, 2,3-butanedione, 1,2-cyclohexanedione, or ninhydrin, preferably under alkaline conditions. Tyrosine residue mimetic can be prepared by reacting tyrosyl with, for example, an aromatic diazonium compound or tetranitromethane. Using N-acetylimidazole and tetranitromethane, O-acetyltyrosyl species and 3-nitro derivatives can be formed, respectively. Cysteine ​​residue mimetic can be prepared by reacting cysteinyl residue with, for example, α-haloacetic acid such as 2-chloroacetic acid or chloroacetamide and corresponding amines; carboxymethyl or carboxyamidemethyl derivatives can be obtained.Cysteine ​​residue mimics can also be prepared by reacting cysteinyl residues with, for example, bromo-trifluoroacetone, α-bromo-β-(5-imidazoyl)propionic acid; chloroacetyl phosphates, N-alkylmaleimides, 3-nitro-2-pyridyl disulfide; methyl 2-pyridyl disulfide; p-chloromercrine benzoate; 2-chloromercrine-4-nitrophenol; or chloro-7-nitrobenzo-oxa-1,3-diazole. Lysine mimics can also be prepared by reacting ricinyl with, for example, succinic acid or other carboxylic acid anhydrides (and by modifying the amino-terminal residue). Lysine and other α-amino-containing residue mimics can also be prepared by reacting imide esters, for example, methyl picoline imidate, pyridoxal phosphate, pyridoxal, chloroborohydride, trinitrobenzenesulfonic acid, O-methylisourea, 2,4, pentanedione, and by transamidase-catalyzed reactions with glyoxylates. Methionine mimics can be prepared, for example, by reaction with methionine sulfoxide. Proline mimics include, for example, pipecolic acid, thiazolidinediocarboxylic acid, 3- or 4-hydroxyproline, dehydroproline, 3- or 4-methylproline, or 3,3,-dimethylproline. Histidine residue mimics can be prepared by reaction with histidyl, for example, diethyl procarbonate or parabromophenacyl bromide. Other mimics include those prepared by hydroxylation of proline and lysine; phosphorylation of hydroxyl groups of ceryl or threonyl residues; methylation of α-amino groups of lysine, arginine, and histidine; acetylation of N-terminal amines; methylation or substitution of main-chain amide residues with N-methylamino acids; or amidation of C-terminal carboxyl groups.

[0225] The residues of the polypeptides of this disclosure, such as amino acids, may also be substituted with amino acids (or peptide mimetic residues) of the opposite chirality. Thus, any naturally occurring amino acid in the L-configuration (which may also be referred to as R or S depending on the structure of the chemical entity) may be referred to as a D-amino acid, but may be substituted with a peptide mimetic amino acid of the same chemical structure type, but with the opposite chirality, which may also be referred to as R-type or S-type.

[0226] This disclosure also provides methods for modifying the polypeptides of this disclosure by natural processes such as post-translational processing (e.g., phosphorylation, acylation, etc.) or by chemical modification techniques. The modifications may be performed anywhere on the polypeptide, including the peptide backbone, amino acid side chains, and amine or carboxyl termini. It will be understood that the same type of modification may be present at several sites of a given polypeptide to the same or varying degrees. Furthermore, a given polypeptide may have many types of modifications. Modifications include transfer RNA-mediated amino acid addition to proteins, such as acetylation, acylation, PEGylation, ADP-ribosylation, amidation, covalent bonding of flavins, covalent bonding of heme moieties, covalent bonding of nucleotides or nucleotide derivatives, covalent bonding of lipids or lipid derivatives, covalent bonding of phosphatidylinositol, crosslinking cyclization, disulfide bond formation, demethylation, covalent crosslink formation, cysteine ​​formation, pyroglutamic acid formation, formylation, γ-carboxylation, glycosylation, GPI anchor formation, hydroxylation, iodization, methylation, myristoylation, oxidation, pegylation, proteolytic processing, phosphorylation, prenylation, racemization, selenoylation, sulfation, and arginylation. See Creighton, TE, *Proteins-Structure and Molecular Properties*, 2nd Ed., WH Freeman and Company, New York (1993); and *Posttranslational Covalent Modification of Proteins*, BC Johnson, Ed., Academic Press, New York, pp. 1-12 (1983).

[0227] Solid-phase chemical peptide synthesis methods can also be used for the synthesis of polypeptides or fragments of the present disclosure. Such methods have been 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, J. Mand, Young, J. D, 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 generally utilize the teachings of HMGeysen 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 all provide peptide synthesis at the tips of numerous “rods” or “pins” bound to a single plate. When using such a system, the plate of rods or pins is inverted and inserted into a second plate in the corresponding well or reservoir, which contains a solution for attaching or fixing the appropriate amino acids to the tips of the pins or rods. By repeating this process, i.e., inverting the tips of the rods and pins and inserting them into the appropriate solution, amino acids are constructed to form the desired peptide. In addition, many available FMOC peptide synthesis systems are available. For example, polypeptide or fragment assembly can be performed on a solid support using Applied Biosystems, Inc.'s Model 431 A™ automated peptide synthesizer.Such apparatus provides an opportunity to readily obtain the peptides of the present disclosure, either by direct synthesis or by the synthesis of a series of fragments that can be conjugated using other known techniques.

[0228] Synthetic polypeptides or their fragments can be recovered and purified by known methods, including ammonium sulfate or ethanol precipitation, acid extraction, anion or cation exchange chromatography, phosphocellulose chromatography, hydrophobic interaction chromatography, affinity chromatography, hydroxyl apatite chromatography, and lectin chromatography. If necessary, a protein refolding step can be used to complete the polypeptide structure. If required, high-performance liquid chromatography (HPLC) can be used in the final purification step.

[0229] This disclosure provides a conditionally active protein variant preparation or formulation comprising at least one protein variant, the formulation being in liquid or dry form. The protein formulation optionally comprises a buffer, a cofactor, a second or additional protein, or one or more excipients. In one embodiment, the formulation is used as a therapeutic conditionally active biological protein that is active under abnormal conditions or non-physiological conditions, such as temperature, pH, or osmotic pressure, oxidative stress, or osmolality, but is low-activity or inactive under normal physiological conditions.

[0230] Standard purification techniques can be used for both recombinant and synthetically synthesized conditionally active biological proteins.

[0231] Identification of reversible or irreversible mutants through mutant screening. The identification of a desired molecule is most directly achieved by measuring protein activity under tolerant and wild-type conditions. Next, the mutant with the largest activity ratio (tolerant / wild-type) can be selected, and point mutation permutations are generated by combining individual mutations using standard methods. Then, the combined permutation protein library is screened for the protein showing the largest difference in activity between tolerant and wild-type conditions.

[0232] Using various methods, such as high-throughput activity assays including fluorescence assays, the activity of the supernatant can be screened to identify protein variants that are sensitive to any desired properties (temperature, pH, etc.). For example, to screen for time-sensitive variants, the enzyme or antibody activity of each individual variant can be determined using commercially available substrates at lower temperatures (e.g., 25°C) and at the temperature at which the original protein functions (e.g., 37°C). The reaction can be initially performed in a multi-well assay format, such as a 96-well assay, and then confirmed using different formats, such as a 14 ml tube format.

[0233] This disclosure further provides a screening assay for identifying enzymes, the assay comprising: (a) providing a plurality of nucleic acids or polypeptides; (b) obtaining polypeptide candidates from thereto be tested for enzyme activity; (c) testing the candidates for enzyme activity; and (d) identifying polypeptide candidates that exhibit increased enzyme activity under abnormal or non-physiological conditions, such as temperature, pH, oxidative stress, gravimetric osmolality, electrolyte concentration, or osmotic pressure, and that exhibit decreased enzyme activity compared to wild-type enzyme proteins under normal physiological conditions.

[0234] In one embodiment, the method further includes a step of modifying at least one nucleic acid or polypeptide before testing a candidate for conditional biological activity; in another embodiment, the test of step (c) further includes a step of testing for an improvement in polypeptide expression in a host cell or host organism; in a further embodiment, the test of step (c) further includes a step of testing enzyme activity in a pH range of about pH 3 to about pH 12; in a further embodiment, the test of step (c) further includes a step of testing enzyme activity in a pH range of about pH 5 to about pH 10; in a further embodiment, the test of step (c) further includes a step of testing enzyme activity in a pH range of about pH 6 to about pH 8; in a further embodiment, the test of step (c) further includes a step of testing enzyme activity at about pH 6.7 and about pH 7.5; in another embodiment, the test of step (c) further includes a step of testing enzyme activity in a temperature range of about 4°C to about 55°C. In another embodiment, the test of step (c) further includes a step of testing enzyme activity within a temperature range of approximately 15°C to approximately 47°C. In another embodiment, the test of step (c) further includes a step of testing enzyme activity within a temperature range of approximately 20°C to approximately 40°C. In another embodiment, the test of step (c) further includes a step of testing enzyme activity at approximately 25°C and approximately 37°C. In another embodiment, the test of step (c) further includes a step of testing enzyme activity under normal osmotic pressure and under abnormal (positive or negative) osmotic pressure. In another embodiment, the test of step (c) further includes a step of testing enzyme activity under normal electrolyte concentrations and under abnormal (positive or negative) electrolyte concentrations. The electrolyte concentration to be tested is selected from one of the concentrations of calcium, sodium, potassium, magnesium, chlorine, bicarbonate, and phosphate, and in another embodiment, the test of step (c) further includes a step of testing enzyme activity that yields a stable reaction product.

[0235] In another embodiment, the Disclosure provides a purified antibody that specifically binds to an enzyme-active polypeptide or fragment thereof of the Disclosure. In one embodiment, the Disclosure provides a fragment of an antibody that specifically binds to an enzyme-active polypeptide.

[0236] Antibodies and antibody-based screening methods This disclosure provides isolated or recombinant antibodies that specifically bind to the enzymes of this disclosure. These antibodies can be used for the isolation, identification, or quantification of the enzymes or related polypeptides of this disclosure. These antibodies can also be used for the isolation of other polypeptides or other related enzymes within the scope of this disclosure. The antibodies can be designed to bind to the active site of the enzyme. Accordingly, this disclosure provides a method for inhibiting an enzyme using the antibodies of this disclosure.

[0237] This antibody can be used in immunoprecipitation, staining, immunoaffinity columns, etc. If necessary, a nucleic acid sequence encoding a specific antigen can be generated by immunization followed by isolation of the polypeptide or nucleic acid, amplification or cloning of the polypeptide, and immobilization on the array of this disclosure. Alternatively, the structure of the antibody produced by the cell to be modified can be modified using the method of this disclosure, for example, to increase or decrease the affinity of the antibody. Furthermore, the ability to produce or modify antibodies may be a phenotype manipulated in the cell by the method of this disclosure.

[0238] Methods for immunization, antibody (polyclonal and monoclonal) production, and isolation are known to those skilled in the art and are described in scientific and patent documents. See, for example, Coligan, CURRENT PROTOCOLS IN IMMUNOLOGY, Wiley / Greene, NY (1991); Stites (eds.), BASIC AND CLINICAL IMMUNOLOGY (7th ed.), Lange Medical Publications, Los Altos, Calif. ("Stites"); Goding, MONOCLONAL ANTIBODIES: PRINCIPLES AND PRACTICE (2nd ed.), Academic Press, New York, NY (1986); Kohler (1975), "Continuous cultures of fused cells secreting antibody of predefined specificity," Nature 256:495; Harlow (1988), ANTIBODIES, A LABORATORY MANUAL, Cold Spring Harbor Publications, New York. Antibodies can also be generated in vitro, for example, using phage display libraries that express recombinant antibody binding sites, in addition to conventional in vivo methods using animals. See, 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 display”, Annu. Rev. Biophys. Biomol. Struct. 26:27-45.

[0239] A polypeptide or peptide can be used to generate an antibody that specifically binds to the polypeptide of the Disclosure, for example, an enzyme. The resulting antibody can be used in an immunoaffinity chromatography procedure to isolate or purify the polypeptide, or to determine whether the polypeptide is present in a biological sample. In such a procedure, a protein preparation, for example, an extract, or a biological sample is contacted with an antibody having the ability to specifically bind to one of the polypeptides of the Disclosure.

[0240] In the immunoaffinity procedure, an antibody is attached to a solid support such as beads or another column matrix. The protein preparation is brought into contact with the antibody under conditions in which the antibody specifically binds to one of the polypeptides of this disclosure. After washing to remove nonspecifically bound proteins, the specifically bound polypeptides are eluted.

[0241] The ability of a protein in a biological sample to bind to an antibody may be determined using any of the various procedures familiar to those skilled in the art. For example, binding can be determined by labeling the antibody with a detectable label such as a fluorescent agent, enzyme label, or radioisotope. Alternatively, the binding of the antibody to the sample may be detected using a secondary antibody having such a detectable label. Detailed assays include ELISA assays, sandwich assays, radioimmunoassays, and Western blotting.

[0242] Polyclonal antibodies produced against the polypeptides of this disclosure can be obtained by directly injecting the polypeptide into an animal or by administering the polypeptide to a non-human animal. The antibodies thus obtained will then bind to the polypeptide itself. In this way, even sequences encoding only fragments of the polypeptide can be used to produce antibodies that can bind to the complete native polypeptide. Such antibodies can then be used to isolate the polypeptide from cells expressing the polypeptide.

[0243] Monoclonal antibodies can be prepared using any technique that yields antibodies produced by serial cell line culture. Examples include the hybridoma method, trioma method, human B-cell hybridoma method, and EBV-hybridoma method (see, for example, Cole (1985) in Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, Inc., pp. 77-96).

[0244] Techniques described for the production of single-chain antibodies (see, for example, U.S. Patent No. 4,946,778) can be adapted for the production of single-chain antibodies against the polypeptides of this disclosure. Alternatively, humanized antibodies or fragments thereof against these polypeptides may be expressed using transgenic mice. Antibodies produced against the polypeptides of this disclosure can be used to screen for similar polypeptides (e.g., enzymes) from other organisms and samples. Such techniques involve contacting polypeptides from organisms with antibodies to detect polypeptides that specifically bind to the antibodies. Any of the procedures described above may be used to detect antibody binding.

[0245] Screening method and "online" monitoring device In carrying out the methods of the disclosure, various instruments and methods can be used, in conjunction with the polypeptides and nucleic acids of the disclosure, for example, to screen polypeptides for enzyme activity, compounds as candidate modulators of enzyme activity, such as activators or inhibitors, antibodies that bind to the polypeptides of the disclosure, to screen nucleic acids that hybridize with the nucleic acids of the disclosure, and to screen cells that express the polypeptides of the disclosure.

[0246] Array, or "biochip" The nucleic acids or polypeptides of this disclosure can be immobilized or applied to an array. The array can be used to screen or monitor a library of compositions (e.g., small molecules, antibodies, nucleic acids, etc.) relating to their binding ability to or their ability to regulate the activity of the nucleic acids or polypeptides of this disclosure. For example, in one embodiment of this disclosure, the parameter being monitored is the transcript expression of an enzyme gene. One or more, or all, transcripts of cells can be measured by hybridization with an array or immobilized nucleic acid on a "biochip" or by hybridization with a sample containing nucleic acids that are representative of or complementary to the cell transcripts. By using a "nucleic acid array" on a microchip, some or all of the transcripts of cells can be quantified simultaneously. Alternatively, an array containing genomic nucleic acids can be used to determine the genotype of a novel modified strain created by the method of this disclosure. Polypeptide arrays can also be used for the simultaneous quantification of multiple proteins. This disclosure can be carried out with any known "array," also known as a "microarray," "nucleic acid array," "polypeptide array," "antibody array," or "biochip," or variations thereof. An array typically consists of multiple "spots" or "target elements," each target element containing a predetermined amount of one or more biomolecules, such as oligonucleotides, immobilized on a predetermined area of ​​the substrate surface for specific binding to a sample molecule, such as an mRNA transcript.

