Conditionally active polypeptide and method for generating the same

Abstract

To provide conditionally active polypeptides and methods for generating them.SOLUTION: Conditionally active polypeptides are obtained by a method comprising the steps of evolving a DNA encoding the parent polypeptide by increasing a net charge of the parent polypeptide using one or more techniques selected from increasing a total number of codons of charged amino acid residues in the DNA and decreasing a total number of codons of uncharged amino acid residues in the DNA to create mutant DNAs; expressing the mutant DNAs to obtain mutant polypeptides; and selecting the conditionally active polypeptide from the mutant polypeptides which exhibits a decrease in activity in a first assay at a first value of a condition compared to the same activity in a second assay at a second value of the same condition.SELECTED DRAWING: None

Description

[Technical field]

[0001] The present disclosure relates to the field of providing improved polypeptides having desired activities. In particular, the present disclosure relates to conditionally active polypeptides and methods of producing conditionally active polypeptides from parent polypeptides, which are more active under certain conditions than under other conditions. [Background technology]

[0002] There is a large body of literature describing how to evolve proteins, particularly enzymes or antibodies, 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 part of the improvement can be attributed to higher kinetic activity, which is generally explained by the Q10 rule (estimated to double metabolic turnover for every 10° C. increase for enzymes).

[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 lower levels compared to the wild-type protein from which they are derived.

[0004] It is desirable to generate conditionally active polypeptides, e.g., polypeptides that are less active or effectively inactive under some conditions and more active under other conditions. It is also desirable to generate polypeptides that are activated or inactivated in certain environments or over time. In addition to temperature, other conditions under which a polypeptide may be evolved or improved to be conditionally active include pH, osmolality, oxidative stress; and electrolyte concentration. In addition to the activity of a polypeptide, it is often desirable to improve other properties during evolution, including chemical resistance and resistance to proteolysis.

[0005] There are many known mutant polypeptides with improved properties that have one or more amino acid substitutions to achieve improved properties. For example, U.S. Patent No. 8,318,469 discloses a mutant Thermus brockianus nucleic acid polymerase with one of two mutations: G43D and F665Y. The mutant polymerase has reduced 5'→3' exonuclease activity compared to a polymerase that does not have the mutation. The 5'→3' exonuclease activity of DNA polymerase enzymes is often undesirable because it can digest nucleic acids (including primers) with unprotected 5' ends.

[0006] US Patent No. 8,536,301 discloses mutant polypeptides that bind with high affinity to the cell surface receptor α5β1 integrin for fibronectin or the cell surface receptors αvβ3 and αvβ5 integrin for vitronectin. The mutant peptides are based on molecular scaffolds into which subsequences containing the RGD integrin binding motif are inserted. The subsequences are about 9-13 amino acids long and have RGD flanked by various amino acids. The molecular scaffolds are preferably based on peptide knottins, such as EETI, AgRP and agatoxin IVB, which have tightly defined three-dimensional conformations.

[0007] US Patent No. 8,921,086 discloses mutant DNA polymerases with improved tolerance of nucleotide analogues in the active site region. The mutant polymerases may have amino acid substitutions at positions 375 and 512 relative to wild-type DNA polymerase. Substitutions at position 375 include E375H, E375S, E375K, E375R, E375A, E375Q, E375W, E375Y and E375F. Substitutions at position 52 include K512W, K512Y, K512F, K512L, K512H, K512D and K512E.

[0008] US Patent No. 6,329,178 discloses mutant DNA polymerases derived from naturally occurring DNA polymerases by mutation of one or more amino acids in the active site. The mutant DNA polymerases have altered fidelity or altered enzymatic activity compared to the naturally occurring DNA polymerases. The naturally occurring DNA polymerases have an active site that includes the amino acid sequence motif: AspTyrSerGlnIleGluLeuArg or LeuLeuVa1AlaLeuAspTyrSerGlnIleGluLeuArg. The mutant DNA polymerases have either (a) two or more amino acid substitutions in the amino acid sequence motif, or (b) amino acid substitutions other than Glu in the amino acid sequence motif.

[0009] Such mutant proteins have altered properties compared to the wild-type proteins from which they are derived, however the altered properties of such mutant proteins do not change under altered conditions.

[0010] Evolving a parent polypeptide to be inactive or virtually inactive (less than 50%, 30% or 10% activity, particularly less than 1% activity) under some conditions, but maintaining equivalent or better activity under other conditions, may require the coexistence of one or more destabilizing mutations and one or more activity-increasing mutations. A need exists for developing conditionally active polypeptides.

[0011] U.S. Patent No. 8,709,755 discloses a method for producing a conditionally active antibody, comprising the steps of evolving DNA encoding a wild-type antibody to produce a mutant antibody, subjecting the mutant antibody and the wild-type antibody to an assay under a first condition and an assay under a second condition, and selecting from at least one mutant antibody a conditionally active antibody that (a) exhibits reduced antigen-binding activity in the assay under the first condition compared to the binding activity of the wild-type antibody in the same assay under the first condition, and (b) exhibits increased antigen-binding activity in the assay under the second condition compared to the activity of the wild-type antibody in the same assay under the second condition.

[0012] When used as a therapeutic antibody, the conditionally active antibody preferably has reduced activity under normal physiological conditions and increased activity under abnormal conditions that may exist at the treatment site, such as the tumor microenvironment. As a result of this preferential action, the conditionally active antibody may be less likely to potentially cause harm to normal tissues / organs where normal physiological conditions exist, and therefore less likely to cause side effects. This may allow for longer treatment periods and / or the use of higher doses of the conditionally active antibody. Summary of the Invention [Means for solving the problem]

[0013] The present disclosure provides new methods for generating conditionally active polypeptides that take advantage of directed mutagenesis for more efficient production. The present disclosure also provides new conditionally active polypeptides generated by this method.

[0014] In one aspect, the invention provides a conditionally active polypeptide obtained by increasing the net charge of a parent polypeptide, said conditionally active polypeptide exhibiting increased activity in a first assay at a first value of a condition compared to the same activity in a second assay at a second value of the same condition. The charged amino acid residues may be selected from aspartic acid, glutamic acid, arginine, lysine and optionally histidine.

[0015] In each of the foregoing embodiments, the net charge may be increased by one or more techniques selected from increasing the total number of charged amino acid residues in the polypeptide, decreasing the total number of uncharged amino acid residues in the polypeptide, or a combination thereof. In each of the foregoing embodiments, the conditions may be selected from pH, temperature, osmolality, osmolality, oxidative stress, electrolyte concentration, protein concentration, and combinations of two or more thereof.

[0016] In each of the foregoing embodiments, the condition can be pH, and the conditionally active polypeptide can exhibit reduced activity at normal physiological pH and increased activity at an abnormal pH that is different from normal physiological pH.

[0017] In each of the foregoing embodiments, the conditional activity may be assayed in the presence of at least one species having a molecular weight of less than 900 amu and a pKa that is at most 2 units or at most 3 units away from the pH at which increased activity is desired. In the foregoing embodiments, the pKa of the species may be between a first value of pH and a second value of pH.

[0018] In each of the foregoing embodiments, the conditional activity may be assayed in the presence of at least one species selected from histidine, histamine, adenosine diphosphate, hydrogen sulfide, adenosine triphosphate, citrate, acetate, lactate, disulfide, ammonium, bicarbonate, dihydrogen phosphate, and combinations thereof.

[0019] In each of the foregoing embodiments, the first assay and the second assay may be performed in an assay solution in the absence of serum.

[0020] In each of the foregoing embodiments, the net charge may be increased by introducing one or more charged amino residues into the parent polypeptide by amino acid residue substitution, amino acid insertion, or a combination thereof.

[0021] In each of the foregoing embodiments, one or more charged amino acid residues may be introduced into the active site of the parent polypeptide. In each of the foregoing embodiments, one or more charged amino acid residues may be introduced into a region outside the active site of the parent polypeptide. In each of the foregoing embodiments, the conditionally active polypeptide may be a conditionally active antibody, and one or more charged amino acid residues may be introduced into at least one of the complementarity determining regions of the conditionally active antibody.

[0022] In each of the foregoing embodiments, the one or more charged amino acid residues may be less than 5 charged amino acid residues, or less than 4 charged amino acid residues, or less than 3 charged amino acid residues, or less than 2 charged amino acid residues. In each of the foregoing embodiments, the one or more charged amino acid residues may be selected from aspartic acid and glutamic acid.

[0023] In each of the foregoing embodiments, the conditionally active polypeptide may be reversibly inactivated at a second value of the condition.

[0024] In each of the foregoing embodiments, the first and second assays may be performed in the presence of a species selected from histidine, histamine, adenosine diphosphate, hydrogen sulfide, adenosine triphosphate, citrate, acetate, lactate, bicarbonate, disulfide, ammonium, dihydrogen phosphate, and combinations thereof.

[0025] In each of the foregoing embodiments, the second value of the condition can be a value of a normal physiological condition that is within the normal range of physiological conditions at the site of administration of the conditionally active polypeptide to the subject or in a tissue or organ at the site of action of the conditionally active polypeptide in the subject, and the first value of the condition can be a value of an abnormal condition that deviates from the normal range of physiological conditions at the site of administration of the conditionally active polypeptide or in a tissue or organ at the site of action of the conditionally active polypeptide.

[0026] In each of the foregoing embodiments, the conditionally active polypeptide can be a protein or a protein fragment. In each of the foregoing embodiments, the conditionally active polypeptide can be an antibody, a single chain antibody, and an antibody fragment, and the activity is an antigen binding activity, an Fc region of an antibody, or an enzyme, and the activity is an enzymatic activity, or a receptor, a regulatory protein, a soluble protein, a cytokine, and a fragment of a receptor, a regulatory protein, a stress protein, a vault-associated protein, a neuronal protein, a gut protein, a growth factor, a mitochondrial protein, a cytoplasmic protein, an animal protein, a structural protein, and a plant protein.

[0027] In yet another aspect, the invention provides a pharmaceutical composition comprising an effective amount of the conditionally active polypeptide of any one of the preceding embodiments and a pharma- ceutically acceptable carrier.

[0028] In each of the foregoing embodiments, the pharmaceutical composition may further comprise at least one anti-cancer agent. In each of the foregoing embodiments, the at least one anti-cancer agent may be a chemotherapeutic agent, a radiotherapeutic agent, a hormonal therapy agent, a toxin, or an immunotherapy agent. In each of the foregoing embodiments, the at least one anti-cancer agent may be a toxin selected from taxanes, maytansinoids, auristatins, calicheamicins, duocarmycins, mmae, hypnotics, duomycins, anthracyclines, CC-1065 analogs, docetaxel, cathepsins, ricin, gelonin, pseudomonas exotoxin, diphtheria toxin, RNase, and toxic radioisotopes. In each of the foregoing embodiments, the conditionally active polypeptide may be conjugated to at least one anti-cancer agent, or at least four anti-cancer agents, or at least six anti-cancer agents.

[0029] In yet another aspect, the invention provides for the use of a conditionally active polypeptide of each of the preceding embodiments for treating a solid tumor, an inflamed joint, or a brain disease or disorder, or for removing senescent cells from a subject.

[0030] In yet another aspect, the invention provides a method of treating a solid tumor, an inflamed joint, or a brain disease or disorder comprising administering a conditionally active polypeptide of each of the preceding embodiments.

[0031] In another aspect, the invention provides a nucleic acid molecule encoding the conditionally active polypeptide of each of the preceding embodiments. In another aspect, the invention provides an expression vector comprising the nucleic acid molecule of the preceding embodiments or a host cell comprising such an expression vector.

[0032] In another aspect, the invention provides a method of diagnosing the presence of cancer in a subject, the method comprising the steps of: (a) contacting a cell or tissue of the subject with a detectably labeled conditionally active polypeptide of each of the preceding embodiments, where the conditionally active polypeptide binds to a target on the cancer cell; and (b) determining whether the conditionally active polypeptide binds to the cell or tissue of the subject.

[0033] In each of the foregoing embodiments, contacting the conditionally active polypeptide with a cell or tissue may occur in vitro. In each of the foregoing embodiments, contacting the conditionally active polypeptide with a cell or tissue may occur in the subject.

[0034] In another aspect, the invention provides a bispecific antibody comprising at least one binding domain containing at least one of the conditionally active polypeptides of the preceding embodiments. In another aspect, the invention provides a chimeric protein containing two different binding or catalytic domains, one or both of the domains comprising at least one of the conditionally active polypeptides of the preceding embodiments. In another aspect, the invention provides an oncolytic virus comprising at least one of the conditionally active polypeptides of the preceding embodiments.

