Conditionally active proteins with pH selectivity

Abstract

To provide a method of producing a conditionally active polypeptide from a parent polypeptide.SOLUTION: The method comprises steps of: (i) evolving the parent polypeptide by introducing mutations into the parent polypeptide to produce mutant polypeptides that have a pI equal to or lower than a pI of the parent polypeptide; (ii) subjecting the mutant polypeptides to a first assay under a normal physiological condition to measure the activity of the mutant polypeptides under the normal physiological condition, and to a second assay under an aberrant condition to measure the activity of the mutant polypeptides under the aberrant condition, where the normal physiological condition and the aberrant condition are the same condition but have different values; and (iii) selecting the conditionally active polypeptide from the mutant polypeptides exhibiting increased activity in the second assay compared to the same activity in the first assay. Conditionally active polypeptides and uses are also provided.SELECTED DRAWING: None

Description

[Technical Field]

[0001] The present disclosure relates to the field of providing polypeptides with conditional activity. Specifically, the present disclosure relates to conditionally active polypeptides with pH-dependent activity and methods of making conditionally active polypeptides from parent polypeptides. [Background technology]

[0002] There is a wealth of literature describing how proteins, particularly enzymes or antibodies, can be evolved for various properties so that they are active or stable under various conditions. For example, enzymes have been evolved to be stable at high temperatures. In situations where enzyme activity improves at high temperatures, a substantial portion of the improvement can be attributed to higher kinetic activity, which is generally explained by the Q10 rule (which estimates that enzyme turnover doubles for every 10°C increase).

[0003] In addition, there are naturally occurring mutations that destabilize a protein under its normal operating conditions, thus reducing protein activity under normal operating conditions. For example, there are known temperature mutants that are active at lower temperatures, but typically at a lower level compared to the wild-type protein from which they are derived.

[0004] To evolve a parent polypeptide that is inactive or virtually inactive (less than 50%, 30%, or 10% activity, and especially 1% activity) under its normal operating conditions but maintains equivalent or better activity under abnormal conditions, it may be necessary for destabilizing mutations to coexist with activity-increasing mutations that do not counteract the destabilizing effect. It is expected that destabilizing mutations will reduce the activity of the polypeptide beyond the effect predicted by standard rules such as Q10, thus creating a conditionally active polypeptide, for example, by allowing the evolution of a polypeptide that is less active or inactive under its normal operating conditions but functions efficiently under abnormal conditions.

[0005] It is desirable to generate conditionally active polypeptides, e.g., polypeptides that are less active or effectively inactive under some conditions and active under others. It is also desirable to generate polypeptides that become activated or inactivated in certain environments or over time. In addition to temperature, other conditions under which polypeptides can be evolved or improved for conditional activity include pH, osmolality, oxidative stress, and electrolyte concentration. When evolving, it is often desirable to improve other properties of a polypeptide in addition to its activity, including chemical resistance and resistance to proteolysis.

[0006] Furthermore, the isoelectric point (pI) of the polypeptide (t 1 / 2 It has been observed that the pI of an antibody is inversely correlated with its half-life. See Momany et al., "Relationship between in vivo degradative rates and isoelectric points of proteins," PNAS, vol., 73, pp. 3093-3097, 1976. This is particularly true for antibodies, since their plasma half-life is positively correlated with their isoelectric point. In particular, antibodies with high pI values ​​not only have faster systemic clearance but also lower bioavailability compared to antibodies with low pI values. A 1-2 unit decrease in the pI of an antibody has been shown to correlate with a decrease in plasma clearance (i.e., a longer half-life). See Ryman, CPT Pharmacometrics Syst.Pharmacol., vol.6, pp. 576-588, 2017.

[0007] Strategies for altering the pI of polypeptides have been described. For example, U.S. Patent No. 9,908,932 discloses a method for shifting the isoelectric point profile of a recombinant protein having seven protein subpopulations with pIs between about 5.45 and about 6.55. The method includes the steps of (a) culturing mammalian cells containing a nucleic acid encoding the recombinant protein in a production bioreactor for a first period under conditions sufficient to produce a product; and (b) incubating the product for a second period of at least 6 hours under conditions sufficient to shift the isoelectric point profile of the product to a more acidic profile. The isoelectric point-shifted product exhibits an increased amount of the fourth and fifth most acidic protein subpopulations of the seven protein subpopulations and a decreased amount of the first and second most basic protein subpopulations of the seven protein subpopulations.

[0008] U.S. Patent No. 9,605,061 discloses a method for modifying the pI of an antibody by introducing at least six amino acid mutations, including substitutions with non-native amino acids, in a constant domain selected from one or both of the heavy and light chain constant domains, where the substituted amino acids have a lower pI than the native amino acids, such that the pI of the mutant antibody is reduced by at least 0.5 logs relative to the pI of the parent antibody.

[0009] US2009 / 0324589 discloses a method for increasing the half-life of an IgG antibody in the blood by controlling the surface charge of the antibody through modification of surface-exposed residues, including those in the variable region. The method comprises the steps of: (a) modifying a nucleic acid encoding a parent polypeptide comprising an FcRn-binding domain to change the charge of at least one amino acid residue exposed on the surface of the parent polypeptide; (b) culturing a host cell to express the modified nucleic acid to express a variant polypeptide; and (c) recovering the expressed variant polypeptide comprising the FcRn-binding domain from the host cell culture.

[0010] There remains a need for conditionally active polypeptides that have higher activity and / or selectivity under specific environments and / or conditions, preferably with increased plasma half-life. Conditionally active polypeptides with increased half-life also act preferentially in locations where abnormal conditions exist, such as the tumor microenvironment, and, due to their increased half-life, provide extended action or a longer overall activity period. Furthermore, these conditionally active polypeptides may cause fewer adverse side effects on normal tissues / organs under normal physiological conditions. Potentially reduced side effects allow for longer treatment periods or higher doses, leading to higher efficacy of the conditionally active polypeptides.

[0011] WO 2010 / 104821 and WO 2011 / 009058 disclose methods for evolving and screening for conditionally active proteins. Summary of the Invention [Problem to be solved by the invention]

[0012] In one embodiment, the disclosure relates to a method for producing a conditionally active polypeptide from a parent polypeptide, comprising: (i) evolving conditionally active polypeptides from a parent polypeptide by introducing one or more mutations into the parent polypeptide to produce one or more variant polypeptides having a pI that is the same as or lower than the pI of the parent polypeptide; (ii) subjecting the one or more variant polypeptides to a first assay under normal physiological conditions that measures the activity of the one or more variant polypeptides under normal physiological conditions, and a second assay under abnormal conditions that measures the activity of the one or more variant polypeptides under abnormal conditions, wherein the normal physiological conditions and the abnormal conditions are under the same conditions but have different values; and (iii) selecting from the one or more variant polypeptides that exhibit increased activity in the second assay compared to the same activity in the first assay.

[0013] In the foregoing embodiments, the conditionally active polypeptide may have a lower pI than the pI of the parent polypeptide.

[0014] In any one of the foregoing embodiments, the conditionally active polypeptide can have a pI below 7.4, or a pI below 7.3, or a pI below 7.2, or a pI below 7.1, or a pI below 7.0.

[0015] In any one of the foregoing embodiments, the one or more mutations may comprise at least one amino acid substitution of an amino acid residue in the parent polypeptide for an amino acid residue in the parent polypeptide that has a higher pI than the pI of the amino acid substituted in the parent polypeptide.

[0016] In any one of the preceding embodiments, the one or more mutations may include 2, 3, 4, 5, 6, 7, 8, 9, or 10 of said substitutions.

[0017] In any one of the foregoing embodiments, the one or more mutations may comprise at least one insertion of an amino acid residue having a pI lower than the pI of the parent polypeptide.

[0018] In any one of the preceding embodiments, the one or more mutations may include 2, 3, 4, or 5 of said insertions.

[0019] In any one of the preceding embodiments, the one or more mutations may comprise a deletion of at least one amino acid residue having a pI higher than the pI of the parent polypeptide.

[0020] In any one of the preceding embodiments, the one or more mutations may include two, three, four, or five of said deletions.

[0021] In any one of the foregoing embodiments, one or more mutations may be located at positions exposed on the surface of the variant polypeptide.

[0022] In any one of the preceding embodiments, the evolving step may comprise introducing one or more additional mutations into the variant polypeptide.

[0023] Any one of the foregoing embodiments may further comprise the step of confirming that at least 50%, or at least 60%, or at least 70%, or at least 80%, or at least 90%, or at least 95%, or at least 98% of the variant polypeptides have a pI that is the same as or lower than the pI of the parent polypeptide.

[0024] The foregoing embodiment may further comprise the step of discarding variant polypeptides having a pI greater than the pI of the parent polypeptide prior to step (ii).

[0025] Any one of the foregoing embodiments can further comprise measuring the pI of the conditionally active polypeptide.

[0026] In any one of the foregoing embodiments, the pI of the conditionally active polypeptide can be at least 0.1, at least 0.2, at least 0.3, at least 0.4, at least 0.5, at least 0.6, at least 0.8, at least 1.0, at least 1.2, at least 1.4, at least 1.5, at least 1.7, at least 2.0, at least 2.5, at least 3.0, at least 3.5, at least 4.0, or at least 5.0 units lower than the pI of the parent polypeptide.

[0027] In any one of the preceding embodiments, the parent polypeptide may be selected from an antibody, an enzyme, a hormone, a growth factor, a cytokine, a regulatory protein, a functional peptide, a biosimilar, an immunomodulator, a receptor, and a ligand.

[0028] In any one of the foregoing embodiments, the parent polypeptide may be an antibody selected from a full-length antibody, a single chain antibody, an antibody fragment, a heavy chain, a light chain, a Fab, and an Fc domain.

[0029] In any one of the foregoing embodiments, the parent polypeptide may be a therapeutic antibody or an antibody candidate being developed for therapeutic use.

[0030] In any one of the preceding embodiments, the parent polypeptide may be an IgG antibody.

[0031] In any one of the preceding embodiments, the one or more mutations may be in the variable region of an IgG antibody.

[0032] In any one of the preceding embodiments, the one or more mutations may be in the constant region of an IgG antibody.

[0033] In any one of the preceding embodiments, the one or more mutations may be in one or more complementarity determining regions of the IgG antibody.

[0034] In any one of the preceding embodiments, the conditions may be selected from pH, temperature, osmolality, osmolality, oxidative stress, and electrolyte concentration.

[0035] In any one of the preceding embodiments, the condition may be pH.

[0036] In the foregoing embodiment, the pH of the first assay may be greater than 7.2 to less than 7.6, and the pH of the second assay may be less than 7.2 or greater than 7.6.

[0037] In any one of the foregoing embodiments, the ratio of the activity of the conditionally active polypeptide in the second assay to the same activity in the first assay may be at least 1.3, 1.5, or at least 1.7, or at least 2.0, or at least 3.0, or at least 4.0, or at least 6.0, or at least 8.0, or at least 10.0, or at least 20.0, or at least 40.0, or at least 60.0, or at least 100.0.

[0038] In any one of the foregoing embodiments, both the first assay and the second assay may be performed in the presence of molecules or ions having a molecular weight of less than 900 a.mu, less than 500 a.mu, less than 200 a.mu, or less than 100 a.mu.

[0039] In the foregoing embodiments, the molecule or ion may be selected from histidine, histamine, hydrogenated adenosine diphosphate, hydrogenated adenosine triphosphate, citrate, bicarbonate, acetate, lactate, disulfide, hydrogen sulfide, ammonium, dihydrogen phosphate, and any combination thereof.

[0040] In the foregoing embodiments, the molecule or ion may be bicarbonate ion having a concentration in the range of about 3 mM to about 200 mM, about 5 mM to about 150 mM, about 5 mM to about 100 mM, about 10 mM to about 100 mM, about 20 mM to about 100 mM, about 25 mM to about 100 mM, about 30 mM to about 100 mM, about 35 mM to about 100 mM, about 40 mM to about 100 mM, or about 50 mM to about 100 mM.

[0041] In the foregoing embodiments, the molecule or ion may be disulfide ion having a concentration ranging from 1 mM to 100 mM, 2 nM to 500 nM, 3 nM to 200 nM, 5 nM to 100 nM.

[0042] In any one of the preceding embodiments, the molecule or ion may be selected from sodium bicarbonate, potassium bicarbonate, sodium disulfide, or potassium disulfide.

[0043] In any one of the foregoing embodiments, the physiological condition may be normal physiological pH, and the abnormal condition is an abnormal pH that is different from normal physiological pH, and the molecule or ion has a pKa between normal physiological pH and the abnormal pH.

[0044] In the foregoing embodiments, the pKa of the molecule or ion may be up to 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.8, or 1.0, 2.0 units away from the abnormal pH.

[0045] In a second aspect, the disclosure relates to a conditionally active polypeptide derived from a parent polypeptide having a pI, wherein the conditionally active polypeptide (a) has a pI that is the same as or lower than the pI of the parent polypeptide, and (b) has a ratio of activity in a second assay under abnormal conditions to the same activity in a first assay under normal physiological conditions of at least 1.3, wherein the activity of the conditionally active polypeptide is measured in the presence of at least one molecule or ion having a molecular weight of less than 900 a.mu.

[0046] In a second embodiment, the molecular weight may be less than 500 a.mu, less than 200 a.mu, or less than 100 a.mu.

[0047] In any one of the foregoing embodiments, the ratio of activity in a second assay under abnormal conditions to the same activity in a first assay under normal physiological conditions may be at least 1.5, at least 1.7, or at least 2.0, or at least 3.0, or at least 4.0, or at least 6.0, or at least 8.0, or at least 10.0, or at least 20.0, or at least 40.0, or at least 60.0, or at least 100.0.

[0048] In any one of the foregoing embodiments, the pI of the conditionally active polypeptide can be at least 0.1, at least 0.2, at least 0.3, at least 0.4, at least 0.5, at least 0.6, at least 0.8, at least 1.0, at least 1.2, at least 1.4, at least 1.5, at least 1.7, at least 2.0, at least 2.5, at least 3.0, at least 3.5, at least 4.0, or at least 5.0 units lower than the pI of the parent polypeptide.

[0049] In any one of the preceding embodiments, the conditionally active polypeptide may be selected from an antibody, an enzyme, a hormone, a growth factor, a cytokine, a regulatory protein, a functional peptide, a biosimilar, an immunomodulator, a receptor, and a ligand.

[0050] In any one of the foregoing embodiments, the conditionally active polypeptide may be an antibody selected from a full-length antibody, a single-chain antibody, an antibody fragment, a heavy chain, a light chain, a Fab, and an Fc domain.

[0051] In the foregoing embodiment, the antibody may be an IgG antibody.

[0052] In any one of the preceding embodiments, the conditions may be selected from pH, temperature, osmolality, osmolality, oxidative stress, and electrolyte concentration.

[0053] In any one of the foregoing embodiments, normal physiological conditions may be a pH in the range of greater than 7.2 to less than 7.6, and abnormal conditions are a pH in the range of 5.5 to less than 7.2.

[0054] In the foregoing embodiments, the molecule or ion can have a pKa between normal physiological pH and abnormal pH.

[0055] In the foregoing embodiments, the pKa of the molecule or ion may be up to 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.8, or 1.0, 2.0 units away from the abnormal pH.

[0056] In any one of the preceding embodiments, the molecule or ion may be selected from histidine, histamine, hydrogenated adenosine diphosphate, hydrogenated adenosine triphosphate, citrate, bicarbonate, acetate, lactate, disulfide, hydrogen sulfide, ammonium, dihydrogen phosphate, and any combination thereof.

[0057] In any one of the foregoing embodiments, the molecule or ion may be bicarbonate ion having a concentration ranging from about 3 mM to about 200 mM, about 5 mM to about 150 mM, about 5 mM to about 100 mM, about 10 mM to about 100 mM, about 20 mM to about 100 mM, about 25 mM to about 100 mM, about 30 mM to about 100 mM, about 35 mM to about 100 mM, about 40 mM to about 100 mM, or about 50 mM to about 100 mM.

[0058] In any one of the foregoing embodiments, the molecule or ion may be disulfide ion having a concentration in the range of 1 mM to 100 mM, 2 nM to 500 nM, 3 nM to 200 nM, 5 nM to 100 nM.

[0059] In any one of the preceding embodiments, the molecule or ion may be selected from sodium bicarbonate, potassium bicarbonate, sodium disulfide, or potassium disulfide.

[0060] In yet another aspect, the disclosure relates to a pharmaceutical composition comprising an effective amount of a conditionally active polypeptide and a pharmaceutically acceptable carrier.

[0061] In yet another aspect, the disclosure relates to the use of the conditionally active polypeptide for the treatment of solid tumors, inflamed joints, or brain diseases or disorders.

[0062] In yet another aspect, the disclosure relates to a method for treating a solid tumor, an inflamed joint, or a brain disease or disorder comprising administering to a patient in need of said treatment a conditionally active polypeptide. [Brief explanation of the drawings]

[0063] [Figure 1] Figure 1 shows the formation of salt bridges in deoxyhemoglobin, where three amino acid residues form two salt bridges, and these salt bridges stabilize the T quaternary structure of deoxyhemoglobin, leading to a reduced affinity for oxygen. DETAILED DESCRIPTION OF THE INVENTION

[0064] definition To facilitate understanding of the examples provided herein, some frequently occurring methods and / or terms are defined here.The definitions of the following terms are incorporated by reference in their entirety from U.S. Pat. No. 8,709,755 B2: "drug," "ambiguous base requirement," "amino acid," "amplification," "chimeric property," "cognate," "comparison window," "conservative amino acid substitution," "corresponding to," "degradation-effective," "defined sequence framework," "defined sequence kernel," "digestion," "directional ligation," "DNA shuffling," "drug" or "drug molecule," "effective amount," "epitope," "enzyme," "evolution" or "evolve," "fragment" or "derivative" or "analog," "global single amino acid substitution," "gene," "genetic instability," "heterologous," "homologous" or "homologous," "industrial application," "identical" or "identity," "identity range," "isolated," "isolated nucleic acid," "ligand," "ligation," "linker" or "spacer," "microenvironment," "molecular property to evolve," "mutation," "N,N,G / T," "normal physiological conditions" or "wild-type operating conditions," "nucleic acid molecule," "nucleic acid molecule," "nucleic acid sequence encoding" or "DNA coding sequence for" or "nucleotide encoding sequence," "nucleic acid encoding an enzyme (protein)" or "DNA encoding an enzyme (protein)" or "polynucleotide encoding an enzyme (protein)," "specific nucleic acid molecular species," "assembling a working nucleic acid sample into a nucleic acid library," "nucleic acid library," "construct," "oligonucleotide" (or synonymously "oligo"), "homologous," "operably linked," "parent polynucleotide set," "patient" or "subject," "physiological condition," "population," "proform," "pseudorandom," "quasi-repeated unit," "random peptide library," "random peptide sequence," "receptor," "recombinant" enzyme," "synthetic" enzyme," "related polynucleotide," "reduced reassortment," "reference sequence," "repeat index (RI)," "restriction site," "selectable polynucleotide," "sequence identity," "similarity," "specifically binds," "specific hybridization," "specific polynucleotide," "stringent hybridization conditions," "substantially identical," "substantially pure enzyme," "substantially pure," "processing," "variable segment," and "variant."