[0247] In implementing the methods of this disclosure, for example, U.S. Patent No. 6,277,628; No. 6,277,489; No. 6,261,776; No. 6,258,606; No. 6,054,270; No. 6,048,695; No. 6,045,996; No. 6,022,963; No. 6,013,440; No. 5,965,452; No. 5,959,098; No. 5,856,174; No. 5,830,645; No. 5,770,456; No. 5,632,957; No. 5,556,752; No. 5,143,854 Any known array and / or method of constructing and using arrays, or modifications thereof, as described in Specification No. 5,807,522; Specification No. 5,800,992; Specification No. 5,744,305; Specification No. 5,700,637; Specification No. 5,556,752; Specification No. 5,434,049, can be incorporated in whole or in part by any known array and / or method of constructing and using arrays, or modifications thereof; also see, for example, International Publication No. 99 / 51773; International Publication No. 99 / 09217; International Publication No. 97 / 46313; International Publication No. 96 / 17958; also see, for example, 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 Publications No. 20010018642, 20010019827, 20010016322, 20010014449, 20010014448, 20010012537, and 20010008765.

[0248] Capillary array The methods of this disclosure may utilize capillary arrays such as GIGAMATRIX® Diversa Corporation, San Diego, Calif. The nucleic acids or polypeptides of this disclosure may be immobilized or applied to the array. The array can be used for screening or monitoring a library of compositions (e.g., small molecules, antibodies, nucleic acids, etc.) relating to their binding ability to or their ability to regulate the activity of the nucleic acids or polypeptides of this disclosure. The capillary array provides another system for holding and screening samples. For example, a sample screening instrument may include a plurality of capillaries formed as adjacent capillaries in an array, where each capillary includes at least one wall defining a lumen for holding a sample. The instrument may further include intervening material positioned between adjacent capillaries in the array, and one or more reference markings formed within the range of the intervening material. A capillary for screening a sample (where the capillary is adapted to be bound in an array of capillaries) may include a first wall defining a lumen for holding the sample and a second wall formed of a filtering material for filtering the excitation energy supplied to the lumen to excite the sample. A polypeptide or nucleic acid, such as a ligand, may be introduced into a first component that enters at least a portion of the capillaries in the capillary array. Each capillary in the capillary array may include at least one wall defining a lumen for holding the first component. Bubbles may be introduced into the capillary after the first component. A second component may be introduced into the capillary, where the second component is separated from the first component by bubbles. The sample of interest can be introduced into the capillaries of a capillary array as a first liquid labeled with detectable particles, where each capillary of the capillary array includes at least one wall defining a lumen for holding the first liquid and the detectable particles, and this at least one wall is coated with a binding material that binds the detectable particles to at least one wall.This method may further include the steps of removing a first liquid from a capillary tube, wherein the bound, detectable particles are retained within the capillary, and introducing a second liquid into the capillary tube. A capillary array may include a plurality of individual capillaries, each having at least one outer wall defining a lumen. The outer wall of a capillary may be one or more walls fused together. Similarly, walls may define a lumen of cylindrical, square, hexagonal, or any other geometric shape, insofar as the wall forms a lumen for holding a liquid or sample. The capillaries of a capillary array may be held together in close proximity to form a planar structure. The capillaries may be joined together by fusion (e.g., if the capillaries are made of glass), bonding, joining, or fastening. A capillary array may be formed of any number of individual capillaries, e.g., in the range of 100 to 4,000,000 capillaries. A capillary array can form a microtiter plate in which approximately 100,000 or more individual capillaries are bonded together.

[0249] In some embodiments, the activity of the conditionally active biological protein of the present invention is inhibited by small molecules under normal physiological conditions, but is not inhibited at all or to a limited extent by the same small molecules under abnormal conditions. For example, the activity of a conditionally active biological protein may be inhibited by oxygen, glucose, or bicarbonate present at specific concentrations in human plasma, but since the concentrations of oxygen, glucose, or bicarbonate may be lower in the tumor microenvironment than in human plasma, the activity of the same protein may be inhibited to a lesser degree or not inhibited at all in the tumor microenvironment.

[0250] In some embodiments, the method of the present invention can simultaneously improve the binding affinity and selectivity of a template polypeptide. For example, starting from a template antibody, the method can produce a conditionally active antibody that has a higher binding affinity to the antigen under abnormal conditions than the template antibody, and a higher ratio of activity under abnormal conditions to activity under normal physiological conditions than the template antibody. In one embodiment, these results are achieved by using combinatorial protein synthesis (CPS) as described in U.S. Patent No. 8,859,467. Specifically, by using CPS, it is possible to simultaneously incorporate mutations that improve both conditional activity and affinity, and thus simultaneously improve both the affinity and selectivity of the selected conditionally active biological protein.

[0251] Assay conditions The normal and abnormal physiological conditions of the assay used in the screening process may be conditions selected from temperature, pH, osmotic pressure, gravimetric osmolality, oxidative stress, and electrolyte concentration, as well as combinations of two or more such conditions. For example, the normal physiological temperature condition may be the normal human body temperature of 37.0°C, while the abnormal temperature condition may be a temperature different from 37.0°C, such as the temperature of the tumor microenvironment, which may be 1-2°C higher than the normal physiological temperature. In another example, the normal and abnormal physiological conditions may also be a normal physiological pH in the range of 7.2-7.6 and an abnormal pH in the range of 6.2-6.8 shown in the tumor microenvironment.

[0252] Both assays under normal and abnormal physiological conditions can be performed in an assay medium. The assay medium may be a solution containing, for example, a buffer and other components. Common buffers that can be used as assay media include citrate buffers such as sodium citrate, phosphate buffers, bicarbonate buffers such as Krebs buffer, phosphate-buffered saline (PBS) buffer, Hanks buffer, Tris buffer, and HEPES buffer. Other buffers known to those skilled in the art as suitable for assays may also be used. These buffers can be used to simulate the compositional characteristics or components of human or animal body fluids such as plasma or lymph.

[0253] The assay solution useful in the method of the present invention may contain at least one component selected from inorganic compounds, ions, and organic molecules, preferably those commonly found in the body fluids of mammals or animals such as humans. Examples of such components include nutritional components and metabolites, as well as any other components that may be found in body fluids. The present invention intends that this component may or may not be part of a buffer system. For example, the assay solution may be a PBS buffer with added bicarbonate ions, where the bicarbonate is not part of the PBS buffer. Alternatively, the bicarbonate ions may be part of a bicarbonate buffer.

[0254] The inorganic compound or ion may be selected from one or more of the following: boric acid, calcium chloride, calcium nitrate, diammonium phosphate, magnesium sulfate, monoammonium phosphate, monopotassium phosphate, potassium chloride, potassium sulfate, copper sulfate, iron sulfate, manganese sulfate, zinc sulfate, magnesium sulfate, calcium nitrate, calcium chelate, copper chelate, iron chelate, manganese chelate, and zinc chelate, ammonium molybdate, ammonium sulfate, calcium carbonate, magnesium phosphate, potassium bicarbonate, potassium nitrate, hydrochloric acid, carbon dioxide, sulfuric acid, phosphoric acid, carbonic acid, uric acid, hydrogen chloride, urea, phosphate ion, sulfate ion, chloride ion, magnesium ion, sodium ion, potassium ion, ammonium ion, iron ion, zinc ion, and copper ion.

[0255] Examples of normal physiological concentrations of some inorganic compounds include uric acid in the range of 2–7.0 mg / dL, calcium ions in the range of 8.2–11.6 mg / dL, chloride ions in the range of 355–381 mg / dL, iron ions in the range of 0.028–0.210 mg / dL, potassium ions in the range of 12.1–25.4 mg / dL, sodium ions in the range of 300–330 mg / dL, carbonic acid in the range of 15–30 mM, citrate ions at approximately 80 μM, histidine ions in the range of 0.05–2.6 mM, histamine in the range of 0.3–1 μM, HAPT ions (hydride-adenosine triphosphate) in the range of 1–20 μM, and HADP ions in the range of 1–20 μM.

[0256] In some embodiments, the ions present in the assay solution under both normal and abnormal physiological conditions are selected from hydroxide ions, halide ions (chloride, bromide, iodide), oxyhalide ions, sulfate ions, magnesium ions, calcium ions, bisulfate ions, carbonate ions, bicarbonate ions, sulfonate ions, oxyhalide ions, nitrate ions, nitrite ions, phosphate ions, hydrogen phosphate ions, dihydrogen phosphate ions, persulfate ions, monopersulfate ions, borate ions, ammonium ions, or organic ions such as carboxylate ions, phenolate ions, sulfonate ions (organic sulfates such as methyl sulfate), vanadate ions, tungstate ions, borate ions, organic boronate ions, citrate ions, oxalate ions, acetate ions, pentaborate ions, histidine ions, and phenolate ions.

[0257] Organic compounds present in assay solutions under both normal and abnormal physiological conditions may be selected from, for example, amino acids such as histidine, alanine, isoleucine, arginine, leucine, asparagine, lysine, aspartic acid, methionine, cysteine, phenylalanine, glutamic acid, threonine, glutamine, tryptophan, glycine, valine, pyrrolicin, proline, selenocysteine, serine, and tyrosine, as well as mixtures thereof.

[0258] Examples of normal physiological concentrations of some amino acids include: alanine at 3.97±0.70 mg / dL, arginine at 2.34±0.62 mg / dL, glutamic acid at 3.41±1.39 mg / dL, glutamine at 5.78±1.55 mg / dL, glycine at 1.77±0.26 mg / dL, histidine at 1.42±0.18 mg / dL, isoleucine at 1.60±0.31 mg / dL, and 1. Examples include leucine at 91±0.34 mg / dL, lysine at 2.95±0.42 mg / dL, methionine at 0.85±0.46 mg / dL, phenylalanine at 1.38±0.32 mg / dL, threonine at 2.02±6.45 mg / dL, tryptophan at 1.08±0.21 mg / dL, tyrosine at 1.48±0.37 mg / dL, and valine at 2.83±0.34 mg / dL.

[0259] Organic compounds present in assay solutions under both normal and abnormal physiological conditions can be selected from non-protein nitrogen-containing compounds such as creatine, creatinine, guanidinoacetic acid, uric acid, allantoin, adenosine, urea, ammonia, and choline. Examples of normal physiological concentrations of some of these compounds include 1.07 ± 0.76 mg / dL of creatine, 0.9–1.65 mg / dL of creatinine, 0.26 ± 0.24 mg / dL of guanidinoacetic acid, 4.0 ± 2.9 mg / dL of uric acid, 0.3–0.6 mg / dL of allantoin, 1.09 ± 0.385 mg / dL of adenosine, 27.1 ± 4.5 mg / dL of urea, and 0.3–1.5 mg / dL of choline.

[0260] Organic compounds present in assay solutions under both normal and abnormal physiological conditions can be selected from organic acids such as citric acid, α-ketoglutaric acid, succinic acid, malic acid, fumaric acid, acetoacetic acid, β-hydroxybutyric acid, lactic acid, pyruvate, α-ketonic acid, acetic acid, and volatile fatty acids. Examples of normal physiological concentrations of some of these organic acids include 2.5 ± 1.9 mg / dL of citric acid, 0.8 mg / dL of α-ketoglutaric acid, 0.5 mg / dL of succinic acid, 0.46 ± 0.24 mg / dL of malic acid, 0.8 to 2.8 mg / dL of acetoacetic acid, 0.5 ± 0.3 mg / dL of β-hydroxybutyric acid, 8 to 17 mg / dL of lactic acid, 1.0 ± 0.77 mg / dL of pyruvate, 0.6 to 2.1 mg / dL of α-ketonic acid, and 1.8 mg / dL of volatile fatty acids.

[0261] Organic compounds present in assay solutions under both normal and abnormal physiological conditions may be selected from sugars (carbohydrates) such as glucose, pentoses, hexoses, xylose, ribose, mannose, and galactose, as well as disaccharides including lactose, GlcNAcβ1-3Gal, Galα1-4Gal, Manα1-2Man, GalNAcβ1-3Gal, and O-, N-, C-, or S-glycosides. Examples of normal physiological concentrations of some of these sugars include 83±4 mg / dL of glucose, 102±73 mg / dL of polysaccharides (as hexoses), 77±63 mg / dL of glucosamine, 0.4-1.4 mg / dL of hexuronate (as glucuronic acid), and 2.55±0.37 mg / dL of pentoses.

[0262] Organic compounds present in assay solutions under both normal and abnormal physiological conditions can be selected from fats or their derivatives, such as cholesterol, lecithin, cephalin, sphingomyelin, and bile acids. Examples of normal physiological concentrations of some of these compounds include 40–70 mg / dL of free cholesterol, 100–200 mg / dL of lecithin, 0–30 mg / dL of cephalin, 10–30 mg / dL of sphingomyelin, and 0.2–0.3 mg / dL of bile acids (as cholic acid).

[0263] The organic compounds present in the assay solutions under both normal and abnormal physiological conditions can be selected from proteins such as fibrinogen, antihemophilic globulin, immunogamma-globulin, immunoeuglobulin, alloaglutinin, β-pseudoglobulin, glycoproteins, lipoproteins, and albumin. For example, the normal physiological concentration of mammalian serum albumin is 3.5–5.0 g / dL. In one embodiment, albumin is bovine serum albumin.

[0264] Organic compounds present in assay solutions under both normal and abnormal physiological conditions can be selected from vitamins such as vitamin A, carotene, vitamin E, ascorbic acid, thiamine, inositol, folic acid, biotin, pantothenic acid, and riboflavin. Examples of normal physiological concentrations of some of these vitamins include vitamin A at 0.019–0.036 mg / dL, vitamin E at 0.90–1.59 mg / dL, inositol at 0.42–0.76 mg / dL, folic acid at 0.00162–0.00195 mg / dL, and biotin at 0.00095–0.00166 mg / dL.

[0265] The concentrations of inorganic compounds, ions, or organic molecules in the assay solution (both under normal physiological conditions and under abnormal conditions) may be within the normal physiological concentration range of inorganic compounds, ions, or organic molecules in human or animal serum. However, concentrations outside the normal physiological range may also be used. For example, the normal range for magnesium ions in human serum is 1.7–2.2 mg / dL, and for calcium it is 8.5–10.2 mg / dL. The magnesium ion concentration in the assay solution may be approximately 0.17 mg / dL to approximately 11 mg / dL. The calcium ion concentration in the assay solution may be approximately 0.85 mg / dL to approximately 51 mg / dL. As a general rule, the concentration of inorganic compounds, ions, or organic molecules in the assay solution may be as low as 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, or 80% of the normal physiological concentration of those inorganic compounds, ions, or organic molecules in human serum, or it may be as high as 1.5, 2, 3, 4, 5, 7, 9, 10, or even 20 times the normal physiological concentration of those inorganic compounds, ions, or organic molecules in human serum. Various components of the assay solution may be used at concentration levels different from their respective normal physiological concentrations.

[0266] The activity of mutant proteins is measured using assays under normal and abnormal physiological conditions. During the assay, both the mutant protein and its binding partner are present in the assay solution. The relationship between the mutant protein and its binding partner can be, for example, antibody-antigen, ligand-receptor, enzyme-substrate, or hormone-receptor. For the mutant protein to express its activity, it must be able to come into contact with and bind to its binding partner. Next, the activity of the mutant protein towards its binding partner is expressed after binding and measured.

[0267] In some embodiments, the ions used in the assay may function in the formation of crosslinks between the mutant protein being screened and its binding partner, particularly those containing charged amino acid residues. Thus, the ions may have the ability to bind to both the mutant protein and its binding partner via hydrogen bonds and / or ionic bonds. This can assist binding between the mutant protein and its binding partner by allowing the ions to reach sites that may be difficult to reach on the macromolecule (mutant protein or its binding partner). In some cases, ions in the assay solution may increase the likelihood of the mutant protein and its binding partner binding to each other. Furthermore, the ions may assist binding between the mutant protein and its binding partner by binding to the macromolecule (mutant protein or its binding partner), either in addition to or instead of the above. This binding may alter the conformation of the macromolecule to a specific conformation that promotes binding to the binding partner, and / or retain the macromolecule in such a conformation.

[0268] It has been observed that ions can assist in the binding of mutant proteins to their binding partners, possibly by forming ionic bonds between the mutant protein and its binding partner. Therefore, compared to the same assay without ions, screening can be far more efficient, and more hits (candidate conditionally active biological proteins) can be identified. Suitable ions can be selected from magnesium ions, sulfate ions, bisulfate ions, carbonate ions, citrate ions, HAPT ions, HADP ions, bicarbonate ions, nitrate ions, nitrite ions, phosphate ions, hydrogen phosphate ions, dihydrogen phosphate ions, persulfate ions, monopersulfate ions, borate ions, lactate ions, citrate ions, histidine ions, histamine ions, and ammonium ions.