[0035] In yet another aspect, the present invention provides a method for preparing a conditionally active polypeptide from a parent polypeptide. In this method, a DNA encoding the parent polypeptide is evolved to increase the net charge of the polypeptide using one or more techniques selected from increasing the total number of codons for charged amino acid residues in the DNA, decreasing the total number of codons for uncharged amino acid residues in the DNA, or a combination thereof to form a mutant DNA. The mutant DNA is expressed to obtain a mutant polypeptide, and a conditionally active polypeptide is selected from the mutant polypeptides. The selected conditionally active polypeptide exhibits increased activity in a first assay at a first value of a condition compared to the same activity in a second assay at a second value of the same condition. The charged amino acid residues are selected from aspartic acid, glutamic acid, arginine, lysine, and histidine.

[0036] In each of the foregoing embodiments, the evolving step may utilize a site-directed mutagenesis technique optionally selected from oligonucleotide-mediated mutagenesis, PCR mutagenesis, and cassette mutagenesis. In each of the foregoing embodiments, the evolving step may utilize codon substitution or codon insertion.

[0037] In each of the foregoing embodiments, the charged amino acid residue may be selected from aspartic acid and glutamic acid.

[0038] In each of the foregoing embodiments, one or more codons may be introduced into a region of DNA encoding an active site of the parent polypeptide. In each of the foregoing embodiments, the parent polypeptide may be a parent antibody and the active site may be a complementarity determining region of the parent antibody.

[0039] In each of the foregoing embodiments, one or more charged amino residues may be introduced into a region outside the active site of the parent polypeptide.

[0040] In each of the foregoing embodiments, the region outside the active site may be an antibody constant region or an antibody framework region.

[0041] In each of the foregoing embodiments, the conditionally active polypeptide can be reversibly inactivated under a second value of the condition.

[0042] In each of the foregoing embodiments, the conditions may be selected from pH, temperature, osmolality, osmolality, oxidative stress, electrolyte concentration, and protein concentration.

[0043] In each of the above embodiments, the first and second assays may be performed in an assay solution that includes a protein found in blood. In each of the foregoing embodiments, the blood protein may be albumin.

[0044] In each of the above embodiments, the first and second assays may be performed in an assay solution in the absence of serum.

[0045] In each of the foregoing embodiments, the value of the second condition can be a value of a normal physiological condition that is within the normal range of physiological conditions at the site of administration of the conditionally active polypeptide to a subject or in a tissue or organ at the site of action of the conditionally active polypeptide in a subject, and the value of the first condition can be a value of an abnormal condition that deviates from the normal range of physiological conditions at the site of administration of the conditionally active polypeptide or in a tissue or organ at the site of action of the conditionally active polypeptide.

[0046] In each of the foregoing embodiments, the condition may be pH, and the first and second assays may be performed in an assay medium containing at least one species having a molecular weight less than 900 a.mu and a pKa up to 3 units away from the first value of pH.

[0047] In each of the foregoing embodiments, the condition may be pH, and the first and second assays may be performed in an assay medium comprising at least one species having a molecular weight of less than 900 amu and a pKa between a first value of pH and a second value of pH.

[0048] In each of the foregoing embodiments, the first and second assays may be performed in an assay medium comprising a species selected from histidine, histamine, hydrogenated adenosine diphosphate, hydrogenated adenosine triphosphate, citrate, acetate, lactate, hydrogen sulfide, disulfide, ammonium, bicarbonate, dihydrogen phosphate, and combinations thereof.

[0049] In each of the foregoing embodiments, the selecting step may further comprise selecting the conditionally active polypeptide based on a property selected from affinity, expression level, and humanization.

[0050] In each of the foregoing embodiments, the expression step may utilize phage display or a eukaryotic production host. In each of the foregoing embodiments, the expression step may be performed in a eukaryotic production host and the selected conditionally active polypeptide may be expressed in the same eukaryotic production host. [Brief description of the drawings]

[0051] [Figure 1] 1 shows the selectivity of the conditionally active antibody prepared in Example 9 at pH 6.0 compared to pH 7.4. [Diagram 2] 1 shows the antigen binding activity of several different conditionally active antibodies assayed in various buffer solutions. [Diagram 3] 1 shows the effect of changing the composition of the Krebs buffer on the binding activity of a conditionally active antibody. [Figure 4] As described in Example 12, the binding activity of three different conditionally active antibodies was dependent on the presence and concentration of bicarbonate at pH 7.4. [Diagram 5] FIG. 1 shows the structure of a chimeric antigen receptor (CAR). [Figure 6] Diagram showing the formation of salt bridges in deoxyhemoglobin, where three amino acid residues form two salt bridges, which stabilize the T quaternary structure of deoxyhemoglobin, leading to a reduced affinity for oxygen. [Figure 7]1 shows the activity of conditionally active antibodies against Ror2 in various buffer solutions. [Figure 8] 1 shows the activity of conditionally active antibodies against Axl in various buffer solutions. [Figure 9A] 1 shows the results of cell killing of A549 cells using wild-type and conditionally active anti-Axl antibodies at pH 6.0 and pH 7.4 at various antibody concentrations. [Figure 9B] 1 shows the results of cell killing of A549 cells using wild-type and conditionally active anti-Axl antibodies at pH 6.0 and pH 7.4 at various antibody concentrations. [Figure 9C] 1 shows the results of cell killing of A549 cells using wild-type and conditionally active anti-Axl antibodies at pH 6.0 and pH 7.4 at various antibody concentrations. [Figure 9D] 1 shows the results of cell killing of A549 cells using wild-type and conditionally active anti-Axl antibodies at pH 6.0 and pH 7.4 at various antibody concentrations. [Figure 9E] 1 shows the results of cell killing of A549 cells using wild-type and conditionally active anti-Axl antibodies at pH 6.0 and pH 7.4 at various antibody concentrations. [Figure 10A] 1 shows the binding affinity of conditionally active anti-Axl antibodies to human Axl and cynomolgus Axl in various buffers and at various pH levels. [Figure 10B] 1 shows the binding affinity of conditionally active anti-Axl antibodies to human Axl and cynomolgus Axl in various buffers and at various pH levels. [Figure 10C] 1 shows the binding affinity of conditionally active anti-Axl antibodies to human Axl and cynomolgus Axl in various buffers and at various pH levels. [Figure 10D] 1 shows the binding affinity of conditionally active anti-Axl antibodies to human Axl and cynomolgus Axl in various buffers and at various pH levels. [Figure 11A] 1 shows cell killing of various cell lines at various pH levels by a conditionally active anti-Axl antibody conjugated to duomycin. [Figure 11B] 1 shows cell killing of various cell lines at various pH levels by a conditionally active anti-Axl antibody conjugated to duomycin. [Figure 11C] 1 shows cell killing of various cell lines at various pH levels by a conditionally active anti-Axl antibody conjugated to duomycin. [Figure 11D] 1 shows cell killing of various cell lines at various pH levels by a conditionally active anti-Axl antibody conjugated to duomycin. [Figure 11E] 1 shows cell killing of various cell lines at various pH levels by a conditionally active anti-Axl antibody conjugated to duomycin. [Figure 11F] 1 shows cell killing of various cell lines at various pH levels by a conditionally active anti-Axl antibody conjugated to duomycin. [Figure 11G] 1 shows cell killing of various cell lines at various pH levels by a conditionally active anti-Axl antibody conjugated to duomycin. [Figure 11H] 1 shows cell killing of various cell lines at various pH levels by a conditionally active anti-Axl antibody conjugated to duomycin. [Figure 12] 1 shows cell killing of A549 cells at various pH levels by a conditionally active anti-Axl antibody conjugated to duomycin. [Figure 13] 1 shows the effect of treatment with a conditionally active anti-Axl antibody conjugated to duomycin on tumor volume in xenografted mice. [Figure 14A] 1 shows the abundance of conditionally active anti-Axl antibody conjugated to duomycin detected in the blood of cynomolgus monkeys over time following injection of anti-Axl antibody / duomycin conjugate. [Figure 14B] 1 shows the abundance of conditionally active anti-Axl antibody conjugated to duomycin detected in the blood of cynomolgus monkeys over time following injection of anti-Axl antibody / duomycin conjugate. [Figure 15A]1 shows the time course of aspartate transaminase (AST) detected in the blood of cynomolgus monkeys, starting immediately before injection (pre(D-3)) and continuing through 3 days after injection (post(D-3)) of a conditionally active anti-Axl antibody conjugated to duomycin. [Figure 15B] 1 shows the time course of alanine aspartate transaminase (ALT) detected in the blood of cynomolgus monkeys, starting immediately before injection (pre(D-3)) and continuing through 3 days after injection (post(D-3)) of a conditionally active anti-Axl antibody conjugated to duomycin. [Figure 16] 1 shows lymphocyte counts over time in the blood of cynomolgus monkeys following injection of a conditionally active anti-Axl antibody conjugated to duomycin. [Figure 17A] 2 shows the effect on tumor volume in xenografted mice following treatment with a conditionally active anti-Ror2 antibody conjugated to paclitaxel as described in Example 21. [Figure 17B] 2 shows the effect on tumor volume in xenografted mice following treatment with a conditionally active anti-Ror2 antibody conjugated to paclitaxel as described in Example 21. [Figure 17C] 2 shows the effect on tumor volume in xenografted mice following treatment with a conditionally active anti-Ror2 antibody conjugated to paclitaxel as described in Example 21. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0052] definition In order to facilitate understanding of the examples provided herein, some frequently occurring methods and / or terms are provided herein.The definitions of the following terms are incorporated by reference 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", "degradatively 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 sequence encoding"", "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", "process", "variable segment", and "variant".

[0053] The term "about" as used herein in connection with a measured quantity relates to the normal variation in the measured quantity that would be expected by a person of ordinary skill in the art making the measurement and exercising a level of care appropriate to the purpose of the measurement and the precision of the measuring equipment used. Unless otherwise specified, "about" relates to a variation of + / - 10% of the given value.

[0054] The term "activity" as used herein refers to any function that a protein may perform, including catalysis of a reaction and binding to a partner. For an enzyme, the activity may be enzymatic activity. For an antibody, the activity may be binding activity between the antibody and its antigen (i.e., binding activity). For a receptor or ligand, the activity may be binding activity between the receptor and its ligand.

[0055] The term "adoptive cell therapy" as used herein refers to the transfer of immune cells with anti-tumor activity to a cancer patient. Adoptive cell therapy is a treatment method that involves identifying lymphocytes with anti-tumor activity in vitro, expanding these cells to large numbers in vitro, and injecting them into a cancer-bearing host. Of particular interest in the present invention is autologous adoptive cell therapy, i.e., adoptive cell therapy in which the immune cells with anti-tumor activity transferred to a cancer patient are derived from the same patient.

[0056] The term "antibody" as used herein refers to intact immunoglobulin molecules and fragments of immunoglobulin molecules capable of binding an epitope of an antigen, such as Fab, Fab', (Fab')2, Fv, and SCA fragments. These antibody fragments retain some ability to selectively bind to the antigen (e.g., polypeptide antigen) of the antibody from which they are derived, and can be generated using methods well known in the art (see, e.g., Harlow and Lane, supra), and are further described below. Antibodies can be used for the isolation of preparative quantities of antigens by immunoaffinity chromatography. Various other uses of such antibodies are in the diagnosis and / or staging of disease (e.g., neoplasia), as well as therapeutic applications for treating diseases such as neoplasia, autoimmune diseases, AIDS, cardiovascular diseases, infectious diseases, and the like. Chimeric, human-like, humanized, or fully human antibodies are particularly useful for administration to human patients.

[0057] A Fab fragment consists of a monovalent antigen-binding fragment of an antibody molecule and can be produced by digestion of whole antibody molecules with the enzyme papain to produce a fragment consisting of an intact light chain and a portion of the heavy chain.

[0058] A Fab' fragment of an antibody molecule can be obtained by treating a whole antibody molecule with pepsin, followed by reduction, to yield a molecule consisting of an intact light chain and a portion of the heavy chain, resulting in two Fab' fragments per antibody molecule thus treated.

[0059] The (Fab')2 fragment of an antibody can be obtained by treating the whole antibody molecule with the enzyme pepsin without subsequent reduction. The (Fab')2 fragment is a dimer of two Fab' fragments held together by two disulfide bonds.

[0060] An Fv fragment is defined as a genetically engineered fragment containing the variable region of the light chain and the variable region of the heavy chain expressed as two chains.