[0065] The definitions of the following terms are incorporated by reference in their entirety from WO2017 / 078839: "about," "activity," "antibody," "antibody-dependent cell-mediated cytotoxicity" or "ADCC," "antigen" or "Ag," "antisense RNA," "biosimilar," "follow-on biological," "biosimilar antibody," "cancer" or "cancerous," "chimeric antigen receptor" or "CAR" or "CARs," "cytokine" or "cytokines," "electrolyte," "full-length antibody," "growth factor," "hormone," "immunomodulator," "individual" or "subject," "library," "ligand," "receptor," "microRNA" or "miRNA," "multispecific antibody," "nanoparticle," "naturally occurring," "recombinant antibody," "regulatory protein," "small interfering RNA" or "siRNA," "therapeutic protein," "therapeutically effective amount," "tumor microenvironment," and "wild-type."

[0066] As used herein, the term "surface-exposed amino acid residue" of a polypeptide refers to an amino acid residue of a polypeptide, at least a portion of whose side chain is in contact with a liquid (e.g., blood, human serum, cytoplasm, etc.) when the polypeptide is present in a liquid. Surface-exposed amino acid residues of a polypeptide can be determined by X-ray crystallography. Potential surface-exposed amino acid residues can also be determined using coordinates from a three-dimensional model of an antibody using a computer program, such as the InsightII program (Accelrys). Other software available for such purposes includes, for example, SYBYL Biopolymer Module software (Tripos Associates). If the algorithm requires user-input size parameters, the "size" of the probe used in the calculation can be set to a radius of approximately 1.4 Angstroms (Å) or less. For example, a method for determining surface-exposed amino acid residues and regions using software for personal computers is described by Pacios (Pacios, Comput. Chem, vol. 18, pp. 377-386, 1994; J. Mol. Model., vol. 1, pp. 46-53, 1995).

[0067] The term "conditionally active polypeptide" refers to a variant or mutant of a parent polypeptide that is more active under at least one condition (e.g., abnormal conditions) than the parent polypeptide and less active under a second condition (e.g., normal physiological conditions) than the parent polypeptide, or refers to a variant or mutant of a parent polypeptide that is more active under at least one condition (e.g., abnormal conditions) than under a second condition (e.g., normal physiological conditions). In one embodiment, the conditionally active polypeptide has at least 1.3, at least 1.5, at least 2.0, or at least 2.5, or at least 3.0, or at least 5.0, or at least 10, or at least 15, or at least 20, or at least 60, or at least 80, or at least 100 times more activity under the second abnormal conditions than under the first normal physiological conditions. The conditionally active polypeptide may exhibit activity at one or more selected biological locations and / or may exhibit increased or decreased activity at another biological location. For example, in some embodiments, a conditionally active polypeptide is substantially inactive at body temperature but active at lower temperatures. Conditionally active polypeptides include conditionally active proteins, protein fragments, antibodies, antibody fragments, enzymes, enzyme fragments, receptors and receptor fragments, cytokines and fragments thereof, hormones and fragments thereof, ligands and fragments thereof, regulatory proteins and fragments thereof, growth factors and fragments thereof, and polypeptides comprising stress proteins, fornix-associated proteins, neuronal proteins, gut proteins, growth factors, mitochondrial proteins, cytoplasmic proteins, animal proteins, structural proteins, plant proteins, and fragments of any of these proteins. Each of the conditionally active polypeptides described herein is preferably a conditionally active biological polypeptide.

[0068] As used herein with respect to a polypeptide, "increasing plasma half-life" or "prolonging plasma half-life" refers to the reduction in plasma residence time (t 1 / 2 ) or a decrease in plasma clearance (CL), which can be expressed as the area under the concentration curve (AUC) over time.

[0069] As used herein, the term "isoelectric point" or "pI" of a polypeptide refers to the pH at which the polypeptide has no net charge. The pI of a polypeptide can be determined experimentally or calculated based on the amino acid sequence of the polypeptide. For example, the pI can be determined by isoelectric focusing of the polypeptide, as known to those skilled in the art. Theoretical calculations of pI can be performed using amino acid sequence analysis software (e.g., GENETYX) based on the amino acids of the polypeptide.

[0070] The term "isoelectric point variant" or "pI variant" of a parent polypeptide refers to a variant polypeptide of a parent polypeptide that has a decreased pI relative to the parent polypeptide from which it is derived, obtained either by substituting amino acid residues with higher pIs for amino acid residues with lower pIs, by deleting amino acid residues that are higher than the pI of the parent polypeptide, or by inserting amino acid residues that are lower than the pI of the parent polypeptide. The present invention extends to both conditionally active pI variants as well as conditionally active polypeptides that have the same pI as the parent polypeptide.

[0071] The terms "parent polypeptide" and "parent protein," as used herein, refer to a polypeptide or protein that can be evolved to generate a conditionally active polypeptide using the methods described herein. A parent polypeptide can be a protein that does not occur in nature. For example, a therapeutic polypeptide or protein or a mutant or variant polypeptide can be used as a parent polypeptide. Examples of parent polypeptides include antibodies, antibody fragments, enzymes, enzyme fragments, cytokines and fragments thereof, hormones and fragments thereof, ligands and fragments thereof, receptors and fragments thereof, regulatory proteins and fragments thereof, and growth factors and fragments thereof.

[0072] The term "pH-dependent" as used herein refers to a polypeptide that has different properties or activities at different pH values.

[0073] The term "polypeptide" as used herein refers to an amino acid polymer in which monomers are linked together by peptide or disulfide bonds. A polypeptide can be a full-length naturally occurring amino acid chain or a fragment thereof, a mutant or variant thereof, e.g., a selected region of the amino acid chain that is of interest in a binding interaction. A polypeptide can also be a synthetic amino acid chain, or a combination of a naturally occurring amino acid chain or a fragment thereof with a synthetic amino acid chain. A fragment refers to an amino acid sequence that is a portion of a full-length protein and is typically about 8 to about 500 amino acids in length, 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 length. In addition, amino acids other than naturally occurring amino acids, such as β-alanine, phenylglycine, and homoarginine, can be included in polypeptides. Commonly occurring non-genetically encoded amino acids can also be included in polypeptides. Amino acids can be either D- or L-optical isomers. D-isomers are preferred for use in certain contexts, as discussed in more detail below. In addition, other peptidomimetics are also useful, for example, in linker sequences of polypeptides (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 to encompass structures comprising two or several polypeptide chains held together by covalent or noncovalent bonds.

[0074] The term "small molecule" typically refers to a molecule having a molecular weight of less than 900 a.mu, or more preferably less than 500 a.mu, or more preferably less than 200 a.mu, or even more preferably less than 100 a.mu. The same molecular weight ranges apply to ions used in these assays. In the assays and environments of the present invention, small molecules or ions may often exist as a mixture of the molecule and its deprotonated ion, depending primarily on the pH of the assay or environment.

[0075] The present disclosure provides a method for generating a conditionally active polypeptide from a parent polypeptide, the method comprising: (i) evolving the parent polypeptide by introducing one or more mutations into the parent polypeptide to produce one or more variant polypeptides having a pI that is the same as or lower than the pI of the parent polypeptide; (ii) subjecting the one or more variant polypeptides to a first assay under normal physiological conditions to measure the activity of the one or more variant polypeptides under normal physiological conditions and a second assay under abnormal conditions to measure the activity of the one or more variant polypeptides under abnormal conditions, where the normal physiological conditions and the abnormal conditions are the same conditions but have different values; and (iii) selecting a conditionally active polypeptide from the one or more variant polypeptides that exhibits increased activity in the second assay under abnormal conditions compared to the same activity in the first assay under normal physiological conditions.

[0076] A. Mutations that affect the pI of a polypeptide The parent polypeptide may be mutated to generate a conditionally active polypeptide having the same or a reduced pI compared to the pI of the parent polypeptide. Preferably, the conditionally active polypeptide will have a reduced pI compared to the pI of the parent polypeptide. Alternatively, or in addition, the conditionally active polypeptide may have a pI of less than 7.4, or less than 7.3, or less than 7.2, or less than 7.1, or less than 7.0. The conditionally active polypeptide will also exhibit pH selectivity, i.e., greater activity at the pH where activity is desired than at normal physiological pH, which may range, for example, from greater than 7.2 to less than 7.6.

[0077] Any suitable mutagenesis technique can be used, including amino acid residue substitution, deletion, insertion, and combinations thereof. Amino acid residue substitution involves replacing a native amino acid residue in a parent polypeptide with another amino acid residue having a lower pI than the amino acid of the replaced native amino acid residue. The pIs of amino acids are shown in Table 1. While this table shows the pI of amino acids as individual molecules rather than as amino acid residues located in the polypeptide, the effect of the pI of an amino acid residue in the case of a portion of a polypeptide follows the trend of the amino acid pIs in Table 1. Thus, for example, replacing a native amino acid residue with a higher pI with an amino acid residue having a lower pI than the higher pI of the native amino acid will lower the pI of the polypeptide. In practice, a native amino acid residue in a parent polypeptide may be replaced with another amino acid residue having a lower pI than the native amino acid. For example, if the native amino acid residue is a basic amino acid residue, it may be substituted with a weakly acidic amino acid residue or a strongly acidic amino acid residue to lower the pI of the polypeptide. In another example, if the native amino acid residue is a residue of a weakly acidic amino acid, it may be substituted with a residue of a strongly acidic amino acid to lower the pI of the polypeptide. [Table 1]

[0078] In some embodiments, two or more amino acid residues are substituted into the parent polypeptide to affect the pI of the parent polypeptide. For example, two, three, four, five, six, seven, eight, nine, ten, or more amino acid residue substitutions may be introduced into the parent polypeptide. In some embodiments, each substitution replaces an amino acid residue with a lower pI with an amino acid residue with a higher pI. In other embodiments, only some of the substitutions replace an amino acid residue with a lower pI with an amino acid residue with a higher pI. In the latter case, the combination of substitutions is selected so that the overall effect of the multiple substitutions is either to maintain the pI of the variant polypeptide the same as that of the parent polypeptide, or to provide a pI for the variant polypeptide that is lower than that of the parent polypeptide.

[0079] In some embodiments, the amino acid residue substitutions used to affect pI may be conservative substitutions, non-conservative substitutions, or a combination thereof.

[0080] Deleting amino acid residues that decrease the pI of a parent polypeptide includes deleting amino acid residues with a pI higher than the pI of the parent polypeptide. For example, if the pI of a parent polypeptide is about 7.2, deleting any one or more basic amino acid residues from the parent polypeptide will decrease the pI of the parent polypeptide. In some embodiments, at least 2, 3, 4, 5, or more amino acid residues are deleted from the parent polypeptide. In some embodiments, each deletion deletes an amino acid residue with a pI lower than the pI of the parent polypeptide. In other embodiments, only some of the deletions delete amino acid residues with a pI higher than the pI of the parent polypeptide. In the latter case, the combination of deletions is selected so that the combined effect of the multiple deletions is to maintain or decrease the pI of the polypeptide.

[0081] Inserting amino acid residues that lower the pI of a parent polypeptide includes inserting amino acid residues having a pI lower than the pI of the parent polypeptide. For example, if the pI of a parent polypeptide is about 7.2, inserting any one or more residues of weakly acidic or strongly acidic amino acids into the parent polypeptide will result in a decrease in the pI of the parent polypeptide. In some embodiments, at least 2, 3, 4, 5, or more amino acid residues are inserted into the parent polypeptide. In some embodiments, each insertion inserts an amino acid residue having a pI lower than the pI of the parent polypeptide. In other embodiments, only some of the insertions insert amino acid residues having a pI lower than the pI of the parent polypeptide. In the latter case, the combination of insertions is selected so that the combined effect of the multiple insertions maintains or decreases the pI of the polypeptide.

[0082] In some embodiments, a combination of two or more of the above amino acid residue substitutions, deletions, and insertions is used to affect the pI of the parent polypeptide. For example, a variant polypeptide may contain one or more amino acid residue substitutions and one or more amino acid residue deletions. In another embodiment, a variant polypeptide may have one or more amino acid residue substitutions and one or more amino acid residue insertions. In yet another example, a variant polypeptide may have one or more amino acid residue deletions and one or more amino acid residue insertions. In yet another example, a variant polypeptide may have all of one or more amino acid residue substitutions, one or more amino acid residue deletions, and one or more amino acid residue insertions. In either case, all or only some of the insertions, substitutions, and / or deletions may use amino acid residues that have a pI lower than either the pI of the polypeptide or the pI of the amino acid being substituted. In each case, the combination of insertions, substitutions, and / or deletions is selected so that the combined effect of the multiple insertions, substitutions, and / or deletions is to maintain or lower the pI of the polypeptide.

[0083] Desirable variant polypeptides will have a pI that is the same as or lower than the pI of the parent polypeptide. Some variant polypeptides will have a pI that is the same as the pI of the parent polypeptide. Other variant polypeptides may have a pI that is at least 0.1, or at least 0.2, or at least 0.3, or at least 0.4, or at least 0.5, or at least 0.6, or at least 0.8, or at least 1.0, or at least 1.2, or at least 1.4, or at least 1.5, or at least 1.7, or at least 2.0, or at least 2.5, or at least 3.0, or at least 3.5, or at least 4.0, or at least 5.0 units lower than the pI of the parent polypeptide.

[0084] In some embodiments, the native amino acid residue that is substituted, the amino acid residue that is deleted, and / or the position at which the amino acid residue is inserted is exposed on the surface of the parent polypeptide, it being understood that mutation of an exposed amino acid residue or position is unlikely to disrupt the three-dimensional structure of the parent polypeptide.

[0085] Amino acid residue substitutions, insertions, and deletions can be made in the nucleotide sequence encoding the parent polypeptide, for example, by site-directed mutagenesis (Kunkel et al., Proc. Natl. Acad. Sci. USA 82:488-492 (1985)) or overlap extension PCR. When the parent polypeptide is an antibody, mutations can also be achieved by affinity maturation of the antibody, or chain shuffling of the antibody heavy or light chain; or selection based on antigen panning using a phage display library (Smith et al., Methods Enzymol. 217:228-257 (1993)). These mutagenesis methods can be performed alone or in appropriate combinations.

[0086] In some further embodiments, in addition to mutations that maintain or decrease the pI relative to the pI of the parent polypeptide, the variant polypeptide may also be subjected to additional mutagenesis at positions other than those having mutations introduced to maintain or decrease the pI. Additional mutagenesis may be performed using any known mutagenesis method, such as, for example, comprehensive positional evolution, comprehensive positional deletion, comprehensive positional insertion, or a combination thereof, as described in detail in US2013 / 0116125.

[0087] For example, a parent polypeptide may be mutated to contain one or more amino acid residue substitutions that maintain or decrease the pI relative to the pI of the parent polypeptide. For example, the mutated polypeptide may also be subjected to global positional evolution, including global positional substitutions, global positional deletions, and global positional insertions at positions other than those that have one or more mutations already introduced to decrease the pI of the polypeptide.

[0088] The pI of any polypeptide can be measured by isoelectric focusing gel electrophoresis (e.g., capillary isoelectric focusing gel electrophoresis) or other methods (see, e.g., the methods described in Righetti et al., Methods Biochem. Anal. 54:379-409, 2011; Friedman et al., Methods Enzymol. 463:515-540, 2009; Koshel et al., Proteomics 12:2918-2926, 2012; Sommer et al., Electrophoresis 30:742-757, 2009; Shimura et al., Electrophoresis 30:11-28, 2009). Additionally, there are methods that can estimate or calculate the pI of a polypeptide based on its amino acid sequence, such as those described in Ribeiro J M. Sillero A., Computers in Biology & Medicine 20(4):235-42, 1990; Ribeiro J M. Sillero A., Computers in Biology & Medicine 21(3):131-41, 1991; and Sillero A. Ribeiro J M., Analytical Biochemistry 179(2):319-25, 1989.

[0089] Any protein, including antibodies, enzymes, hormones, growth factors, cytokines, regulatory proteins, functional peptides, biosimilars, immunomodulators, therapeutic proteins, receptors, and ligands, can be used as the parent polypeptide of the present invention. The parent polypeptide can also be a fragment of any of the above proteins. For example, the parent polypeptide can be 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, the parent polypeptide can be a stress protein, fornix-associated protein, neuronal protein, gut protein, growth factor, mitochondrial protein, cytosolic protein, animal protein, structural protein, plant protein, or a fragment of any of these proteins.