[0269] It has been found that ions function to facilitate binding between the mutant protein and its binding partner at a pH close to the ion's pKa. Such ions are preferably relatively small compared to the size of the mutant protein.

[0270] In one embodiment, if the abnormal condition is a pH different from the normal physiological pH under normal physiological conditions, suitable ions for increasing the hit count of candidate conditionally active biological proteins can be selected from ions having a pKa close to the abnormal pH being tested in the assay. For example, the pKa of an ion may differ from the abnormal pH by 1 pH unit, 0.8 pH units, 0.6 pH units, 0.5 pH units, 0.4 pH units, 0.3 pH units, 0.2 pH units, or 0.1 pH units.

[0271] Exemplary pKas of ions useful in the present invention (these pKas may vary slightly with temperature) are as follows: ammonium ion has a pKa of approximately 9.24, dihydrogen phosphate has a pKa of 7.2, acetic acid has a pKa of approximately 4.76, histidine has a pKa of approximately 6.04, bicarbonate ion has a pKa of approximately 6.4, citrate has a pKa of 6.4, lactate ion has a pKa of approximately 3.86, histamine has a pKa of approximately 6.9, HATP has a pKa of 6.95 (HATP3-⇔ATP4-+H+), and HADP has a pKa of 6.88 (HADP3-⇔ADP4-+H+).

[0272] In one embodiment, conditionally active biological proteins are assayed and selected in the presence of hydrogen sulfide. Hydrogen sulfide has a pKa of 7.05. In some embodiments, different hydrogen sulfide concentrations may be used for assays corresponding to normal physiological conditions and assays corresponding to abnormal physiological conditions. Alternatively, the assay media for both normal and abnormal physiological conditions may have approximately the same hydrogen sulfide concentration, with some differences in the values ​​for specific conditions; for example, this assay may be performed at different pH levels. The hydrogen sulfide concentration used in the assay may be 100 μm to about 100 mM. Preferably, the assay media has a hydrogen sulfide concentration of 1 to 10 mM, or 1 to 5 mM, or 1 to 3 mM. Assays performed in the presence of hydrogen sulfide are well known.

[0273] In certain embodiments, once the pH of an abnormal condition (i.e., abnormal pH) is known, ions suitable for increasing the hit of candidate conditionally active biological proteins can be selected from ions having a pKa at or close to the abnormal pH. For example, candidate ions may provide pKas that differ from the abnormal pH by about 1 pH unit, 0.8 pH units, 0.6 pH units, 0.5 pH units, 0.4 pH units, 0.3 pH units, 0.2 pH units, or 0.1 pH units.

[0274] As mentioned above, ions are most effective at assisting the binding of mutant proteins to their binding partners at or near their pKa. For example, in assay solutions with a pH of 7.2–7.6, bicarbonate ions (with a pKa of approximately 6.4) are known to be less effective at assisting the binding of mutant proteins to their binding partners. As the pH of the assay solution is reduced to 6.7, and even to approximately 6.4, bicarbonate ions become progressively more effective at assisting the binding of mutant proteins to their binding partners. As a result, an assay at pH 6.4 may identify more hits compared to an assay at pH 7.2–7.6. Similarly, histidine is less effective at assisting the binding of mutant proteins to their binding partners at pH 7.4. As the pH of the assay solution is reduced to 6.7, and even to approximately 6.0, histidine becomes progressively more effective at assisting the binding of mutant proteins to their binding partners, and it also becomes possible to identify more hits in the pH range of approximately 6.2–6.4.

[0275] In this invention, it was unexpectedly discovered that when the pH of the assay solution under normal physiological conditions (i.e., normal physiological pH) differs from the pH of the assay solution under abnormal conditions (i.e., abnormal pH), ions with pKa values ​​ranging from approximately the midpoint between normal physiological pH and abnormal pH to approximately abnormal pH can significantly assist the binding of the mutant protein being screened to its binding partner. As a result, this screening assay is far more efficient in finding more hit or candidate conditional biological proteins that are highly active under abnormal conditions.

[0276] In some embodiments, the pKa may differ from the abnormal pH by at least 1 pH unit. If the abnormal pH is acidic, the pKa of a suitable ion may range from (abnormal pH-1) to the midpoint between the abnormal pH and the normal physiological pH. If the abnormal pH is basic, the pKa of a suitable ion may range from (abnormal pH+1) to the midpoint between the abnormal pH and the normal physiological pH. The ions may be selected from those described herein. However, many more ions not expressly described herein may also be used. Once the abnormal pH and normal physiological pH of the screening assay are selected, those skilled in the art will understand that, using the guidelines of the present invention, any ion having a suitable pKa to increase the efficiency of the screening in terms of identifying more hits with high activity under abnormal conditions can be selected.

[0277] For example, if the abnormal pH for a given exemplary screening is 8.4 and the normal physiological pH is 7.4, then any ion with a pKa in the range of approximately 7.9 (midpoint) to 9.4 (i.e., 8.4+1) can be used for screening. Some ions with pKas in this range include tricine (pKa 8.05), hydrazine (pKa 8.1), bicine (pKa 8.26), N-(2-hydroxyethyl)piperazine-N'-(4-butanesulfonic acid) (pKa 8.3), N-tris[hydroxymethyl]methyl-3-aminopropanesulfonic acid (pKa 8.4), and taurine (pKa 9.06). In another example, if the abnormal pH for a given exemplary screening is 6 and the normal physiological pH is 7.4, then any ion with a pKa in the range of approximately 5 (i.e., 6-1) to 6.7 (midpoint) can be used for screening. Some ions with pKas in this range include malate (pKa 5.13), pyridine (pKa 5.23), piperazine (pKa 5.33), cacodylate (pKa 6.27), succinate (pKa 5.64), 2-(N-morpholino)ethanesulfonic acid (pKa 6.10), citrate (pKa 6.4), histidine (pKa 6.04), and bis-tris (6.46). Those skilled in the art will be able to identify known chemical compounds, including both inorganic and organic compounds, that can be converted to ions with pKas within this range by referring to a vast number of chemical manuals and texts. Among chemical compounds with suitable pKas, those with smaller molecular weights may be preferred.

[0278] Therefore, unexpectedly, the present invention found that the production of the ultimately identified conditionally active biological protein depends not only on the generation of the correct protein mutant from the wild-type protein, but also on the use of suitable pKa ions in the assay solution. Since ions can facilitate the efficient selection of highly active mutants from large libraries, the present invention suggests that, in addition to generating large libraries of mutant proteins (e.g., by CPE and CPS), efforts should be focused on finding suitable ions (with appropriate pKa) to use in the assay solution. Furthermore, without suitable ions, the efficiency of screening may be lower, and the likelihood of finding highly active mutants may decrease. Consequently, multiple screening rounds may be required to obtain the same number of highly active mutants without suitable ions.

[0279] The ions in the assay solution may be formed in situ from the components of the assay solution, or they may be directly incorporated into the assay solution. For example, carbon dioxide and bicarbonate ions may be provided by dissolving CO2 from air into the assay solution. In another example, sodium dihydrogen phosphate may be added to the assay solution to provide dihydrogen phosphate ions.

[0280] The concentration of this component in the assay solution (for both assays under normal physiological conditions and assays under abnormal conditions) may be the same as, or substantially the same as, the concentration of the same component typically found in the body fluids of mammals such as humans. In other embodiments, the concentration of the component may be higher, particularly in the case of a component that is an ion capable of assisting the binding between the mutant protein and its binding partner, because it has been observed that higher concentrations of such ions may lead to the formation of ionic bonds between the mutant protein and its binding partner, and in particular, facilitate binding, which may increase the likelihood of finding more hits or candidate conditionally active proteins.

[0281] In some embodiments, the ion concentration in the assay solution may be positively correlated with the likelihood of finding more hits using the assay, particularly when concentrations higher than normal physiological concentrations are used. For example, human serum has a bicarbonate ion concentration of approximately 15–30 mM. In one example, when the bicarbonate ion concentration in the assay solution was increased from 3 mM to 10 mM, 20 mM, 30 mM, 50 mM, and 100 mM, the number of hits in the assay also increased with increasing bicarbonate concentration. Based on this, the assay solution can use bicarbonate concentrations in the range of approximately 3 mM to 200 mM, or approximately 5 mM to 150 mM, or approximately 5 mM to 100 mM, or approximately 10 mM to 100 mM, or approximately 20 mM to 100 mM, or approximately 25 mM to 100 mM, or approximately 30 mM to 100 mM, or approximately 35 mM to 100 mM, or approximately 40 mM to 100 mM, or approximately 50 mM to 100 mM.

[0282] In another embodiment, the citrate concentration in the assay solution may be approximately 30 μM to approximately 120 μM, or approximately 40 μM to approximately 110 μM, or approximately 50 μM to approximately 110 μM, or approximately 60 μM to approximately 100 μM, or approximately μM to approximately 90 μM, or approximately μM.

[0283] Conditionally active biological proteins have been shown to have a higher proportion of charged amino acid residues compared to their original wild-type proteins. For example, three positively charged amino acid residues: lysine, arginine, and histidine; and two negatively charged amino acid residues: aspartate and glutamate. In selected conditionally active biological proteins, these charged amino acids occupy a larger proportion compared to the wild-type protein (from which mutant proteins are developed, and from which conditionally active biological proteins are selected). A higher proportion of charged amino acid residues can lead to the formation of more hydrogen / ionic bonds with ions, which may relate to the use of ions in the assay solution.

[0284] In one embodiment, normal physiological conditions are a normal physiological pH in the range of 7.2 to 7.6, and abnormal conditions are an abnormal pH in the range of 6.2 to 6.8. The assay solution for the assay under normal physiological conditions has a normal physiological pH and 50 mM bicarbonate ions. The assay solution for the assay under abnormal conditions has an abnormal pH and 50 mM bicarbonate ions. Since the pKa of bicarbonate ions is approximately 6.4, bicarbonate ions can assist in the binding of mutant proteins to their binding partners at an abnormal pH of 6.2 to 6.8, for example, pH 6.4.

[0285] In yet another embodiment, normal physiological conditions are a normal physiological pH in the range of 7.2–7.6, and abnormal conditions are an abnormal pH in the range of 6.2–6.8. The assay solution for the assay under normal physiological conditions has a normal physiological pH and 80 μM citrate ions. The assay solution for the assay under abnormal conditions has an abnormal pH and 80 μM citrate ions. Since the citrate ion has a pKa of 6.4, the citrate ion can effectively assist in the binding between the mutant protein and its binding partner in the assay solution under abnormal conditions of pH 6.2–6.8. Therefore, more candidate conditionally active biological proteins that have higher binding activity under pH 6.4 conditions and lower activity under pH 7.2–7.8 conditions can be identified. Other ions, including acetate, histidine, bicarbonate, HATP, and HADP, function similarly, and assay solutions containing these ions can effectively screen for mutant proteins that have higher binding activity at pH around the ion's pKa and lower binding activity at pH different from the ion's pKa (e.g., normal physiological pH).

[0286] In yet another embodiment, normal physiological conditions are a normal physiological temperature of 37°C, and abnormal conditions are abnormal temperatures of 38-39°C (temperatures in some tumor microenvironments). The assay solution for the assay under normal physiological conditions has a normal physiological temperature and 70 mM bicarbonate ions. The assay solution for the assay under abnormal conditions has an abnormal temperature and 70 mM bicarbonate ions.

[0287] In yet another embodiment, normal physiological conditions are specific electrolyte concentrations in normal human serum, and abnormal conditions are the same electrolyte concentrations at different abnormal concentrations that can occur at different locations in an animal or human or are caused by the state of an animal or human that changes the normal physiological electrolyte concentrations in human serum.

[0288] The binding between the mutant protein and / or its binding partner can also be affected in several other ways. Typically, this effect will be exerted by including one or more additional components in the assay medium. These additional components can be designed to interact with either or both of the mutant protein and the binding partner. In addition, these additional components can affect binding using combinations of two or more interactions and combinations of two or more types of interactions.

[0289] In one embodiment, the binding interaction of interest is between an antibody and an antigen. In this embodiment, one or more additional components are included in the assay medium and can affect the antibody, the antigen, or both. In this way, the desired binding interaction can be enhanced.

[0290] In addition to ions that can form ionic bonds with the mutant protein and / or its binding partner to assist in the binding between the mutant protein and the binding partner, the present invention also includes other components that can be utilized to assist in the binding between the mutant protein and its binding partner. In one embodiment, molecules that can form hydrogen bonds with the mutant protein and / or its binding partner can be utilized. In another embodiment, molecules having hydrophobic interaction capabilities with the mutant protein and / or its binding partner can be used. In yet another embodiment, molecules having van der Waals interaction capabilities with the mutant protein and / or its binding partner are contemplated.

[0291] As used herein, the term "hydrogen bond" refers to a relatively weak non-covalent interaction between a hydrogen atom covalently bonded to an electronegative atom such as carbon, nitrogen, oxygen, sulfur, chlorine, or fluorine (a hydrogen bond donor) and a lone pair of electrons on an electron-donating atom such as nitrogen, oxygen, sulfur, chlorine, or fluorine (a hydrogen bond acceptor).

[0292] Components capable of forming hydrogen bonds with mutant proteins and / or their binding partners include organic and inorganic molecules with polar bonds. Mutant proteins and / or their binding partners typically contain amino acids capable of forming hydrogen bonds. Preferred amino acids have side chains with polar groups capable of forming hydrogen bonds. Non-limiting examples of preferred amino acids include glutamine (Gln), glutamic acid (Glu), arginine (Arg), asparagine (Asn), aspartic acid (Asp), lysine (Lys), histidine (His), serine (Ser), threonine (Thr), tyrosine (Tyr), cysteine ​​(Cys), methionine (Met), and tryptophan (Trp).

[0293] These amino acids can function as both hydrogen donors and hydrogen acceptors. For example, the oxygen atom of an -OH group, as may be found in Ser, Thr, and Tyr; the oxygen atom of an -C=O group, as may be found in Glu and Asp; the sulfur atom of an -SH group or -SC-, as may be found in Cys and Met; the nitrogen atom of an -NH3+ group, as may be found in Lys and Arg; and the nitrogen atom of an -NH- group, as may be found in Trp, His, and Arg, can all function as hydrogen acceptors. In addition, any group in this list that contains a hydrogen atom (e.g., -OH, -SH, NH3+, and -NH-) can also function as a hydrogen donor.

[0294] In some embodiments, the backbone of the mutant protein and / or its binding partner may also be involved in the formation of one or more hydrogen bonds. For example, the backbone may have a repeating -(C=O)-NH- structure, as in the peptide bond. The oxygen and nitrogen atoms of this structure may function as hydrogen acceptors, while the hydrogen atom may participate in hydrogen bonding.

[0295] Examples of inorganic compounds having at least one polar bond involving a hydrogen or oxygen atom that can be used for hydrogen bonding include H2O, NH3, H2O2, hydrazine, carbonates, sulfates, and phosphates. Organic compounds such as alcohols, phenols, thiols, aliphatic amines, amides, epoxides, carboxylic acids, ketones, aldehydes, ethers, esters, organic chlorides, and organofluorines. Compounds capable of forming hydrogen bonds are well known in the chemical literature, for example, those discussed in "The Nature of the Chemical Bond," by Linus Pauling, Cornell University Press, 1940, pages 284-334 (this disclosure is incorporated herein by reference in its entirety with respect to its enumeration of compounds having hydrogen bonding interaction ability).