[0061] Single chain antibodies ("SCAs" or scFvs) are engineered single chain molecules comprising the variable region of a light chain and the variable region of a heavy chain linked by a suitable flexible polypeptide linker, and may include additional amino acid sequences at the amino and / or carboxyl termini. For example, single chain antibodies may include a tethering segment for linking to an encoding polynucleotide. A functional single chain antibody generally includes a sufficient portion of the variable region of the light chain and sufficient region of the variable region of the heavy chain so that it retains the properties of a full-length antibody for binding to a specific target molecule or epitope.

[0062] The term "antibody-dependent cell-mediated cytotoxicity" or "ADCC" refers to a form of cytotoxicity in which secreted immunoglobulins bind to Fc receptors (FcRs) present on certain cytotoxic cells (e.g., natural killer (NK) cells, neutrophils, and macrophages), thereby enabling these cytotoxic effector cells to specifically bind to antigen-bearing target cells and subsequently kill those target cells with cytotoxins. Ligand-specific high-affinity IgG antibodies against the surface of target cells stimulate the cytotoxic cells and are necessary for such killing. Lysis of target cells is extracellular, requires direct cell-to-cell contact, and does not involve complement.

[0063] The ability of any particular antibody to mediate target cell lysis by ADCC can be assayed. To assess ADCC activity, the antibody of interest is added to target cells presenting a target ligand in conjunction with immune effector cells, and activation of the immune effector cells by antigen-antibody complexes can result in cytolysis of the target cells. Cytolysis is generally detected by the release of label (e.g., radioactive substrates, fluorescent dyes, or natural intracellular proteins) from the lysed cells. Effector cells useful for such assays include peripheral blood mononuclear cells (PBMCs) and natural killer (NK) cells. Specific examples of in vitro ADCC assays are described in Bruggemann et al, 1987, J. Exp Med, vol. 166, page 1351; Wilkinson et al, 2001, J Immunol. Methods, vol. 258, page 183; Patel et al, 1995 J. Immunol. Methods, vol. 184, page 29. Alternatively, or additionally, ADCC activity of an antibody of interest may be assessed in vivo, e.g., in an animal model such as that disclosed in Clynes et al., 1998, PNAS USA, vol. 95, p. 652.

[0064] The term "antigen" or "Ag" as used herein is defined as a molecule capable of eliciting an immune response. This immune response may include either or both of antibody production or activation of specific immunologically competent cells. Those skilled in the art will appreciate that any macromolecule may serve as an antigen, including virtually any protein or peptide. It is readily apparent that antigens may be generated, synthesized, or derived from biological samples. Such biological samples may include, but are not limited to, tissue samples, tumor samples, cells, or biological fluids.

[0065] The term "antisense RNA" as used herein refers to an RNA molecule capable of forming a duplex with a second RNA molecule by complementarity or partial complementarity with the second RNA molecule. The antisense RNA molecule may be complementary to the translated or untranslated region of the second RNA molecule. The antisense RNA does not need to be completely complementary to the second RNA molecule. The antisense RNA may or may not be the same length as the second RNA molecule; the antisense RNA molecule may be either longer or shorter than the second RNA molecule. When the second RNA molecule is an mRNA, the binding of the antisense RNA will completely or partially prevent the mRNA from being translated into a functional protein product.

[0066] The terms "biosimilar" or "follow-on product" are used in a manner consistent with the working definition published by the US Food and Drug Administration (FDA), which defines a biosimilar as a product that is "highly similar" to the reference product (albeit with minor differences in clinically inactive ingredients). In practice, there may be no clinically meaningful differences between the reference and biosimilar products in terms of safety, purity, and potency (Article 262 of the Public Health Act (PHS)). A biosimilar may also fulfil one or more of the guidelines adopted on 30 May 2012 by the Committee for Medicinal Products for Human Use (CHMP) of the European Medicines Agency and published by the European Union as "Guideline on similar biological medicinal products containing monoclonal antibodies - non-clinical and clinical issues" (document reference EMA / CHMP / BMWP / 403543 / 2010). For example, a "biosimilar antibody" refers to a generic version of a new drug antibody (reference antibody) that is typically produced by another company. Differences between a biosimilar antibody and a reference antibody can include post-translational modifications, for example, by adding other biochemical groups to the antibody, such as phosphates, various lipids and carbohydrates; by post-translational proteolytic cleavage; by changing the chemical nature of amino acids (e.g., formylation); or by many other mechanisms. Other post-translational modifications can be the result of manipulations in the manufacturing process - for example, glycation can occur when the formulation is exposed to reducing sugars. In some cases, storage conditions can be permissive for certain degradation pathways to occur, such as oxidation, deamidation, or aggregation. All of these formulation variations can be included in a biosimilar antibody.

[0067] The terms "cancer" and "cancerous" refer to or describe the physiological condition in mammals that is typically characterized by unregulated cell growth / proliferation. A "tumor" contains one or more cancerous cells. Examples of cancer include, but are not limited to, carcinoma, lymphoma, blastoma, sarcoma, and leukemia or lymphoid malignancies. More particular examples of such cancers include squamous cell carcinoma (e.g., epithelial squamous cell carcinoma), lung cancer (including small cell lung cancer, non-small cell lung cancer ("NSCLC"), lung adenocarcinoma, and lung squamous cell carcinoma), peritoneal cancer, hepatocellular carcinoma, gastric cancer (including gastrointestinal cancer), pancreatic cancer, glioblastoma, cervical cancer, ovarian cancer, liver cancer, bladder cancer, hepatoma, breast cancer, colon cancer, rectal cancer, colorectal cancer, endometrial or uterine cancer, salivary gland cancer, kidney or renal cancer, prostate cancer, vulvar cancer, thyroid cancer, liver cancer, anal cancer, penile cancer, and head and neck cancer.

[0068] The term "chimeric antigen receptor" or "CAR" or "CARs" as used herein refers to an engineered receptor that grafts antigen specificity onto cytotoxic cells, such as T cells, NK cells, and macrophages. The CAR of the present invention may comprise at least one antigen-specific targeting region (ASTR), an extracellular spacer domain (ESD), a transmembrane domain (TM), one or more costimulatory domains (CSD), and an intracellular signaling domain (ISD). In some embodiments, the ESD and / or CSD are optional. In one embodiment, the ASTR is bispecific and can recognize two different antigens or epitopes. After the ASTR specifically binds to the target antigen, the ISD activates the intracellular signaling of the cytotoxic cell. For example, the ISD can redirect T cell specificity and cytotoxicity to a target of choice in an MHC-independent manner, depending on the antigen-binding properties of the CAR. MHC-independent antigen recognition allows CAR-expressing cytotoxic cells to recognize antigens independent of antigen processing, thus circumventing a major tumor escape mechanism. Furthermore, when expressed in T cells, CARs advantageously do not dimerize with endogenous T cell receptor (TCR) α and β chains.

[0069] The term "complementarity determining region" or "CDR" as used herein refers to the antibody region in which the amino acid residues involved in antigen binding are located. CDRs have the highest variability in the amino acid alignment of antibody variable domains. Databases such as the Kabat database can be used to identify CDRs, which are defined as including, for example, amino acid residues 24-34 (L1), 50-56 (L2) and 89-97 (L3) of the light chain variable domain, and 31-35 (H1), 50-65 (H2) and 95-102 (H3) of the heavy chain variable domain (Kabat et al., The Journal of Immunology, vol. 147, pp. 1709-1719, 1991, Sequences of Proteins of Immunological Interest, Fifth Edition, USDepartment of Health and Human Services, NIH Publication No. 91-3242). Alternatively, the CDRs can be defined as residues from the "hypervariable loops" (residues 26-33 (L1), 50-52 (L2) and 91-96 (L3) in the light chain variable domain and 26-32 (H1), 53-55 (H2) and 96-101 (H3) in the heavy chain variable domain (Chothia and Lesk, J. Mol. Biol 1987;196:901-917). Typically, the numbering of amino acid residues in the variable domain is performed according to the method described in Kabat et al., supra. Phrases such as "Kabat position", "Kabat residue" and "according to Kabat" are used herein to refer to the hypervariable loops as defined by Kabat et al. for heavy chain variable domains or light chain variable domains. al. Using the Kabat numbering system, the actual linear amino acid sequence of a variable domain may contain fewer or additional amino acids corresponding to a shortening of, or insertion into, the framework (FR) or CDR of the variable domain. For example, a heavy chain variable domain may include an amino acid insertion after residue 52 of CDR H2 (residues 52a, 52b and 52c according to Kabat) and an inserted residue after heavy chain FR residue 82 (e.g., residues 82a, 82b, 82c, etc. according to Kabat).The Kabat numbering of residues can be determined for a given antibody by alignment of the antibody's sequence with a "standard" Kabat numbered sequence at regions of homology.

[0070] The term "conditionally active polypeptide" refers to a variant or mutant of a parent polypeptide that is more active than the parent polypeptide under at least one condition and less active than the parent polypeptide under a second condition, or a variant or mutant of a parent polypeptide where the variant or mutant polypeptide is at least 1.3 times more active under a first condition than under a second condition. The conditionally active polypeptide may exhibit activity at one or more selected locations in the organism and / or may exhibit increased or decreased activity at another location in the organism. For example, in one embodiment, the evolved conditionally active polypeptide is effectively 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, and growth factors and fragments thereof. Each of the conditionally active polypeptides described herein is preferably a conditionally active biological polypeptide.

[0071] The term "cytokine" or "cytokines," as used herein, refers to a general class of biomolecules that cause / affect cells of the immune system. This definition is intended to include, but is not limited to, biomolecules that act locally or at other locations distant from the site of secretion through the blood circulation to regulate or modulate an individual's immune response. Exemplary cytokines include, but are not limited to, interferon-α (IFN-α), interferon-β (IFN-β), and interferon-γ (IFN-γ), interleukins (e.g., IL-1 through IL-29, specifically, IL-2, IL-5, IL-6, IL-7, IL-10, IL-12, IL-15, and IL-18), tumor necrosis factors (e.g., TNF-α and TNF-β), erythropoietin (EPO), MIP3a, monocyte chemotactic protein (MCP)-1, intracellular adhesion molecules (ICAMs), macrophage colony-stimulating factor (M-CSF), granulocyte colony-stimulating factor (G-CSF), and granulocyte-macrophage colony-stimulating factor (GM-CSF).

[0072] As used herein, the term "electrolyte" is used to define a mineral in blood or other bodily fluids that carries an electrical charge. For example, in one embodiment, there may be "electrolyte concentrations" that differ between normal and abnormal physiological conditions. Exemplary electrolytes include, but are not limited to, ionized calcium, sodium, potassium, magnesium, chloride, citrate, lactate, bicarbonate, and phosphate.

[0073] The term "full-length antibody" refers to an antibody that contains an antigen-binding variable region (V H Or V L), and a light chain constant domain (CL) and heavy chain constant domains CH1, CH2 and CH3. The constant domains may be native sequence constant domains (e.g., human native sequence constant domains) or amino acid sequence variants thereof. Full length antibodies can be assigned to different "classes" depending on the amino acid sequence of the constant domain of their heavy chain. There are five major classes of full length antibodies: IgA, IgD, IgE, IgG, and IgM, some of which can be further divided into "subclasses" (isotypes), e.g., IgG1, IgG2, IgG3, IgG4, IgA, and IgA2. The heavy chain constant domains that correspond to the different antibody classes are called α, δ, ε, γ, and μ, respectively.

[0074] The term "growth factor" as used herein refers to a polypeptide molecule capable of causing cell differentiation. Examples of growth factors include, but are not limited to, epidermal growth factor (EGF), transforming growth factor-α (TGFα), transforming growth factor-β (TGF-β), human endothelial cell growth factor (ECGF), granulocyte-macrophage colony-stimulating factor (GM-CSF), bone morphogenetic protein (BMP), nerve growth factor (NGF), vascular endothelial growth factor (NEGF), fibroblast growth factor (FGF), insulin-like growth factor (IGF), cartilage-derived morphogenetic protein (CDMP), and platelet-derived growth factor (PDGF).