[0090] In one exemplary embodiment, the parent polypeptide is an antibody. The parent antibody may be a therapeutic antibody or a candidate antibody being developed for therapeutic use. Examples of parent antibodies include rituximab (Rituxan®, IDEC / Genentech / Roche), a chimeric anti-CD20 antibody approved for the treatment of non-Hodgkin's lymphoma (see, e.g., U.S. Pat. No. 5,736,137); HuMax-CD20, an anti-CD20 antibody currently being developed by Genmab; AME-133 (Applied Molecular Evolution), hA20 (Immunomedics, Inc.), HumaLYM (Intracel), and PRO70769 (PCT / US2003 / 040426, entitled "Immunoglobulin Variants and Uses Thereof"). Many antibodies targeting members of the epidermal growth factor receptor family, including EGFR (ErbB-1), Her2 / neu (ErbB-2), Her3 (ErbB-3), and Her4 (ErbB-4), can benefit from the pI bioengineered constant regions of the present invention. For example, the pI bioengineered constant regions of the present invention may be used in trastuzumab (Herceptin®, Genentech), a humanized anti-Her2 / neu antibody approved for treating breast cancer (see, e.g., U.S. Pat. No. 5,677,171); pertuzumab (rhuMab-2C4, Omnitarg®), currently being developed by Genentech; the anti-Her2 antibody described in U.S. Pat. No. 4,753,894; cetuximab (Erbitux®, Imclone) (U.S. Pat. No. 4,943,533; PCT WO 96 / 40210), a chimeric anti-EGFR antibody in clinical trials for various cancers; ABX-EGF (U.S. Pat. No. 6,235,883), currently being developed by Abgenix-Immunex-Amgen; and HuMax-EGF, currently being developed by Genmab. (U.S. Patent No. 10 / 172,317), 425, EMD55900 and EMD7200 (Merck KGaA) (U.S. Patent No. 5,558,864;Murthy et al. 1987, Arch Biochem Biophys. 252(2):549-60; Rodeck et al., 1987, J Cell Biochem.35(4):315-20; Kettleborough et al., 1991, Protein Eng.4(7):773-83);ICR62 (Institute of Cancer Research) (PCT WO 95 / 20045; Modjtahedi et al., 1993, J. Cell Biophys. 1993, 22(1-3):129-46; Modjtahedi et al., 1993, Br J Cancer.1993, 67(2):247-53; Modjtahedi et al, 1996, Br J Cancer, 73(2):228-35; Modjtahedi et al, 2003, Int J Cancer, 105(2):273-80); TheraCIM hR3 (YM Biosciences, Canada and Centro de Immunologia Molecular, Cuba (U.S. Pat. No. 5,891,996; U.S. Pat. No. 6,506,883; Mateo et al, 1997, Immunotechnology, 3(1):71-81); mAb-806 (Ludwig Institute for Cancer Research, Memorial Sloan-Kettering) (Jungbluth et al. 2003, Proc Natl Acad Sci USA. 100(2):639-44); KSB-102 (KS Biomedix); MR1-1 (WAX, National Cancer Institute) (PCT WO 0162931A2);and SC100 (Scancell) (PCT WO 01 / 88138). In another preferred embodiment, the pI bioengineered constant regions of the present invention find use in alemtuzumab (Campath®, Millenium), a humanized monoclonal antibody currently approved for the treatment of B-cell chronic lymphocytic leukemia. The bioengineered constant regions of the present invention may find use in a variety of antibodies substantially similar to other clinical products and candidates, including, but not limited to: muromonab-CD3 (Orthoclone OKT3®), an anti-CD3 antibody developed by Ortho Biotech / Johnson & Johnson; ibritumomab tiuxetan (Zevalin)®, an anti-CD20 antibody developed by IDEC / Schering AG; gemtuzumab ozogamicin (Mylotarg®), an anti-CD33 (p67 protein) antibody developed by Celltech / Wyeth; alefacept (Amevive®), an anti-LFA-3 Fc fusion developed by Biogen; abciximab (ReoPro®), an anti-LFA-3 Fc fusion developed by Novartis, developed by Centocor / Lilly; basiliximab (Simulect®), developed by Novartis; palivizumab (Synagis®), developed by MediImmune; infliximab (Remicade®), an anti-TNFalpha antibody developed by Centocor; adalimumab (Humira®), an anti-TNFalpha antibody developed by Abbott; Humicade™, an anti-TNFalpha antibody developed by Celltech; etanercept (Enbrel®), an anti-TNFalpha Fc fusion developed by Immunex / Amgen; ABX-CBL, an anti-CD147 antibody developed by Abgenix; ABX-IL8, an anti-IL8 antibody developed by Abgenix;ABX-MA1, an anti-MUC18 antibody developed by Abgenix; Pemtumomab (R1549, 90Y-muHMFG1), an anti-MUC1 antibody being developed by Antisoma; Therex (R1550), an anti-MUC1 antibody developed by Antisoma; AngioMab (AS1405), developed by Antisoma; HuBC-1, developed by Antisoma; Thioplatin (AS1407), developed by Antisoma; Antegren® (natalizumab), an anti-alpha-4-beta-1 (VLA-4) and alpha-4-beta-7 antibody developed by Biogen; VLA-1 mAb, an anti-VLA-1 integrin antibody developed by Biogen; LTBR mAb, an anti-lymphotoxin beta receptor (LTBR) antibody developed by Biogen; CAT-152, an anti-TGF-132 antibody developed by Cambridge Antibody Technology; J695, a Cambridge Antibody Anti-IL-12 antibody developed by Technology and Abbott; CAT-192, an anti-TGFβ1 antibody developed by Cambridge Antibody Technology and Genzyme; CAT-213, an anti-Eotaxin1 antibody developed by Cambridge Antibody Technology; LymphoStat-B™, an anti-Blys antibody developed by Cambridge Antibody Technology and Human Genome Sciences Inc.; TRAIL-R1mAb, an anti-TRAIL-R1 antibody developed by Cambridge Antibody Technology and Human Genome Sciences, Inc.; Avastin™ (bevacizumab, rhuMAb-VEGF), an anti-VEGF antibody developed by Genentech; an anti-HER receptor family antibody developed by Genentech; Anti-Tissue Factor (ATF), an anti-tissue factor antibody developed by Genentech;Xolair™ (Omalizumab), an anti-IgE antibody developed by Genentech; Raptiva™ (Efalizumab), an anti-CD11a antibody developed by Genentech and Xoma; MLN-02 antibody (formerly LDP-02) developed by Genentech and Millenium Pharmaceuticals; HuMax CD4, an anti-CD4 antibody developed by Genmab; HuMax-IL15, an anti-IL15 antibody developed by Genmab and Amgen; HuMax-Inflam, developed by Genmab and Medarex; HuMax-Cancer, an anti-Heparanase I antibody developed by Genmab, Medarex, and Oxford Pharma Sciences; HuMax-Lymphoma, developed by Genmab and Amgen; HuMax-TAC, developed by Genmab; IDEC-131, an anti-CD40L antibody developed by IDEC Pharmaceuticals; IDEC-151 (Clenoliximab), an anti-CD4 antibody developed by IDEC Pharmaceuticals; IDEC-114, an anti-CD80 antibody developed by IDEC Pharmaceuticals; IDEC-152, an anti-CD23 antibody developed by IDEC Pharmaceuticals; anti-macrophage migration factor (MIF) antibodies developed by IDEC Pharmaceuticals; BEC2, an anti-idiotypic antibody developed by Imclone; IMC-1C11, an anti-KDR antibody developed by Imclone; DC101, an anti-flk-1 antibody developed by Imclone; an anti-VE-cadherin antibody developed by Imclone; CEA-Cide™ (labetuzumab), an anti-carcinoembryonic antigen (CEA) antibody developed by Immunomedics; LymphoCide™ (Epratuzumab), an anti-CD22 antibody developed by Immunomedics; AFP-Cide developed by Immunomedics;MyelomaCide, developed by Immunomedics; LkoCide, developed by Immunomedics; ProstaCide, developed by Immunomedics; MDX-010, an anti-CTLA4 antibody developed by Medarex; MDX-060, an anti-CD30 antibody developed by Medarex; MDX-070, developed by Medarex; MDX-018, Osidem™ (IDM-1), an anti-Her2 antibody developed by Medarex and Immuno-Designed Molecules; HuMax™-CD4, an anti-CD4 antibody developed by Medarex and Genmab; HuMax-IL15, an anti-IL15 antibody developed by Medarex and Genmab; CNTO 148, an anti-TNFα antibody developed by Medarex and Centocor / J&J; CNTO 1275, an anti-cytokine antibody developed by Centocor / J&J; MOR101 and MOR102, anti-intercellular adhesion molecule-1 (ICAM-1) (CD54) antibodies developed by MorphoSys; MOR201, an anti-fibroblast growth factor receptor 3 (FGFR-3) antibody developed by MorphoSys; Nuvion® (visilizumab), an anti-CD3 antibody developed by Protein Design Labs; HuZAF™, an anti-gamma interferon antibody developed by Protein Design Labs; an anti-α5β1 integrin antibody developed by Protein Design Labs; an anti-IL-12 antibody developed by Protein Design Labs; ING-1, an anti-Ep-CAM antibody developed by Xoma Antibodies; MLN01, an anti-Beta2 integrin antibody developed by Xoma; pI-ADC antibody developed by Seattle Genetics; all references cited above in this paragraph are expressly incorporated herein by reference.

[0091] The parent antibody can be a full-length antibody, antibody fragment, single-chain antibody, Fab, or Fc domain. The parent antibody can be an IgG antibody. Among several antibody isotypes, IgG antibodies have a sufficiently large molecular weight that their primary metabolic pathway does not involve renal excretion. IgG is known to be recycled via a salvage pathway via FcRn, resulting in a long in vivo half-life. IgG antibodies are thought to be primarily metabolized via metabolic pathways in endothelial cells (He et al., J. Immunol., vol. 160, pp. 1029-1035, 1998). Specifically, when IgG antibodies are nonspecifically internalized by endothelial cells, they are recycled by binding to FcRn, while IgG antibodies that do not bind to FcRn are thought to be degraded. The plasma half-life of IgG antibodies is inversely correlated with their pI, as described, for example, in WO2007 / 114319 and WO2009 / 041643.

[0092] Among IgG antibodies, the parent antibody can be an IgG1 antibody, a common isotype for therapeutic antibodies, for various reasons, including high effector function. However, the heavy chain constant region of IgG1 has a higher pI than that of IgG2 (8.10 vs. 7.31). By introducing some IgG2 amino acid residues at specific positions into the IgG1 backbone by amino acid residue substitution, the resulting IgG1 has a lower pI and exhibits a longer half-life in plasma than the parent IgG1 antibody. For example, IgG1 has glycine at position 137, while IgG2 has glutamic acid (a strongly acidic amino acid) at the same position. Substituting glutamic acid for glycine at position 137 of IgG1 reduces the pI of the mutant IgG1 antibody and increases its half-life.

[0093] In some embodiments, one or more mutations or combinations of mutations described herein to maintain or decrease the pI relative to the pI of the parent polypeptide are located in the antibody heavy chain, the antibody light chain, or both the antibody heavy and light chains. B. Mutations in the antibody heavy chain

[0094] In some embodiments, the mutations or combinations of mutations described herein for the purpose of maintaining or decreasing the pI of the polypeptide are made at least in the CH1 region of the heavy chain of an IgG antibody. In these embodiments, the mutations can be independently selected from mutations at positions 119, 131, 133, 137, 138, 164, 192, 193, 196, 199, 203, 205, 208, 210, 214, 217, and 219 in the CH1 region, and any combination of mutations at these positions. Mutations can be introduced by substitution, deletion, or insertion at one of these 17 positions, or at all possible combinations and subcombinations of these 17 positions. For example, a mutant antibody can have 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, or 17 CH1 substitutions at one or more of these 17 positions. Thus, any single mutation or combination of mutations in the CH1 region is possible. Additionally, one or more mutations in the CH1 region may optionally be combined with one or more other mutations, or with combinations of mutations in any of the CH2, CH3, hinge, and LC regions, in which case the combination of mutations in the CH1, CH2, CH3, hinge, and LC regions may be selected to reduce the pI of the variant antibody compared to the pI of the parent antibody.

[0095] Useful substitutions that can decrease the pI of the heavy chain include substitutions of aspartic acid or glutamic acid residues at one or more of positions 121, 124, 129, 132, 134, 126, 152, 155, 157, 159, 101, 161, 162, 165, 176, 177, 178, 190, 191, 194, 195, 197, 212, 216 and 218 in the CH1 region of an IgG antibody heavy chain.

[0096] Specific substitutions in the CH1 region of an antibody heavy chain include, but are not limited to, a non-native glutamic acid at position 119, a non-native cysteine ​​at position 131, a non-native arginine, lysine, or glutamine at position 133, a non-native glutamic acid at position 137, a non-native serine at position 138, a non-native glutamic acid at position 164, a non-native asparagine at position 192, a non-native phenylalanine at position 193, a non-native lysine at position 196, a non-native threonine at position 199, a non-native aspartic acid at position 203, a non-native glutamic acid or glutamine at position 205, a non-native aspartic acid at position 208, a non-native glutamic acid or glutamine at position 210, a non-native threonine at position 214, a non-native arginine at position 217, and a non-native cysteine ​​at position 219, and any combination thereof.

[0097] In some embodiments, the mutations are made in the hinge region of the antibody heavy chain, including positions 221, 222, 223, 224, 225, 233, 234, 235, and 236. Specifically, 1, 2, 3, 4, or 5 mutations, and particularly 1, 2, 3, 4, or 5 substitutions, can be made at positions 221-225. Again, all possible combinations are contemplated, either alone or in combination with other mutations in other regions.

[0098] Specific mutations in the hinge region of an antibody heavy chain can include, but are not limited to, a deletion at position 221, a non-native valine or threonine at position 222, a deletion at position 223, a non-native glutamic acid at position 224, a deletion at position 225, a deletion at position 235, a deletion or non-native alanine at position 236, and any combination thereof. In some cases, only one or more of these mutations are introduced in the hinge region of an antibody heavy chain; in other embodiments, one or more of these mutations are combined with one or more mutations in other regions of the parent antibody in any combination.

[0099] In some embodiments, mutations can be made in the CH2 region of the antibody heavy chain, including positions 274, 296, 300, 309, 320, 322, 326, 327, 334, and 339. Specifically, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 mutations can be made in any combination among these 10 positions in the CH2 region of the antibody heavy chain.

[0100] Specific substitutions in the CH2 region of the antibody heavy chain can include, but are not limited to, a non-native glutamine or glutamic acid at position 274, a non-native phenylalanine at position 296, a non-native phenylalanine at position 300, a non-native valine at position 309, a non-native glutamic acid at position 320, a non-native glutamic acid at position 322, a non-native glutamic acid at position 326, a non-native glycine at position 327, a non-native glutamic acid at position 334, a non-native threonine at position 339, and any combination of these substitutions, further in combination with one or more other mutations in other regions of the antibody.

[0101] The mutations can be independently selected from mutations at positions 355, 359, 362, 384, 389, 392, 397, 418, 419, 444, and 447 in the CH3 region of the antibody heavy chain. The variant antibody can have 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or 11 mutations at these positions in the CH3 region, in any combination. Furthermore, as described herein, any one or more mutations in the CH3 region can be combined with one or more other mutations in the CH2, CH1, hinge, and LC regions of the antibody.

[0102] Specific substitutions in the CH3 region of the antibody heavy chain can include, but are not limited to, a non-native glutamine or glutamic acid at position 355, a non-native serine at position 384, a non-native asparagine or glutamic acid at position 392, a non-native methionine at position 397, a non-native glutamic acid at position 419, a non-native glutamic acid at position 359, a non-native glutamic acid at position 362, a non-native glutamic acid at position 389, a non-native glutamic acid at position 418, a non-native glutamic acid at position 444, and a non-native aspartic acid at position 447, as well as any combination of two or more of these substitutions.

[0103] Thus, all possible combinations of the following antibody heavy chain deletions or substitutions can be made, with each mutation optionally included or excluded: non-native glutamic acid at position 119, non-native cysteine ​​at position 131, non-native arginine, lysine, or glutamine at position 133, non-native glutamic acid at position 137, non-native serine at position 138, non-native glutamic acid at position 164, non-native asparagine at position 192, non-native phenylalanine at position 193, non-native glutamic acid at position 196 ... lysine at position 199, non-native threonine at position 199, non-native aspartic acid at position 203, non-native glutamic acid or glutamine at position 205, non-native aspartic acid at position 208, non-native glutamic acid or glutamine at position 210, non-native threonine at position 214, non-native arginine at position 217 and non-native cysteine ​​at position 219, a deletion at position 221, a non-native valine or threonine at position 222, a deletion at position 223, a non-native glutamic acid at position 224, and a non-native cysteine ​​at position 225. 225 deletion, position 235 deletion, position 221 deletion, non-native valine or threonine at position 222, position 223 deletion, non-native glutamic acid at position 224, position 225 deletion, position 235 deletion, non-native glutamine or glutamic acid at position 274, non-native phenylalanine at position 296, non-native phenylalanine at position 300, non-native valine at position 309, non-native glutamic acid at position 320, non-native glutamic acid at position 322, non-native glutamic acid at position 326, non-native glycine at position 327, non-native glutamic acid at position 334, non-native threonine at position 339, non-native glutamine or glutamic acid at position 355, non-native serine at position 384, non-native asparagine or glutamic acid at position 392, non-native methionine at position 397, non-native glutamic acid at position 419, non-native glutamic acid at position 359, non-native glutamic acid at position 362, non-native glutamic acid at position 389, non-native glutamic acid at position 418,a non-native glutamic acid at position 444, and a non-native aspartic acid at position 447.

[0104] C. Antibody Light Chain Mutations In some embodiments, the mutations can be in the light chain of an IgG antibody. The mutations can be located at positions independently selected from light chain positions 126, 145, 152, 156, 169, 199, 202, and 207. The antibody can have 1, 2, 3, 4, 5, 6, 7, or 8 mutations in the light chain at these positions in any combination. Furthermore, any single or combination of light chain mutations can be combined with any one or more of the heavy chain mutations of the above antibodies.

[0105] Specific substitutions in the antibody light chain may include, but are not limited to, a non-native glutamine or glutamic acid at position 126, a non-native glutamine, glutamic acid, or threonine at position 145, a non-native aspartic acid at position 152, a non-native glutamic acid at position 156, a non-native glutamine or glutamic acid at position 169, a non-native glutamic acid at position 199, a non-native glutamic acid at position 202, and a non-native glutamic acid at position 207.

[0106] D. Heavy and Light Chain Mutations In some embodiments, the variant antibody may have mutations in both the heavy and light chains, as described above. In some embodiments, the variant antibody may have mutations only in the heavy chain, in which case the variant antibody will have the light chain of the parent antibody. In some embodiments, the variant antibody may have mutations only in the light chain, in which case the variant antibody will have the heavy chain of the parent antibody.

[0107] Thus, any possible combination of the following mutations in the heavy and light chains of the antibody can be made, with each mutation optionally included or excluded: a) Heavy chain: non-native glutamic acid at position 119; non-native cysteine ​​at position 131; non-native arginine, lysine, or glutamine at position 133; non-native glutamic acid at position 137; non-native serine at position 138; non-native glutamic acid at position 164; non-native asparagine at position 192; non-native phenylalanine at position 193, non-native phenylalanine at position 196, non-native cysteine ​​at position 131; non-native arginine, lysine, or glutamine at position 133; non-native glutamic acid at position 137; non-native serine at position 138; non-native glutamic acid at position 164; non-native asparagine at position 192; non-native phenylalanine at position 193, non-native cysteine ​​at position 194, non-native cysteine ​​at position 195, non-native cysteine ​​at position 196, non-native cysteine ​​at position 197, non-native cysteine ​​at position 198, non-native cysteine ​​at position 199, non-native cysteine ​​at position 200, non-native cysteine ​​at position 201, non-native cysteine ​​at position 202, non-native cysteine ​​at position 203, non-native cysteine ​​at position 204, non-native cysteine ​​at position 205, non-native cysteine ​​at position 206, non-native cysteine ​​at position 207, non-native cystein a non-native lysine, a non-native threonine at position 199, a non-native aspartic acid at position 203, a non-native glutamic acid or glutamine at position 205, a non-native aspartic acid at position 208, a non-native glutamic acid or glutamine at position 210, a non-native threonine at position 214, a non-native arginine at position 217, and a non-native cysteine ​​at position 219, a deletion at position 221, a non-native valine or threonine at position 222, a deletion at position 223, a non-native glutamic acid at position 224, and a non-native valine or threonine at position 225. deletion at position 225, deletion at position 235, deletion at position 221, non-native valine or threonine at position 222, deletion at position 223, non-native glutamic acid at position 224, deletion at position 225, and deletion at position 235, non-native glutamine or glutamic acid at position 274, non-native phenylalanine at position 296, non-native phenylalanine at position 300, non-native valine at position 309, non-native glutamic acid at position 320, non-native glutamic acid at position 322, non-native glutamic acid at position 326, non-native glycine at position 327, non-native glutamic acid at position 334, non-native threonine at position 339, non-native glutamine or glutamic acid at position 355, non-native serine at position 384, non-native asparagine or glutamic acid at position 392, non-native methionine at position 397, non-native glutamic acid at position 419, non-native glutamic acid at position 359, non-native glutamic acid at position 362, non-native glutamic acid at position 389, non-native glutamic acid at position 418,a non-native glutamic acid at position 444, and a deletion or non-native aspartic acid at position 447. Light chain: b) a non-native glutamine or glutamic acid at position 126, a non-native glutamine, glutamic acid, or threonine at position 145, a non-native aspartic acid at position 152, a non-native glutamic acid at position 156, a non-native glutamine or glutamic acid at position 169, a non-native glutamic acid at position 199, a non-native glutamic acid at position 202, and a non-native glutamic acid at position 207. E. Mutations in antibody constant regions

[0108] In some embodiments, one or more mutations may be located in the heavy chain constant region, the light chain constant region, or both of an antibody, such as an IgG antibody. For example, there may be 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 mutations in the heavy chain constant region and / or the light chain constant region of the antibody.