[0296] In some embodiments, alcohols include methanol, ethanol, propanol, isopropanol, butanol, pentanol, l-hexanol, 2-octanol, l-decanol, cyclohexanol, and higher alcohols; diols, such as ethylene glycol, propylene glycol, glycerol, diethylene glycol, and polyalkylene glycols. Suitable phenols include hydroquinone, resorcinol, catechol, phenol, o-, m-, and p-cresol, thymol, α and β-naphthol, pyrogallol, guaiacol, and phloroglucinol. Suitable thiols include methanethiol, ethanethiol, 1-propantheol, 2-propantheol, butanethiol, tert-butyl mercaptan, pentanethiols, hexanethiol, thiophenol, dimercaptosuccinic acid, 2-mercaptoethanol, and 2-mercaptonidole. Suitable amines include methylamine, ethylamine, propylamine, isopropylamine, aniline, dimethylamine and methylethylamine, trimethylamine, aziridine, piperidine, N-methylpiperidine, benzidine, cyclohexylamine, ethylenediamine, hexamethylenediamine, o-, m-, and p-toluidine and N-phenylpiperidine. Suitable amides include ethaneamide, N,N-dimethylacetamide, N,N-dimethylformamide, N,N-dimethylmethoxyacetamide and N-methyl-Np-cyanoethylformamide. Suitable epoxides include ethylene oxide, propylene oxide, tert-butyl hydroperoxide, styrene oxide, epoxide glycidol, cyclohexene oxide, di-tert-butylperoxide, cumene hydroperoxide or ethylbenzene hydroperoxide, isobutylene oxide, and 1,2-epoxyoctane.Examples of carboxylic acids include terephthalic acid, isophthalic acid, phthalic acid, salicylic acid, benzoic acid, acetic acid, lauric acid, adipic acid, lactic acid, citric acid, acrylic acid, glycine, hexahydrobenzoic acid, o-, m-, and p-toluic acid, nicotinic acid, isonicotinic acid, and para-aminobenzoic acid. Examples of ketones include acetone, 3-propanone, butanone, pentanone, methyl ethyl ketone, diisobutyl ketone, ethyl butyl ketone, methyl isobutyl ketone, methyl tert-butyl ketone, cyclohexanone, acetone, methyl ethyl ketone, methyl propyl ketone, methyl butyl ketone, methyl amyl ketone, methylhexyl ketone, diethyl ketone, ethyl butyl ketone, dipropyl ketone, diisobutyl ketone, diacetone alcohol, phorone, isophorone, cyclohexanone, methylcyclohexanone, and acetophenone. Examples of aldehydes include formaldehyde, acetaldehyde, propionaldehyde, butyraldehyde, benzaldehyde, cinnamaldehyde, isobutyraldehyde, valeraldehyde, octaldehyde, benzaldehyde, cinnamaldehyde, cyclohexanone, salicylaldehyde, and furfural. Examples of esters include ethyl acetate, methyl acetate, ethyl formate, butyl acetate, ethyl lactate, ethyl butyrate, propyl acetate, ethyl formate, propyl formate, butyl formate, amyl formate, methyl acetate, ethyl acetate, propyl acetate, butyl acetate, amyl acetate, methylisoamyl acetate, methoxybutyl acetate, hexyl acetate, cyclohexyl acetate, benzyl acetate, methyl propionate, ethyl propionate, butyl propionate, amyl propionate, methyl butyrate, ethyl butyrate, butyl butyrate, amyl butyrate, methyl acetoacetate, and ethyl acetoacetate. Examples of ethers that can be used in the present invention include dimethyl ether, methyl ethyl ether, diethyl ether, methyl propyl ether, and dimethoxyethane. The ethers may also be cyclic, such as ethylene oxide, tetrahydrofuran, and dioxane.

[0297] Examples of organic chlorides include chloroform, pentachloroethane, dichloromethane, trichloromethane, carbon tetrachloride, tetrachloromethane, tetrachloroethane, pentachloroethane, trichloroethylene, tetrachloroethylene, and ethylene dichloride. Examples of organic fluorine compounds include fluoromethane, difluoromethane, trifluoromethane, trifluoroethane, tetrafluoroethane, pentafluoroethane, difluoropropane, trifluoropropane, tetrafluoropropane, pentafluoropropane, hexafluoropropane, and heptafluoropropane.

[0298] Hydrogen bonds can be classified into strong, moderate, or weak based on their strength (Jeffrey, George A.; An introduction to hydrogen bonding, Oxford University Press, 1997). Strong hydrogen bonds have a donor-acceptor distance of 2.2–2.5 Å and an energy range of 14–40 kcal / mol. Moderate hydrogen bonds have a donor-acceptor distance of 2.5–3.2 Å and an energy range of 4–15 kcal / mol. Weak hydrogen bonds have a donor-acceptor distance of 3.2–4.0 Å and an energy range of <4 kcal / mol. Some examples of hydrogen bonds, along with their energy levels, are FH···:F (38.6 kcal / mol), OH···:N (6.9 kcal / mol), OH···:O (5.0 kcal / mol), NH···:N (3.1 kcal / mol), and NH···:O (1.9 kcal / mol). For further details, see Perrin et al. "Strong" hydrogen bonds in chemistry and biology, Annual Review of Physical Chemistry, vol. 48, pages 511-544, 1997; Guthrie, "Short strong hydrogen bonds: can they explain enzymic catalysis?" Chemistry & Biology March 1996, 3: 163-170.

[0299] In some embodiments, the components used in the present invention can form strong hydrogen bonds with mutant proteins and / or their binding partners. These components tend to have atoms with high electronegativity. The atoms known to have the highest electronegativity are, in order, F>O>Cl>N. Therefore, the present invention preferably uses organic compounds containing fluorine, a hydroxyl group, or a carbonyl group in the formation of hydrogen bonds. In one embodiment, organofluorine may be used in the present invention to form strong hydrogen bonds.

[0300] In another embodiment, a component having hydrophobic interaction ability with the mutant protein and / or its binding partner is used. Such a component includes an organic compound having a hydrophobic group.

[0301] As used herein, the term “hydrophobic interaction” refers to a reversible attractive interaction between a hydrophobic compound or a hydrophobic region of one compound and another hydrophobic compound or a hydrophobic region of another compound. This type of interaction is described in “Hydrophobic Interactions,” A. Ben-Nairn (1980), Plenum Press, New York (this disclosure is incorporated herein by reference in relation to its description of hydrophobic interactions).

[0302] Hydrophobic materials, due to their nonpolar nature, are subject to the repulsive force of water molecules. When relatively nonpolar molecules or groups in an aqueous solution associate with other nonpolar molecules rather than with water, this is called a "hydrophobic interaction."

[0303] Mutant proteins and their binding partners typically contain amino acids with hydrophobic interacting ability. These amino acids can typically be characterized by having at least one side chain with a nonpolar group that has hydrophobic interacting ability. Examples of hydrophobic amino acids include alanine (Ala), isoleucine (Ile), leucine (Leu), phenylalanine (Phe), valine (Val), proline (Pro), glycine (Gly), and to a lesser degree, methionine (Met) and tryptophan (Trp).

[0304] Components having hydrophobic interaction ability with mutant proteins and / or their binding partners include organic compounds that are hydrophobic molecules or molecules containing at least one hydrophobic moiety. In some embodiments, these hydrophobic components may be hydrocarbons selected from aromatic hydrocarbons, substituted aromatic hydrocarbons, polycyclic aromatic hydrocarbons, aromatic or non-aromatic heterocyclics, cycloalkanes, alkanes, alkenes, and alkynes. Hydrophobic groups may include aromatic groups, alkyl, cycloalkyl, alkenyl, and alkynyl groups. The terms “alkyl,” “alkenyl,” and “alkynyl,” as used herein, refer to unsaturated aliphatic groups having 1 to 30 carbon atoms, including linear alkenyl / alkynyl groups, branched alkenyl / alkynyl groups, cycloalkenyl (alicyclic) groups, alkyl-substituted cycloalkyl groups, and cycloalkyl-substituted alkenyl / alkynyl groups. Such hydrocarbon moieties may also be substituted on one or more carbon atoms.

[0305] It can be understood that the strength of hydrophobic interactions is based on the amount of available "hydrophobic substances" that can interact with each other. Therefore, hydrophobic interactions can be regulated, for example, by increasing the amount of hydrophobic moieties in molecules involved in hydrophobic interactions and / or increasing their "hydrophobic" properties. For example, a hydrophobic moiety (which may in its original form include a hydrocarbon chain) can be modified to increase its hydrophobicity (its ability to increase the strength of hydrophobic interactions involving that moiety) by adding a hydrophobic side chain to one of the carbons in its carbon skeleton. In preferred embodiments of the present invention, this may include, for example, the addition of various steroid compounds and / or derivatives thereof, such as sterol compounds, and more specifically, various polycyclic compounds including cholesterol. Generally, the side chain may be linear, aromatic, aliphatic, cyclic, polycyclic, or any other variety of hydrophobic side chains as intended by those skilled in the art.

[0306] Components capable of van der Waals interactions with mutant proteins and / or their binding partners are, though not necessarily, typically compounds with polar moieties. As used herein, “van der Waals interaction” refers to interatomic, interpartinochemical, intermolecular, and surface-to-atomic forces resulting from the interplay of fluctuating polarities of adjacent atoms, parts, or molecules as a result of dipole interactions and / or quantum dynamics.

[0307] In this invention, van der Waals interaction is an attractive force between a mutant protein or binding partner and its constituent components. Van der Waals interaction can arise from three sources. Firstly, some molecules / parts can be permanent electric dipoles, even if electrically neutral. Due to a fixed distortion in the electron charge distribution in the structure of some molecules / parts, one side of the molecule / part is always somewhat positive and the other side is somewhat negative. The tendency of such permanent dipoles to align with each other results in a net attractive force. This is the interaction between two permanent dipoles (Kaesom force).

[0308] Second, the presence of molecules that are permanent dipoles can temporarily distort the electronic charges of other adjacent polar or nonpolar molecules, thereby inducing further polarization. Additional attractive forces arise when permanent dipoles interact with adjacent induced dipoles. This is the interaction between a permanent dipole and the corresponding induced dipole, and it can be referred to as the Debye force. Third, even if the molecules involved are not permanent dipoles (e.g., the organic liquid benzene), there is a force that attracts between molecules due to two instantaneously induced dipoles in the molecule. This is the interaction between two instantaneously induced dipoles, and it can be referred to as the London dispersion force.

[0309] There are a number of amino acids with van der Waals interaction capabilities in the mutant protein and / or binding partner. These amino acids can have polar side chains, including glutamine (Gln), asparagine (Asn), histidine (His), serine (Ser), threonine (Thr), tyrosine (Tyr), cysteine (Cys), methionine (Met), and tryptophan (Trp). These amino acids can also have side chains with nonpolar groups, including alanine (Ala), isoleucine (Ile), leucine (Leu), phenylalanine (Phe), valine (Val), proline (Pro), and glycine (Gly).

[0310] The components with van der Waals interaction capabilities between the mutant protein and / or its binding partner include polar or nonpolar inorganic compounds soluble in the assay solution. The assay solution is generally an aqueous solution, so these polar or nonpolar inorganic compounds are preferably soluble in water. Materials preferred for van der Waals interactions are polar ones with dipole-dipole interaction capabilities. For example, AlF3 has polar Al-F bonds and is soluble in water (about 0.67 g / 100 ml water at 20 °C). HgCl2 has polar Hg-Cl bonds and is soluble in water at 7.4 g / 100 ml at 20 °C. PrCl2 has polar Pr-Cl bonds and is soluble in water at about 1 g / 100 ml at 20 °C.

[0311] Suitable polar compounds having van der Waals interaction ability include alcohols, thiols, ketones, amines, amides, esters, ethers, and aldehydes. Suitable examples of these compounds are described above in relation to hydrogen bonding. Suitable nonpolar compounds having van der Waals interaction ability include aromatic hydrocarbons, substituted aromatic hydrocarbons, polycyclic aromatic hydrocarbons, aromatic or non-aromatic heterocycles, cycloalkanes, alkanes, alkenes, and alkynes.

[0312] By utilizing hydrogen bonding components, hydrophobic components, and van der Waals components, the binding of mutant proteins to their binding partners can be influenced in several ways. In one embodiment, a crosslink can be formed between the mutant protein and its binding partner through hydrogen bonding, hydrophobic interactions, and / or van der Waals interactions. Such a crosslink can bring the mutant protein and its binding partner closer together to promote binding, and / or position the mutant protein and / or its binding partner in a way that promotes binding to each other.

[0313] In another embodiment, hydrogen bonding and / or hydrophobic interactions can increase the likelihood of a mutant protein binding to its binding partner by, for example, assembling or associating the protein and its binding partner in a manner that increases the likelihood of binding. Therefore, by using one or more of these interactions individually or in combination, it may be possible to bring the mutant protein and its binding partner closer together or to position them in a manner that promotes binding, for example, by causing the binding sites to be attracted closer to each other, or by positioning the unbinding portions of the molecules further apart, thereby positioning the binding sites closer to each other.

[0314] In another embodiment, hydrogen bonding and / or hydrophobic interactions may affect the conformation of the mutant protein and / or its binding partner, resulting in a conformation that further facilitates the binding of the mutant protein to its binding partner. Specifically, binding or interaction with one or more amino acids of the mutant protein and / or its binding partner may cause one or more conformational changes in the mutant protein or its binding partner that are favorable to the mutant protein / binding partner binding reaction.

[0315] The present invention performs two pairs of assays, one to determine the decrease in the activity of a mutant protein in an assay under normal physiological conditions compared to the original protein from which the mutant protein originated under normal physiological conditions, and the second assay to determine the increase in the activity of a mutant protein in an assay under abnormal conditions compared to the original protein from which the mutant protein originated under abnormal conditions. In some cases, the original protein from which the mutant protein originates may be a wild-type protein. In other cases, the original protein from which the mutant protein originates may itself be a mutant protein prepared using one or more mutation techniques described in other parts of this specification.

[0316] The conditions used in the assay pair of the present invention can be selected from temperature, pH, osmotic pressure, gravimetric osmolality, oxidative stress, electrolyte concentration, and the concentration of any other components of the assay solution or medium. Thus, a particular component of the assay medium may be used at substantially the same concentration in both pairs of assays. In such cases, the components are typically present for the purpose of simulating a specific environment in humans or animals, such as serum, tumor microenvironment, muscle environment, nervous environment, or any other environment that may be faced at the administration point, through which the administered treatment may pass, or at the treatment point. An important aspect of selecting one or more components to simulate these environments is that it may thereby improve the outcome of the selection process performed using the assay pair. For example, simulating a specific environment makes it possible to evaluate the various effects that specific components of that environment have on mutant proteins during the selection process. Components of a specific environment may, for example, alter or bind to mutant proteins, inhibit the activity of mutant proteins, or inactivate mutant proteins.

[0317] In some embodiments, one or more components of the assay solution are preferably small molecules such as hydrogen sulfide, bicarbonate, histamine, lactic acid, and acetic acid. In one embodiment, the small molecule components are preferably present in the assay solution at a concentration of about 100 μm to about 100 mM, more preferably about 0.5 to about 50 mM, or about 1 to about 10 mM.

[0318] The concentration of a component in the assay solution may be the same as, or substantially the same as, the concentration of the same component typically found in the body fluids of mammals such as humans. This can be referred to as the normal physiological concentration of the component in the body fluid. In other embodiments, the concentration of a particular component in the assay solution may be lower or higher than the concentration of the same component typically found in the body fluids of mammals such as humans.

[0319] In another embodiment, the components may be present at substantially different concentrations in each pair of assays. In such cases, the concentration of the components is the condition that distinguishes the assay solution of an assay under normal physiological conditions from the assay solution of an assay under abnormal conditions; therefore, the presence, absence, or concentration of the components becomes the condition being assayed. Accordingly, the conditionally active biological proteins produced by this embodiment of the method of the present invention can be selected with respect to activity that is at least partially dependent on the concentration of the components.

[0320] In some embodiments, a component may be present in one pair of assay solutions but not in the other pair. For example, the lactate concentration in the assay solution under abnormal conditions may be set to a level that simulates the lactate concentration in the tumor microenvironment. Lactate may not be present in the assay solution pair under normal physiological conditions.

[0321] In one embodiment, the normal physiological condition is a first lactate concentration representative of the normal physiological condition, and the abnormal condition is a second lactate concentration representative of the abnormal condition present at a specific location within the body.

[0322] In another example, glucose may be absent from the assay solution under abnormal conditions to simulate the absence of glucose as may be found in the tumor microenvironment, while glucose may be set to a level that simulates plasma glucose concentration in the pair of assay solutions under normal physiological conditions. This feature allows conditionally active biological proteins to be preferentially delivered to a location or environment in an inactive or minimally active state during transport, and then activated when they reach an environment where the concentrations of the components in the assay solution under abnormal conditions are present.