[0075] The term "hormones" as used herein refers to substances often identified as mediators, typically released by cells or glands in one part of an organism to serve as messengers to other parts of the organism. Exemplary hormones include endocrine hormones, which are released directly into the bloodstream, and exocrine hormones (or ectohormones), which are secreted directly into ducts from which they flow by diffusion into the bloodstream or from cell to cell in a process known as paracrine signaling. Vertebrate hormones can be divided into three chemical classes: peptide hormones, lipid and phospholipid derived hormones, and monoamines. Peptide hormones consist of a polypeptide chain. Examples of peptide hormones include insulin and growth hormone. Lipid and phospholipid derived hormones are derived from lipids and phospholipids such as linoleic acid and arachidonic acid. The major class are the steroid hormones, which are derived from cholesterol and eicosanoids. Examples of steroid hormones are testosterone and cortisol. Monoamines are derived from aromatics such as phenylalanine, tyrosine, and tryptophan by the action of aromatic amino acid decarboxylase enzymes. Examples of monoamines are thyroxine and adrenaline.

[0076] The term "immunomodulatory agent" as used herein refers to an agent whose action on the immune system results in an immediate or delayed increase or decrease in the activity of at least one pathway involved in the immune response. Such responses can be naturally occurring as part of the innate or adaptive immune system or both, or can be artificially elicited. Examples of immunomodulatory agents include cytokines, stem cell growth factors, lymphotoxins, such as tumor necrosis factor (TNF), and hematopoietic factors, such as interleukins (e.g., interleukin-1 (IL-1), IL-2, IL-3, IL-6, IL-10, IL-12, IL-18, and IL-21), colony stimulating factors (e.g., granulocyte-colony stimulating factor (G-CSF) and granulocyte-macrophage colony stimulating factor (GM-CSF)), interferons (e.g., interferon-α, -β, and -γ), stem cell growth factor, also referred to as "S1 factor", erythropoietin, and thrombopoietin. Examples of suitable immunomodulatory moieties include IL-2, IL-6, IL-10, IL-12, IL-18, IL-21, interferons, TNF (e.g., TNF-α), and the like.

[0077] An "individual" or "subject" is a mammal, including, but not limited to, domesticated animals (e.g., cows, sheep, cats, dogs, and horses), primates (e.g., humans and non-human primates such as monkeys), rabbits, and rodents (e.g., mice and rats).

[0078] The term "library" as used herein refers to a collection of proteins as a single pool. Libraries may be made using recombinant DNA technology. For example, a collection of cDNAs or any other protein-encoding DNA may be inserted into an expression vector to make a protein library. A collection of cDNAs or protein-encoding DNA may also be inserted into a phage genome to make a bacteriophage display library of wild-type proteins. A collection of cDNAs may be made from a particular cell population or tissue sample, such as by the method disclosed by Sambrook et al. (Molecular Cloning, Cold Spring Harbor Laboratory Press, 1989). Collections of cDNAs from particular cell types are also commercially available from suppliers such as Stratagene®. A library of wild-type proteins as used herein is not a collection of biological samples.

[0079] The term "ligand" as used herein refers to a molecule that is recognized by and specifically binds to a particular receptor at one or more binding sites. Examples of ligands include, but are not limited to, agonists and antagonists of cell membrane receptors, toxins and venoms, viral epitopes, hormones, hormone receptor peptides, enzymes, enzyme substrates, cofactors, drugs (e.g., opiates, steroids, etc.), lectins, saccharides, polynucleotides, nucleic acids, oligosaccharides, proteins, and monoclonal antibodies. Typically, a ligand comprises two structural moieties: a first moiety that is involved in the binding of the ligand to its receptor and a second moiety that is not involved in such binding.

[0080] The term "receptor" as used herein refers to a molecule that has affinity for a given ligand. Receptors can be naturally occurring or synthetic molecules. Receptors may be used unmodified or as aggregates with other species. Receptors can be covalently or non-covalently bound to binding members directly or through specific binding substances. Examples of receptors include, but are not limited to, antibodies and antisera, including monoclonal antibodies that exhibit reactivity with specific antigenic determinants (such as those found on viruses, cells, or other materials), cell membrane receptors, glycoconjugates and glycoproteins, enzymes, and hormone receptors. Binding of a ligand to its receptor indicates the formation of a complex by combination of the ligand and its receptor molecule through specific molecular recognition, which can be detected by various ligand-receptor binding assays known to those skilled in the art.

[0081] The term "microRNA" or "miRNA" as used herein refers to an unprocessed or processed RNA transcript from a miRNA gene. Unprocessed microRNA gene transcripts typically comprise RNA transcripts of about 70-100 nucleotides in length. Transcribed microRNAs can be processed by digestion with an RNase (e.g., Dicer, Argonaute, or RNaseIII) to an active 19-25 nucleotide RNA molecule. This active 19-25 nucleotide RNA molecule is also referred to as a "processed" microRNA gene transcript or a "mature" microRNA.

[0082] The term "multispecific antibody" as used herein is an antibody that has binding specificities for at least two different epitopes. An exemplary multispecific antibody can bind to both BBB-R and a brain antigen. Multispecific antibodies can be prepared as full-length antibodies or antibody fragments (e.g., F(ab')2 bispecific antibodies). Engineered antibodies that contain two, three or more (e.g., four) functional antigen binding sites are also contemplated (see, e.g., U.S. Patent Application Publication No. 2002 / 0004587 A1).

[0083] The term "mutagenic primer" as used herein refers to an oligonucleotide primer used in a linear cyclic amplification reaction (e.g., polymerase chain reaction or PCR), where the primer does not exactly match the template DNA sequence. The mismatched nucleotide of the mutagenic primer is referred to as a mutation site for the mutagenic primer. Thus, during the amplification reaction, the mismatched nucleotide of the primer is incorporated into the amplification product, resulting in the synthesis of a mutant DNA strand containing the mutagenic primer. The mutagenic primer thus allows the introduction of specific mutations into the template DNA sequence.

[0084] The term "nanoparticle" as used herein refers to a microscopic particle with a size in nanometers (nm) and a maximum linear dimension of less than about 1000 nm, or less than about 500 nm, or less than about 200 nm, or less than about 100 nm, or less than about 50 nm. As used herein, linear dimension refers to the distance measured in a straight line between any two points on the nanoparticle. The nanoparticles of the present invention may be irregular, oval, spindle, rod, disk, pancake, cylinder, red blood cell-like, spherical, or approximately spherical in shape, so long as their shape and size allow for binding interactions. The nanoparticles of the present invention are preferably made of biocompatible materials (polymers or lipids).

[0085] The term "naturally occurring" as used herein refers to the fact that an object is found in nature. For example, a polypeptide or polynucleotide sequence that exists in an organism (including viruses) that can be isolated from a source in nature and has not been intentionally modified by humans in a laboratory is naturally occurring. A polypeptide excised from a larger polypeptide is not a naturally occurring polypeptide because the end groups of the excised polypeptide will differ from those of the larger naturally occurring polypeptide in the excised form due to the end groups no longer being attached to adjacent polypeptides. In general, the term naturally occurring refers to an entity as it exists in a non-pathological (unaffected) individual, such as may be typical for that species.

[0086] The term "net charge" of a polypeptide is the ratio of the total number of charged amino acid residues in the polypeptide to the total number of amino acid residues in the polypeptide. Charged amino acid residues include aspartic acid, glutamic acid, arginine, lysine, and optionally histidine. Uncharged amino acid residues include the other 15 naturally occurring amino acids. The net charge of a polypeptide can be increased by either increasing the total number of charged amino acid residues in the polypeptide, decreasing the total number of uncharged amino acid residues in the polypeptide, or a combination thereof.

[0087] The term "oxidative stress" as used herein refers to an imbalance between the production of reactive oxygen radicals (e.g., superoxide anion (O2"), nitric oxide, hydroxyl radical (OH) and hydrogen peroxide (H2O2)) generated, such as as a by-product of metabolic processes that utilize molecular oxygen, and the ability of living systems to readily detoxify the reactive intermediates or repair the resulting damage (see, e.g., Coyle et al., Science 262:689-695 (1993)). Oxidative stress can refer to the condition of animal cells or tissues in vitro or in vivo. Disruption of the normal redox state within living cells can cause toxic effects through the production of peroxides and free radicals that damage all components of the cell, including proteins, lipids and DNA.

[0088] In humans, oxidative stress is involved in many diseases such as atherosclerosis, Parkinson's disease, heart failure, myocardial infarction, Alzheimer's disease, fragile X syndrome, and chronic fatigue syndrome, but short-term oxidative stress may also be important in preventing aging by inducing mitohormesis.Reactive oxygen species can be beneficial because they are used by the immune system as a way to attack and kill pathogens.Reactive oxygen species are also used in cell signaling.This is called redox signaling.

[0089] In chemical terms, oxidative stress is an increase in the cellular reduction potential (less negative) or a decrease in the reducing capacity of cellular redox couples such as glutathione. The effect of oxidative stress depends on the magnitude of this change, which allows the cell to overcome small disturbances and regain its original state. However, more severe oxidative stress can cause cell death, and even moderate oxidation can trigger apoptosis, while more intense stress can cause necrosis.

[0090] 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 or protein using the methods of the present invention. A parent polypeptide protein can be a wild-type protein or a non-wild-type protein. For example, a therapeutic polypeptide or protein or a mutant or variant polypeptide or protein can be used as a parent polypeptide or protein. Examples of parent polypeptides and proteins 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.

[0091] The term "pH dependent" as used herein refers to a polypeptide that has different properties or activities at different pH values.

[0092] The term "polypeptide" as used herein refers to a polymer in which the monomers are amino acids and are linked together by peptide or disulfide bonds. A polypeptide can be a full-length naturally occurring amino acid chain or a fragment, a mutant or variant thereof, e.g., a selected region of an 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, typically about 8 to about 500 amino acids long, preferably about 8 to about 300 amino acids long, more preferably about 8 to about 200 amino acids long, and even more preferably about 10 to about 50 or 100 amino acids long. In addition, amino acids other than naturally occurring amino acids, e.g., β-alanine, phenylglycine, and homoarginine, can be included in proteins. Commonly found 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 include structures that include two or several polypeptide chains held together by covalent or non-covalent bonds.

[0093] The term "prodrug" as used herein refers to a precursor compound that undergoes metabolic activation in vivo to produce an active drug. A thorough discussion is provided in T. Higuchi and V. Stella, "Pro-drugs as Novel Delivery Systems," V.14 of the ACS Symposium Series, and Edward B. Roche, ed., "Bioreversible Carriers in Drug Design," American Pharmaceutical Association and Pergamon Press (1987).

[0094] The term "recombinant antibody" as used herein refers to an antibody (e.g., a chimeric, humanized, or human antibody or antigen-binding fragment thereof) expressed by a host cell containing nucleic acid encoding the antibody. Examples of "host cells" for producing recombinant antibodies include: (1) mammalian cells, such as Chinese hamster ovary (CHO), COS, myeloma cells (including Y0 and NS0 cells), baby hamster kidney (BHK), Hela and Vero cells; (2) insect cells, such as sf9, sf21 and Tn5; (3) plant cells, such as plants belonging to the genus Nicotiana (e.g., Nicotiana tabacum); (4) yeast cells, such as those belonging to the genus Saccharomyces (e.g., Saccharomyces cerevisiae) or those belonging to the genus Aspergillus (e.g., Aspergillus niger); (5) bacterial cells, such as Escherichia coli cells or Bacillus subtilis cells. subtilis) cells etc.

[0095] The term "regulatory protein" as used herein refers to any protein that increases or decreases the activity of another polypeptide or RNA molecule; increases or decreases the abundance of another polypeptide or RNA molecule; changes the interaction between another polypeptide or RNA molecule and other polypeptides, DNA or RNA molecules, or any other binding substrate; and / or changes the cellular location of another polypeptide or RNA molecule. When a regulatory protein increases or decreases the rate of transcription of a gene, it is often referred to as a transcription factor that exerts an effect on the promoter or enhancer region of the gene. Examples of transcription factors include mammalian transcription factors such as NFkB, NF1, cyclic AMP response element binding protein (CREB), MyoD1, homeobox transcription factors, Spl, oncogenes and jun, Mep-1, GATA-1, Isl-1, LFB1, NFAT, Pit-1, OCA-B, Oct-1 and Oct-2, yeast A / alpha, cErb-A, myc, mad and max, p53, mdml, and others as described in Latchman, 1998, Eukaryotic Transcription Factors, 3rd. Ed., Academic Press: New York. Fusion protein derivatives of these or other transcription factors may also be used, in which at least the DNA-binding motif of the fusion protein, which provides the binding specificity, is fused to a small molecule regulator binding site.