[0109] The mutations in the heavy and / or light chain constant regions are sufficient to reduce the pI of the parent antibody by at least 0.2, 0.4, 0.6, 0.8, 1.0, 1.2, 1.4, 1.6, 1.8, 2.0, 2.5, 3.0, 3.5, 4.0, 4.5 or 5.0 units compared to the pI of the parent antibody. F. Parent antibody variable region

[0110] In some embodiments, one or more mutations are located in the heavy chain variable region, the light chain variable region, or both of an antibody, such as an IgG antibody. In one embodiment, the mutations are located, for example, in one or more of the heavy and / or light chain CDR1, CDR2, and CDR3, and / or one or more framework regions (FR), such as FR1, FR2, FR3, and FR4, of the antibody. Introducing mutations in the variable region may be advantageous compared to mutations in the constant region, as mutations in the constant region may lead to increased immunogenicity.

[0111] In one example, the mutations are located at positions within the variable region that are not masked by antigen binding. Positions that are not masked by antigen binding are positions that remain exposed on the surface of an antigen-binding antibody. Alternatively, mutations may be made at positions that do not substantially disrupt antigen binding.

[0112] Methods for identifying CDRs and FRs are known (Kabat et al., Sequence of Proteins of Immunological Interest (1987), National Institute of Health, Bethesda, Md.; Chothia et al., Nature, vol. 342, p. 877, 1989). Amino acid residues that can be modified in FRs include residues that directly bind to antigens via non-covalent bonds (Amit et al., Science, 233:747-53, 1986), residues that have some effect on the CDR structure (Chothia et al., J. Mol., J. Biol., 196:901-917, 1987), and residues involved in the interaction between the heavy chain variable region and the light chain variable region (Published Application EP 239400A1).

[0113] The mutagenesis methods described above produce a collection of one or more variant polypeptides. These variant polypeptides may have a pI that is the same as or lower than the pI of the parent polypeptide. Among the variant polypeptides that can be generated using the various mutagenesis methods described above, some may have a pI that is the same as or higher than the pI of the parent polypeptide. This may be caused, for example, by additional mutations introduced by global positional evolution, global positional deletion, global positional insertion, or a combination thereof, which may overcome the effects of mutations introduced to maintain or decrease the pI. Thus, in some embodiments, the present invention confirms that a particular variant polypeptide, or at least a substantial portion of the variant polypeptides, has a pI that is lower than the pI of the parent polypeptide. This can be achieved by isoelectric focusing gel electrophoresis, which shows that a significant portion of the variant polypeptides are concentrated in a region having a pH lower than the pI of the parent polypeptide. For example, at least 50%, or at least 60%, or at least 70%, or at least 80%, or at least 90%, or at least 95%, or at least 98% of the variant polypeptides may have a pI that is lower than the pI of the parent polypeptide.

[0114] In some embodiments, the present invention optionally filters the mutant polypeptides to remove some or all of the mutant polypeptides having a pI equal to or higher than the pI of the parent polypeptide. Filtration of these mutant polypeptides can also be achieved by isoelectric focusing gel electrophoresis, whereby mutant polypeptides focused in a region having a pH lower than the pI of the parent polypeptide can be collected and used for further processing. Mutant polypeptides focused in a region having a pH equal to or higher than the pI of the parent polypeptide can be removed. G. Screening for Conditionally Active Polypeptides

[0115] The mutant polypeptides are screened for conditional activity. In this manner, polypeptides can be identified that have both conditional activity and a lower pI than the parent polypeptide, a desirable combination of characteristics, particularly for antibodies such as therapeutic antibodies.

[0116] A method for screening mutant polypeptides to select a conditionally active polypeptide is described in WO 2017 / 078839. Conditionally active polypeptides can optionally be identified by screening for increased activity under abnormal conditions that deviate from normal physiological conditions, compared to the same activity of the same mutant polypeptide under corresponding normal physiological conditions. (a) screening for increased activity under abnormal conditions that deviate from normal physiological conditions, compared to the same activity of the parent polypeptide under the same abnormal conditions; (b) screening for a decrease in activity under normal physiological conditions compared to the same activity of the parent polypeptide under the same normal physiological conditions; or Selection can be made by combining the above (a) and (b).

[0117] A conditionally active polypeptide can be selective, i.e., the ratio of activity under abnormal conditions to activity under normal physiological conditions is at least about 1.3, at least about 1.5, or at least about 1.7, or at least about 2.0, or at least about 3.0, or at least about 4.0, or at least about 6.0, or at least about 8.0, or at least about 10.0, or at least about 20.0, or at least about 40.0, or at least about 60.0, or at least about 100.0.

[0118] The abnormal condition and the normal physiological condition are different values ​​of the same condition. For example, the abnormal condition and the normal physiological condition may be two different temperatures or two different pH values. The conditions may be selected from temperature, pH, osmolality, osmolality, oxidative stress, electrolyte concentration, and combinations of two or more such conditions.

[0119] For example, a normal physiological condition of temperature may be normal human body temperature of 37.0° C., while an abnormal condition of temperature may be a temperature different from 37.0° C., such as the temperature in a tumor microenvironment, which may be 1-2° C. higher than normal physiological temperature. In another example, a normal physiological condition may be a normal human physiological pH in the range of 7.2-7.8, or 7.2-7.6, and an abnormal pH such as in the range of 5.5-7.2, 6-7, or 6.2-6.8 as may be found in a tumor microenvironment.

[0120] Both assays under normal and abnormal physiological conditions can be performed in an assay medium. The assay medium can be, for example, a solution that can contain buffers and other components. Common buffers that can be used in the assay medium include citrate buffers such as sodium citrate, phosphate buffers, bicarbonate buffers such as Krebs buffer, phosphate-buffered saline (PBS) buffer, Hank's buffer, Tris buffer, HEPES buffer, and the like. Other buffers known to those skilled in the art that are suitable for assays can also be used. These buffers can be used to mimic the compositional properties or components of human or animal body fluids, such as plasma or lymph.

[0121] 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, such as humans or animals. 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 contemplates 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, where the bicarbonate is not part of the PBS buffer. Alternatively, the bicarbonate is a component of a Krebs buffer.

[0122] While a component is present in substantially the same concentration in both assay solutions (for the first and second conditions), the two assay solutions may differ in other respects, such as pH, temperature, electrolyte concentration, or osmolality, and thus the component is used as a constant rather than a difference between the first and second conditions, or between the two conditions, normal physiological conditions and abnormal conditions, respectively.

[0123] In some embodiments, a component is present in both assay solutions at a concentration that is close to or the same as the normal physiological concentration of that component in a mammal, particularly a human.

[0124] The inorganic compound or ion may be selected from one or more of 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 chelates, copper chelates, iron chelates, manganese chelates and zinc chelates, ammonium molybdate, ammonium sulfate, calcium carbonate, magnesium phosphate, sodium disulfide, potassium disulfide, sodium bicarbonate, potassium bicarbonate, potassium nitrate, hydrochloric acid, carbon dioxide, sulfuric acid, phosphoric acid, carbonic acid, uric acid, hydrogen chloride, urea, phosphorus ions, sulfate ions, chloride ions, magnesium ions, sodium ions, potassium ions, ammonium ions, iron ions, zinc ions and copper ions.

[0125] Examples of normal physiological concentrations of some inorganic compounds include uric acid in the concentration range of 2-7.0 mg / dL, calcium ion in the concentration range of 8.2-11.6 mg / dL, chloride ion in the concentration range of 355-381 mg / dL, iron ion in the concentration range of 0.028-0.210 mg / dL, potassium ion in the concentration range of 12.1-25.4 mg / dL, sodium ion in the concentration range of 300-330 mg / dL, carbonate in the concentration range of 15-30 mM, citrate ion at approximately 80 μM, histidine ion in the range of 0.05-2.6 mM, histamine in the range of 0.3-1 μM, HAPT ion (hydrogenated adenosine triphosphate) in the range of 1-20 μM, and HADP ion in the range of 1-20 μM.

[0126] In some embodiments, the ions present in the assay solutions in both the first and second conditions, or the normal and abnormal physiological conditions, respectively, are selected from hydroxide, halide (chloride, bromide, iodide), oxyhalide, sulfate, magnesium, calcium, bisulfate, carbonate, bicarbonate, sulfonate, oxyhalide, nitrate, nitrite, phosphate, hydrogen phosphate, dihydrogen phosphate, persulfate, monopersulfate, borate, ammonium, or organic ions, such as carboxylate, phenolate, sulfonate (organic sulfates such as methyl sulfate), vanadate, tungstate, borate, organoborate, citrate, oxalate, acetate, pentaborate, histidine, and phenolate.

[0127] The organic compounds present in both assay solutions under the first and second conditions, or under normal and abnormal physiological conditions, respectively, may be selected from amino acids such as histidine, alanine, isoleucine, arginine, leucine, asparagine, lysine, aspartic acid, methionine, cysteine, phenylalanine, glutamic acid, threonine, glutamine, tryptophan, glycine, valine, pyrrolysine, proline, selenocysteine, serine, tyrosine, and mixtures thereof.

[0128] Examples of normal physiological concentrations of some of the amino acids include 3.97 ± 0.70 mg / dL alanine, 2.34 ± 0.62 mg / dL arginine, 3.41 ± 1.39 mg / dL glutamic acid, 5.78 ± 1.55 mg / dL glutamine, 1.77 ± 0.26 mg / dL glycine, 1.42 ± 0.18 mg / dL histidine, 1.60 ± 0.31 mg / dL isoleucine, and 1.77 ± 0.26 mg / dL urea. These included 91±0.34 mg / dL leucine, 2.95±0.42 mg / dL lysine, 0.85±0.46 mg / dL methionine, 1.38±0.32 mg / dL phenylalanine, 2.02±6.45 mg / dL threonine, 1.08±0.21 mg / dL tryptophan, 1.48±0.37 mg / dL tyrosine, and 2.83±0.34 mg / dL valine.

[0129] The organic compounds present in both assay solutions under the first and second conditions, or under normal and abnormal physiological conditions, respectively, 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 creatine, 0.9-1.65 mg / dL creatinine, 0.26±0.24 mg / dL guanidinoacetic acid, 4.0±2.9 mg / dL uric acid, 0.3-0.6 mg / dL allantoin, 1.09±0.385 mg / dL adenosine, 27.1±4.5 mg / dL urea, and 0.3-1.5 mg / dL choline.

[0130] The organic compounds present in the 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, pyruvic acid, α-ketonic acids, acetic acid, and volatile fatty acids. Examples of normal physiological concentrations of some of these organic acids include 2.5±1.9 mg / dL citric acid, 0.8 mg / dL α-ketoglutaric acid, 0.5 mg / dL succinic acid, 0.46±0.24 mg / dL malic acid, 0.8-2.8 mg / dL acetoacetic acid, 0.5±0.3 mg / dL β-hydroxybutyric acid, 8-17 mg / dL lactic acid, 1.0±0.77 mg / dL pyruvic acid, 0.6-2.1 mg / dL α-ketonic acids, and 1.8 mg / dL volatile fatty acids.

[0131] Organic compounds present in assay solutions under both normal and abnormal physiological conditions can 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 glucose, 102±73 mg / dL polysaccharides (as hexoses), 77±63 mg / dL glucosamine, 0.4-1.4 mg / dL hexuronate (as glucuronic acid), and 2.55±0.37 mg / dL pentoses.

[0132] The organic compounds present in the assay solutions under both normal and abnormal physiological conditions can be selected from lipids 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 free cholesterol, 100-200 mg / dL lecithin, 0-30 mg / dL cephalin, 10-30 mg / dL sphingomyelin, and 0.2-0.3 mg / dL bile acids (as cholic acid).

[0133] 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, immune gamma globulin, immune euglobulin, isoagglutinin, beta-pseudoglobulin, glycoprotein, lipoprotein, and albumin. For example, the normal physiological concentration of mammalian serum albumin is 3.5-5.0 g / dL. In one embodiment, the albumin is bovine serum albumin.

[0134] The organic compounds present in the assay solutions under both normal and abnormal physiological conditions may be selected from vitamins such as vitamin A, carotene, vitamin E, ascorbic acid, thiamine, inositol, folic acid, biotin, pantothenic acid, riboflavin, etc. Examples of normal physiological concentrations of some of these vitamins include vitamin A from 0.019 to 0.036 mg / dL, vitamin E from 0.90 to 1.59 mg / dL, inositol from 0.42 to 0.76 mg / dL, folic acid from 0.00162 to 0.00195 mg / dL, and biotin from 0.00095 to 0.00166 mg / dL.

[0135] The concentration of an inorganic compound, ion, or organic molecule in the assay solution (for both assays under normal and abnormal physiological conditions) can be within the normal physiological range of that inorganic compound, ion, or organic molecule in human or animal serum. However, concentrations outside the normal physiological range can also be used. For example, the normal range for magnesium ions in human serum is 1.7 to 2.2 mg / dL, and for calcium ions is 8.5 to 10.2 mg / dL. The magnesium ion concentration in the assay solution can be from about 0.17 mg / dL to about 11 mg / dL. The calcium ion concentration in the assay solution can be from about 0.85 mg / dL to about 51 mg / dL. As a general rule, the concentration of an inorganic compound, ion, or organic molecule in the assay solution may be as low as 5%, or 10%, or 20%, or 30%, or 40%, or 50%, or 60%, or 70%, or 80% of the normal physiological concentration of that inorganic compound, ion, or organic molecule in human serum, or as high as 1.5, or 2, or 3, or 4, or 5, or 7, or 9, or 10, or even 20 times the normal physiological concentration of that inorganic compound, ion, or organic molecule in human serum. Various components of the assay solution may be used at concentration levels different from their respective normal physiological concentrations.

[0136] The activity of the variant polypeptide is measured using assays under normal physiological conditions and abnormal conditions. During the assay, both the variant polypeptide and its binding partner are present in the assay solution. The relationship between the variant polypeptide and its binding partner can be, for example, antibody-antigen, ligand-receptor, enzyme-substrate, or hormone-receptor. For the variant polypeptide to exhibit its activity, it must be able to contact and bind to its binding partner. The activity of the variant polypeptide toward its binding partner is then exhibited and measured following binding between the variant polypeptide and its binding partner.

[0137] In some embodiments, ions used in the assay may function to form bridges between the mutant polypeptide being screened and its binding partner, particularly those containing charged amino acid residues. Thus, ions may be capable of binding to both the mutant polypeptide and its binding partner through hydrogen and / or ionic bonds. This may aid in binding between the mutant polypeptide and its binding partner by allowing ions to access sites that may be difficult for large molecules (either the mutant polypeptide or its binding partner) to access. In some cases, ions in the assay solution may increase the likelihood that the mutant polypeptide and its binding partner will bind to each other. Furthermore, ions may additionally or alternatively aid in binding between the mutant polypeptide and its binding partner by binding to the large molecule (either the mutant polypeptide or its binding partner). This binding may alter the conformation of the large molecule to and / or maintain the large molecule in a particular conformation that facilitates binding to the binding partner.

[0138] It has been observed that ions can assist in binding between a mutant polypeptide and its binding partner, possibly by forming ionic bonds between the mutant polypeptide and its binding partner. Thus, screening can be much more efficient and more hits (candidate conditionally active polypeptides) can be identified compared to the same assay without ions. Suitable ions can be selected from magnesium, sulfate, bisulfate, carbonate, citrate, HAPT, HADP, bicarbonate, nitrate, nitrite, phosphate, hydrogen phosphate, dihydrogen phosphate, persulfate, monopersulfate, borate, lactate, citrate, histidine, histamine, and ammonium.

[0139] Ions have been found to function to support binding between a variant polypeptide and its binding partner at a pH near the pKa of the ion, and such ions are preferably relatively small compared to the size of the variant polypeptide.

[0140] In one embodiment, when the abnormal condition is a pH different from normal physiological pH under normal physiological conditions, ions suitable for increasing the number of hits for candidate conditionally active polypeptides can be selected from ions having a pKa close to the abnormal pH tested in the assay. For example, the pKa of the ion can be up to 2 pH units away from the abnormal pH, up to 1 pH unit away from the abnormal pH, up to 0.8 pH units away from the abnormal pH, up to 0.6 pH units away from the abnormal pH, up to 0.5 pH units away from the abnormal pH, up to 0.4 pH units away from the abnormal pH, up to 0.3 pH units away from the abnormal pH, up to 0.2 pH units away from the abnormal pH, or up to 0.1 pH units away from the abnormal pH.

[0141] Exemplary pKas of ions useful in the present invention (which may vary very slightly at different temperatures) are as follows: ammonium ion has a pKa of about 9.24, dihydrogen phosphate has a pKa of about 7.2, acetate has a pKa of about 4.76, histidine has a pKa of about 6.04, bicarbonate ion has a pKa of about 6.4, citrate has a pKa of 6.4, lactate ion has a pKa of about 3.86, histamine has a pKa of about 6.9, and HATP has a pKa of 6.95 (HATP 3- ⇔ATP 4- +H + ), and HADP has a pKa of 6.88 (HADP 3- ⇔ADP 4- +H + ).

[0142] In one embodiment, conditionally active polypeptides are assayed and selected in the presence of disulfide. Disulfide has a pKa of 7.05. In some embodiments, different disulfide concentrations can be used in assays corresponding to normal physiological conditions and assays corresponding to abnormal physiological conditions. Alternatively, the assay media for both normal and abnormal physiological conditions can have approximately the same disulfide concentration, but differ in some specific conditional values; for example, the assays can be performed at different pHs. The disulfide concentration used in the assay can be between 1 mM and 100 mM. Preferably, the assay media has a disulfide concentration of 2 to 500 nM, or 3 to 200 nM, or 5 to 100 nM. In some aspects, the disulfide concentration can be between 1 mM and 20 mM, or 2 mM and 10 mM. Assays performed in the presence of disulfide are known.

[0143] In certain embodiments, once the pH of the abnormal condition (i.e., the abnormal pH) is known, ions suitable for increasing candidate conditionally active polypeptide hits may be selected from ions having a pKa at or near the abnormal pH; for example, candidate ions may have a pKa that is up to 4 pH units away from the abnormal pH, up to 3 pH units away from the abnormal pH, up to 2 pH units away from the abnormal pH, up to 1 pH unit away from the abnormal pH, up to 0.8 pH units away from the abnormal pH, up to 0.6 pH units away from the abnormal pH, up to 0.5 pH units away from the abnormal pH, up to 0.4 pH units away from the abnormal pH, up to 0.3 pH units away from the abnormal pH, up to 0.2 pH units away from the abnormal pH, or up to 0.1 pH unit away from the abnormal pH.