[0323] For example, the tumor microenvironment typically has both lower glucose and higher lactate concentrations compared to human serum. Normal physiological glucose concentrations in serum range from approximately 2.5 mM to 10 mM. On the other hand, glucose concentrations in the tumor microenvironment are typically extremely low, ranging from 0.05 mM to 0.5 mM. In one embodiment, the assay solution for an assay under normal physiological conditions has a glucose concentration in the range of approximately 2.5 mM to 10 mM, while the assay solution for an assay under abnormal conditions has a glucose concentration in the range of approximately 0.05 mM to 0.5 mM. The conditionally active biological protein thus produced exhibits higher activity in a low-glucose environment than in a high-glucose environment. This conditionally active biological protein is functional in the tumor microenvironment but may exhibit low activity while passing through the bloodstream.

[0324] The normal physiological concentration of lactate in serum is in the range of approximately 1 mM to 2 mM. On the other hand, in the tumor microenvironment, the lactate concentration is typically in the range of 10 mM to 20 mM. In one embodiment, the assay solution for an assay under normal physiological conditions has a lactate concentration in the range of approximately 1 mM to 2 mM, while the assay solution for an assay under abnormal conditions has a lactate concentration in the range of approximately 10 mM to 20 mM. The conditionally active biological protein thus produced exhibits higher activity in a high-lactate environment than in a low-lactate environment. Therefore, this conditionally active biological protein is functional in the tumor microenvironment but may exhibit low activity while passing through the bloodstream.

[0325] Similarly, it is known that lactate concentrations are higher than normal (abnormal) in muscle pain. Therefore, when searching for mutant proteins that can be activated in a muscle pain environment, the pair of assays under abnormal conditions can be performed in the presence of higher lactate concentrations to simulate the muscle pain environment, while the pair of assays under normal physiological conditions can be performed with lower lactate concentrations or in the absence of lactate. In this way, mutant proteins whose activity increases with increasing lactate concentration in a muscle pain environment can be selected. Such conditionally active biological proteins may be useful, for example, as anti-inflammatory agents.

[0326] In another embodiment, two or more components may be used in both pairs of assay solutions. In this type of assay, conditionally active biological proteins can be selected using features of both types of assays described above. Alternatively, the selectivity for conditionally active biological proteins can be increased by using two or more components. For example, returning to the tumor microenvironment, the pair of assays under abnormal conditions can be performed in an assay medium having both high lactate and low glucose concentrations, while the pair of assays under the corresponding normal physiological conditions can be performed in an assay medium having both relatively low lactate and relatively high glucose concentrations.

[0327] The present invention aims to enable the selection of conditionally active biological proteins that exhibit higher activity at a certain concentration of a component compared to different concentrations of the same component, by using each component individually or in combination, selected from inorganic compounds, ions, and organic molecules.

[0328] Assays that rely on different concentrations of one or more metabolites as a condition to distinguish between normal and abnormal environments can be particularly suitable for selecting conditionally active biological proteins that are more active in the tumor microenvironment than in plasma, because the tumor microenvironment typically contains many metabolites at different concentrations compared to the same metabolites in plasma.

[0329] Kinoshita et al., “Absolute Concentrations of Metabolites in Human Brain Tumors Using In Vitro Proton Magnetic Resonance Spectroscopy,” NMR IN BIOMEDICINE, vol.10, pp.2-12, 1997, compared metabolites in normal brains and brain tumors. This group found that N-acetylaspartate is present at a concentration of 5000-6000 μM in normal brains, but this concentration is only 300-400 μM in glioblastomas, 1500-2000 μM in astrocytomas, and 600-1500 μM in undifferentiated astrocytomas. Furthermore, inositol is present at a concentration of 1500-2000 μM in a normal brain, but this concentration is 2500-4000 μM in glioblastoma, 2700-4500 μM in astrocytoma, and 3800-5800 μM in undifferentiated astrocytoma. Phosphorylethanolamine is present at a concentration of 900-1200 μM in a normal brain, but this concentration is 2000-2800 μM in glioblastoma, 1170-1370 μM in astrocytoma, and 1500-2500 μM in undifferentiated astrocytoma. Glycine is present at a concentration of 600-1100 μM in a normal brain, but this concentration is 4500-5500 μM in glioblastoma, 750-1100 μM in astrocytoma, and 1900-3500 μM in undifferentiated astrocytoma. Alanine is present in normal brain tissue at concentrations of 700–1150 μM, but this concentration is 2900–3600 μM in glioblastoma, 800–1200 μM in astrocytoma, and 300–700 μM in undifferentiated astrocytoma. These metabolites can also have different concentrations in the blood; for example, N-acetylaspartate is present at a concentration of approximately 85,000 μM in the blood; inositol at a concentration of approximately 21,700 μM; glycine at a concentration of approximately 220–400 μM; and alanine at a concentration of approximately 220–300 μM.

[0330] Therefore, these metabolites, including at least N-acetylaspartate, inositol, glycine, and alanine, can be used at different concentrations in the assay solution to select conditionally active biological proteins that are active in brain tumors but not in blood or normal brain tissue. For example, to select conditionally active biological proteins that are active in the glioblastoma tumor microenvironment but not active or at least have low activity in blood or normal brain tissue, an assay solution with a concentration of 85,000 μM N-acetylaspartate may be used in the assay pair under normal physiological conditions, while an assay solution with a concentration of 350 μM N-acetylaspartate may be used in the assay pair under abnormal conditions.

[0331] Mayers et al., “Elevated circulating branched-chain amino acids are an early event in pancreatic adenocarcinoma development,” Nature Medicine, vol.20, pp.1193-1198, 2014, investigated the concentrations of various different metabolites, including branched-chain amino acids, in the pre-diagnostic plasma of pancreatic patients. They found that several metabolites were present in the bloodstream at different concentrations in pancreatic tumor patients compared to the same metabolites in the blood of people without pancreatic cancer. Mayers et al. also found that pancreatic cancer patients had a significant increase in branched-chain amino acids in their plasma compared to normal subjects. Branched-chain amino acids present at elevated concentrations included isoleucine, leucine, and valine (Table 1 of Mayers). Other metabolites shown in Figure 1 of Mayers are present at significantly different concentrations in the plasma of pancreatic cancer patients compared to normal healthy individuals. These metabolites include at least acetylglycine, glycine, phenylalanine, tyrosine, 2-aminoadipic acid, taurodeoxycholate / taurochenodeoxycholate, aconitate, isocitrate, lactate, α-glycerophosphate, and urate. Therefore, based on the finding that certain metabolites exist at different concentrations in the plasma of pancreatic cancer patients and normal healthy patients, it can be predicted that the tumor microenvironment of pancreatic cancer will also have different concentrations of these metabolites than those that may exist in the pancreatic microenvironment of healthy patients.

[0332] Therefore, in one embodiment, one or more of these metabolites may be used in an assay solution under normal physiological conditions in an amount that approximates the concentration of these metabolites in the plasma of a healthy person (i.e., the normal physiological concentration of the metabolite). For example, the known normal physiological concentrations in the plasma of a healthy person are approximately 1.60 ± 0.31 mg / dL for isoleucine, approximately 1.91 ± 0.34 mg / dL for leucine, and approximately 2.83 ± 0.34 mg / dL for valine. An assay solution under normal physiological conditions may have one or more of these branched amino acids at normal physiological concentrations within these ranges. An assay solution under abnormal conditions may have the same branched amino acid at a concentration approximately 5 times, 10 times, 20 times, 50 times, 70 times, 100 times, 150 times, 200 times, or 500 times higher than the normal physiological concentration of the corresponding branched amino acid in a healthy person. Based on the findings of Mayers et al., the higher concentrations of these branched amino acids found in plasma by Mayers et al. are likely to originate from the tumor microenvironment and be diluted in the bloodstream, thus reflecting the expectation that concentrations of these branched amino acids are significantly elevated in the pancreatic tumor microenvironment. Similarly, assays under abnormal conditions may reflect concentrations of other metabolites in the blood of pancreatic cancer patients, even if the concentration of a particular metabolite is significantly lower in cancer patients compared to normal individuals. In this way, screening can simulate real-world environments, thereby ensuring that the most active variants for that specific environment are selected.

[0333] In some other embodiments, the assay solution under normal physiological conditions may contain one or more branched amino acids at concentrations that mimic the concentrations in the plasma of pancreatic cancer patients and thus simulate the actual plasma environment of these patients. In such embodiments, the assay solution under abnormal conditions may have the same branched amino acids at concentrations about 2, 3, 4, 5, 7, 8, 10, 15, 20, or 50 times higher than the concentrations of the corresponding branched amino acids in the plasma of pancreatic cancer patients, reflecting the fact that these higher concentrations occur in the tumor microenvironment and that the concentrations in the bloodstream correspond to a dilution of the actual concentrations in the tumor microenvironment. Similarly, other metabolites may also have different concentrations in the assay solutions under normal physiological conditions and abnormal conditions, reflecting actual differences expected from the data collected regarding bloodstream. In some cases, a deficiency of a specific metabolite may be observed in the bloodstream of pancreatic patients. In such cases, the concentration reflecting the measured bloodstream concentration can be used in assays under normal physiological conditions, while even lower concentrations can be used in assays under abnormal conditions, taking into account the expectation that the metabolite will be consumed in the tumor microenvironment. Thus, conditionally active biological proteins selected using the assay solution may exhibit higher activity in the pancreatic tumor microenvironment compared to the plasma of pancreatic cancer patients.

[0334] In some embodiments, the entire plasma of a pancreatic cancer patient may be used in the present invention. For example, in one embodiment, one or both of the assays under normal physiological conditions and abnormal conditions may use a simulated version of one or more components of the plasma of a pancreatic cancer patient in the assay solution. In an exemplary embodiment, the assay solution under normal physiological conditions has a pH in the range of 7.2 to 7.6 and is supplemented with 30 wt.% of the plasma of a pancreatic cancer patient, while the assay solution under abnormal conditions has a pH in the range of 6.4 to 6.8 and is supplemented with 30 wt.% of the plasma of a pancreatic cancer patient. In this embodiment, the plasma of the pancreatic cancer patient is present both to (1) ensure that the conditionally active biological protein is not activated in the blood at pH 7.2 to 7.6, and to (2) ensure that the conditionally active biological protein can also be activated at pH 6.2 to 6.8 in the tumor microenvironment, even in the presence of this metabolite composition found in the blood of a pancreatic cancer patient. This allows the treatment to be tailored to the pancreatic cancer patient.

[0335] In another exemplary embodiment, the assay solution under normal physiological conditions has a pH in the range of 7.2 to 7.6 and is supplemented with 30 wt.% of plasma from pancreatic cancer patients, while the assay solution under abnormal conditions has a pH in the range of 6.4 to 6.8 and is not supplemented with any plasma from pancreatic cancer patients.

[0336] In each of the several types of assays discussed above, the same components selected from inorganic compounds, ions, and organic molecules may be used. For example, in the case of lactic acid, it may be used at substantially the same concentration in both the normal physiological conditions and the abnormal conditions of the assay solution pair. The difference between normal physiological conditions and abnormal conditions will then be one or more other aspects, such as temperature, pH, or the concentration of another component. In different embodiments, lactic acid may be used as one of the factors distinguishing normal physiological conditions from abnormal conditions, thereby reflecting the fact that lactic acid is present at a higher concentration in the abnormal tumor microenvironment compared to normal physiological conditions (non-tumor microenvironment).

[0337] In some embodiments, two or more components are added to the assay solutions for both normal and abnormal conditions at substantially the same concentrations. For example, both citrate and bovine serum albumin (BSA) are added to these assay solutions. In both assay solutions, the citrate concentration may be about 80 μM, and the BSA concentration may be about 10–20%. More specifically, the assay solution for the pair of assays under normal physiological conditions may have a pH in the range of 7.2–7.6, with a citrate concentration of about 80 μM and a BSA concentration of about 10–20%. The assay solution for the pair of assays under abnormal conditions may have a pH in the range of 6.4–6.8, with a citrate concentration of about 80 μM and a BSA concentration of about 10–20%.

[0338] In one embodiment, human serum may be added to both assay solutions under normal physiological conditions and abnormal conditions at substantially the same concentration. Since human serum contains numerous inorganic compounds, ions, and organic molecules (including proteins), the assay solution may have a number of components selected from inorganic compounds, ions, and organic molecules that are present at substantially the same concentration between the two assay solutions.

[0339] In some other embodiments, at least one of two or more components is added to the assay solutions under normal physiological conditions and abnormal conditions at different concentrations. For example, both lactate and bovine serum albumin (BSA) are added to the assay solution. The lactate concentration may differ between the assay solutions under normal physiological conditions and abnormal conditions, while the BSA may be at the same concentration in both assay solutions. Lactate may be at a concentration in the range of 30–50 mg / dL in the assay solution under abnormal conditions and at a concentration in the range of 8–15 mg / dL in the assay solution under normal physiological conditions. On the other hand, BSA may be at the same concentration in both assay solutions, such as about 10–20%. By using these assay solutions, the conditionally active biological proteins thus selected are more active at high lactate concentrations of 30–50 mg / dL compared to low lactate concentrations of 8–15 mg / dL in the presence of BSA.

[0340] In some embodiments, the assay solution may be designed to select a conditionally active biological protein having activity dependent on two or more conditions. In one exemplary embodiment, the conditionally active biological protein may have activity dependent on both pH and lactate. The assay solution for selecting such a conditionally active biological protein may be an assay solution under normal physiological conditions with a pH of 7.2–7.6 and a lactate concentration in the range of 8–15 mg / dL. The assay solution under abnormal conditions may be a assay solution with a pH of 6.4–6.8 and a lactate concentration in the range of 30–50 mg / dL. Optionally, both the assay solutions under normal physiological conditions and abnormal conditions may also contain ions that assist in the binding of the mutant protein to its binding partner, thereby increasing the hit count of the candidate biologically active protein.

[0341] In yet another exemplary embodiment, a conditionally active biological protein may have pH, glucose, and lactate-dependent activity. The assay solution for selecting such a conditionally active biological protein may be an assay solution under normal physiological conditions with a pH of 7.2–7.6, a glucose concentration in the range of 2.5–10 mM, and a lactate concentration in the range of 8–15 mg / dL. The assay solution under abnormal conditions may be a pH of 6.4–6.8, a glucose concentration in the range of 0.05–0.5 mM, and a lactate concentration in the range of 30–50 mg / dL. Optionally, both the normal and abnormal assay solutions may also contain ions that assist in the binding of the mutant protein to its binding partner, thereby increasing the number of candidate biologically active proteins that bind to the binding partner at pH 6.4–6.8. The conditionally active biological proteins selected using this assay solution exhibit higher activity in an environment with pH 6.4–6.8, glucose concentration 0.05–0.5 mM, and lactate concentration 30–50 mg / dL compared to an environment with pH 7.2–7.6, glucose concentration 2.5–10 mM, and lactate concentration 8–15 mg / dL.

[0342] Two or more components selected from inorganic compounds, ions, and organic molecules are used to create an assay solution with abnormal conditions that simulate the environment of the site / location (i.e., target site) where the selected conditionally active biological protein will be delivered. In some embodiments, at least three components present in the target site environment may be added to the assay solution, or at least four components present in the target site environment may be added to the assay solution, or at least five components present in the target site environment may be added to the assay solution, or at least six components present in the target site environment may be added to the assay solution.

[0343] In one embodiment, bodily fluids collected from a target site may be used directly as the assay solution for an assay under abnormal conditions. For example, synovial fluid may be collected from a subject, preferably a subject with a joint disease requiring treatment. The collected synovial fluid may be optionally diluted and used as the assay solution in a pair of assays under abnormal conditions to select a conditionally active biological protein. By optionally diluting the collected synovial fluid and using it as the assay solution for an assay under abnormal conditions and as an assay solution simulating human plasma for an assay under normal physiological conditions, the selected conditionally active biological protein (e.g., TNF-α) may be more active in the joint than in other locations or organs. For example, a subject with inflamed joints (such as arthritis) may be treated with TNF-α. However, TNF-α typically has severe side effects that damage other tissues and organs. Conditionally active TNF-α, which is highly active in synovial fluid but inactive or low in the blood, may deliver TNF-α activity to the joint while reducing or potentially eliminating the side effects of TNF-α on other parts of the body.