[0096] The term "site-directed mutagenesis" as used herein refers to a process of generating a predetermined mutation at a defined site in a parent polypeptide. Defined site refers to a site selected according to the needs of the study. A predetermined mutation can be a substitution of an amino acid residue at the defined site with a specific amino acid, an insertion of a specific amino acid residue at the defined site, or a deletion of a specific amino acid at the defined site.

[0097] The term "small interfering RNA" or "siRNA" as used herein refers to an RNA or RNA-like molecule that can interact with and cause the destruction of an mRNA molecule that shares sequence homology with the siRNA (Elbashir et al., Genes Dev, vol. 15, pp. 188-200, 2001). It is believed that siRNAs can be incorporated into a ribonucleoprotein complex known as the RNA-induced silencing complex (RISC). RISC uses the siRNA sequence to identify mRNA molecules that are at least partially complementary to the incorporated siRNA strand, and then cleaves or inhibits the translation of these target mRNA molecules. Typical siRNAs are double-stranded nucleic acid molecules with each strand of about 19 to about 28 nucleotides (i.e., about 19, 20, 21, 22, 23, 24, 25, 26, 27, or 28 nucleotides). siRNAs can also be single-stranded RNA, but with less efficiency compared to double-stranded siRNAs. Single-stranded siRNAs have a length of about 19 to about 49 nucleotides. Single-stranded siRNA has a 5' phosphate or is phosphorylated in situ or in vivo at the 5' position. Single-stranded siRNA can be chemically or by in vitro transcription synthesis and endogenously expressed from an expression vector or expression cassette. The 5' phosphate group can be added via kinase or can be the result of nuclease cleavage of RNA.

[0098] The term "small molecule" refers to a molecule or ion that typically has a molecular weight 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. In the assays and environments of the present invention, small molecules may often exist as a mixture of the molecule and a deprotonated ion of the molecule, depending primarily on the pH of the assay or environment.

[0099] The term "therapeutic protein" as used herein refers to any protein and / or polypeptide that can be administered to a mammal to produce a biological or medical response in a tissue, system, animal or human that is being sought, for example, by a researcher or clinician. A therapeutic protein may produce more than one biological or medical response. Examples of therapeutic proteins include antibodies, enzymes, hormones, cytokines, regulatory proteins, and fragments thereof.

[0100] The term "therapeutically effective amount" as used herein means any amount that results in, but is not limited to, a cure, prevention, or amelioration of a disease, disorder, or side effect, or a slowing of the rate of progression of a disease or disorder, when compared to a corresponding subject not receiving such amount. The term also includes within its scope an amount effective to enhance normal physiological function, as well as an amount effective to cause a patient to have physiological function that enhances or supplements the therapeutic effect of a second pharmaceutical agent.

[0101] The term "tumor microenvironment" as used herein refers to the microenvironment within and around a solid tumor that supports tumor cell growth and metastasis. The tumor microenvironment includes surrounding blood vessels, immune cells, fibroblasts, other cells, soluble factors, signaling molecules, extracellular matrix, and mechanical cues that can promote neoplastic transformation, support tumor growth and invasion, protect the tumor from host immunity, develop therapeutic resistance, and provide a niche in which latent metastases can grow. A tumor and its surrounding microenvironment are intimately related and constantly interact. A tumor can affect its microenvironment by releasing extracellular signals, promoting tumor angiogenesis, and inducing peripheral immune tolerance, while immune cells in the microenvironment can affect the growth and evolution of cancerous cells. Swarts et al. “Tumor Microenvironment Complexity: Emerging Roles in Cancer Therapy,” Cancer Res, vol., 72, pages 2473-2480, 2012; Weber et al., “The tumor microenvironment,” Surgical Oncology, vol. 21, pages 172-177, 2012; Blagosklonny, “Antiangiogenic therapy and tumor progression,” Cancer See Cell, vol. 5, pages 13-17, 2004; Siemann, “Tumor microenvironment,” Wiley, 2010; and Bagley, “The tumor microenvironment,” Springer, 2010.

[0102] As used herein, the term "wild type" means that the polynucleotide does not contain any mutations. "Wild type protein", "wild-type protein", "wild-type biological protein", or "wild-type biological protein" may refer to a protein that can be isolated from nature, that can be active at an activity level found in nature and that can contain an amino acid sequence found in nature. The terms "parent molecule" and "target protein" also encompass wild-type proteins.

[0103] A. Conditionally Active Polypeptides The present disclosure provides a conditionally active polypeptide and a method for generating a conditionally active polypeptide from a parent polypeptide. The conditionally active polypeptide has an increased net charge compared to the parent polypeptide. The increase in net charge can be achieved by introducing one or more charged amino acid residues into the parent polypeptide or deleting one or more uncharged amino acid residues from the parent polypeptide, or both, to increase the net charge of the parent polypeptide. The charged amino acid residues are selected from aspartic acid, glutamic acid, arginine, lysine, and histidine. In one embodiment, the mutation can be a substitution that replaces one or more uncharged amino acid residues of the parent polypeptide with one or more charged amino acid residues. In another embodiment, the mutation can be an insertion that inserts one or more additional charged amino acid residues into the parent polypeptide. A combination of such substitutions and insertions can also be utilized. In another embodiment, the mutation can be a deletion of one or more uncharged amino acid residues from the parent polypeptide.

[0104] The charged amino acid residues introduced into the parent polypeptide are D (aspartic acid or Asp), E (glutamic acid or Glu), R (arginine or Arg), K (lysine or Lys) and H (histidine or His). D and E are negatively charged amino acid residues. R, K and H are positively charged amino acid residues. Aspartic acid, glutamic acid, lysine and arginine are typically charged at normal physiological pH. Histidine may also be charged at normal physiological pH.

[0105] A conditionally active polypeptide having an increased number of charged amino acid residues exhibits (a) a decrease in activity in a first assay at a first value of a condition compared to the same activity of the parent polypeptide in the first assay, and (b) an increase in the same activity in a second assay at a second value of the same condition compared to the same activity of the parent polypeptide in the second assay.

[0106] In some embodiments, a conditionally active polypeptide exhibits increased activity in a first assay at a first value of a condition, as compared to the same activity in a second assay at a second value of the same condition.

[0107] In one aspect, the conditionally active polypeptide can be a light chain variable region (SEQ ID NOs: 2-5) of an antibody against an Axl protein generated from a parent light chain variable region having SEQ ID NO: 1. In another aspect, the conditionally active polypeptide can be a heavy chain variable region (SEQ ID NOs: 7-9) of an antibody against an Axl protein generated from a parent heavy chain variable region having SEQ ID NO: 6. In yet another aspect, the conditionally active antibody can contain both a light chain variable region selected from SEQ ID NOs: 2-5 and a heavy chain variable region selected from SEQ ID NOs: 7-9.

[0108] In another embodiment, the conditionally active polypeptide is a light chain variable region (SEQ ID NOs: 11-13) of an antibody against Ror2 protein generated from a parent light chain variable region having SEQ ID NO: 10. In another embodiment, the conditionally active polypeptide is a heavy chain variable region (SEQ ID NOs: 15-18) of an antibody against Ror2 protein generated from a parent heavy chain variable region having SEQ ID NO: 14. In yet another embodiment, the conditionally active antibody may contain both a light chain variable region selected from SEQ ID NOs: 11-13 and a heavy chain variable region selected from SEQ ID NOs: 15-18.

[0109] Conditionally active antibodies against Axl or Ror2 are further described in Examples 15 to 22 of the present disclosure. The conditionally active light and heavy chain variable regions contain amino acid substitutions in which charged amino acid residues, specifically E and D, are introduced into the complementarity determining regions of the parent light and heavy chain variable regions.

[0110] The conditions under which the assay is performed are the same conditions but with different first and second values ​​or ranges. For example, the conditions are such that the second normal physiological pH (normal physiological conditions) is 7.0-7.8 or 7.2-7.6 and the first abnormal pH (as abnormal conditions) is 5.8-7.0 or 6.2-6.8. The conditions can also be, but are not limited to, temperature, concentration of electrolytes, concentration of organic molecules or osmotic pressure.

[0111] In one aspect, the condition is pH, and the conditionally active polypeptide has a pH-dependent activity in the presence of a species having a pKa within 2 pH units or 3 pH units of the pH at which activity is desired. In another aspect, the invention relates to a conditionally active polypeptide having a 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. In another aspect, the invention relates to a conditionally active polypeptide having a pH-dependent activity in the presence of a species selected from disulfide, bicarbonate, histidine, citrate, acetate, lactate, and mixtures thereof.

[0112] Species present in the assay medium that have a large 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. As a result, the pKa of a species that affects the activity of a conditionally active polypeptide may play a role in determining the extent of the effect that species may have on a particular activity of the polypeptide and / or at a particular pH.

[0113] A pH-dependent conditionally active polypeptide is more active at a first pH than at a second, different pH (both activities are measured in assays in the presence of one or more of the species listed above). 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.

[0114] The ratio of activity at a first pH to the same activity at a second pH in the same assay medium can be 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.

[0115] 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 that have at least two ionized states: an uncharged or low-charged state and a charged or high-charged state, may dissociate to a greater extent at a particular pH depending on the pKa value, thereby increasing the likelihood of interacting with charged amino acid residues present in the conditionally active polypeptide. This factor may be used to enhance the selectivity and / or pH-dependent activity of the conditionally active polypeptide.

[0116] Without being bound by theory, it is believed that the charged amino acid residues introduced into the parent polypeptide change the activity of the conditionally active polypeptide at different pHs by changing the charge on the conditionally active polypeptide when the pH of the assay medium is changed. For example, the negatively charged amino acid residues (e.g., E and D) of the conditionally active polypeptide are uncharged or less charged when the pH of the assay medium is in the acidic pH range, but are negatively charged when the pH of the assay medium is in the neutral or basic pH range. It is believed that the change in charge on the conditionally active polypeptide allows for different interactions of the polypeptide with one of the species discussed above at different pHs. In one embodiment, the conditionally active polypeptide is more likely to interact with a charged species in an assay medium at one pH, but less likely to interact with the same charged species in the same assay medium at another different pH. This is therefore believed to affect the activity of the conditionally active polypeptide.

[0117] The nature of the charge on the conditionally active polypeptide may be one factor used to determine the species suitable for affecting the activity of the conditionally active polypeptide. In some embodiments, the conditionally active polypeptide may have more positively charged amino acid residues: lysine, arginine, and histidine compared to the parent polypeptide. Thus, the conditionally active polypeptide may be selected to have a desired level of interaction with a particular species present in the environment in which activity is desired and / or to have a desired level of interaction with a particular species present in the environment in which reduced activity is desired. Similarly, the conditionally active polypeptide may have more negatively charged amino acid residues: aspartic acid and glutamic acid compared to the parent polypeptide.

[0118] The location of charged amino acid residues on a pH-dependent conditionally active polypeptide can also affect activity. For example, the proximity of charged amino acid residues to a binding site of a conditionally active polypeptide can be used to affect the activity of the polypeptide.

[0119] In some embodiments, the activity of a pH-dependent conditionally active polypeptide can be blocked or prevented when a charged environmental species interacts with the conditionally active polypeptide. For example, a charged amino acid that interacts with a charged environmental species can have an allosteric effect on the binding site of the conditionally active polypeptide.

[0120] In other embodiments, when a charged environmental species interacts with a conditionally active polypeptide, it may be possible that salt bridges can form between different moieties on the polypeptide, particularly between charged or polar moieties. The formation of salt bridges is known to stabilize polypeptide structures (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 minor structural fluctuations, 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). "Breathing" of protein structure is important for protein function and its binding with its partners because this structural fluctuation allows the conditionally active protein to efficiently recognize and bind to its partners (Karplus, et al., "Molecular dynamics and protein functions," PNAS, vol. 102, pp. 6679-6685, 2015). The formation of a salt bridge can make the binding site on the conditionally active polypeptide, especially the binding pocket, less accessible to the partner, possibly because the salt bridge can directly block the partner from reaching the binding site. Even a salt bridge that is far from the binding site can change the conformation of the binding site due to allosteric effects, inhibiting binding. Thus, after the salt bridge stabilizes (fixes) the structure of the conditionally active polypeptide, the polypeptide can become less active in terms of binding to its partners, resulting in reduced activity.