[0144] As previously mentioned, ions are most effective at supporting binding between a mutant polypeptide and its binding partner at a pH at or near the ion's pKa. For example, in assay solutions with a pH of 7.2 to 7.6, bicarbonate ions (which have a pKa of approximately 6.4) have been found to be less effective at supporting binding between a mutant polypeptide and its binding partner. As the pH of the assay solution is lowered to 6.7 or even to approximately 6.0, bicarbonate ions become increasingly effective at supporting binding between a mutant polypeptide and its binding partner. As a result, assays at pH 6.0 may identify more hits than assays at pH 7.2 to 7.6. Similarly, histidine is less effective at supporting binding between a mutant polypeptide and its binding partner at pH 7.4. As the pH of the assay solution is lowered to 6.7 or even to approximately 6.0, histidine becomes increasingly effective at supporting binding between a mutant polypeptide and its binding partner, and more hits may be identified, for example, at a pH in the range of approximately 6.2 to 6.4.

[0145] It has been found that when the pH of an assay solution under normal physiological conditions (i.e., normal physiological pH) differs from the pH of an assay solution under abnormal conditions (i.e., abnormal pH), ions with pKas ranging from approximately the midpoint between normal physiological pH and abnormal pH to approximately the abnormal pH can significantly support the binding between the mutant polypeptide being screened and its binding partner. As a result, this screening assay is much more efficient in finding more hit or candidate conditional polypeptides that are highly active under abnormal conditions.

[0146] In some embodiments, the pKa may even be at least one pH unit away from the abnormal pH. When the abnormal pH is an acidic pH, the pKa of a suitable ion may range from (abnormal pH -1) to the midpoint between the abnormal pH and normal physiological pH. When the abnormal pH is a basic pH, the pKa of a suitable ion may range from (abnormal pH +1) to the midpoint between the abnormal pH and normal physiological pH. Ions may be selected from those described herein. However, many more ions not explicitly described herein may also be used. It is understood that once the abnormal pH and normal physiological pH of a screening assay have been selected, one of skill in the art may use the guidelines described herein to select any ion with a suitable pKa to increase the efficiency of the screen in identifying more hits with high activity under abnormal conditions.

[0147] For example, if the abnormal pH for one exemplary screen is 8.4 and the normal physiological pH is 7.4, any ion with a pKa in the range of about 7.9 (midpoint) to 9.4 (i.e., 8.4 + 1) can be used for screening. Some ions with pKas in this range include those derived from 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 one exemplary screen is 6 and the normal physiological pH is 7.4, any ion with a pKa in the range of about 5 (i.e., 6 - 1) to 6.7 (midpoint) can be used for screening. Some ions with pKas in this range include those derived from 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 (pKa 6.46). Those skilled in the art can consult numerous chemistry manuals and textbooks to identify known chemical compounds, including both inorganic and organic chemical compounds, that can be converted into ions with pKas within this range. Among chemical compounds with suitable pKas, those with smaller molecular weights may be preferred.

[0148] Thus, the present invention unexpectedly found that the generation of the ultimately identified conditionally active polypeptide not only depends on the generation of the correct polypeptide variant, but also on the use of ions with suitable pKa in the assay solution. Because ions can facilitate the efficient selection of highly active variants from a large library, the present invention believes that in addition to generating a large library of variant polypeptides (e.g., by CPE and CPS), efforts should be directed toward finding suitable ions (with appropriate pKa) to use in the assay solution. Furthermore, the absence of suitable ions may result in low screening efficiency and a reduced likelihood of finding highly active variants. Consequently, multiple screening rounds may be required to obtain the same number of highly active variants without suitable ions.

[0149] Ions in the assay solution may be formed in situ from components of the assay solution or may be included directly in the assay solution. For example, CO2 from the air may be dissolved in the assay solution to provide carbonate and bicarbonate ions. In another example, sodium dihydrogen phosphate may be added to the assay solution to provide dihydrogen phosphate ions.

[0150] The concentration of this component in the assay solution (for both assays under normal and abnormal physiological conditions) may be the same or substantially the same as the concentration of the same component typically found in a naturally occurring bodily fluid of a mammal, such as a human. In other embodiments, the concentration of the component may be higher, particularly for components that are ions that may function to assist in binding between the variant polypeptide and its binding partner, since it has been observed that higher concentrations of such ions may result in the formation of ionic bonds between the variant polypeptide and its binding partner, effectively facilitating binding and increasing the likelihood of finding more hit or candidate conditionally active polypeptides.

[0151] In some embodiments, the concentration of ions in the assay solution can be positively correlated with the likelihood of finding more hits using the assay, especially when concentrations above normal physiological concentrations are used. For example, human serum has a bicarbonate ion concentration of approximately 15-30 mM. In one example, as 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 each increase in bicarbonate concentration. Taking this into consideration, the assay solution can have a bicarbonate concentration in the range of about 3 mM to about 200 mM, or about 5 mM to about 150 mM, or about 5 mM to about 100 mM, or about 10 mM to about 100 mM, or about 20 mM to about 100 mM, or about 25 mM to about 100 mM, or about 30 mM to about 100 mM, or about 35 mM to about 100 mM, or about 40 mM to about 100 mM, or about 50 mM to about 100 mM.

[0152] In another embodiment, the citrate concentration in the assay solution can be about 30 μM to about 120 μM, or about 40 μM to about 110 μM, or about 50 μM to about 110 μM, or about 60 μM to about 100 μM, or about μM to about 90 μM, or about μM.

[0153] In one embodiment, normal physiological conditions are normal physiological pH in the range of 7.2 to 7.6, and abnormal conditions are abnormal pH in the range of 5.5 to 7.2, 6 to 7, or 6.2 to 6.8. Assay solutions for assays under normal physiological conditions have normal physiological pH and 50 mM bicarbonate. Assay solutions for assays under abnormal conditions have abnormal pH and 50 mM bicarbonate. Because the pKa of bicarbonate is approximately 6.4, bicarbonate can support binding between the mutant polypeptide and its binding partner at abnormal pHs of 6.0 to 6.4, e.g., pH 6.0 or 6.2.

[0154] In yet another embodiment, the normal physiological conditions are normal physiological pH in the range of 7.2 to 7.6, and the abnormal conditions are abnormal pH in the range of 5.5 to 7.2, 6 to 7, or 6.2 to 6.8. The assay solution for the assay under normal physiological conditions has normal physiological pH and 80 μM citrate ion. The assay solution for the assay under abnormal conditions has abnormal pH and 80 μM citrate ion. Because citrate ion has a pKa of 6.4, it can effectively support the binding between the mutant polypeptide and the binding partner in the assay solution under abnormal conditions of pH 6.0 to 6.4. Therefore, more candidate conditionally active polypeptides having higher binding activity under pH 6.0 to 6.4 and lower activity under pH 7.2 to 7.8 can be identified. Other ions, including acetate, histidine, bicarbonate, HATP, and HADP, function similarly, and assay solutions containing such ions allow for the effective screening of variant polypeptides that have higher binding activity at pH values ​​around the ion's pKa and lower binding activity at pH values ​​different from the ion's pKa (e.g., normal physiological pH).

[0155] In yet another embodiment, the normal physiological conditions are a normal physiological temperature of 37°C and the abnormal conditions are an abnormal temperature of 38-39°C (temperatures present in some tumor microenvironments). The assay solution for assays under normal physiological conditions has a normal physiological temperature and 20 mM bicarbonate. The assay solution for assays under abnormal conditions has an abnormal temperature and 20 mM bicarbonate.

[0156] In yet another embodiment, the normal physiological condition is a particular concentration of an electrolyte in normal human serum, and the abnormal condition is a concentration of the same electrolyte at a different abnormal concentration that may exist in a different location in the animal or human or may be caused by a condition in the animal or human that alters the normal physiological electrolyte concentration in human serum.

[0157] Binding between a variant polypeptide and / or its binding partner can also be affected in a number of other ways. Typically, this can be done by including one or more additional components in the assay solution. These additional components can be designed to interact with either the variant polypeptide, the binding partner, or both. In addition, these additional components can affect binding using a combination of two or more interactions and a combination of two or more types of interactions.

[0158] In one embodiment, the binding interaction of interest is between an antibody and an antigen. In this embodiment, one or more additional components may be included in the assay solution to affect the antibody, the antigen, or both. In this manner, the desired binding interaction may be enhanced.

[0159] In addition to ions that can form ionic bonds with a variant polypeptide and / or its binding partner to aid in binding between the variant polypeptide and its binding partner, the present invention also encompasses other components that can be utilized to aid in binding between a variant polypeptide and its binding partner. In one embodiment, molecules that can form hydrogen bonds with a variant polypeptide and / or its binding partner can be utilized. In another embodiment, molecules capable of hydrophobic interactions with a variant polypeptide and / or its binding partner can be used. In yet another embodiment, molecules capable of van der Waals interactions with a variant polypeptide and / or its binding partner are contemplated.

[0160] "Hydrogen bond" refers to a relatively weak, non-covalent interaction between hydrogen covalently bonded to an electronegative atom such as carbon, nitrogen, oxygen, sulfur, chlorine, or fluorine (hydrogen bond donor) and the unshared pair of electrons of an electron-donating atom such as nitrogen, oxygen, sulfur, chlorine, or fluorine (hydrogen bond acceptor).

[0161] Components capable of forming hydrogen bonds with a variant polypeptide and / or its binding partner include organic molecules with polar bonds as well as inorganic molecules. A variant polypeptide and / or a binding partner of a variant polypeptide typically contains amino acids capable of forming hydrogen bonds. Suitable amino acids have side chains with polar groups capable of forming hydrogen bonds. Non-limiting examples of suitable 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).

[0162] These amino acids can function as both hydrogen donors and hydrogen acceptors, for example, the oxygen atom of an -OH group, as found in Ser, Thr, and Tyr; the oxygen atom of a -C=O group, as found in Glu and Asp; the sulfur atom of an -SH group or -SC-, as found in Cys and Met; and the sulfur atom of an -NH3 group, as found in Lys and Arg. + Nitrogen atoms of groups, as well as nitrogen atoms of -NH- groups such as may be found in Trp, His, and Arg, can all function as hydrogen acceptors. Also, groups in this list that contain a hydrogen atom (e.g., -OH, -SH, NH3 + and -NH-) can also function as a hydrogen donor.

[0163] In some embodiments, the backbone of the variant polypeptide and / or its binding partner may also participate in the formation of one or more hydrogen bonds. For example, the backbone may have a repeating structure of -(C=O)-NH-, as in a peptide bond. The oxygen and nitrogen atoms of this structure may function as hydrogen acceptors, while the hydrogen atoms may participate in hydrogen bonds.

[0164] Inorganic compounds having at least one polar bond involving a hydrogen or oxygen atom that can be used for hydrogen bonding include, for example, HO, NH, HO, hydrazine, carbonates, sulfates, and phosphates. Organic compounds include alcohols; phenols; thiols; aliphatic amines, amides; epoxides, carboxylic acids; ketones, aldehydes, ethers, esters, organic chlorides, and organic fluorines. Compounds capable of forming hydrogen bonds are well known in the chemical literature, such as those discussed in "The Nature of the Chemical Bond," Linus Pauling, Cornell University Press, 1940, pages 284-334.

[0165] In some embodiments, alcohols include methanol, ethanol, propanol, isopropanol, butanol, pentanol, 1-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-propanethiol, 2-propanethiol, butanethiol, tert-butyl mercaptan, pentanethiols, hexanethiol, thiophenol, dimercaptosuccinic acid, 2-mercaptoethanol, and 2-mercaptoindole. Suitable amines include methylamine, ethylamine, propylamine, isopropylamine, aniline, dimethylamine, methylethylamine, trimethylamine, aziridine, piperidine, N-methylpiperidine, benzidine, cyclohexylamine, ethylenediamine, hexamethylenediamine, o-, m-, and p-toluidine, and N-phenylpiperidine. Suitable amides include ethanamide, 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-butyl peroxide, 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, methyl hexyl ketone, diethyl ketone, ethyl butyl ketone, dipropyl ketone, diisobutyl ketone, diacetone alcohol, phorone, isophorone, cyclohexanone, methylcyclohexanone, and acetophenone. Aldehydes include formaldehyde, acetaldehyde, propionaldehyde, butyraldehyde, benzaldehyde, cinnamaldehyde, isobutyraldehyde, valeraldehyde, octaldehyde, benzaldehyde, cinnamaldehyde, cyclohexanone, salicylic aldehyde, and furfural.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, methyl isoamyl 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. Ethers that can be used in the present invention include dimethyl ether, methyl ethyl ether, diethyl ether, methyl propyl ether, and dimethoxyethane. Ethers can also be cyclic, such as ethylene oxide, tetrahydrofuran, and dioxane.

[0166] The organic chlorides include chloroform, pentachloroethane, dichloromethane, trichloromethane, carbon tetrachloride, tetrachloromethane, tetrachloroethane, pentachloroethane, trichloroethylene, tetrachloroethylene, and ethylene dichloride.The organic fluorines include fluoromethane, difluoromethane, trifluoromethane, trifluoroethane, tetrafluoroethane, pentafluoroethane, difluoropropane, trifluoropropane, tetrafluoropropane, pentafluoropropane, hexafluoropropane, and heptafluoropropane.

[0167] Hydrogen bonds can be classified as strong, medium, or weak based on the strength of the bond (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. Medium 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 more information, 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.

[0168] In some embodiments, components used in the present invention can form strong hydrogen bonds with the variant polypeptide and / or its binding partner. 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, hydroxyl groups, or carbonyl groups in forming hydrogen bonds. In one embodiment, organic fluorines may be used in the present invention to form strong hydrogen bonds.

[0169] In another embodiment, moieties capable of hydrophobic interaction with the variant polypeptide and / or its binding partner are utilized, including organic compounds with hydrophobic groups.

[0170] "Hydrophobic interaction" refers to a reversible attractive interaction between a hydrophobic compound or a hydrophobic region of a compound and another hydrophobic compound or region of another compound. This type of interaction is described in "Hydrophobic Interactions," A. Ben-Nairn (1980), Plenum Press, New York.

[0171] Hydrophobic materials, due to their non-polar nature, experience the repulsive force of water molecules. When relatively non-polar molecules or groups in aqueous solution associate with other non-polar molecules or groups rather than with water, this is called a "hydrophobic interaction."

[0172] The variant polypeptides and their binding partners typically contain amino acids capable of hydrophobic interaction. These amino acids can typically be characterized by having at least one side chain with a non-polar group capable of hydrophobic interaction. Hydrophobic amino acids include, for example, alanine (Ala), isoleucine (Ile), leucine (Leu), phenylalanine (Phe), valine (Val), proline (Pro), glycine (Gly), and, to a lesser extent, methionine (Met), and tryptophan (Trp).

[0173] Components capable of hydrophobic interaction with the variant polypeptide and / or its binding partner include organic compounds that are hydrophobic molecules or molecules containing at least one hydrophobic moiety. In some embodiments, these hydrophobic components can be hydrocarbons selected from aromatic hydrocarbons, substituted aromatic hydrocarbons, polycyclic aromatic hydrocarbons, aromatic or non-aromatic heterocycles, cycloalkanes, alkanes, alkenes, and alkynes. Hydrophobic groups can 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 straight-chain alkenyl / alkynyl groups, branched-chain 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.

[0174] It can be understood that the strength of hydrophobic interactions is based on the amount of available "hydrophobes" that can interact with each other. Thus, hydrophobic interactions can be adjusted, for example, by increasing the amount and / or "hydrophobicity" of the hydrophobic moiety in the molecule involved in the hydrophobic interaction. For example, a hydrophobic moiety (which may comprise a hydrocarbon chain in its native form) can be modified to increase its hydrophobicity (its ability to increase the strength of the hydrophobic interaction involving that moiety) by adding a hydrophobic side chain to one of the carbons of its carbon skeleton. In preferred embodiments, this can include the addition of various steroid compounds and / or their derivatives, such as sterol-based compounds, and more particularly various polycyclic compounds, including cholesterol. Generally, the side chain can be linear, aromatic, aliphatic, cyclic, polycyclic, or any other type of hydrophobic side chain as contemplated by those skilled in the art.

[0175] The types of components capable of van der Waals interactions with the variant polypeptide and / or its binding partner are usually, but not necessarily, compounds with polar moieties. As used herein, "van der Waals interactions" refers to the attractive forces between atoms, moieties, molecules, and surfaces that arise due to the interrelationship of the fluctuating polarities of adjacent atoms, moieties, molecules as a result of dipole-dipole interactions and / or quantum dynamics.

[0176] Van der Waals interactions in the present invention are attractive forces between a variant polypeptide or binding partner and a component. Van der Waals interactions can arise from three sources. First, some molecules / moieties can be permanent electric dipoles, even though they are electrically neutral. Due to a fixed distortion of the electronic charge distribution in the structure of some molecules / moieties, one side of the molecule / moiety is always somewhat positive and the other side 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 (Keesom force).

[0177] Second, the presence of a molecule that is a permanent dipole can temporarily distort the electronic charges of other adjacent polar or nonpolar molecules, thereby inducing further polarization. An additional attractive force results when a permanent dipole interacts with an adjacent induced dipole. This is the interaction between a permanent dipole and a corresponding induced dipole and can be referred to as a Debye force. Third, even if the molecules involved are not permanent dipoles (e.g., the organic liquid benzene), there is still an attractive force between the molecules due to the two momentarily induced dipoles in the molecule. This is the interaction between two momentarily induced dipoles and can be referred to as a London dispersion force.

[0178] The variant polypeptides and / or binding partners contain a number of amino acids capable of van der Waals interactions. These amino acids may 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 may also have side chains with nonpolar groups, including alanine (Ala), isoleucine (Ile), leucine (Leu), phenylalanine (Phe), valine (Val), proline (Pro), and glycine (Gly).

[0179] Components capable of van der Waals interactions with the variant polypeptide and / or its binding partner include polar or non-polar inorganic compounds soluble in the assay solution. The assay solution is generally an aqueous solution, and therefore these polar or non-polar inorganic compounds are preferably soluble in water. Preferred materials for van der Waals interactions are polar, such that they are capable of dipole-dipole interactions. For example, AlF3 has a polar Al-F bond and is soluble in water (approximately 0.67 g / 100 ml water at 20°C). HgCl2 has a polar Hg-Cl bond and is soluble in water at 7.4 g / 100 ml at 20°C. PrCl2 has a polar Pr-Cl bond and is soluble in water at approximately 1 g / 100 ml at 20°C.

[0180] Suitable polar compounds capable of van der Waals interactions include alcohols, thiols, ketones, amines, amides, esters, ethers, and aldehydes. Suitable examples of these compounds are described above in relation to hydrogen bonding. Suitable non-polar compounds capable of van der Waals interactions include aromatic hydrocarbons, substituted aromatic hydrocarbons, polycyclic aromatic hydrocarbons, aromatic or non-aromatic heterocycles, cycloalkanes, alkanes, alkenes, and alkynes.