[0344] The development of conditionally active biological proteins that exhibit activity dependent on multiple conditions will lead to improved selectivity of these proteins to target sites in the body. Ideally, at locations where only some of the conditions are present, the conditionally active biological protein will be inactive or at least significantly less active. In one embodiment, a conditionally active biological protein that is active at pH 6.4–6.8, glucose concentration 0.05–0.5 mM, and lactate concentration 30–50 mg / dL can be specifically delivered to the tumor microenvironment because all of these conditions are present in the tumor microenvironment. Since other tissues or organs may only have one or two of these conditions, this conditionally active biological protein is insufficient to fully activate in other tissues or organs. For example, post-exercise muscles may have a low pH in the range of 6.4–6.8. However, it may not have the other conditions to be assayed. Therefore, the conditionally active biological protein will be inactive or at least significantly less active in post-exercise muscles.

[0345] In some embodiments, a step may be taken to confirm that the activity of a conditionally active biological protein truly depends on the conditions used to select the conditionally active biological protein. For example, a conditionally active biological protein is selected to depend on three conditions: pH 6.4–6.8, glucose concentration 0.05–0.5 mM, and lactate concentration 30–50 mg / dL. The selected conditionally active biological protein is then tested individually under each of these three conditions and in an environment including pairs of the three conditions, to confirm that the conditionally active biological protein is inactive or has low activity in these tests.

[0346] In some embodiments, certain components of serum are intentionally minimized or excluded from the assay medium. For example, during antibody screening, serum components that bind to or adsorb antibodies can be minimized or excluded from the assay medium. Such bound antibodies can produce false positives, potentially including conjugated mutant antibodies that simply bind to components present in serum under various different conditions rather than being conditionally active. Therefore, by using careful selection of assay components such that components that could potentially bind to mutants in the assay are minimized or excluded, the number of non-functional mutants that could be inadvertently identified as positive for conditional activity due to binding to assay components other than the desired binding partner can be reduced. For example, in some embodiments screening mutant proteins that tend to bind to components in human serum, BSA may be used in the assay solution to reduce or eliminate the possibility of false positives caused by mutant proteins that bind to components in human serum. Other similar substitutions can also be made in detailed examples to achieve the same goal.

[0347] Screening format The screening process of the present invention may be any preferred method known to those skilled in the art. Examples include ELISA, enzyme activity assays, in vitro (organ, etc.) real tissue screening, tissue slides, whole animals, cell lines, and the use of 3D systems. For example, a preferred cell-based assay is described in International Publication No. 2013 / 040445, a tissue-based assay is described in U.S. Patent No. 7,993,271, a whole-animal-based screening method is described in U.S. Patent Application Publication No. 2010 / 0263599, and a 3D system-based screening method is described in U.S. Patent Application Publication No. 2011 / 0143960.

[0348] In some embodiments, the screening environment is either an environment close to the cell membrane, such as inside, on, or outside the cell membrane, or an environment within the joint. Several factors that may affect binding affinity during screening in the cell membrane environment include receptor expression, internalization, and antibody-drug conjugate (ADC) titer.

[0349] In some embodiments, the development process can produce mutant proteins that simultaneously possess other desired properties in addition to the conditional activity properties discussed above. Suitable other desired properties that can be developed include binding affinity, expression, and humanization. Therefore, using the present invention, it is possible to produce conditionally active biological proteins that also have improved at least one or more of these other desired properties.

[0350] In some embodiments, the present invention produces a conditionally active biological protein. The selected conditionally active biological protein can be further mutated, for example, in a second development step using one of the mutagenesis techniques disclosed herein, thereby improving other properties of the selected conditionally active biological protein, such as binding affinity, expression, or humanization. After this second development step, the mutant protein can be screened for both conditional activity and the improved properties.

[0351] In some embodiments, after developing a wild-type protein to produce a mutant protein, a first conditionally active biological protein is selected that exhibits both (a) decreased activity compared to the wild-type protein in an assay under normal physiological conditions, and (b) increased activity compared to the wild-type protein in an assay under abnormal conditions. The first conditionally active biological protein is then subjected to one or more additional development, expression, and selection steps, and at least a second conditionally active biological protein may be selected that exhibits both (a) decreased activity compared to the wild-type protein in an assay under normal physiological conditions, and (b) increased activity compared to the wild-type protein in an assay under abnormal conditions, as well as a larger ratio of activity under abnormal conditions to activity under normal physiological conditions compared to the first conditionally active biological protein and / or the wild type.

[0352] In certain embodiments, the present invention aims to produce conditionally active biological proteins that have a large activity ratio between activity under abnormal conditions and activity under normal physiological conditions (for example, greater selectivity between abnormal and normal physiological conditions). The ratio of activity under abnormal conditions to activity under normal physiological conditions, i.e., selectivity, may be at least about 2:1, or at least about 3:1, or at least about 4:1, or at least about 5:1, or at least about 6:1, or at least about 7:1, or at least about 8:1, or at least about 9:1, or at least about 10:1, or at least about 11:1, or at least about 12:1, or at least about 13:1, or at least about 14:1, or at least about 15:1, or at least about 16:1, or at least about 17:1, or at least about 18:1, or at least about 19:1, or at least about 20:1, or at least about 30:1, or at least about 40:1, or at least about 50:1, or at least about 60:1, or at least about 70:1, or at least about 80:1, or at least about 90:1, or at least about 100:1.

[0353] In one embodiment, the conditionally active biological protein is an antibody whose ratio of activity under abnormal conditions to activity under normal physiological conditions may be at least about 5:1, or at least about 6:1, or at least about 7:1, or at least about 8:1, or at least about 9:1, or at least about 10:1. In one embodiment, a tumor site is targeted using the conditionally active biological protein, where the conditionally active biological protein is active at the tumor site and significantly less active or inactive at non-tumor sites (under normal physiological conditions).

[0354] In one embodiment, a conditionally active biological protein is an antibody intended to be conjugated with another drug, such as those disclosed in other parts of this specification. This conditionally active antibody may have a higher ratio of activity under abnormal conditions to activity under normal physiological conditions. For example, a conditionally active antibody conjugated with another drug may have a ratio of activity under abnormal conditions to activity under normal physiological conditions of at least about 10:1, or at least about 11:1, or at least about 12:1, or at least about 13:1, or at least about 14:1, or at least about 15:1, or at least about 16:1, or at least about 17:1, or at least about 18:1, or at least about 19:1, or at least about 20:1. This may be particularly important if the conjugating drug is, for example, toxic or radioactive, as it is desirable to concentrate such conjugated drug at the disease or treatment site.

[0355] Manipulation of conditionally activated antibodies The conditionally activated antibodies of the present invention can be manipulated by one or more antibody manipulation techniques described herein. Non-limiting examples of antibody manipulation techniques include antibody conjugation, manipulation of multispecific antibodies, and manipulation of the Fc region of antibodies.

[0356] Conjugation of conditionally activated antibodies The conditionally activated antibodies provided by the present invention can be conjugated to molecules. Since these conditionally activated antibodies preferentially act in, for example, the brain, synovial fluid, tumor microenvironment, or stem cell niche, the conditionally activated antibodies can be conjugated to molecules for the purpose of transporting the molecules to one of the brain, synovial fluid, or tumor microenvironment. In some embodiments, the conjugated molecules have some degree of toxicity, and this toxicity can be reduced by conjugation with the conditionally activated antibody during transport through the body. Therefore, this can influence the preferential action of such toxic agents at the disease or treatment site.

[0357] Conjugation of a conditionally active antibody with a molecule such as a therapeutic or diagnostic agent can be by covalent or non-covalent bonding. Covalent conjugation may be direct or via a linker. In certain embodiments, direct conjugation is achieved by protein fusion (i.e., gene fusion of two genes encoding a conditionally active antibody and a neuropathic drug, and expression as a single protein). In certain embodiments, direct conjugation is achieved by the formation of a covalent bond between a reactive group on the conditionally active antibody and a corresponding group or acceptor on the molecule. In certain embodiments, direct conjugation is achieved by modifying (e.g., by gene modification) one of the two molecules to be conjugated to contain a reactive group (as a non-limiting example, a sulfhydryl group or a carboxyl group) that forms a covalent bond with the other molecule to be conjugated under appropriate conditions. As one non-limiting example, a molecule (e.g., an amino acid) having a desired reactive group (e.g., a cysteine ​​residue) can be introduced into a conditionally active antibody to form a disulfide bond with a molecule such as a neuropathic drug. Methods for covalently conjugating nucleic acids with proteins are known in the art (i.e., photocrosslinking, see, for example, Zatsepin et al. Russ. Chem. Rev., 74:77-95 (2005)).

[0358] Non-covalent conjugations can be formed by any non-covalent means, including hydrophobic bonds, ionic bonds, and electrostatic interactions, as will be readily apparent to those skilled in the art.

[0359] Conjugation may also be carried out using various linkers. For example, a conditionally active antibody and a neuroleptic drug can be conjugated using various bifunctional protein coupling agents, such as N-succinimidyl-3-(2-pyridyldithio)propionate (SPDP), succinimidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxylate (SMCC), iminothiolane (IT), bifunctional derivatives of imide esters (such as dimethylHCl adipimidoate), active esters (such as disuccinimidyl suberate), aldehydes (such as glutaraldehyde), bisazide compounds (such as bis(p-azidobenzoyl)hexanediamine), bisdiazonium derivatives (such as bis-(p-diazoniumbenzoyl)-ethylenediamine), diisocyanates (such as toluene 2,6-diisocyanate), and bisactive fluorine compounds (such as 1,5-difluoro-2,4-dinitrobenzene). Peptide linkers consisting of 1 to 20 amino acids linked by peptide bonds may also be used. In certain such embodiments, the amino acids are selected from 20 naturally occurring amino acids. In certain other such embodiments, one or more amino acids are selected from glycine, alanine, proline, asparagine, glutamine, and lysine.

[0360] The linker may be a "cleavable linker" that facilitates the release of the nerve agent upon delivery to the treatment or disease site. For example, acid-unstable linkers, peptidase-sensitive linkers, photodissociative linkers, dimethyl linkers, or disulfide-containing linkers may be used (Chari et al., Cancer Res., 52:127-131 (1992); U.S. Patent No. 5,208,020). Some examples of crosslinking reagents for antibody conjugation include BMPS, EMCS, GMBS, HBVS, LC-SMCC, MBS, MPBH, SBAP, SIA, SLAB, SMCC, SMPB, SMPH, sulfo-EMCS, sulfo-GMBS, sulfo-KMUS, sulfo-MBS, sulfo-SIAB, sulfo-SMCC, and sulfo-SMPB, and SVSB (succinimidyl-(4-vinylsulfone)benzoate).

[0361] The conjugated therapeutic agent may be toxic to the body, such as radioactive particles, chemotherapeutic drugs, or cytotoxins (i.e., cytotoxins). By delivering the conjugated therapeutic agent to the disease site using the conditionally activated antibody of the present invention, the toxic effects of these therapeutic agents in areas of the body where their activity is undesirable can be significantly reduced. Techniques for conjugating radioactive particles to antibodies are known in the art. Ibritumomab tiuxetan (Zevalin®) and tocitumomab (Bexxar®) are examples of monoclonal antibodies conjugated with radioactive particles. Both are antibodies against the CD20 antigen conjugated with different radioactive particles. Similarly, techniques for conjugating chemotherapeutic drugs to antibodies are also known in the art. There are at least two commercially available antibodies conjugated with chemotherapeutic drugs: brentuximab vedotin (Adcetris®) and ado-trastuzumab emtansine (Kadcyla®). Techniques for conjugating cytotoxins to antibodies are also known in the art. For example, denileukin diphthitox (Ontak®, an anticancer drug) consists of an immune system protein known as interleukin-2 (IL-2) attached to a toxin derived from a pathogen that causes diphtheria.

[0362] It is intended that any type of radioactive particle, chemotherapeutic agent, and cytotoxin can be conjugated to the conditionally activated antibody of the present invention in order to reduce the side effects of these agents while they are being delivered to the therapeutic or diseased site.

[0363] In some embodiments, the radioactive particles conjugated to a conditionally active antibody include particles impregnated with one or more radioisotopes and possess sufficient radioactivity for localized ablation of cells. Such particles may include glass, metal, resin, albumin, or polymer. The metal in the radioactive particles may be selected from iron, gadolinium, and calcium. Examples of one or more radioisotopes in radioactive particles are selected from gallium-67 (67Ga), yttrium-90 (90Y), gallium-68 (68Ga), thallium-201 (201T1), strontium-89 (89Sr), indium-III (111In), iodine-131 (131I), samarium-153 (153Sm), technetium-99m (99mTc), rhenium-186 (186Re), rhenium-188 (188Re), copper-62 (62Cu), and copper-64 (64Cu). Preferably, the radioisotopes in the composition emit beta rays, gamma rays, and / or positrons.

[0364] In some embodiments, the chemotherapeutic agent conjugated to the conditionally activated antibody is selected from anthracyclines, topoisomerase I and / or II inhibitors, spindle toxin plant alkaloids, alkylating agents, antimetabolites, ellipticin, and harmine.

[0365] Anthracyclines (or anthracycline antibiotics) are derived from bacteria of the genus Streptomyces. These compounds are used to treat a wide variety of cancers, including hepatocellular carcinoma, leukemia, lymphoma, and breast, uterine, ovarian, and lung cancer. Examples of anthracyclines include, but are not limited to, doxorubicin, daunorubicin, epirubicin, idarubicin, barurubicin, pirarubicin, zorubicin, akurarubicin, detrubicin, carminomycin, morpholinodoxorubicin, morpholinodaunorubicin, methoxymorpholinyldoxorubicin, and pharmaceutically acceptable salts.

[0366] Topoisomerases are essential enzymes that maintain the topology of DNA. Inhibition of type I or type II topoisomerases disrupts proper DNA superhelical formation, thereby interfering with both DNA transcription and replication. Some type I topoisomerase inhibitors include camptothecin derivatives. Camptothecin derivatives include camptothecin analogs such as irinotecan, topotecan, hexatecan, siratecan, lutortecan, calenitecan (BNP1350), gimatecan (ST1481), berotecan (CKD602), or pharmaceutically acceptable salts thereof. Examples of type II topoisomerase inhibitors include, but are not limited to, amsacrine, etoposide, etoposide phosphate, and teniposide. The roots of American Mayapple (Podophyllum peltatum) contain semi-synthetic derivatives of epipodophyllotoxins, which are naturally occurring alkaloids.

[0367] Spindle toxins, plant alkaloids, originate from plants and inhibit cell division by interfering with microtubule function, which is essential for cell division. Examples of these alkaloids include, but are not limited to, vinca alkaloids (such as vinblastine, vincristine, vindesine, vinorelbine, and vinpocetine) and taxanes. Examples of taxanes include, but are not limited to, paclitaxel, docetaxel, larotaxel, cabazitaxel, ortataxel, tesetaxel, and pharmaceutically acceptable salts thereof.

[0368] Examples of alkylating agents, though not limited to them, include mechloretamine, cyclophosphamide, chlorambucil, ifosfamide, and platinum compounds, such as oxaliplatin, cisplatin, or carboplatin.

[0369] Antimetabolites are chemicals that inhibit the use of metabolites that are part of normal metabolism. The presence of antimetabolites alters cell growth and cell division. Purines or pyrimidine analogs interfere with the incorporation of nucleotides into DNA, thereby halting DNA synthesis and, consequently, cell division. They also affect RNA synthesis. Examples of purine analogs include azathioprine, mercaptopurine, thioguanine, fludarabine, pentostatin, and cladribine. Examples of pyrimidine analogs include 5-fluorouracil (5FU) (which inhibits thymidylate synthase), floxuridine (FUDR), and cytosine arabinoside (cytarabine).

[0370] Antifolic acid agents are chemotherapeutic drugs that weaken the function of folic acid. A well-known example is methotrexate, a folic acid analog that inhibits the enzyme dihydrofolate reductase (DHFR) and thus prevents the formation of tetrahydrofolate. This leads to the inhibition of DNA, RNA, and protein production (since tetrahydrofolate is also involved in the synthesis of the amino acids serine and methionine). Other antifolic acid agents include, but are not limited to, trimethoprim, larcitrexed, pyrimethamine, and pemetrexed.