[0121] One known example of how polypeptides and their structures are stabilized by salt bridges is hemoglobin. Structural and chemical studies have revealed that salt bridges involve at least two pairs of chemical groups: the amino terminus and side chains of histidines β146 and α122, which have pKa values ​​close to 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 6). At high pH, ​​the side chain of histidine β146 is not protonated and no salt bridge is formed. However, as the pH decreases, the side chain of histidine β146 becomes protonated and a salt bridge forms between histidine β146 and aspartic acid β94, which stabilizes the quaternary structure of deoxyhemoglobin, leading to an increased tendency for oxygen to be released in actively metabolizing tissues (lower pH). Hemoglobin exhibits a pH-dependent binding activity for oxygen, where at low pH, the binding activity for oxygen decreases due to the formation of a salt bridge. On the other hand, at high pH, ​​the binding activity for oxygen increases due to the absence of the salt bridge.

[0122] Similarly, small molecules such as bicarbonate 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 lower than its pKa of 6.4, bicarbonate is protonated and therefore uncharged. Uncharged bicarbonate cannot form salt bridges and therefore has little effect on the binding of a conditionally active polypeptide to its partner. Thus, a conditionally active polypeptide has high binding activity with its partner at low pH. On the other hand, at a pH higher than the pKa of bicarbonate, bicarbonate is ionized by losing a proton and therefore becomes negatively charged. The negatively charged bicarbonate forms salt bridges between positively charged or polar moieties on the conditionally active polypeptide, stabilizing the structure of the conditionally active polypeptide. This can block or reduce the binding of the conditionally active polypeptide to its partner. Thus, the conditionally active polypeptide has low activity at high pH. Thus, a conditionally active polypeptide has a conditionally active activity in the presence of bicarbonate, with high binding activity at low pH compared to high pH.

[0123] In the absence of species such as bicarbonate in the assay medium, the 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 (fix) the structure of the polypeptide. Thus, the partner will have equal access to the binding site on the conditionally active polypeptide at any pH, resulting in comparable activity at the first and second pH.

[0124] In other embodiments, interaction between a small molecule or ion and a conditionally active polypeptide may alter the structure of the polypeptide in a manner that increases its activity. For example, the altered structure may improve the binding affinity of the conditionally active polypeptide by altering the position, steric hindrance, or binding energy of the binding site required for binding affinity. In such cases, it may be desirable to select a small molecule that binds to the conditionally active polypeptide at the pH where activity is desired.

[0125] Although salt bridges (ionic bonds) are the strongest and most common way in 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 polypeptides may also contribute to stabilizing (fixing) the structure of the conditionally active polypeptide. Such other interactions include hydrogen bonds, hydrophobic interactions, and van der Waals interactions.

[0126] In some embodiments, to select suitable compounds or ions, the conditionally active polypeptide is compared to the parent polypeptide from which it was evolved to determine whether the conditionally active polypeptide has a higher percentage of negatively charged amino acid residues or positively charged amino acid residues. Compounds with suitable charges at the second pH, respectively, can then be selected to affect the activity of the conditionally active polypeptide. For example, when the conditionally active polypeptide has a higher percentage of positively charged amino acid residues than the parent polypeptide, suitable small molecules typically must be negatively charged to interact with the conditionally active polypeptide at the second pH. On the other hand, when the conditionally active polypeptide has a higher percentage of negatively charged amino acid residues than the parent polypeptide, suitable small molecules typically must be positively charged to interact with the conditionally active polypeptide at the second pH.

[0127] 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 a small molecule or ion and a target polypeptide. The target polypeptide can be, for example, an antigen for a conditionally active antibody or a ligand for a conditionally active receptor.

[0128] A suitable small molecule may be any inorganic or organic molecule that transitions from an uncharged or low-charged state at a first pH to a charged or high-charged state at a second pH. Thus, the small molecule must typically have a pKa between the first and second pHs. For example, bicarbonate has a pKa of 6.4. Thus, at a higher pH, such as pH 7.4, the negatively charged bicarbonate may bind to the charged amino acid residues of the conditionally active polypeptide, reducing its activity. On the other hand, at a lower pH, such as pH 6.0, the low-charged bicarbonate may not bind to the conditionally active polypeptide in the same amount, allowing for a higher activity of the conditionally active polypeptide.

[0129] Disulfides have a pKa of 7.05. Thus, at higher pH, such as pH 7.4, more negatively charged disulfides may bind to the positively charged amino acid residues of the conditionally active polypeptide, reducing its activity. On the other hand, at lower pH, such as pH 6.2-6.8, less charged hydrogen sulfide / disulfide does not bind to the conditionally active polypeptide at the same level, thus allowing for higher activity of the conditionally active polypeptide.

[0130] Small molecules having a pKa between the first and second pH are preferred for use in the present invention. Preferred species are selected from disulfide, bicarbonate, histidine, histamine, citrate, acetate and lactate. Each of these small molecules has a pKa between 6.2 and 7.0. In addition, other small molecules such as tricine (pKa 8.05) and bicine (pKa 8.26) may also be used. For other suitable small molecules, reference may be made to textbooks using the principles of the present application, such as CRC Handbook of Chemistry and Physics, 96th Edition, by CRC press, 2015; Chemical Properties Handbook, McGraw-Hill Education, 1998.

[0131] The concentration of the small molecule in the assay medium or environment is preferably at or close to the physiological concentration of the small molecule 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 physiological concentration of disulfide is also low. The concentration of disulfide in the assay medium may be 3-100 mM, or 5-80 mM, or 10-50 mM, or 10-30 mM.

[0132] In the present invention, the conditionally active polypeptide is selected and utilized at a concentration such that the normal physiological concentration of the particular species in the environment can have a significant effect on the activity of the conditionally active polypeptide at the pH range of interest. Thus, for many therapeutic treatments, it may be advantageous for the conditionally active polypeptide to have low activity at about pH 7.2-7.4 of blood or human serum to allow delivery of the therapeutic treatment via the bloodstream while minimizing or preventing activation of the conditionally active polypeptide. As a result, it may be advantageous for such treatments to select a small molecule with a pKa below pH 7.2-7.4 to ensure that there is a sufficient amount of ionized small molecule at bloodstream pH to have a significant effect on the activity of the conditionally active polypeptide. At the same time, the pKa of the small molecule must be at or above the pH at which activity of the conditionally active polypeptide is desired to ensure activation of the conditionally active polypeptide by protonating the small molecule and freeing up a binding site on the conditionally active polypeptide.

[0133] Small molecules preferably have a low molecular weight and / or a relatively small conformation to ensure maximum access to small pockets on the target polypeptide or conditionally active polypeptide by minimizing steric hindrance. Thus, small molecules typically have 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, both disulfides and bicarbonates have low molecular weights and small structures that provide access to pockets on the target polypeptide or conditionally active polypeptide, as shown in Examples 13 and 14 below.

[0134] The small molecules may be present in the assay or environment at substantially the same concentration, e.g., about 20 μM for bicarbonate. In some embodiments, the small molecules may be present at different concentrations in different environments, and therefore it may be desirable to mimic this in the assay. For example, disulfide has a higher concentration in the tumor microenvironment than in human serum. Thus, one assay may mimic a tumor microenvironment with an acidic pH and a higher concentration of disulfide, while a second assay may mimic human serum with a neutral or slightly basic pH and a lower concentration of disulfide. The acidic pH may be in the range of 6.0-6.8, while the neutral or slightly basic pH may be about 7.4. The higher disulfide in the first assay may be 30 μM, while the lower disulfide in the second buffer may be 10 μM or less, or 5 μM.

[0135] In some embodiments, the conditionally active polypeptide is pH dependent in the presence of two or more different small molecules, for example, a combination of bicarbonate and histidine.

[0136] In the absence of a small molecule, the conditionally active polypeptide may lose its pH dependence, such that in the absence of a small molecule, the conditionally active polypeptide may have similar activity between a first pH and a second pH in the absence of the small molecule.

[0137] In some embodiments, the first pH is an acidic pH while the second pH is a basic or neutral pH. In other embodiments, the first pH is a basic pH while the second pH is an acidic or neutral pH. For example, the first pH may 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 second pH may be a pH in the range of about 7.0 to 7.8, or about 7.2 to 7.6.

[0138] Conditionally active polypeptides that are more active at acidic pH and less active at basic or neutral pH can target tumor microenvironments that are acidic at a pH of about 5.5-7.2, or about 6.2-6.8.

[0139] In other embodiments, the first pH at which the pH-dependent polypeptide is more active may be a basic pH, such as 7.6 to 7.9, such as that of 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 second pH may be the pH of blood, about 7.2 to 7.6, at which the conditionally active polypeptide is less active. These conditionally active polypeptides may be suitable for targeting joint diseases, particularly joint inflammation.

[0140] In other embodiments, the conditionally active polypeptide may be designed to target the brain. There is a pH difference on either side of the blood-brain barrier, with the pH on the brain side being about 0.2 pH units lower than the blood pH. Thus, the first pH of the brain, where the conditionally active polypeptide is more active, may be about 7.0-7.2 (brain pH), while the second pH may be about 7.4 (blood pH).

[0141] 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.

[0142] The conditionally active polypeptide may be a fragment of a 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 an Fc fragment of an antibody.

[0143] The Fc fragment may be used as a parent polypeptide to generate a conditionally active Fc fragment that has a higher complement binding activity at a first pH than the same complement binding activity at a second pH. The binding of the Fc fragment to complement can be used to effect antibody-dependent cell-mediated cytotoxicity. The first pH may be acidic, such as the pH of the tumor microenvironment, ranging from 5.5 to 7.2 or 6.2 to 6.8, while the second pH is in the range of 7.2 to 7.6. The first pH is different from the pH of lysosomes, which typically have a pH of about 4.0. Furthermore, lysosomes are where the Fc fragment is targeted for degradation like any other polypeptide. Lysosomes lack complement, and cell-mediated cytotoxicity cannot occur via lysosomes.

[0144] The conditionally active polypeptide may have two functional domains, at least one of which, preferably both, may have pH-dependent activity.These two functional domains may be simultaneously evolved and selected to identify both functional domains in the same mutant polypeptide.Alternatively, these two functional domains may be independently evolved and selected to identify pH-dependent activity separately.If the two functional domains are not in the same mutant polypeptide, they may be fused together as a chimeric polypeptide with both separately identified functional domains.

[0145] A conditionally active polypeptide is reversibly or irreversibly inactivated under a second condition or normal physiological conditions, but is active under a first condition or abnormal condition at the same or equivalent level as the second condition or normal physiological conditions. Such conditionally active polypeptides and methods for producing these polypeptides are described in U.S. Pat. No. 8,709,755 B2. Conditionally active polypeptides are particularly valuable for the development of novel therapeutic agents that are active in a host for a short or limited period of time. This is particularly valuable when limited activity is required to perform a desired therapy, while the prolonged action of a given dose of therapeutic agent may be harmful to the host. Examples of beneficial applications include high doses of local or systemic treatments, as well as high concentrations of local treatments. Inactivation under the second condition or normal physiological conditions can be determined by a combination of administration of the polypeptide and the rate of inactivation. This condition-based inactivation is particularly important for enzymatic therapeutic agents whose catalytic activity causes substantial harm in a relatively short period of time.

[0146] Conditionally active polypeptides are activated or inactivated reversibly or irreversibly over time, or are activated or inactivated only when in a particular microenvironment within the body, including a particular organ within the body (e.g., a tumor microenvironment, synovial fluid, bladder, or kidney). In some embodiments, the conditionally active polypeptide is an antibody or antibody fragment against one or more target proteins (antigens) as described herein.

[0147] The conditionally active polypeptide can be an isolated polypeptide having a pH-dependent activity, in the presence of a species selected from bicarbonate, histidine, histamine, disulfide, citrate, lactate, and combinations thereof, the activity at a first pH is at least about 1.3 times that at a second pH. The same activity is not pH-dependent in the absence of a small molecule. In some embodiments, the activity at a first pH is at least about 1.5 times, or at least about 1.7 times, or at least about 2.0 times, or at least about 3.0 times, or at least about 4.0 times, or at least about 6.0 times, or at least about 8.0 times, or at least about 10.0 times, or at least about 20.0 times, or at least about 40.0 times, or at least about 60.0 times, or at least about 100.0 times that at a second pH. The first pH can be an abnormal pH in the range of about 5.5 to 7.2 or about 6.2 to 6.8, and the second pH can be a normal physiological pH in the range of about 7.2 to 7.6.