[0181] Hydrogen-bonding components, hydrophobic components, and van der Waals components can be used to influence binding between a variant polypeptide and its binding partner in several ways. In one embodiment, bridges may be formed between a variant polypeptide and its binding partner by hydrogen bonding, hydrophobic interactions, and / or van der Waals interactions. Such bridges may bring the variant polypeptide and the binding partner into close proximity to each other, facilitating binding, and / or may position the variant polypeptide and / or the binding partner relative to each other to facilitate binding.

[0182] In another embodiment, hydrogen bonding and / or hydrophobic interactions may increase the likelihood that a variant polypeptide will bind to its binding partner, e.g., by causing the polypeptide and binding partner to assemble or associate with one another in a manner that increases the likelihood of binding. Thus, one or more of these interactions, alone or in combination, may be used to assemble the variant polypeptide and binding partner closer together or position them in a manner that promotes binding, e.g., by drawing the binding sites closer to one another or by positioning the non-binding portions of the molecule further apart, thereby positioning the binding sites closer to one another.

[0183] In yet another embodiment, hydrogen bonding and / or hydrophobic interactions may affect the conformation of the variant polypeptide and / or its binding partner, resulting in a conformation that is more conducive to binding between the variant polypeptide and its binding partner. Specifically, binding or interaction with one or more amino acids of the variant polypeptide and / or binding partner may result in one or more conformational changes in the variant polypeptide or binding partner that are favorable to the variant polypeptide / binding partner binding reaction.

[0184] The present invention performs two pairs of assays, one that determines a decrease in activity of a variant polypeptide in an assay under normal physiological conditions compared to the parent polypeptide from which the variant polypeptide was derived under normal physiological conditions, and a second that determines an increase in activity of a variant polypeptide in an assay under abnormal conditions compared to the parent polypeptide from which the variant polypeptide was derived under abnormal conditions. The examples described in WO2017 / 078839 illustrate the selection of conditionally active polypeptides that are more active at the abnormal pH where activity is desired than at normal physiological pH.

[0185] In one embodiment, the mutant polypeptide is subjected to an assay under normal physiological conditions and an assay under abnormal conditions, and a conditionally active polypeptide is selected from mutant polypeptides that have increased activity in an assay under abnormal conditions compared to the same activity of the mutant polypeptide in an assay under normal physiological conditions.

[0186] The conditions used in the assay pairs of the present invention can be selected from temperature, pH, osmolality, 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 can be used at substantially the same concentration in both assay pairs. In such cases, the component is typically present to mimic a particular environment in a human or animal, such as serum, a tumor microenvironment, a synovial fluid environment, a neural environment, or any other environment that may be encountered at the point of administration, through which an administered therapy may pass, or at the point of treatment. An important aspect of selecting one or more components that mimic these environments is that it can improve the results of a selection process performed using the assay pair. For example, mimicking a particular environment allows for the evaluation of different effects of particular components of that environment on a mutant polypeptide during the selection process. A particular environmental component may, for example, alter or bind to a mutant polypeptide, inhibit the activity of the mutant polypeptide, inactivate the mutant polypeptide, etc.

[0187] In some embodiments, one or more components of the assay solution are preferably small molecules or ions, such as disulfide, hydrogen sulfide, histidine, histamine, citrate, bicarbonate, lactate, and acetate. In one embodiment, the small molecule or ion component is preferably present in the assay solution at a concentration of about 100 μm to about 100 mM, or more preferably about 0.5 to about 50 mM, or about 1 to about 10 mM.

[0188] 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 a naturally occurring bodily fluid of a mammal, such as a human. This may be referred to as the normal physiological concentration of the component in the bodily 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 a naturally occurring bodily fluid of a mammal, such as a human.

[0189] In another embodiment, the components may be present at substantially different concentrations in each of the assay pairs. In such cases, the presence, absence, or concentration of the components becomes the condition being assayed, since it is the concentration of the components that is the condition that distinguishes between assay solutions for assays under normal physiological conditions and those under abnormal conditions. Thus, the conditionally active polypeptides produced by this embodiment of the method of the invention can be selected for an activity that is at least partially dependent on the concentration of the components.

[0190] In some embodiments, a component may be present in one pair of assay solutions but completely absent from the other pair of assay solutions. For example, the lactate concentration in the abnormal condition assay solution may be set to a level that mimics the lactate concentration in the tumor microenvironment. Lactate may be absent from the normal physiological condition assay solution pair.

[0191] In one embodiment, the normal physiological condition is a first lactate concentration representative of normal physiological conditions, and the abnormal condition is a second lactate concentration representative of abnormal conditions present at a particular location in the body.

[0192] In another example, glucose may be absent in the abnormal conditions assay solution to mimic the absence of glucose as may be found in a tumor microenvironment, while glucose may be set to a level that mimics plasma glucose concentrations in the pair of normal physiological conditions assay solutions. This feature can be used to preferentially deliver a conditionally active polypeptide to a location or environment that is inactive or minimally active during transport, and activate the conditionally active polypeptide upon reaching an environment where concentrations of the components in the abnormal conditions assay solution are present.

[0193] For example, the tumor microenvironment typically has both low glucose concentrations and high lactate concentrations compared to human serum. Normal physiological glucose concentrations range from about 2.5 mM to about 10 mM in serum. On the other hand, glucose concentrations in the tumor microenvironment are typically very low, ranging from 0.05 mM to 0.5 mM. In one embodiment, assay solutions for assays under normal physiological conditions have a glucose concentration ranging from about 2.5 mM to about 10 mM, while assay solutions for assays under abnormal conditions have a glucose concentration ranging from about 0.05 mM to about 0.5 mM. The conditionally active polypeptides thus produced have higher activity in low-glucose environments (in the tumor microenvironment) than in high-glucose environments (in normal tissue or blood). While the conditionally active polypeptides are functional in the tumor microenvironment, they may have reduced activity while passing through the bloodstream.

[0194] Normal physiological concentrations of lactate in serum range from about 1 mM to about 2 mM. On the other hand, lactate concentrations in tumor microenvironments typically range from 10 mM to 20 mM. In one embodiment, assay solutions for assays under normal physiological conditions have lactate concentrations ranging from about 1 mM to about 2 mM, while assay solutions for assays under abnormal conditions have lactate concentrations ranging from about 10 mM to about 20 mM. The conditionally active polypeptides thus prepared have higher activity in high lactate environments (tumor microenvironments) than in low lactate environments (normal tissues or blood). Therefore, these conditionally active polypeptides are functional in tumor microenvironments but may have reduced activity while passing through the bloodstream.

[0195] Similarly, it is known that muscle pain results in higher-than-normal (abnormal) lactate concentrations. Therefore, when searching for mutant polypeptides that may be active in a muscle pain environment, an abnormal condition assay pair can be performed in the presence of higher lactate concentrations to mimic the muscle pain environment, while a normal physiological condition assay pair can be performed with lower lactate concentrations or in the absence of lactate. In this way, mutant polypeptides whose activity improves with increasing lactate concentrations in a muscle pain environment can be selected. Such conditionally active polypeptides may be useful, for example, as anti-inflammatory agents.

[0196] In another embodiment, two or more components may be used in both pairs of assay solutions. In this type of assay, conditionally active polypeptides may be selected using features of both of the two types of assays described above. Alternatively, two or more components may be used to increase the selectivity of the conditionally active polypeptide. For example, returning to the tumor microenvironment, the abnormal condition assay pair may be performed in an assay medium comprising both a high lactate concentration and a low glucose concentration, while the corresponding normal physiological condition assay pair may be performed in an assay medium comprising both a relatively low lactate concentration and a relatively high glucose concentration.

[0197] The present invention contemplates that components selected from inorganic compounds, ions, and organic molecules may be used alone or in combination to select conditionally active polypeptides that are more active at a given concentration of the component compared to a different concentration of the same component.

[0198] Assays that rely on differential concentrations of one or more metabolites as conditions that distinguish between a normal environment (normal physiological conditions) and an abnormal environment (abnormal conditions) can be particularly suitable for selecting conditionally active polypeptides that are more active in the tumor microenvironment than in plasma, because the tumor microenvironment typically has many metabolites that have different concentrations compared to the concentrations of the same metabolites in plasma.

[0199] 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 brain and brain tumors. This group found that N-acetylaspartic acid has a concentration of 5000-6000 μM in normal brain, but this concentration is only 300-400 μM in glioblastoma, 1500-2000 μM in astrocytoma, and 600-1500 μM in anaplastic astrocytoma. Furthermore, inositol has a concentration of 1500-2000 μM in normal brain, but this concentration is 2500-4000 μM in glioblastoma, 2700-4500 μM in astrocytoma, and 3800-5800 μM in anaplastic astrocytoma. Phosphorylethanolamine has a concentration of 900-1200 μM in normal brain, but this concentration is 2000-2800 μM in glioblastoma, 1170-1370 μM in astrocytoma, and 1500-2500 μM in anaplastic astrocytoma. Glycine has a concentration of 600-1100 μM in normal brain, but this concentration is 4500-5500 μM in glioblastoma, 750-1100 μM in astrocytoma, and 1900-3500 μM in anaplastic astrocytoma. Alanine has a concentration of 700-1150 μM in normal brain, but this concentration is 2900-3600 μM in glioblastoma, 800-1200 μM in astrocytoma, and 300-700 μM in anaplastic astrocytoma. These metabolites may also have different concentrations in the blood; for example, N-acetylaspartic acid has a blood concentration of approximately 85000 μM; inositol has a blood concentration of approximately 21700 μM; glycine has a blood concentration of approximately 220-400 μM; and alanine has a blood concentration of approximately 220-300 μM.

[0200] Therefore, these metabolites, including at least N-acetylaspartic acid, inositol, glycine, and alanine, can be used at different concentrations in assay solutions to select conditionally active polypeptides that are active in brain tumors but not in blood or normal brain tissue. For example, to select conditionally active polypeptides that are active in the tumor microenvironment of glioblastoma but not active, or at least less active, in blood or normal brain tissue, an assay solution containing 85,000 μM N-acetylaspartic acid can be used in an assay pair under normal physiological conditions, and an assay solution containing 350 μM N-acetylaspartic acid can be used in an assay pair under abnormal conditions.

[0201] 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, examined the concentrations of various different metabolites, including branched-chain amino acids, in the prediagnostic plasma of pancreatic patients. They found that patients with pancreatic tumors had several metabolites present at different concentrations in their bloodstream compared to the concentrations of the same metabolites in the blood of people without pancreatic cancer. Mayers et al. (supra) also found that pancreatic cancer patients had significantly elevated branched-chain amino acids in their plasma compared to normal subjects. Branched-chain amino acids present at elevated concentrations include isoleucine, leucine, and valine (Table 1 in Mayers et al., supra). Other metabolites, shown in Figure 1 in Mayers et al. (supra), were 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 are present 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 may be present in the pancreatic microenvironment of healthy patients.

[0202] Thus, 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 individual (i.e., the normal physiological concentration of the metabolites). For example, known normal physiological concentrations in the plasma of a healthy individual are about 1.60±0.31 mg / dL for isoleucine, about 1.91±0.34 mg / dL for leucine, and about 2.83±0.34 mg / dL for valine. The assay solution under normal physiological conditions may have normal physiological concentrations of one or more of these branched-chain amino acids within these ranges. The assay solution under abnormal conditions may have a concentration of the same branched-chain amino acid that is about 5-fold, or about 10-fold, or about 20-fold, or about 50-fold, or about 70-fold, or about 100-fold, or about 150-fold, or about 200-fold, or about 500-fold higher than the normal physiological concentration of the corresponding branched-chain amino acid in a healthy individual. This may reflect the fact that the higher concentrations of these branched-chain amino acids found in plasma by Mayers et al. (supra) arise from the tumor microenvironment and are diluted in the bloodstream, and therefore, based on the findings of Mayers et al. (supra), one would expect the concentrations of these branched-chain amino acids to be significantly elevated in the pancreatic tumor microenvironment. Similarly, assays under abnormal conditions may reflect the concentrations of other metabolites in the blood of pancreatic cancer patients, even if the concentrations of certain metabolites are significantly lower in cancer patients compared to normal individuals. In this way, one can ensure that the screen mimics the actual environment, thereby selecting the most active variants for the particular environment of interest.

[0203] 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 to mimic the actual plasma environment of these patients. In such embodiments, the assay solution under abnormal conditions may have a concentration of the same branched amino acid that is about 2-fold, or about 3-fold, or about 4-fold, or about 5-fold, or about 7-fold, or about 8-fold, or about 10-fold, or about 15-fold, or about 20-fold, or about 50-fold higher than the concentration of the corresponding branched amino acid in the plasma of pancreatic cancer patients, to reflect the fact that these higher concentrations occur in the tumor microenvironment and that concentrations in the bloodstream represent 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, which may reflect actual differences expected from data collected on the bloodstream. In some cases, a particular metabolite may be deficient in the bloodstream of a pancreatic patient, in which case a concentration reflective of the measured bloodstream concentration can be used in the assay for normal physiological conditions, and a lower concentration can be used in the assay for abnormal conditions to take into account the likelihood that the metabolite will be consumed in the tumor microenvironment. Conditionally active polypeptides selected using this assay solution may be more active in the pancreatic cancer microenvironment than in the plasma of pancreatic cancer patients.

[0204] In some embodiments, the present invention may utilize whole plasma from a pancreatic cancer patient. For example, in one embodiment, assay solutions for one or both of the assays under normal physiological conditions and abnormal conditions may include a simulation of one or more components of plasma from a pancreatic cancer patient. In an exemplary embodiment, the assay solution for normal physiological conditions has a pH in the range of 7.2-7.6 and is spiked with 30 wt.% plasma from a pancreatic cancer patient, while the assay solution for abnormal conditions has a pH in the range of 6.2-6.8 and is spiked with 30 wt.% plasma from a pancreatic cancer patient. In this embodiment, the plasma from the pancreatic cancer patient is present to both (1) ensure that the conditionally active polypeptide is not activated in blood at a pH of 7.2-7.6, and (2) also ensure that the conditionally active polypeptide can be activated in the tumor microenvironment at a pH of 5.5-7.2, 6-7, or 6.2-6.8, even in the presence of this metabolic composition found in the blood of pancreatic cancer patients. This allows treatment to be tailored to the pancreatic cancer patient.

[0205] 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 added with 30 wt.% plasma from a pancreatic cancer patient, while the assay solution under abnormal conditions has a pH in the range of 5.5 to 7.2 or 6.2 to 6.8 and is not added with any plasma from a pancreatic cancer patient.

[0206] Each of the several types of assays discussed above may use the same component selected from inorganic compounds, ions, and organic molecules. For example, in the case of lactate, lactate may be used at substantially the same concentration in a pair of assay solutions for both normal and abnormal physiological conditions. The normal and abnormal physiological conditions will in turn differ in one or more other aspects, such as temperature, pH, concentration of other components, etc. In different embodiments, lactate may be used as one of the factors distinguishing between normal and abnormal physiological conditions, thereby reflecting that lactate is at a higher concentration in an abnormal tumor microenvironment compared to normal physiological conditions (non-tumor microenvironment).

[0207] In some embodiments, two or more components are added to both the assay solutions under normal physiological conditions and those under abnormal conditions at substantially the same concentrations. For example, both citrate and bovine serum albumin (BSA) are added to these assay solutions. The citrate concentration in both assay solutions may be about 80 μM, and the BSA concentration may be about 10-20%. More specifically, the assay solution for the assay pair under normal physiological conditions may have a pH in the range of 7.2-7.6, with citrate at a concentration of about 80 μM and BSA at a concentration of about 10-20%. The assay solution for the assay pair under abnormal conditions may have a pH in the range of 6.2-6.8, with citrate at a concentration of about 80 μM and BSA at a concentration of about 10-20%.

[0208] In one embodiment, serum may be added to both the assay solutions under normal physiological conditions and those under abnormal conditions at substantially the same concentration. Because serum contains numerous inorganic compounds, ions, and organic molecules (including polypeptides), the assay solution may contain multiple components selected from inorganic compounds, ions, and organic molecules present at substantially the same concentrations between the two assay solutions. The assay solution may contain 5-30 vol.%, or 7-25 vol.%, or 10-20 vol.%, or 10-15 vol.% serum. In some other embodiments, the assay solutions under both normal physiological conditions and those under abnormal conditions are serum-free. The serum may be human serum, bovine serum, or serum from any other mammal. In some other embodiments, the assay solution is serum-free.

[0209] Assay solutions under normal and abnormal physiological conditions may have different pHs, and the pH of such assay solutions may be adjusted using CO and O levels in the buffer using bicarbonate.

[0210] In some other embodiments, at least one of two or more components is added at different concentrations to the assay solutions under normal and abnormal physiological conditions. For example, both lactate and bovine serum albumin (BSA) are added to the assay solutions. The lactate concentration may differ between the assay solutions under normal and abnormal physiological conditions, while BSA may have the same concentration in both assay solutions. Lactate may have a concentration in the range of 30-50 mg / dL in the assay solution under abnormal physiological conditions and a concentration in the range of 8-15 mg / dL in the assay solution under normal physiological conditions. On the other hand, BSA has the same concentration in both assay solutions, such as about 10-20%. The conditionally active polypeptide thus selected using these assay solutions is 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.

[0211] In some embodiments, assay solutions may be designed to select for conditionally active polypeptides whose activity is dependent on two or more conditions. In one exemplary embodiment, a conditionally active polypeptide may have activity that is dependent on both pH and lactate. An assay solution for selecting such conditionally active polypeptides may have an assay solution with a pH of 7.2-7.6 and a lactate concentration of 8-15 mg / dL for normal physiological conditions. An assay solution for abnormal conditions may have a pH of 6.2-6.8 and a lactate concentration of 30-50 mg / dL. Optionally, both assay solutions for normal and abnormal physiological conditions may also contain ions that support binding between the mutant polypeptide and its binding partner, thereby increasing the number of hits for candidate biologically active polypeptides.

[0212] In yet another exemplary embodiment, the conditionally active polypeptide may have activity dependent on pH, glucose, and lactate. An assay solution for selecting such conditionally active polypeptides may have a pH of 7.2-7.6, a glucose concentration ranging from 2.5-10 mM, and a lactate concentration ranging from 8-15 mg / dL for normal physiological conditions. An assay solution for abnormal conditions may have a pH of 6.2-6.8, a glucose concentration ranging from 0.05-0.5 mM, and a lactate concentration ranging from 30-50 mg / dL. Optionally, both assay solutions for normal and abnormal physiological conditions may also contain ions that support binding between the mutant polypeptide and its binding partner, thereby increasing the number of candidate biologically active polypeptides that bind to their binding partner at pH 6.2-6.8. Conditionally active polypeptides selected using such assay solutions are more active in an environment of pH 6.2-6.8, glucose concentration 0.05-0.5 mM, and lactate concentration 30-50 mg / dL than in an environment of pH 7.2-7.6, glucose concentration 2.5-10 mM, and lactate concentration 8-15 mg / dL.