[0371] Other chemotherapeutic agents, such as ellipticin and harmine, can also be conjugated to conditionally activated antibodies. Ellipticin and its derivatives, such as 9-hydroxyellipticinium, N2-methyl-9-hydroxyellipticinium, 2-(diethyiamino-2-ethyl)9-hydroxyellipticinium acetate, 2-(diisopropylamino-ethyl)9-hydroxyellipticinium acetate, and 2-(β-piperidino-2-ethyl)9-hydroxyellipticinium, are all effective chemotherapeutic agents.

[0372] Harmine is a natural plant alkaloid product isolated from the seeds of Peganum harmala. Harmine-based chemotherapeutic agents include harmine, harmanine, harmol, harmarol, and harman, as well as quinazoline derivatives: bacicin and bacicinone.

[0373] In some embodiments, cytotoxins conjugated to conditionally activated antibodies include taxol, cytochalasin B, gramicidin D, ethidium bromide, emetine, mitomycin, etoposide, tenoposide, vincristine, vinblastine, colchicine, doxorubicin, daunorubicin, anthracinedione, mitoxantrone, mitramycin, actinomycin D, 1-dehydrotestosterone, glucocorticoids, procaine, tetracaine, lidocaine, propranolol, and puromycin, as well as their analogs or homologs. Other toxins include, for example, lysine, CC-1065 and its analogs, and duocalmycins. Further toxins include diptheria toxin and snake venom (e.g., cobra venom).

[0374] In some embodiments, the conditionally activated antibodies of the present invention may be conjugated to a diagnostic agent. Diagnostic agents used in the present invention may include any diagnostic agents known in the art, as provided, for example, in the following literature: Armstrong et al, “Diagnostic Imaging,” 5th Ed., Blackwell Publishing (2004); Torchilin, VP, Ed., “Targeted Delivery of Imaging Agents,” CRC Press (1995); Vallabhajosula, S., “Molecular Imaging: Radiopharmaceuticals for PET and SPECT,” Springer (2009). Diagnostic agents can be detected in a variety of ways, including, but not limited to, the use of agents that provide and / or enhance detectable signals, including gamma-ray emission signals, radioactive signals, echogenic signals, optical signals, fluorescent signals, absorptive signals, magnetic signals, or tomographic signals. While not limited to specific techniques, imaging techniques for diagnostic agents include single-photon emission computed tomography (SPECT), magnetic resonance imaging (MRI), optical imaging, positron emission tomography (PET), computed tomography (CT), X-ray imaging, and gamma-ray imaging.

[0375] In some embodiments, the diagnostic agent may include, for example, a chelator bound to a metal ion, which is used in various imaging diagnostic methods. Examples of chelators include, but are not limited to, ethylenediaminetetraacetic acid (EDTA), [4-(1,4,8,11-tetraazacyclotetradeca-1-yl)methylbenzoic acid (CPTA), cyclohexanediaminetetraacetic acid (CDTA), ethylenebis(oxyethylenenitrilo)tetraacetic acid (EGTA), diethylenetriaminepentaacetic acid (DTPA), citric acid, hydroxyethylethylenediaminetriacetic acid (HEDTA), iminodiacetic acid (IDA), triethylenetetraaminehexaacetic acid (TTHA), 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetra(methylenephosphonic acid) (DOTP), 1,4,8,11-tetraazacyclododecane-1,4,8,11-tetraacetic acid (TETA), 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA), and their derivatives.

[0376] Radioisotopes can be incorporated into some of the diagnostic agents described herein and may include radionuclides that emit gamma rays, positrons, β and α particles, or X-rays. Suitable radionuclides include, but are not limited to, Ac, As, At, nB, 128Ba, 212Bi, 75Br, 77Br, 14C, 109Cd, 62Cu, 64Cu, 67Cu, 18F, 67Ga, 68Ga, 3H, 123I, 125I, 130I, 131I, 111In, 177Lu, 13N, 150, 32P, 33P, 212Pb, 103Pd, 186Re, 188Re, 47Sc, 153Sm, 89Sr, 99mTc, 88Y, and 90Y. In certain embodiments, the radioactive agents may include mIn-DTPA, 99mTc(CO)3-DTPA, 99mTc(CO)3-ENPy2, 62 / 64 / 67Cu-TETA, 99mTc(CO)3-IDA, and 99mTc(CO)3 triamines (cyclic or linear). In other embodiments, these agents may include DOTA and various analogues having 111In, 177Lu, 153Sm, 88 / 90Y, 62 / 64 / 67Cu, or 67 / 68Ga. In some embodiments, liposomes can be radiolabeled by incorporating chelate-bound lipids, such as DTPA-lipids, as provided in the following literature: Phillips et al, Wiley Interdisciplinary Reviews: Nanomedicine and Nanobiotechnology, vol.1, pages 69-83 (2008); Torchilin, VP & Weissig, V., Eds. Liposomes 2nd Ed.: Oxford Univ. Press (2003); Elbayoumi, TA & Torchilin, VP, Eur. J. Nucl. Med. Mol. Imaging, 33:1196-1205 (2006); Mougin-Degraef, M. et al, Int'l J. Pharmaceutics, 344:110-17 (2007).

[0377] In other embodiments, the diagnostic agent may include optical agents such as fluorescent agents, phosphorescent agents, and chemiluminescent agents. Numerous agents (e.g., dyes, probes, labels, or indicators) are known in the art and can be used in the present invention. (See, for example, Invitrogen, “The Handbook—A Guide to Fluorescent Probes and Labeling Technologies,” Tenth Edition (2005)). Fluorescent agents may include various organic and / or inorganic small molecules or various fluorescent proteins and their derivatives. For example, examples of fluorescent agents include, but are not limited to, cyanine, phthalocyanine, porphyrin, indocyanine, rhodamine, phenoxazine, phenylxanthene, phenothiazine, phenoselenazine, fluorescein, benzoporphyrin, squaline, dipyrrolopyrimidone, tetracene, quinoline, pyrazine, choline, crokonium, acridone, phenantholidine, rhodamine, acridine, anthraquinone, chalcogenopyrilium analogs, chlorine, naphthalocyanine, methine dyes, indolenium dyes, azo compounds, azulene, azaazulene, triphenylmethane dyes, indole, benzoindole, indocarbocyanine, benzoindocarbocyanine, and BODIPY® derivatives having the general structure of 4,4-difiuoro-4-bora-3a,4a-diaza-s-indacene, and / or any conjugates and / or derivatives thereof.Other drugs that can be used include, but are not limited to, fluorescein, fluorescein-polyaspartate conjugate, fluorescein-polyglutamate conjugate, fluorescein-polyarginine conjugate, indocyanine green, indocyanine-dodecaaspartate conjugate, indocyanine (NIRD)-polyaspartate conjugate, isosulfan blue, indole disulfonate, benzoindole disulfonate, bis(ethylcarboxymethyl)indocyanine, bis(pentylcarboxymethyl)indocyanine, polyhydroxyindole sulfonate, polyhydroxybenzoindole sulfonate, rigid heteroatom indole disulfonate, indocyanine bispropanoic acid, indocyanine bishexanoic acid, 3,6-dicyano-2, 5-[(N,N,N',N'-tetrakis(carboxymethyl)amino]pyrazine, 3,6-[(N,N,N',N'-tetrakis(2-hydroxyethyl)amino]pyrazine-2,5-dicarboxylic acid, 3,6-bis(N-azatedino)pyrazine-2,5-dicarboxylic acid, 3,6-bis(N-morpholino)pyrazine-2,5-dicarboxylic acid, 3,6-bis(N-piperazino)pyrazine-2, Examples include 5-dicarboxylic acid, 3,6-bis(N-thiomorpholino)pyrazine-2,5-dicarboxylic acid, 3,6-bis(N-thiomorpholino)pyrazine-2,5-dicarboxylic acid S-oxide, 2,5-dicyano-3,6-bis(N-thiomorpholino)pyrazine S,S-dioxide, indocarbocyanine tetrasulfonate, chloroindocarbocyanine, and 3,6-diaminopyrazine-2,5-dicarboxylic acid.

[0378] In further embodiments, the diagnostic agent may include contrast agents generally known in the art, such as complexes of superparamagnetic iron oxide (SPIO), gadolinium, or manganese (see, for example, Armstrong et al, “Diagnostic Imaging,” 5th Ed., Blackwell Publishing (2004)). In some embodiments, the diagnostic agent may include magnetic resonance (MR) contrast agents. Exceptional magnetic resonance contrast agents include, but are not limited to, paramagnetic agents and superparamagnetic agents. Exceptional paramagnetic agents include, but are not limited to, gadopentetic acid, gadoteric acid, gadodiamide, gadolinium, gadoteridol, mangahodipel, gadovercetamide, ammonium iron citrate, gadopentic acid, gadobutrol, or gadoxetic acid. Exceptional superparamagnetic agents include, but are not limited to, superparamagnetic iron oxide and ferristen. In certain embodiments, the diagnostic agent may include, for example, a radiographic contrast agent as provided in the following literature: HS Thomsen, R. Muller and R. F Mattrey, Eds., “Trends in Contrast Media,” Berlin: Springer-Verlag, 1999; P. Dawson, D. Cosgrove and R. Grainger, Eds., “Textbook of Contrast Media” (ISIS Medical Media 1999); Torchilin, VP, Curr. Pharm. Biotech., vol.1, pages 183-215 (2000); Bogdanov, AA et al, Adv. Drug Del. Rev., Vol.37, pages 279-293 (1999); Sachse, A. et al, Investigative Radiology, vol.32, pages 44-50 (1997).Examples of X-ray contrast agents include, without limitation, iopamidol, iomeprole, iohexol, iopentol, iopromide, iosimide, ioversol, iotrolane, iotasul, iodixanol, iodecimol, ioglucamide, iogrunide, ioglamide, iosalcol, ioxiran, iopamilon, metrizamide, iobitridol, and iosimenol. In specific embodiments, examples of X-ray contrast agents include iopamidol, iomeprole, iopromide, iohexol, iopentol, ioversol, iobitridol, iodixanol, iotrolane, and iosimenol.

[0379] In some embodiments, conditionally activated antibodies may be conjugated to proteins, such as interleukins, cytokines, enzymes, growth factors, or other antibodies. Some examples of such proteins include, for example, tumor necrosis factor, α-interferon (EFN-α), β-interferon (IFN-β), nerve growth factor (NGF), platelet-derived growth factor (PDGF), tissue plasminogen activator (TPA), apoptotic agents (e.g., TNF-α, TNF-β, AIM I as disclosed in International Publication No. 97 / 33899), AIM II (see International Publication No. 97 / 34911), and Fas ligand (Takahashi et al., J.Immunol., vol.6, pages...). Examples include VEGI (International Publication No. 99 / 23105), antithrombotic agents or anti-angiogenic agents (e.g., angiostatins or endostatins); or biological reaction modifiers, such as lymphokines (e.g., interleukin-1 ("IL-I"), interleukin-2 ("IL-2"), interleukin-6 ("IL-6"), granulocyte-macrophage colony-stimulating factor ("GM-CSF"), and granulocyte colony-stimulating factor ("G-CSF")); or growth factors (e.g., growth hormone ("GH")).

[0380] In some embodiments, conditionally activated antibodies for crossing the blood-brain barrier (BBB) ​​may be conjugated to drugs for the treatment of neurological disorders. These drugs, transported together with the antibodies across the BBB, may remain in the brain to treat the neurological disorder. Neurological disorders refer to diseases or disorders affecting and / or having a CNS etiology. Exemplary CNS diseases or disorders include, but are not limited to, neuropathy, amyloidosis, cancer, eye diseases or disorders, viral or microbial infections, inflammation, ischemia, neurodegenerative diseases, epileptic seizures, behavioral disorders, and lysosomal storage disorders.

[0381] For the purposes of this application, the CNS will be understood to include the eye, which is normally separated from the rest of the body by the blood-retinal barrier. Specific examples of neurological disorders include, but are not limited to, neurodegenerative diseases such as Lewy body dementia, post-polio syndrome, Shy-Drager syndrome, olivopontocerebellar atrophy, Parkinson's disease, multiple system atrophy, striatonigral degeneration, tauopathies such as Alzheimer's disease and supranuclear palsy, prion diseases such as bovine spongiform encephalopathy, scrapie, Creutzfeldt-Jakob syndrome, kuru, Gerstmann-Streussler-Scheinker disease, chronic wasting disease, and fatal familial insomnia, bulbar palsy, motor neuron diseases, and neurological disorders. Examples include heterogeneous disorders of the brain, such as Canavan disease, Huntington's disease, neuronal ceroid lipofuscinosis, Alexander disease, Tourette syndrome, Menkes's curly hair syndrome, Cockayne syndrome, Haller-Vorden-Spatz syndrome, Lafora disease, Rett syndrome, hepatolenticular degeneration, Lesch-Nyhan syndrome, and Unverlicht-Lundborg syndrome; dementia (including, but not limited to, Pick's disease and spinocerebellar ataxia); and cancer (including, for example, cancers of the CNS and / or brain, including brain metastases arising from cancer elsewhere in the body).

[0382] Drugs for the treatment of neurological disorders may include, but are not limited to, antibodies, peptides, proteins, natural ligands for one or more CNS targets, modified forms of natural ligands for one or more CNS targets, aptamers, inhibitory nucleic acids (i.e., small inhibitory RNA (siRNA) and small hairpin RNA (shRNA)), ribozymes, and small molecules, or any of the aforementioned active fragments. Examples of neuropathy-causing drugs include, but are not limited to, antibodies, aptamers, proteins, peptides, inhibitory nucleic acids and small molecules, and any of the aforementioned active fragments, either themselves or those that specifically recognize and / or act upon (i.e., inhibit, activate, or detect) CNS antigens or target molecules, such as, but are not limited to, amyloid precursor proteins or portions thereof, amyloid-beta, β-secretase, γ-secretase, τ, α-synuclein, parkin, huntingtin, DR6, presenilin, ApoE, glioma or other CNS cancer markers, and neurotrophins. Non-limiting examples of neurotoxic drugs and the disorders for which they may be used for treatment include: anti-BACE1 antibodies for the treatment of Alzheimer's disease, acute and chronic brain injury, and stroke; anti-Aβ antibodies for the treatment of Alzheimer's disease; neurotrophins for the treatment of stroke, acute brain injury, and spinal cord injury; brain-derived neurotrophic factor (BDNF) and fibroblast growth factor 2 (FGF-2) for the treatment of chronic brain injury (neurogenesis); anti-epidermal growth factor receptor (EGFR) antibodies for the treatment of brain cancer; glial cell line-derived neurotrophic factor (GDNF) for the treatment of Parkinson's disease; brain-derived neurotrophic factor (BDNF) for the treatment of amyotrophic lateral sclerosis and depression; lysosomal enzymes for the treatment of lysosomal storage disorders in the brain; ciliary neurotrophic factor (CNTF) for the treatment of amyotrophic lateral sclerosis; neureglin-1 for the treatment of schizophrenia; and anti-HER2 antibodies (e.g., trastuzumab) for the treatment of brain metastases from HER2-positive cancer.

[0383] In some embodiments, a conditionally active antibody may be conjugated onto the Fc region of the antibody. The conjugate molecules, compounds, or drugs described above may be conjugated onto the Fc region as described in U.S. Patent No. 8,362,210. For example, the Fc region may be conjugated with a cytokine or toxin that is delivered to a site where the conditionally active antibody exhibits preferential activity. Methods for conjugating polypeptides onto the Fc region of an antibody are known in the art. For example, U.S. Patent Nos. 5,336,603, 5,622,929, 5,359,046, 5,349,053, 5,447,851, 5,723,125, 5,783,181, 5,908,626, 5,844,095, and 5,112,946. European Patent No. 307,434; European Patent No. 367,166; European Patent No. 394,827; International Publication Brochures No. 91 / 06570, 96 / 04388, 96 / 22024, 97 / 34631, and 99 / 04813; Ashkenazi See et al., Proc. Natl. Acad. Sci. USA, vol. 88, pages 10535-10539, 1991; Traunecker et al., Nature, vol. 331, pages 84-86, 1988; Zheng et al., J. Immunol., vol. 154, pages 5590-5600, 1995; and ViI et al., Proc. Natl. Acad. Sci. USA, vol. 89, pages 11337-11341, 1992.