[0148] With a selected conditionally active polypeptide, it is possible to identify or synthesize a nucleic acid, e.g., a DNA sequence, that encodes the conditionally active polypeptide. The nucleic acid can be used recombinantly to produce the conditionally active polypeptide in large quantities. For example, the nucleic acid can be inserted into an expression vector. In this case, the expression vector has a promoter operably linked to the nucleic acid to ensure high expression levels of the conditionally active polypeptide. The expression vector can be introduced into a host cell, such as a yeast, insect cell production host, or mammalian cell production host. Examples of host cells include 3T3 mouse fibroblast cells; BHK21 Syrian hamster fibroblast cells; MDCK, canine epithelial cells; Hela human epithelial cells; PtK1 rat kangaroo epithelial cells; SP2 / 0 mouse plasma cells; and NS0 mouse plasma cells; HEK293 human embryonic kidney cells; COS monkey kidney cells; CHO, CHO-S Chinese hamster ovary cells; R1 mouse embryo cells; E14.1 mouse embryo cells; H1 human embryo cells; H9 human embryo cells; and PER C.6, human embryo cells.

[0149] B. Engineering Conditionally Active Polypeptides The conditionally active polypeptide may be engineered by one or more of the protein engineering techniques described herein. Non-limiting examples of protein engineering techniques include conjugating the conditionally active polypeptide to a nucleic acid, conjugating the conditionally active polypeptide to a nanoparticle, engineering the conditionally active polypeptide in a chimeric antigen receptor, and engineering a masked conditionally active polypeptide.

[0150] The conditionally active polypeptide of the present invention can be conjugated to a nucleic acid molecule, such as a DNA or RNA molecule, via a linker. The conditionally active polypeptide can facilitate delivery of the nucleic acid molecule to a target location in a subject that has a condition in which the conditionally active polypeptide is more active than other locations where the condition does not exist. For example, the conditionally active polypeptide can be a conditionally active antibody that has a higher binding activity to its antigen under conditions in a tumor microenvironment than conditions in other locations, such as human serum. Using this effect, the nucleic acid molecule can be delivered to the tumor microenvironment by conjugating the nucleic acid molecule to the conditionally active polypeptide and administering the conjugate to a subject.

[0151] In some embodiments, the nucleic acid molecule may be an agent that regulates the expression of a gene at a target location. Aberrant gene expression is associated with many diseases. Correcting aberrant gene expression may therefore contribute to the control or even cure of these diseases. For example, aberrant gene expression is characteristic of the majority of cancer cells, where some genes, such as many cancer genes, have elevated expression levels (e.g., epidermal growth factor receptor 2 (HER2) is overexpressed in breast cancer cells). Selective inhibition of the constitutively elevated expression of cancer genes provides an opportunity to inhibit the proliferation of cancer cells.

[0152] Nucleic acid molecules that can inhibit gene expression include antisense RNA, small interfering RNA (siRNA), microRNA, oligo-DNA, and oligonucleotide mimetics with uncharged achiral polyamide backbones linking the nucleobases (Pooga et al., Curr Cancer Drug Targets, 1(3):231-9, 2001; Pandey et al., Expert Opin Biol Ther., 9(8):975-89, 2009).

[0153] Antisense RNA is a short RNA molecule that can bind to a specific complementary region of an mRNA by base pairing to inhibit the expression of the mRNA in a sequence-specific manner. Antisense RNA can contain RNase H, which can cleave the mRNA at the site of binding to the antisense RNA or physically block translation or other steps in mRNA processing and protein synthesis.

[0154] Small interfering RNA (siRNA) is typically a short double-stranded RNA segment with at least a portion of its sequence complementary to the mRNA sequence to be blocked from translation. siRNA functions by post-transcriptional mechanisms of gene silencing using chromatin remodeling, inhibition of protein translation, or direct mRNA degradation, and is ubiquitous in eukaryotic cells (Caplen, "Gene therapy progress and prospects. Downregulating gene expression: the impact of RNA interference," Gene Ther., 11(16):1241-1248, 2004) and Bertrand et al., "Comparison of antisense oligonucleotides and siRNAs in cell culture and in vivo," Biochem Biophys Res Commun., 296(4):1000-1004, 2002).

[0155] In particular, through RISC, siRNAs can initiate a potent cascade of sequence-specific degradation of mRNAs that share homology with the siRNA (Fire et al., "Potent and specific genetic interference by double-stranded RNA in Caenorhabditis elegans," Nature 391:806-811, 1998). When siRNAs are introduced into cells, they are processed by an RNase III enzyme called Dicer, which cleaves long siRNAs into short 21-23 nucleotide duplexes with symmetric 2-3 nucleotide 3' overhangs and 5' phosphate and 3' hydroxyl groups (Tuschl et al., "Targeted mRNA degradation by double-stranded RNA in vitro," Genes Dev. 13:3191-3197, 1999; Hamilton and Baulcombe, "A species of small antisense RNA in posttranscriptional gene silencing in plants," Science, 286:950-952, 1999). Thus, effective siRNAs only require small segments of contiguous complementary sequence to pair with mRNA to initiate siRNA-mediated silencing (Jackson and Linsley, "Noise amidst the silence: off-target effects of siRNAs?" Trends Genet., 20:521-524, 2004). siRNAs do not integrate into the genome and therefore offer increased safety compared to plasmid or viral vehicles.

[0156] MicroRNAs (miRNAs) are a class of naturally occurring small non-coding RNA molecules 21–25 nucleotides long. MicroRNAs are partially complementary to the mRNA molecules on which they act. The main function of microRNAs is to reduce gene expression by translational repression, mRNA cleavage, and deadenylation. A central online repository of miRNA species, sequence data, annotations, and target predictions is called miRBase and is maintained by the Sanger Institute, UK. When microRNA genes are transcribed by RNA polymerase II, pri-miRNAs with 5' caps and polyA tails are produced. In the nucleus, pri-miRNAs are processed by the microprocessor complex, consisting of the RNaseIII enzyme Drosha and the double-stranded RNA Pasha / DGCR8, to generate pre-miRNAs. These pre-miRNAs are transported to the cytoplasm by the karyopherin exportin (Exp5) and Ran-GTP complexes, where Ran GTPase binds Exp5 to form nuclear heterotrimers with the pre-miRNAs. Further processing of these pre-miRNAs by the RNase III enzyme Dicer produces mature microRNAs.

[0157] Another class of nucleic acids that can be delivered by conditionally active polypeptides are oligonucleotide mimetics that contain an uncharged, achiral polyamide backbone to which the nucleobases are linked. These oligonucleotide mimetics are often referred to as peptide nucleic acids (PNAs). More specifically, PNAs are DNA analogs in which N-(2-aminoethyl)glycine polyamides replace the phosphate-ribose ring backbone, and methylene carbonyl linkers attach natural and unnatural nucleobases to the central amine of N-(2-aminoethyl)glycine. Despite the radical changes in backbone structure, PNAs have the ability to bind sequence-specifically to DNA and mRNA according to Watson-Crick base pairing rules.

[0158] PNAs bind to complementary DNA / RNA with higher affinity than natural nucleic acids, due in part to the lack of negative charges in the backbone, resulting in reduced charge-charge repulsion, as well as favorable geometric factors. PNA-DNA / mRNA complexes are highly stable in biological fluids, leading to inhibition of transcription and translation of target genes by specifically hybridizing with DNA or mRNA. Generally, PNAs are synthesized using well-known solid-phase peptide synthesis protocols. Kim et al.,J.Am.Chem.Soc.,115,6477-6481,1993;Hyrup et al.,J.Am.Chem.Soc.,116,7964-7970,1994;Egholm et al.,Nature,365,566-568,1993;Dueholm et al.,New J.Chem.,21,19-31,1997;Wittung et al.,J.Am.Chem.Soc.,118,7049-7054,1996;Leijon et al.,Biochemistry,33,9820-9825,1994,Orum et al. al.,BioTechniques,19,472-480,1995;Tomac et al. (See, e.g., et al., J. Am. Chem. Soc., 118, 5544-5552, 1996.) In contrast to DNA, which is depurinated when treated with strong acid and hydrolyzed in alkaline hydroxides, PNA is completely acid-stable and quite stable to weak bases.

[0159] Another class of nucleic acid that can be delivered by conditionally active polypeptides is oligo-DNA. Oligo-DNA is a short single-stranded segment of DNA that can selectively inhibit the expression of genes containing sequences complementary to the oligo-DNA upon entry into cellular plasma. For antisense applications, oligo-DNA interacts with target mRNA or pre-mRNA to form a duplex and inhibit its translation or processing, resulting in inhibition of protein biosynthesis. For antigen applications, oligo-DNA must enter the cell nucleus and form a triplex with double-stranded genomic DNA to inhibit gene transcription, thus leading to less mRNA produced and less protein gene product produced.

[0160] A further class of nucleic acids that can be delivered by conditionally active polypeptides are spherical nucleic acids (SNAs, see Zhang, J Am Chem Soc., 134(40):16488-16491, 2012). SNAs include densely functionalized and highly oriented nucleic acids covalently attached to the surface of metallic, semiconducting, or insulating inorganic or polymeric core materials. SNAs may also be coreless hollow structures composed almost entirely of nucleic acid molecules. Such spherical nucleic acids have the ability to circumvent the subject's natural defenses against exogenous nucleic acids. Spherical nucleic acids exploit the unique properties resulting from their densely packed, highly oriented nucleic acid shell to provide protection and efficient delivery of nucleic acids. Such shells create regions of high local salt concentration, which, in combination with steric inhibition, serve to reduce nuclease activity and protect nucleic acids from enzymatic degradation. In addition, these spherical nucleic acids recruit scavenger proteins from the natural extracellular environment to their surface, which facilitates endocytosis.

[0161] After entering the cytoplasm, spherical nucleic acid can inhibit the expression of target genes by either antisense or siRNA pathways.As a result, spherical nucleic acid offers several advantages over viral vectors and many other synthetic systems, including low toxicity, low immunogenicity, resistance to enzymatic degradation, and more sustained gene knockdown.Conditionally active polypeptides, particularly conditionally active antibodies, can deliver spherical nucleic acid to target locations, such as diseased or inflamed tissues (e.g., tumors and inflamed joints).

[0162] The conditionally active polypeptide can also be conjugated to a nanoparticle via a linker to facilitate delivery of the nanoparticle to a target location that has the condition in which the conditionally active polypeptide is more active. Nanoparticles are known vehicles for toxins, radiopharmaceuticals or other therapeutic agents that can be encapsulated in the nanoparticle.

[0163] The therapeutic agent encapsulated in the nanoparticles may be a protein that dedifferentiates tumor cells and thus may reverse tumor cells back to normal cells (Friedmann-Morvinski and Verma, “Dedifferentiation and reprogramming: origins of cancer stem cells,” EMBO Reports, 15(3):244-253, 2014). By linking the nanoparticles to a conditionally active antibody, the linked nanoparticles and the encapsulated therapeutic agent may be selectively delivered to the environment in which the conditionally active antibody is most active.

[0164] The present invention may use several types of nanoparticles with different configurations. Nanoparticles may be made of various biocompatible materials, including biostable polymers, biodegradable polymers, fullerenes, lipids, or combinations thereof. Biostable polymers refer to polymers that do not degrade in vivo. Biodegradable polymers refer to polymers that can be degraded after delivery to a patient. For example, if the polymer is exposed to bodily fluids such as blood, the polymer may be gradually absorbed and / or excreted by enzymes in the body. Methods for making nanoparticles with different degradation rates are known to those skilled in the art, see, for example, U.S. Pat. Nos. 6,451,338, 6,168,804, and 6,258,378.

[0165] Exemplary nanoparticles of the present invention include liposomes, polymersomes, and polymer particles. Liposomes refer to compartments that are completely surrounded by a bilayer, typically composed of phospholipids. Liposomes can be prepared according to standard techniques known to those skilled in the art. One technique is by suspending a suitable lipid, such as phosphatidylcholine, in an aqueous medium, followed by sonication of the mixture. Another technique is by rapidly mixing a solution of lipid in ethanol-water, for example by injecting the lipid with a needle into a stirred ethanol-water solution. In some embodiments, liposomes can also contain other amphiphilic substances, such as sphingomyelin or poly(ethylene glycol) (PEG), in addition to or instead of the lipid.

[0166] Polymersomes contain diblock or triblock copolymers that can be modified to form bilayer structures similar to liposomes. Depending on the length and composition of the block copolymer, polymersomes can be substantially more robust than liposomes. In addition, the chemistry of each block of the block copolymer can be controlled, allowing the composition of the polymersome to be tailored to suit the desired application. For example, the membrane thickness of the polymersome, i.e., the thickness of the bilayer structure, can be controlled by varying the chain length of the individual blocks in the block copolymer. Adjusting the glass transition temperature of each block of the copolymer can affect the fluidity and therefore the permeability of the polymersome membrane. By varying the properties of the copolymer, it is even possible to modify the release mechanism of the encapsulated drug.