[0213] The two or more components selected from inorganic compounds, ions, and organic molecules are intended to create an abnormal condition assay solution that mimics the environment of the location / site (i.e., target site) to which the selected conditionally active polypeptide is to be delivered. In some embodiments, at least three components in the environment of the target site may be added to the assay solution, or at least four components in the environment of the target site may be added to the assay solution, or at least five components in the environment of the target site may be added to the assay solution, or at least six components in the environment of the target site may be added to the assay solution.

[0214] In one embodiment, bodily fluid collected from a target site (where a conditionally active polypeptide may be more active) may be used directly as an 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 in need of treatment. The collected synovial fluid may be optionally diluted and used as an assay solution in an assay pair under abnormal conditions to select a conditionally active polypeptide. By using the collected synovial fluid, optionally diluted, as an 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 polypeptide (e.g., TNF-α) may be more active in the joint than in other locations or organs. For example, a subject with an inflamed joint (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 not or less active in the blood, may deliver the activity of TNF-α to the joints while reducing or potentially eliminating the side effects of TNF-α on other parts of the body.

[0215] The development of conditionally active polypeptides with activity dependent on multiple conditions will result in improved selectivity of the conditionally active polypeptide for target sites within a subject's body. Ideally, the conditionally active polypeptide is inactive, or at least significantly less active, at other locations where only some of the conditions are present. In one embodiment, a conditionally active polypeptide active at a pH of 6.2-6.8, a glucose concentration of 0.05-0.5 mM, and a lactate concentration of 30-50 mg / dL can be delivered specifically to the tumor microenvironment, since all of these conditions are present in the tumor microenvironment. Other tissues or organs may only have one or two of these conditions, but not all three, which may be insufficient to fully activate the conditionally active polypeptide in other tissues or organs. For example, post-exercise muscle may have a low pH in the range of 6.2-6.8, but may not have other assay conditions. Thus, the conditionally active polypeptide is inactive, or at least less active, in post-exercise muscle.

[0216] In some embodiments, a step can be performed to confirm that the activity of the conditionally active polypeptide is indeed dependent on the conditions used to select the conditionally active polypeptide. For example, a conditionally active polypeptide can be selected that is dependent on three conditions: pH 6.2-6.8, glucose concentration 0.05-0.5 mM, and lactate concentration 30-50 mg / dL. The selected conditionally active polypeptide can then be tested under each of these three conditions individually and in an environment containing a pair of the three conditions to confirm that the conditionally active polypeptide is inactive or has low activity under these test conditions or environments.

[0217] In some embodiments, certain serum components are intentionally minimized or excluded from the assay medium. For example, when screening for antibodies, serum components that bind to or adsorb the antibody can be minimized or excluded from the assay medium. Such bound antibodies can generate false positives, thereby including binding mutant antibodies that are not conditionally active but rather simply bind to components present in serum under a variety of different conditions. Thus, by using careful selection of assay components that minimize or eliminate components that could potentially bind to the variants in the assay, the number of non-functional variants that may falsely be identified as positive for conditional activity due to binding to components in the assay other than the desired binding partner can be reduced. For example, in some embodiments screening for variant polypeptides that tend to bind to components in human serum, BSA can be used in the assay solution to reduce or eliminate the possibility of false positives caused by variant polypeptides binding to components in human serum. Other similar substitutions can also be made in the detailed examples to achieve the same goal.

[0218] In some embodiments, the assay conditions mimic the environment near the cell membrane, such as inside, on, or outside the cell membrane, or the environment of the joint. Some factors that may affect binding activity during screening in the cell membrane environment include receptor expression, internalization, antibody-drug conjugate (ADC) potency, etc.

[0219] The assay format can be any suitable assay 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, suitable cell-based assays are described in WO 2013 / 040445, tissue-based assays are described in U.S. Pat. No. 7,993,271, whole animal-based screening methods are described in U.S. Pat. App. Pub. No. 2010 / 0263599, and 3D system-based screening methods are described in U.S. Pat. App. Pub. No. 2011 / 0143960.

[0220] In some embodiments, the evolution process may generate variant polypeptides that may simultaneously possess other desirable properties in addition to the conditionally active properties discussed above. Suitable other desirable properties that may be evolved include binding activity, expression, humanization, etc. Thus, the present invention may be used to generate conditionally active polypeptides that also have improved at least one or more of these other desirable properties.

[0221] In some embodiments, the selected conditionally active polypeptides can be further mutated, e.g., in a second evolution step, using one of the mutagenesis techniques disclosed herein, to improve another property of the selected conditionally active polypeptide, such as binding activity, expression, humanization, etc. After this second evolution step, the mutant polypeptides can be screened for both conditional activity and the improved property.

[0222] In some embodiments, after evolving a parent polypeptide to generate variant polypeptides, a first conditionally active polypeptide is selected that exhibits both an increased first activity in an assay under abnormal conditions compared to the first activity under normal physiological conditions. The first conditionally active polypeptide can then be subjected to one or more additional evolution, expression, and selection steps to select at least a second conditionally active polypeptide that either (1) exhibits an increased second activity in an assay under abnormal conditions compared to the second activity in an assay under normal physiological conditions, or (2) exhibits a greater ratio of the first activity under abnormal conditions to the first activity under normal physiological conditions compared to the first conditionally active polypeptide and / or the parent polypeptide. The second conditionally active polypeptide can have both a higher first activity and a higher second activity under abnormal conditions compared to their respective activities under normal physiological conditions, and a lower first activity and a lower second activity under normal physiological conditions compared to the parent polypeptide.

[0223] In certain embodiments, the present invention aims to produce conditionally active polypeptides that have a large ratio of activity under abnormal conditions to activity under normal physiological conditions (e.g., greater selectivity between abnormal and normal physiological conditions). The ratio of activity under abnormal conditions to activity under normal physiological conditions, i.e., selectivity, can 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.

[0224] In one embodiment, the conditionally active polypeptide is an antibody, which may have a ratio of activity under abnormal conditions to activity under normal physiological conditions of 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 15:1, or at least about 20:1, or at least about 40:1, or at least about 80:1. In one embodiment, the conditionally active polypeptide is used to target tumor sites, where the conditionally active polypeptide is active at tumor sites (in the tumor microenvironment) and is significantly less active or inactive at non-tumor sites (normal physiological conditions).

[0225] In one embodiment, the conditionally active polypeptide is an antibody intended to be conjugated to another agent, such as those disclosed elsewhere herein. 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 to another agent 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 when the conjugated agent is, for example, toxic or radioactive, because it is desirable to localize the conjugated agent at the disease or treatment site (where the abnormal condition exists).

[0226] H. Confirmation of the pI of the Conditionally Active Polypeptide In some embodiments, the pI of the selected conditionally active polypeptide is confirmed to be lower than the pI of the parent polypeptide using any of the techniques described above. For example, the pI of the conditionally active polypeptide may be at least 0.1, or at least 0.2, or at least 0.3, or at least 0.4, or at least 0.5, or at least 0.6, or at least 0.8, or at least 1.0, or at least 1.2, or at least 1.4, or at least 1.5, or at least 1.7, or at least 2.0, or at least 2.5, or at least 3.0, or at least 3.5, or at least 4.0, or at least 5.0 units lower than the pI of the parent polypeptide.

[0227] I. Conditionally Active Polypeptides In another aspect, the present invention provides a conditionally active polypeptide having a pI lower than the pI of the parent polypeptide, wherein the activity of the conditionally active polypeptide is increased under abnormal conditions that deviate from normal physiological conditions compared to the same activity of the same mutant polypeptide under corresponding normal physiological conditions, and in some cases, (a) the activity is increased under abnormal conditions that deviate from normal physiological conditions compared to the same activity of the parent polypeptide under the same abnormal conditions; (b) the activity is decreased under normal physiological conditions compared to the same activity of the parent polypeptide under the same normal physiological conditions; or (c) A combination of (a) and (b) above.

[0228] Conditionally active polypeptides are reversibly or irreversibly inactivated under normal physiological conditions but are active under abnormal conditions. In some embodiments, these conditionally active polypeptides may have activity under abnormal conditions that is equal to or greater than the same activity of the parent polypeptide under normal physiological conditions.

[0229] Conditionally active polypeptides are particularly valuable for the development of therapeutic agents that are active within a host for a limited period of time. This is particularly valuable when limited activity is required to achieve the desired treatment, whereas prolonging the therapeutic effect would be detrimental to the host. Examples of beneficial applications include local or systemic treatments, as well as regional treatments. One advantage of conditionally active polypeptides is that higher doses can be used in therapeutic applications, potentially reducing adverse side effects. Inactivation under normal physiological conditions can be used to reduce adverse side effects.

[0230] Inactivation under normal physiological conditions is determined by a combination of the dosage of the polypeptide and the rate of inactivation, which is particularly important for enzyme therapies that can cause substantial negative side effects in a relatively short period of time.

[0231] Conditionally active polypeptides may also be activated or inactivated time-reversibly or irreversibly, or may be activated or inactivated only when located in a particular microenvironment within the body, including a particular organ within the body. Exemplary microenvironments may include, but are not limited to, tumors, synovial fluid, and the microenvironments of the bladder or kidney.

[0232] In some embodiments, the conditionally active polypeptide is an antibody or antibody fragment directed against one or more target antigens described herein.

[0233] As discussed above, the abnormal conditions and normal physiological conditions can be conditions selected from temperature, pH, osmolality, osmolality, oxidative stress, electrolyte concentration, and combinations of two or more such conditions. In one embodiment, the condition is pH, and the conditionally active polypeptide has a pH-dependent activity. Specifically, the conditionally active polypeptide has increased activity at abnormal pH compared to that at normal physiological pH.

[0234] In one aspect, the invention relates to conditionally active polypeptides that have pH-dependent activity in the presence of a species having a pKa within 0.5, 1, 1.5, 2, 2.5, 3, or 4 units of the pH at which activity is desired. In another aspect, the invention relates to conditionally active polypeptides that have pH-dependent activity in the presence of a species having a pKa of about 4 to about 10, or about 4.5 to about 9.5, or about 5 to about 9, or about 5.5 to about 8, or about 6.0 to about 7.0.

[0235] Species present in the assay medium that have a significant effect on the activity of a conditionally active polypeptide tend to be species that have at least two ionization states: an uncharged or low-charged state and a charged or high-charged state. Therefore, the pKa of a species that has an effect on the activity of a conditionally active polypeptide can be useful in determining the extent to which that species has an effect on the polypeptide's activity at a particular pH.

[0236] In another aspect, the invention relates to a conditionally active polypeptide that has pH-dependent activity in the presence of a species selected from histidine, histamine, hydrogenated adenosine diphosphate, hydrogenated adenosine triphosphate, citrate, bicarbonate, acetate, lactate, disulfide, hydrogen sulfide, ammonium, dihydrogen phosphate, and any combination thereof. In some embodiments, the pH-dependent conditionally active polypeptide has higher activity at a second pH than at a first, different pH, and both activities are measured in the assay in the presence of these species. To determine the pH dependence of a conditionally active polypeptide, the same activity of the polypeptide is assayed at two different pH values ​​in the same assay medium.

[0237] The ratio of the activity at a second pH to the same activity at a first pH in the same assay medium is referred to as the selectivity of the pH-dependent, conditionally active polypeptide. A pH-dependent, conditionally active polypeptide has a selectivity of at least about 1.3, or at least about 1.5, or at least about 1.7, or at least about 2.0, or at least about 3.0, or at least about 4.0, or at least about 6.0, or at least about 8.0, or at least about 10.0, or at least about 20.0, or at least about 40.0, or at least about 60.0, or at least about 100.0.

[0238] pH-dependent conditionally active polypeptides are often observed to have an increased number (or percentage) of charged amino acid residues compared to the amino acid residues of the parent polypeptide from which the conditionally active polypeptide is derived. There are three positively charged amino acid residues: lysine, arginine, and histidine; and two negatively charged amino acid residues: aspartic acid and glutamic acid. In some embodiments, these charged amino acid residues are present in excess in the pH-dependent conditionally active polypeptide compared to the parent polypeptide from which the pH-dependent conditionally active polypeptide is derived. As a result, the pH-dependent conditionally active polypeptide, having an increased number of charged amino acid residues compared to the parent polypeptide, is more likely to interact with charged species in the assay medium. This, in turn, affects the activity of the conditionally active polypeptide.

[0239] It has also been observed that pH-dependent conditionally active polypeptides typically have different activities in the presence of different species in the assay medium. Species with at least two ionized states, an uncharged or low-charged state and a charged or high-charged state, can dissociate to a greater extent at a particular pH depending on their pKa values, thereby increasing the likelihood of interaction with charged amino acid residues present in the conditionally active polypeptide. This feature can be used to enhance the selectivity and / or pH-dependent activity of the conditionally active polypeptide.

[0240] The nature of the charge on the conditionally active polypeptide can be one factor used to determine suitable species for affecting the activity of the conditionally active polypeptide. In some embodiments, the conditionally active polypeptide can have more positively charged amino acid residues, such as lysine, arginine, and histidine, than the parent polypeptide. Alternatively, the conditionally active polypeptide can have more negatively charged amino acid residues, such as aspartic acid and glutamic acid, than the parent polypeptide. In this manner, the conditionally active polypeptide can be selected to have a desired level of interaction with particular species present in the environment in which its activity is desired and / or to have a desired level of interaction with particular species present in the environment in which reduced activity is desired.

[0241] The location of charged amino acid residues on a pH-dependent conditionally active polypeptide can also affect the activity of the conditionally active polypeptide. For example, the proximity of charged amino acid residues to the binding site of a conditionally active polypeptide can affect the binding activity of the polypeptide.

[0242] In some embodiments, the interaction of a charged environmental species with the conditionally active polypeptide may prevent or impede the activity of the pH-dependent conditionally active polypeptide. For example, a charged amino acid that interacts with a charged environmental species may exert an allosteric effect on the binding site of the conditionally active polypeptide.

[0243] In other embodiments, interactions between charged environmental species and the conditionally active polypeptide can result in the formation of salt bridges between different moieties on the polypeptide, particularly between charged and polar moieties. Salt bridge formation is known to stabilize polypeptide structure (Donald, et al., "Salt Bridges: Geometrically Specific, Designable Interactions," Proteins, 79(3):898-915, 2011; Hendsch, et al., "Do salt bridges stabilize proteins? A continuum electrostatic analysis," Protein Science, 3:211-226, 1994). Salt bridges can stabilize or fix protein structures that undergo constant, small structural changes, commonly referred to as "breathing" (Parak, "Proteins in action: the physics of structural fluctuations and conformational changes," Curr Opin Struct Biol., 13(5):552-557, 2003). Protein structural "breathing" is important for protein function and binding because structural fluctuations can enable proteins to efficiently recognize and bind to their partners (Karplus, et al., "Molecular dynamics and protein functions," PNAS, vol. 102, pp. 6679-6685, 2015). Forming salt bridges can make the binding site, especially the binding pocket, on a conditionally active polypeptide less accessible to its partner. This is likely because the salt bridge directly blocks the partner's access to the binding site or reduces the "breathing" of the protein structure. Even if the salt bridge is located far from the binding site, the allosteric effect of the salt bridge can cause a conformational change in the binding site that inhibits binding. Therefore, after the salt bridge stabilizes (locks) the structure of a conditionally active polypeptide, the polypeptide may become less active in binding to its partner, resulting in reduced activity.

[0244] Hemoglobin is known as one polypeptide whose structure is stabilized by salt bridges. Structural and chemical studies have revealed that salt bridges in hemoglobin involve at least two pairs of chemical groups: the amino terminus and side chains of histidines β146 and α122 (which have pKa values ​​near pH 7). In deoxyhemoglobin, the terminal carboxylate group of β146 forms a salt bridge with a lysine residue in the α subunit of the other αβ dimer. This interaction fixes the side chain of histidine β146 in a position where it can participate in a salt bridge with the negatively charged aspartic acid 94 of the same chain, provided that the imidazole group of the histidine residue is protonated (Figure 1). At high pH, ​​the side chain of histidine β146 is unprotonated, and a salt bridge cannot be formed. However, as the pH decreases, the side chain of histidine β146 becomes protonated, forming a salt bridge between histidine β146 and aspartic acid β94, thereby stabilizing the quaternary structure of deoxyhemoglobin and increasing its propensity for oxygen release in actively metabolizing tissues (lower pH). Hemoglobin exhibits a pH-dependent binding activity for oxygen; as a result, at low pH, its binding activity for oxygen decreases due to the formation of salt bridges. On the other hand, at high pH, ​​its binding activity for oxygen increases due to the absence of these salt bridges.

[0245] Similarly, ions such as bicarbonate ions can reduce the binding activity of a conditionally active polypeptide to its partner by forming salt bridges in the conditionally active polypeptide. For example, at a pH greater than the pKa of bicarbonate ions, bicarbonate ions become negatively charged. Negatively charged bicarbonate ions can form salt bridges between positively charged or polar moieties on the conditionally active polypeptide. These salt bridges prevent or reduce binding between the conditionally active polypeptide and its partner. At pHs below 6.4, bicarbonate ions are protonated and neutralized. Uncharged bicarbonate cannot form salt bridges and thus would not affect binding between the conditionally active polypeptide and its partner. In this scenario, the conditionally active polypeptide may have higher binding activity with its partner at a pH below 6.4, the pKa of bicarbonate, than at a pH greater than 6.4. In this example, the conditionally active polypeptide is conditionally active in the presence of bicarbonate ions, i.e., it exhibits pH-dependent activity.

[0246] In the absence of species such as bicarbonate in the assay medium, a conditionally active polypeptide may lose its conditional activity. This is likely due to the absence of salt bridges on the conditionally active polypeptide that stabilize (lock) the structure of the polypeptide. Thus, in the absence of bicarbonate, binding partners may have similar access to binding sites on the conditionally active polypeptide at all pHs, resulting in similar activity at all pHs and eliminating conditional activity.

[0247] In other embodiments, interaction of a small molecule or ion with a conditionally active polypeptide may alter the structure of the polypeptide in a manner that alters its activity. For example, the structural change may improve the binding affinity of the conditionally active polypeptide by changing the position, steric hindrance, or binding energy for the binding site. In such cases, it may be desirable to select a small molecule or ion that binds to the conditionally active polypeptide at the pH where activity is desired.

[0248] Although salt bridges (ionic bonds) are the strongest and most common way by which compounds and ions affect the activity of conditionally active polypeptides, it should be understood that other interactions between such compounds and ions and the conditionally active polypeptide may also contribute to stabilizing or fixing the structure of the conditionally active polypeptide. Such other interactions include hydrogen bonds, hydrophobic interactions, and van der Waals interactions.

[0249] In some embodiments, to select a suitable compound or ion, a conditionally active polypeptide is compared to the parent polypeptide from which it was evolved to determine whether the conditionally active polypeptide has a higher proportion of negatively or positively charged amino acid residues. Compounds or ions with appropriate charges at the desired pH and at normal physiological pH can then be selected based on their size and pKa value and used to affect the activity of the conditionally active polypeptide. For example, if the conditionally active polypeptide has a higher proportion of positively charged amino acid residues than the parent polypeptide, a suitable small molecule or ion should typically be negatively charged at normal physiological pH and neutral at the desired pH to interact with the conditionally active polypeptide. On the other hand, if the conditionally active polypeptide has a higher proportion of negatively charged amino acid residues than the parent polypeptide, a suitable small molecule or ion should typically be positively charged at normal physiological pH and neutral at the desired pH to interact with the conditionally active polypeptide.