[0384] In one embodiment, the conditionally active antibody used in the conjugation disclosed herein preferably has a ratio of activity under abnormal conditions to activity under normal physiological conditions of at least about 10:1, or at least about 12:1, or at least about 14:1, or at least about 16:1, or at least about 18:1, or at least about 20:1, or at least about 22:1, or at least about 24:1, or at least about 26:1.

[0385] In some embodiments, a conditionally active antibody may be covalently attached to a conjugate drug via an intermediate linker having at least two reactive groups, one of which reacts with the conditionally active antibody and the other with the conjugate drug. The linker (which may include any compatible organic compound) can be selected so as not to adversely affect the reactivity and / or selectivity of the conditionally active antibody or the conjugate drug when it reacts with the conditionally active antibody or the conjugate drug. Furthermore, the attachment of the linker to the conjugate drug must not destroy the activity of the conjugate drug.

[0386] Suitable linkers for oxidative conditionally active antibodies include those containing a group selected from primary amines, secondary amines, hydrazines, hydrazides, hydroxylamines, phenylhydrazines, semicarbazides, and thiosemicarbazides. Suitable linkers for reductive conditionally active antibodies include those having a specific reactive group that reacts with the sulfhydryl group of the reductive conditionally active antibody. Such reactive groups are not limited to, but include reactive haloalkyl groups (e.g., including haloacetyl groups), p-mercury benzoate groups, and groups having Michael-type addition reaction ability (e.g., maleimides and groups of the type described by Mitra and Lawton, J. Amer. Chem. Soc. Vol. 101, pages 3097-3110, 1979).

[0387] Manipulation of multispecific conditionally activated antibodies A multispecific antibody is an antibody that possesses multiepitope specificity. Multispecific antibodies include, but are not limited to, antibodies in which VHVL units contain heavy chain variable domains (VH) and light chain variable domains (VL) that have multiepitope specificity; antibodies having two or more VL and VH domains in which each VHVL unit binds to a different epitope; antibodies having two or more single variable domains in which each single variable domain binds to a different epitope; antibodies containing one or more antibody fragments; and antibodies containing antibody fragments that are covalently or noncovalently linked.

[0388] To construct multispecific antibodies, including bispecific antibodies, antibody fragments having at least one free sulfhydryl group are obtained. These antibody fragments may be obtained from full-length conditionally active antibodies. Enzymatic digestion of conditionally active antibodies can yield antibody fragments. Exemplary enzymatic digestion methods include pepsin, papain, and Lys-C. Exemplary antibody fragments include, but are not limited to, Fab, Fab', F(ab')2, Fv, diabody (Db); tandem diabody (taDb), linear antibody (see U.S. Patent No. 5,641,870, Example 2; Zapata et al., Protein Eng., vol. 8, pages 1057-1062 (1995)); one-arm antibody, single variable domain antibody, minibody (Olafsen et al (2004), Protein Eng. Design & Sel., vol. 17, pages 315-323), single-chain antibody molecules, fragments produced by Fab expression libraries, anti-idiotype (anti-Id) antibodies, complementarity-determining regions, and epitope-binding fragments. Antibody fragments may also be produced using DNA recombination techniques. The DNA encoding the antibody fragment can be cloned into a plasmid expression vector or phagemide vector and directly expressed in Escherichia coli (E. coli). Methods for antibody enzyme digestion, DNA cloning, and recombinant protein expression are well known to those skilled in the art.

[0389] The antibody fragment may be purified using conventional techniques, and upon reduction, free thiol groups are generated. The antibody fragment having free thiol groups reacts with a crosslinking agent, such as bismaleimide. The crosslinked antibody fragment is purified and then reacted with a second antibody fragment having free thiol groups. The final product of the two crosslinked antibody fragments is purified. In certain embodiments, each antibody fragment is a Fab, and the final product of two Fabs linked via bismaleimide is referred to herein as bismaleimide-(thio-Fab)2, or bis-Fab. Such multispecific antibodies and antibody analogs, including bis-Fab, can be used to rapidly synthesize a number of antibody fragment combinations, or structural variants of native antibodies or specific antibody fragment combinations.

[0390] Multispecific antibodies can be synthesized with modifying crosslinkers so that additional functional moieties can be added to the multispecific antibody. The modifying crosslinker allows for the binding of any sulfhydryl-reactive moiety. In one embodiment, N-succinimidyl-S-acetylthioacetate (SAT A) is bound to bismaleimide to form bismaleimide acetylthioacetate (BMata). After deprotection of the masked thiol group, any functional group having a sulfhydryl-reactive (or thiol-reactive) moiety can bind to the multispecific antibody.

[0391] Examples of thiol-reactive reagents include multifunctional linker reagents, scavengers, i.e., affinity labeling reagents (e.g., biotin-linker reagents), detection labels (e.g., fluorophore reagents), immobilization reagents (e.g., SEPHAROSE®, polystyrene, or glass), or drug-linker intermediates. An example of a thiol-reactive reagent is N-ethylmaleimide (NEM). Such multispecific antibodies or antibody analogs having a modifying crosslinking agent may be further reacted with a drug partial reagent or other label. The reaction of a multispecific antibody or antibody analog with a drug-linker intermediate yields a multispecific antibody-drug conjugate or antibody-analog-drug conjugate, respectively.

[0392] Many other techniques for producing multispecific antibodies can also be used in this invention. The following are some of the documents describing these techniques: (1) Recombinant co-expression of two immunoglobulin heavy-light chain pairs with different specificities, Milstein and Cuello, Nature, vol. 305, page 537 (1983), International Publication No. 93 / 08829, and Traunecker et al., EMBO J., vol. 10, page 3655 (1991); (2) U.S. Patent No. 5,731,168 regarding the "knob-in-hole" operation; (3) International Publication No. 2009 / 089004A1 regarding the production of antibody Fc-heterodimer molecules by manipulating the electrostatic steering effect; (4) U.S. Patent No. 4,676,980 regarding the crosslinking of two or more antibodies or fragments, and Brennan et al., Science, vol. 229, page 81 (1985); (5) Production of bispecific antibodies using a leucine zipper, Kostelny et al. (6) On the production of bispecific antibody fragments using "Diabody" technology, Hollinger et al., Proc. Natl. Acad. Sci. USA, vol. 90, pages 6444-6448 (1993); (7) On the use of single-chain Fv(sFv) dimers, Gruber et al., J. Immunol., vol. 152, page 5368 (1994); (8) On the preparation of triplicate antibodies, Tutt et al., J. Immunol. 147:60 (1991); and (9) On engineered antibodies having three or more functional antigen-binding sites, including "octopus antibody" or "dual variable domain immunoglobulin" (DVD), U.S. Patent Application Publication No. 2006 / 0025576A1 and Wu et al. Nature Biotechnology, vol.25, pages 1290-1297(2007).

[0393] In one embodiment, a conditionally active antibody for crossing the blood-brain barrier (BBB) ​​is manipulated to produce a multispecific antibody (e.g., a bispecific antibody). This multispecific antibody includes a first antigen-binding site that binds to the BBB-R and a second antigen-binding site that binds to a brain antigen. At least the first antigen-binding site for the BBB-R is conditionally active. A brain antigen is an antigen expressed in the brain that can be targeted by an antibody or small molecule. Examples of such antigens include, but are not limited to, β-secretase 1 (BACE1), amyloid β (Aβ), epidermal growth factor receptor (EGFR), human epidermal growth factor receptor 2 (HER2), tau, apolipoprotein E4 (ApoE4), α-synuclein, CD20, huntingtin, prion protein (PrP), leucine-rich repeat kinase 2 (LRRK2), parkin, presenilin 1, presenilin 2, γ-secretase, death receptor 6 (DR6), amyloid precursor protein (APP), p75 neurotrophin receptor (p75NTR), and caspase 6. In one embodiment, the antigen is BACE1.

[0394] Multispecific antibodies can exhibit high selectivity in preferred target tissues containing all or most of the targets (antigens) to which the multispecific antibody can bind. For example, a bispecific antibody can provide selectivity for target cells by showing a higher preference for target cells that express both antigens recognized by the bispecific antibody compared to non-target cells that express only one of the antigens. Therefore, due to the dynamics of this system, in equilibrium, there are bispecific antibodies that bind more to target cells than to non-target cells.

[0395] Manipulation of bispecific conditionally activated antibodies against immunoeffector cell surface antigens and target antigens The bispecific conditionally activated antibody of the present invention can attract immune effector cells to disease sites where a target antigen is present. The bispecific conditionally activated antibody is an antibody that can specifically bind to two different antigens: an immune effector cell surface antigen and a target antigen. The bispecific antibody may be a full-length antibody containing two arms, one arm which binds to the immune effector cell surface antigen and the other arm which binds to the target antigen. The bispecific antibody may also be an antibody fragment containing only a heavy chain variable domain (VH) and a light chain variable domain (VL). In one embodiment, the antibody fragment contains at least two VHVL units, one of which binds to the immune effector cell surface antigen and the other arm which binds to the target antigen. In another embodiment, the antibody fragment contains at least two single variable domains (VH or VL), one of which binds to the immune effector cell surface antigen and the other arm which binds to the target antigen. In some embodiments, the bispecific conditionally activated antibody contains two scFvs, one which binds to the immune effector cell surface antigen and the other arm which binds to the target antigen.

[0396] The immune effector cells that are attracted can be drawn to affected cells or tissues containing the target antigen due to their binding activity to both the immune effector cells and the target antigen on the affected cells or tissues. Since immune effector cells have the ability to suppress or even destroy affected cells or tissues, the attracted immune effector cells can then attack the affected cells or tissues, thus aiding in the healing of the disease. For example, immune effector cells can destroy tumor cells or infected cells. Examples of immune effector cells include natural killer cells, macrophages, lymphokine-activated killer (LAK) cells, and T cells.

[0397] A bispecific conditionally activated antibody has two binding activities, one for an immunoeffector cell surface antigen and one for a target antigen. In one embodiment, both binding activities are conditional, meaning that the binding activity of the bispecific conditionally activated antibody to the immunoeffector cell surface antigen and the target antigen is lower than that of the wild-type antibody under normal physiological conditions and higher than that of the wild-type antibody under abnormal conditions. In another embodiment, only one of the two binding activities is conditional, meaning that either the binding activity of the bispecific conditionally activated antibody to the immunoeffector cell surface antigen or the binding activity of the bispecific conditionally activated antibody to the target antigen is conditional. In this case, either the binding activity of the bispecific conditionally activated antibody to the immunoeffector cell surface antigen or the binding activity of the bispecific conditionally activated antibody to the target antigen is lower than the corresponding activity of the wild-type antibody under normal physiological...

Claims

1. A method for producing a conditionally active biological protein from a parent biological protein, wherein the conditionally active biological protein exhibits both (a) decreased binding activity in an assay at a normal physiological pH range of 7.2 to 7.6 compared to the parent biological protein, and (b) increased binding activity in an assay at an abnormal pH of 6.0 compared to the parent biological protein, and the method is i. The process of selecting a biologically related protein; ii. The process of developing DNA that encodes a philobiological protein using one or more developmental techniques for creating mutant DNA; iii. A step of expressing mutant DNA to obtain at least one mutant protein that exhibits both (a) decreased binding activity in assays at normal physiological pH compared to the parental biological protein, and (b) increased binding activity in assays at abnormal pH compared to the parental biological protein; and iv. Select a conditionally active biological protein from mutant proteins that exhibit both (a) decreased binding activity in assays at normal physiological pH compared to the parent biological protein, and (b) increased binding activity in assays at abnormal pH compared to the parent biological protein. Includes, A method wherein assays at normal physiological pH and at abnormal pH are performed in an assay solution containing at least one ion having a pKa within one pH unit of the abnormal pH, and at least one ion having a pKa within one pH unit of the abnormal pH is present at the same concentration in the assay solution under both normal physiological pH and abnormal pH conditions.

2. The method according to claim 1, wherein at least one ion having a pKa within 1 pH unit of an abnormal pH is present in the assay solution at a normal physiological concentration of mammalian body fluids.

3. The method according to claim 1, wherein the assay solution does not contain human serum.

4. The selection step includes selecting a conditionally active biological protein that exhibits both (a) decreased binding activity in the assay at normal physiological pH compared to a reference protein, and (b) increased binding activity in the assay at abnormal pH compared to a reference protein. The method according to claim 1, wherein the reference protein has substantially the same binding activity in both normal physiological pH and abnormal pH assays.

5. The method according to claim 1, wherein the conditionally active biological protein is an antibody or an antibody fragment.

6. The method according to claim 1, wherein the philobiological protein is a wild-type antibody or antibody fragment.

7. The method according to claim 1, wherein the philobiological protein is selected from a protein library, which is a collection of proteins encoded by cDNA.

8. The method according to claim 1, wherein the parent biological protein is selected from a bacteriophage display library.

9. The method according to claim 1, wherein the philobiological protein is selected from a collection of recombinant proteins.

10. The method according to claim 1, wherein at least one ion having a pKa within 1 pH unit of an abnormal pH has a concentration in the range of 5% to 500% of the normal physiological concentration, or 10% to 400% of the normal physiological concentration, or 30% to 300% of the normal physiological concentration, or 50% to 200% of the normal physiological concentration, or 75% to 150% of the normal physiological concentration.

11. The method according to claim 1, wherein at least one ion having a pKa within 1 pH unit of an abnormal pH has a concentration in the range of 5% to 500% of the normal physiological concentration, or 10% to 400% of the normal physiological concentration, or 30% to 300% of the normal physiological concentration, or 50% to 200% of the normal physiological concentration, or 75% to 150% of the normal physiological concentration.

12. The method according to claim 1, wherein the pKa of at least one ion is within 0.8 pH units of the abnormal pH, or within 0.6 pH units of the abnormal pH, or within 0.5 pH units of the abnormal pH, or within 0.4 pH units of the abnormal pH, or within 0.3 pH units of the abnormal pH, or within 0.2 pH units of the abnormal pH.

13. The method according to claim 1, wherein the philobiological protein selected in step (i) is a fragment of a wild-type protein.

14. The method according to claim 1, wherein the parental biological protein selected in step (i) is produced using mutagenesis technology.

15. The method according to claim 1, wherein the conditionally active biological protein selected in step (iv) is an antibody or an antibody fragment.

16. The method according to claim 1, further comprising the step of developing a conditionally active biological protein selected in step (iv) in order to produce an improved conditionally active biological protein having at least one improved property.

17. The method according to claim 16, wherein at least one improved property is an increased ratio of binding activity in an assay under abnormal pH to binding activity under normal physiological pH, compared to a conditionally active biological protein selected in step (iv).

18. The method according to claim 16, wherein at least one improved characteristic is selected from expression level and humanization.

19. The method according to claim 16, wherein the improved conditionally active biological protein has an increased ratio of binding activity in an assay under abnormal pH to binding activity under normal physiological pH, compared to the selected conditionally active biological protein used in step (i).

20. The method according to claim 1, further comprising the step of conjugating a conditionally active biological protein selected in step (iv) with a drug selected from cytokines, interleukins, enzymes, hormones, growth factors, cytotoxic agents, chemotherapeutic agents, radioactive particles, and diagnostic agents.

21. The method according to claim 1, wherein the conditionally active biological protein selected in step (iv) is a conditionally active antibody, and the method further comprises a step of genetically modifying the conditionally active antibody selected in step (iv) to be a bispecific antibody or antibody fragment.

22. The method according to claim 21, wherein the gene manipulation step involves genetically modifying a bispecific antibody to bind to both an immunoeffector cell surface antigen and a different target antigen.

23. The method according to claim 22, wherein the immune effector cells are selected from natural killer cells, macrophages, lymphokine-activated killer cells, and T cells.

24. The method according to claim 23, wherein the immune effector cell is a T cell and the immune effector cell surface antigen is CD3.

25. The method according to any one of claims 22 to 24, wherein the target antigen is an antigen specific to diseased cells or affected tissue.

26. The method according to claim 25, wherein the affected tissue is tumor tissue.

27. The method according to any one of claims 22 to 26, further comprising the step of genetically modifying a conditionally active biological antibody selected in step (iv) to include at least one of a masking portion and a cleavable portion.

28. The method according to claim 27, wherein one or both of the masking portion and the cleavable portion are adapted to be cleaved from a conditionally activated biological antibody when in proximity to diseased cells or tissue.

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