[0250] In other embodiments, the activity of the conditionally active polypeptide is controlled by the interaction of a small molecule or ion with a target polypeptide that is a binding partner of the conditionally active polypeptide. In this case, the same principles discussed above are equally applicable, except that the goal is to create an interaction between the small molecule or ion and the target polypeptide. The target polypeptide can be, for example, an antigen for a conditionally active antibody or a ligand for a conditionally active receptor.

[0251] A suitable small molecule or ion can be any inorganic or organic compound or ion that transitions from an uncharged or low-charged state at the desired pH to a charged or high-charged state at normal physiological pH. Therefore, the small molecule or ion should typically have a pKa between the desired pH and normal physiological pH. For example, bicarbonate has a pKa of 6.4. Thus, at a pH such as 7.4, negatively charged bicarbonate may bind to charged amino acid residues in a conditionally active polypeptide, reducing activity. On the other hand, at a lower pH, such as pH 6.0, low-charged bicarbonate may not bind to the conditionally active polypeptide in the same amount, potentially allowing for higher activity of the conditionally active polypeptide.

[0252] Disulfides have a pKa of 7.05. Therefore, at normal physiological pH, such as pH 7.4, more negatively charged disulfides may bind to the positively charged amino acid residues of a conditionally active polypeptide, reducing its activity. On the other hand, at lower pH, such as pH 6.2-6.8, less charged hydrogen sulfide / disulfides do not bind to the conditionally active polypeptide at the same level, thereby allowing for greater activity of the conditionally active polypeptide.

[0253] Small molecules or ions with a pKa between the desired pH and normal physiological pH are preferred for use in the present invention. Preferred species are selected from disulfide, hydrogen sulfide, histidine, histamine, citrate, bicarbonate, acetate, and lactate. These small molecules or ions each have a pKa of 6.2 to 7.0. Additionally, other small molecules such as tricine (pKa 8.05) and bicine (pKa 8.26) may also be used. Other suitable small molecules or ions can be found using the principles of the present application in textbooks such as the CRC Handbook of Chemistry and Physics, 96th Edition, by CRC press, 2015, and Chemical Properties Handbook, McGraw-Hill Education, 1998.

[0254] The concentration of the small molecule or ion in the assay medium or environment is preferably at or near the physiological concentration of the small molecule or ion in the subject. For example, the physiological concentration of bicarbonate in human serum is in the range of 15-30 mM. Thus, the concentration of bicarbonate in the assay medium may be 10 mM-40 mM, or 15 mM-30 mM, or 20 mM-25 mM, or about 20 mM. The concentration of disulfide in the assay medium may be 3-500 nM, or 5-200 nM, or 10-100 nM, or 10-50 nM.

[0255] In the present invention, a conditionally active polypeptide is selected and utilized at a concentration such that the normal physiological concentration of a particular species in the environment can have a significant effect on the activity of the conditionally active polypeptide in the desired pH range. Therefore, for many therapeutic treatments, it would be advantageous to have low activity for the conditionally active polypeptide in blood or human serum around pH 7.2-7.4 to enable delivery of the therapeutic treatment via the bloodstream while minimizing or preventing the conditionally active polypeptide from activation. Therefore, for such treatments, selecting a small molecule or ion with a pKa below pH 7.2-7.4 would be advantageous to ensure a sufficient amount of ionization of the small molecule at bloodstream pH to significantly affect the activity of the conditionally active polypeptide. At the same time, the pKa of the small molecule or ion must be above the pH at which activity of the conditionally active polypeptide is desired to ensure activation of the conditionally active polypeptide by protonation of the small molecule or ion to a free binding site on the conditionally active polypeptide.

[0256] The small molecule or ion preferably has a low molecular weight and / or a relatively small conformation to ensure maximum access to the small pocket on the target polypeptide or conditionally active polypeptide by minimizing steric hindrance. Thus, the small molecule or ion typically has a molecular weight of less than 900 a.mu, or more preferably less than 500 a.mu, or more preferably less than 200 a.mu, or even more preferably less than 100 a.mu. For example, hydrogen sulfide, disulfide, and bicarbonate all have low molecular weights and compact structures that allow them to access the pocket on the target polypeptide or conditionally active polypeptide, as shown in Examples 13 and 14 below.

[0257] Small molecules or ions may be present in assays used to select condition activity or environments at substantially the same concentration, e.g., about 20 μM for bicarbonate. In some embodiments, small molecules or ions may be present at different concentrations in different environments, and therefore, it may be desirable to simulate this in the assay. For example, disulfide has a higher concentration in the tumor microenvironment than in human serum. Thus, a first assay may simulate human serum with a neutral or slightly basic pH and a lower disulfide concentration, while a second assay may simulate a tumor microenvironment with an acidic pH and a higher disulfide concentration. The acidic pH may be in the range of 6.0 to 6.8, while the neutral or slightly basic pH may be about 7.4. The lower disulfide concentration for the first assay simulating human serum may be 10 μM or less, or 5 μM, while the higher disulfide concentration for the second assay simulating the tumor microenvironment may be 30 μM.

[0258] In some embodiments, the conditionally active polypeptide is pH dependent when two or more different small molecules and / or ions are present, such as, for example, a combination of bicarbonate and histidine.

[0259] In the absence of small molecules or ions, a conditionally active polypeptide may lose its pH dependence, and thus, in the absence of small molecules or ions, a conditionally active polypeptide may have similar activity between the abnormal pH at which activity is desired and normal physiological pH.

[0260] In certain embodiments, the abnormal pH at which activity is desired is an acidic pH, while the normal physiological pH is a basic or neutral pH. For example, the abnormal pH can be a pH in the range of about 5.5 to 7.2, or about 6.0 to 7.0, or about 6.2 to 6.8. The normal physiological pH can be a pH in the range above 7.2 to below 7.6. Conditionally active polypeptides that are more active at acidic pH and less active at basic or neutral pH can target tumor microenvironments where the abnormal pH is about 5.5 to 7.2, or about 6.2 to 6.8, or acidic.

[0261] In other embodiments, the abnormal pH at which activity is desired is a basic pH, and the normal physiological pH is an acidic or neutral pH. For example, the abnormal pH at which the pH-dependent polypeptide is more active can be a basic pH, e.g., 7.6 to 7.9, such as in synovial fluid (see Jebens et al., "On the viscosity and pH of synovial fluid and pH of blood," Journal of Bone and Joint Surgery, vol. 41 B, pp. 388-400, 1959). The normal physiological pH can be a blood pH above 7.2 to 7.6, at which the conditionally active polypeptide is less active. These conditionally active polypeptides are believed to be suitable for targeting joint diseases and arthritis.

[0262] In another embodiment, the conditionally active polypeptide can be designed to target the brain. There is a pH difference on both sides of the blood-brain barrier, with the brain pH being approximately 0.2 pH units lower than the pH of blood or human serum. Therefore, the abnormal pH in the brain, at which the conditionally active polypeptide is more active, can be approximately 7.0 to 7.2 (brain pH), whereas normal physiological pH can be greater than 7.2 and less than 7.6.

[0263] The conditionally active polypeptide may be an enzyme, a cytokine, a receptor, particularly a cellular receptor, a regulatory polypeptide, a soluble polypeptide, an antibody, or a hormone.

[0264] The conditionally active polypeptide may be a fragment of the parent polypeptide. For example, the conditionally active polypeptide may be an antibody fragment, a single-chain antibody, an enzyme fragment, a receptor fragment, a cytokine fragment, or a hormone fragment. The antibody fragment may be the Fc fragment of an antibody.

[0265] Fc fragments can be used as parent polypeptides to generate conditionally active Fc fragments. Complement binding of Fc fragments can be used to mediate antibody-dependent cell-mediated cytotoxicity. Abnormal pH can be acidic, ranging from 5.5 to 7.2 or 6.2 to 6.8, such as the pH in the tumor microenvironment, whereas normal physiological pH ranges from above 7.2 to below 7.6. Abnormal pH differs from the pH of lysosomes, which typically have a pH around 4.0. Furthermore, lysosomes are where Fc fragments, like any other polypeptides, are targeted for degradation. Lysosomes lack complement, and cell-mediated cytotoxicity cannot occur via lysosomes.

[0266] A conditionally active polypeptide can have two functional domains, with at least one, and preferably both, functional domains having pH-dependent activity. The two functional domains can be evolved simultaneously and selected to identify both functional domains in the same mutant polypeptide. Alternatively, the two functional domains can be evolved and selected independently. In this case, the two functional domains can be fused to form a chimeric polypeptide.

[0267] In one embodiment, the conditionally active polypeptide exhibits increased activity at abnormal pHs where activity is desired compared to the parent polypeptide in the presence of a factor such as a protein, and decreased activity at normal physiological pH compared to the parent polypeptide. The protein may be a protein present in blood, human serum, or a bodily microenvironment such as a tumor microenvironment, an inflamed region, synovial fluid, or the brain. One suitable protein may be albumin, particularly a mammalian albumin such as bovine albumin or human albumin.

[0268] In one embodiment, a protein such as albumin is present in the assay solution used to screen and select conditionally active polypeptides. In another embodiment, an assay solution having a protein such as albumin can also be used to test the activity of the selected conditionally active polypeptides under the same or different conditions. J. Engineering Conditionally Active Polypeptides

[0269] Selected conditionally active polypeptides of the invention can be further modified using any of the methods described in WO 2017 / 078839 in the sections entitled "Engineering of conditionally active polypeptides," "Engineering masked conditionally active polypeptides," and "Engineering of conditionally active antibodies." Additionally, the conditionally active polypeptides and modified conditionally active polypeptides of the invention can be inserted into viral particles that are oncolytic viruses, as described in WO 2017 / 078839. K. Production of Conditionally Active Polypeptides

[0270] Selected conditionally active polypeptides and modified conditionally active polypeptides of the invention can be produced for therapeutic, prophylactic, diagnostic, and research uses using the methods described in WO 2017 / 078839 in the section entitled "Production of the Conditionally Active polypeptides." G. Pharmaceutical Compositions

[0271] Pharmaceutical compositions comprising conditionally active polypeptides or modified conditionally active polypeptides, as well as uses of such pharmaceutical compositions, are described in WO 2017 / 078839. The present invention extends to pharmaceutical compositions comprising the conditionally active polypeptides or further modified conditionally active polypeptides that can be used in therapeutic, prophylactic and diagnostic applications. L. Uses of Conditionally Active Polypeptides

[0272] The present invention also includes the use of the conditionally active polypeptides for the curative or prophylactic treatment of solid tumors, inflamed joints, or brain diseases or disorders.

[0273] Also included within the scope of the present invention is a method of treating a solid tumor, an inflammatory joint, or a brain disease or disorder by administering a conditionally active polypeptide of the present invention to a patient in need of said treatment.

[0274] It must be noted that as used in this specification and the appended claims, the singular forms "a," "an," and "the" include plural references unless the context clearly dictates otherwise. Furthermore, the terms "a" (or "an"), "one or more," and "at least one" may be used interchangeably herein. The terms "comprising," "including," "having," and "constructed from" may also be used interchangeably.

[0275] Unless otherwise indicated, all numerical values ​​used in the specification and claims expressing quantities of ingredients, properties, such as molecular weights, percentages, ratios, reaction conditions, and the like, are to be understood as being modified in all instances by the term "about," whether or not the term "about" is present. Accordingly, unless indicated to the contrary, the numerical parameters set forth in the specification and claims are approximations and may vary depending upon the desired properties sought to be achieved by the present disclosure. At the very least, and without attempting to limit the application of the doctrine of equivalents to the scope of the claims, each numerical parameter should at least be construed in light of the number of reported significant digits and by applying ordinary rounding techniques. Notwithstanding that the numerical ranges and parameters setting forth the broad scope of the present disclosure are approximations, the numerical values ​​set forth in the specific examples are reported as precisely as possible. However, any numerical value inherently contains certain errors necessarily resulting from the standard deviation found in its respective testing measurements.

[0276] It is to be understood that each component, compound, substituent, or parameter disclosed herein is to be construed as disclosed for use alone or in combination with one or more of any other components, compounds, substituents, or parameters disclosed herein.

[0277] It should also be understood that each amount / value or amount / value range of each component, compound, substituent, or parameter disclosed herein is also to be construed as being disclosed in combination with each amount / value or amount / value range disclosed for any other component(s), compound(s), substituent(s), or parameter(s) disclosed herein, and thus any combination of amounts / values ​​or amount / value ranges of two or more components, compounds, substituents, or parameters disclosed herein are also disclosed in combination with each other for purposes of this description.

[0278] It is further understood that each range disclosed herein should be interpreted as a disclosure of each specific value within the disclosed range, having the same number of significant digits. Thus, a range of 1 to 4 should be interpreted as an explicit disclosure of the values ​​1, 2, 3, and 4. It is further understood that each lower limit of each range disclosed herein should be interpreted as a disclosure in combination with each upper limit of each range and each specific value within each range for the same component, compound, substituent, or parameter. Thus, this disclosure should be interpreted as a disclosure of all ranges obtained by combining each lower limit of each range with each upper limit of each range or each specific value within each range, or by combining each upper limit of each range with each specific value within each range.

[0279] Furthermore, a specific amount / value of a component, compound, substituent, or parameter disclosed in the description or examples should be construed as disclosing either a lower or upper range limit and thus can be combined with any other lower or upper range limit or specific amount / value of the same component, compound, substituent, or parameter disclosed elsewhere in this application to form a range of the component, compound, substituent, or parameter.

[0280] All documents mentioned herein are hereby incorporated by reference in their entirety or in lieu of providing the disclosure to which they are specifically relied upon. Applicants do not intend to offer any disclosed embodiments to the public, and to the extent that any disclosed variations or alternatives may not literally fall within the scope of the claims, they are considered part of this specification under the doctrine of equivalents.

[0281] However, although the foregoing description sets forth numerical features and advantages of the present invention together with details of the structure and function of the invention, it should be understood that the disclosure is merely illustrative and that modifications may be made in details, particularly in terms of shape, size and arrangement of elements, within the scope of the principles of the invention to the fullest extent indicated by the broad and general meaning of the terms used in the appended claims.

Claims

1. 1. A method for producing a conditionally active antibody or antigen-binding antibody fragment thereof from a parent antibody or antigen-binding antibody fragment thereof, comprising: (i) evolving a parent antibody or antigen-binding antibody fragment thereof by introducing one or more mutations into the parent antibody or antigen-binding antibody fragment thereof to produce one or more variant antibodies or antigen-binding antibody fragments thereof that have a lower pI (isoelectric point) than the pI of the parent antibody or antigen-binding antibody fragment thereof; (ii) determining which variant antibodies or antigen-binding antibody fragments have a pI lower than that of the parent antibody or antigen-binding antibody fragment, and discarding those variant antibodies or antigen-binding antibody fragments having a pI that is the same as or higher than that of the parent antibody or antigen-binding antibody fragment, while retaining those variant antibodies or antigen-binding antibody fragments having a pI lower than that of the parent antibody or antigen-binding antibody fragment; (iii) determining the activity of one or more of the retained variant antibodies or antigen-binding antibody fragments thereof in a first assay at pH 7.2 to 7.6 and measuring the same activity of one or more of the retained variant antibodies or antigen-binding antibody fragments thereof in a second assay at pH 6 to 7; and (iv) selecting a conditionally active antibody or antigen-binding antibody fragment thereof from one or more mutant antibodies or antigen-binding antibody fragments thereof that (a) exhibit activity in the second assay that is at least 1.3, or at least 1.5, or at least 1.7, or at least 2.0, or at least 3.0, or at least 4.0, or at least 6.0, or at least 8.0, or at least 10.0 greater than the activity exhibited in the first assay, and (b) have a pI that is at least 0.1, or at least 0.2, or at least 0.3, or at least 0.4, or at least 0.5, or at least 0.6, or at least 0.8, or at least 1.0 units lower than the pI of the parent antibody or antigen-binding antibody fragment thereof. A method comprising:

2. 2. The method of claim 1, wherein the conditionally active antibody or antigen-binding antibody fragment thereof has a pI below 7.4, or a pI below 7.3, or a pI below 7.2, or a pI below 7.1, or a pI below 7.

0.

3. 3. The method of claim 1 or 2, wherein the one or more mutations comprise 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid substitutions of amino acid residues for amino acid residues in the parent antibody or antigen-binding antibody fragment thereof that have a higher pI than the pI of the amino acid substituted into the parent antibody or antigen-binding antibody fragment thereof.

4. 4. The method of any one of claims 1 to 3, wherein the one or more mutations comprise an insertion of 1, 2, 3, 4, or 5 amino acid residues having a lower pI than the pI of the parent antibody or antigen-binding antibody fragment thereof, or an insertion of 1, 2, 3, 4, or 5 amino acid residues having a pI that is at least 0.1, or at least 0.2, or at least 0.3, or at least 0.4, or at least 0.5, or at least 0.6, or at least 0.8, or at least 1.0, or at least 1.2, or at least 1.4, or at least 1.5, or at least 1.7, or at least 2.0 units lower than the pI of the parent antibody or antigen-binding antibody fragment thereof.

5. 5. The method of any one of claims 1 to 4, wherein the one or more mutations comprise deletion of 1, 2, 3, 4, or 5 amino acid residues that have a pI higher than the pI of the parent antibody or antigen-binding antibody fragment thereof.

6. The method of any one of claims 1 to 5, wherein the one or more mutations are located at positions exposed on the surface of the mutant antibody or antigen-binding antibody fragment thereof.

7. both the first assay and the second assay are carried out in the presence of one or more molecules or ions having a molecular weight of less than 900 a.m.u., or less than 500 a.m.u., or less than 200 a.m.u., or less than 100 a.m.u.; the one or more molecules or ions are selected from histidine, histamine, hydrogenated adenosine diphosphate, hydrogenated adenosine triphosphate, citrate, bicarbonate, acetate, lactate, disulfide, hydrogen sulfide, ammonium, dihydrogen phosphate, and any combination thereof; or the one or more molecules or ions are bicarbonate ions having a concentration in the range of 3 mM to 200 mM, or 5 mM to 150 mM, or 5 mM to 100 mM, or 10 mM to 100 mM, or 20 mM to 100 mM, or 25 mM to 100 mM, or 30 mM to 100 mM, or 35 mM to 100 mM, or 40 mM to 100 mM, or 50 mM to 100 mM; or the one or more molecules or ions are disulfide ions having a concentration in the range of 1 mM to 100 mM, or 2 nM to 500 nM, or 3 nM to 200 nM, or 5 nM to 100 nM; or the one or more molecules or ions are selected from sodium bicarbonate, potassium bicarbonate, sodium disulfide, or potassium disulfide; The method according to any one of claims 1 to 6.

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