[0167] Polymersomes can be prepared by a process that involves (i) dissolving a block copolymer in an organic solvent, (ii) applying the resulting solution to a vessel surface, and then (iii) removing the solvent, thereby leaving a thin film of the copolymer on the vessel wall. The thin film is then hydrated to form polymersomes. Alternatively, polymersomes can be created by dissolving the block copolymer in a solvent and then adding a weak solvent for one of the blocks of the copolymer.

[0168] Therapeutic agents can be encapsulated in polymersomes using several techniques. For example, the therapeutic agent may be mixed into water and then used to rehydrate the copolymer thin film. Another example is by osmotically forcing the therapeutic agent into the core of a preformed polymersome, a process known as forced loading. Another example is by using a double emulsion technique, which can produce polymersomes with relatively monodispersity and high loading efficiency. The double emulsion technique involves using microfluidic techniques to generate double emulsions containing water droplets surrounded by an organic solvent layer. This droplet-in-a-drop structure is then dispersed in a continuous aqueous phase. The block copolymer dissolves in the organic solvent and self-assembles on the concentric interface of the double emulsion into a protopolymersome. The final polymersome is formed after complete evaporation of the organic solvent from the shell of the protopolymersome. This technique allows fine tuning of the size of the polymersome. In addition, the inner fluid can be kept completely separated from the outer fluid throughout the entire process, allowing for extremely efficient encapsulation of the therapeutic agent.

[0169] Polymer particles refer to solid or porous particles, in contrast to the shell structures of liposomes and polymersomes. Methods for attaching therapeutic agents to the surface of polymer particle structures or incorporating bioactive agents therein are known to those skilled in the art.

[0170] Polymers that may be used to prepare the nanoparticles of the invention include, but are not limited to, poly(N-acetylglucosamine) (chitin), chitosan, poly(3-hydroxyvalerate), poly(lactide-co-glycolide), poly(3-hydroxybutyrate), poly(4-hydroxybutyrate), poly(3-hydroxybutyrate-co-3-hydroxyvalerate), polyorthoesters, polyanhydrides, poly(glycolic acid), poly(glycolide), poly(L-lactic acid), poly(L-lactide), poly(D,L-lactic acid), poly(D,L-lactide). , poly(L-lactide-co-D,L-lactide), poly(caprolactone), poly(L-lactide-co-caprolactone), poly(D,L-lactide-co-caprolactone), poly(glycolide-co-caprolactone), poly(trimethylene carbonate), polyesteramides, poly(glycolic acid-co-trimethylene carbonate), co-poly(ether esters) (e.g., PEO / PLA), polyphosphazenes, biomolecules (fibrin, fibrin glue, fibrinogen, cellulose, starch, collagen and hyaluronan). acid, elastin and hyaluronic acid, polyurethanes, silicones, polyesters, polyolefins, polyisobutylene and ethylene-alpha-olefin copolymers, acrylic polymers and copolymers other than polyacrylates, halogenated vinyl polymers and copolymers (e.g. polyvinyl chloride), polyvinyl ethers (e.g. polyvinyl methyl ether), halogenated polyvinylidene (e.g. polyvinylidene chloride), polyacrylonitrile, polyvinyl ketones, polyvinyl aromatics (e.g. polystyrene), polyvinyl esters (poly vinyl acetate, acrylonitrile-styrene copolymers, acrylonitrile butadiene styrene (ABS) resins, polyamides (nylon 66 and polycaprolactam, etc.), polycarbonates including tyrosine-based polycarbonates, polyoxymethylenes, polyimides, polyethers, polyurethanes, rayon, rayon triacetate, cellulose, cellulose acetate, cellulose butyrate, cellulose acetate butyrate, cellophane, cellulose nitrate, cellulose propionate, cellulose ethers, and carboxymethyl cellulose.

[0171] In some embodiments, the nanoparticles may also provide tissue selectivity through coating in addition to the selectivity derived from the conditionally active polypeptide. For example, the nanoparticles may be coated with an electrostatically adsorbed poly(glutamic acid)-based peptide coating to alter the outer composition of the core particle. A negatively charged polyglutamic acid-based peptide containing arginine-glycine-aspartic acid (RGD) ligand can increase in vitro gene delivery to endothelial cells compared to coated particles with scrambled sequences that contain RDG instead of RGD. The peptide consists of three components: a stretch of poly(glutamic acid) that provides a negative charge, a polyglycine linker, and a terminal sequence that varies in charge and may alter the biophysical properties and tissue selectivity of the particle. The coating as well as the particle itself are biodegradable through their amide and ester bonds, respectively. See Harris et al. ("Tissue-Specific Gene Delivery via Nanoparticle Coating," Biomaterials, vol. 31, pp. 998-1006, 2010).

[0172] T cells are used by the mammalian immune system to fight against substances or cells that carry foreign antigens. When T cells encounter solid tumors, they often fail to mount an effective response. Even if T cells reach the tumor site, they face a barrage of immunosuppressive factors that allow the cancer cells to escape the immune system. CAR-T technology involves reprogramming natural circulating T cells by inserting a chimeric antigen receptor (CAR) into the T cells using genetic engineering methods to create highly specific CAR-T cells, in which the CAR specifically binds to an antigen on the surface of the target tissue, thereby directing the engineered CAR-T cells to the target tissue. Thus, CAR-T cells can specifically target tumor cells, making them much more effective than natural circulating T cells. CAR-T cells can also be engineered to target other target tissues, such as inflamed joints and brain tissue.

[0173] The CAR of the present invention comprises at least one antigen-specific targeting region (ASTR), an extracellular spacer domain (ESD), a transmembrane domain (TM), one or more costimulatory domains (CSD), and an intracellular signaling domain (ISD). See FIG. 5 and Jensen et al., “Design and implementation of adoptive therapy with chimeric antigen receptor-modified T cells,” Immunol Rev., vol. 257, pp. 127-144, 2014. After the ASTR specifically binds to a target antigen on a tumor or other target tissue, the ISD activates intracellular signaling of the CAR-T cell. For example, the ISD utilizes the antigen-binding properties of antibodies to redirect CAR-T cell specificity and reactivity to a target of choice (e.g., tumor cells or other target cells) in an MHC-unrestricted manner. This MHC-unrestricted antigen recognition allows CAR-T cells to recognize tumor cells and initiate antigen processing, thus circumventing a major mechanism of tumor escape from immune system surveillance. In certain embodiments, the ESD and / or CSD are optional. In another embodiment, the ASTR has bispecificity and is therefore capable of specifically binding to two different antigens or epitopes.

[0174] The conditionally active polypeptide of the present invention may be engineered as an ASTR or a portion thereof, so that the CAR is more active in a specific environment, such as a tumor microenvironment or synovial fluid, in binding to a target antigen, compared to blood or other body sites where a different environment exists. Such a CAR can preferentially deliver T cells to disease sites, thus dramatically reducing the side effects caused by T cells attacking normal tissues. This allows the use of higher doses of T cells, increasing therapeutic efficacy and improving the subject's tolerance to the treatment.

[0175] These CARs are particularly useful for the development of novel therapeutics that require a short or limited time in the subject's body. Beneficial applications include systemic treatments at high dosages, as well as localized treatments at high concentrations. See Maher, "Immunotherapy of Malignant Disease Using Chimeric Antigen Receptor Engrafted T Cells," ISRN Oncology, vol. 2012, article ID 278093, 2012.

[0176] ASTRs may include conditionally active polypeptides, such as antibodies, particularly single chain antibodies, or fragments thereof, that specifically bind to antigens on tumors or other target tissues. Some examples of polypeptides suitable for ASTRs include linked cytokines (which provide for recognition of cells bearing cytokine receptors), affibodies, ligand binding domains from naturally occurring receptors, and soluble protein / peptide ligands for receptors, for example, on tumor cells. Virtually any molecule capable of binding with high affinity to a given antigen can be used for an ASTR.

[0177] In some embodiments, the CAR of the present invention comprises at least two ASTRs that target at least two different antigens or two epitopes on the same antigen. In one embodiment, the CAR comprises three or more ASTRs that target at least three or more different antigens or epitopes. When multiple ASTRs are present in a CAR, the ASTRs may be arranged in tandem and separated by a linker peptide (Figure 5).

[0178] In yet another embodiment, the ASTR comprises a diabody, in which the scFv is generated with a linker peptide that drives dimerization of the scFv because the two variable regions are too short to fold together. Even shorter linkers (1 or 2 amino acids) lead to the formation of trimers, so-called triabodies or tribodies. Tetrabodies can also be used in ASTRs.

[0179] The antigens targeted by the CAR are present on the surface or inside the cells of the tissue targeted for removal, such as tumors, glandular (e.g., prostate) hyperplasia, warts, and unwanted adipose tissue. Surface antigens are more efficiently recognized and bound by the ASTR of the CAR, but intracellular antigens can also be targeted by the CAR. In some embodiments, the target antigen is preferably specific to cancer, inflammatory disease, neuronal disorder, diabetes, cardiovascular disease, or infectious disease. Examples of target antigens include antigens expressed by various immune cells, carcinomas, sarcomas, lymphomas, leukemias, germ cell tumors, blastomas, and cells associated with various hematological, autoimmune, and / or inflammatory diseases.

[0180] Cancer-specific antigens that can be targeted by ASTR include 4-IBB, 5T4, adenocarcinoma antigen, alpha-fetoprotein, BAFF, B lymphoma cells, C242 antigen, CA-125, carbonic anhydrase 9 (CA-IX), C-MET, CCR4, CD152, CD19, CD20, CD200, CD22, CD221, CD23 (IgE receptor), CD28, CD30 (TNFRSF8), CD33, CD4, CD40, and CD44. v6, CD51, CD52, CD56, CD74, CD80, CEA, CTLA-4, DR5, EGFR, EpCAM, CD3, FAP, fibronectin extra domain-B, folate receptor 1, GD2, GD3 ganglioside, glycoprotein 75, GPNMB, HER2 / neu, HGF, human scatter factor receptor kinase, IGF-1 receptor, IGF-I, IgG1, LI-CAM, IL-13, IL-6, insulin-like growth factor I receptor, integrin α5β1, integrin ανβ3, MORAb-009, MS4A1, MUC1, mucin CanAg, N-glycolylneuraminic acid, NPC-1C, PDGF-R a, PDL192, phosphatidylserine, prostate cancer cells, RANKL, RON, ROR1, SCH 900105, SDC1, SLAMF7, TAG-72, tenascin-C, TGFβ2, TGF-β, TRAIL-R1, TRAIL-R2, tumor antigen CTAA16.88, VEGF-A, VEGFR-1, VEGFR2 or vimentin.

[0181] Antigens specific to inflammatory diseases that can be targeted by ASTR include AOC3 (VAP-1), CAM-3001, CCL11 (eotaxin-1), CD125, CD147 (basigin), CD154 (CD40L), CD2, CD20, CD23 (IgE receptor), CD25 (a chain of the IL-2 receptor), CD3, CD4, CD5, IFN-a, IFN-gamma, IgE, IgE Fc region, IL-1, IL-12, IL-23, IL-13, IL-17, IL-17A, IL-22, IL-4, IL-5, IL-6, IL-6 receptor, integrin a4, integrin α4β7, Lama glama, LFA-1 (CD11a), MEDI-528, myostatin, OX-40, and rhuMAb. These include one or more of β7, scleroscin, SOST, TGFβ1, TNF-a, or VEGF-A.

[0182] Antigens specific to neuronal disorders that can be targeted by the ASTR of the present invention include one or more of β-amyloid or MABT5102A. Antigens specific to diabetes that can be targeted by the ASTR of the present invention include one or more of L-Iβ or CD3. Antigens specific to cardiovascular diseases that can be targeted by the ASTR of the present invention include one or more of C5, cardiac myosin, CD41 (integrin α-lib), fibulin II, β chain, ITGB2 (CD18), and sphingosine-1-phosphate.

[0183] Antigens specific to infectious diseases that may be targeted by the ASTRs of the invention include one or more of anthrax toxin, CCR5, CD4, clumping factor A, cytomegalovirus, cytomegalovirus glycoprotein B, endotoxin, Escherichia coli, hepatitis B surface antigen, hepatitis B virus, HIV-1, Hsp90, influenza A hemagglutinin, lipoteichoic acid, Pseudomonas aeruginosa, rabies virus glycoprotein, respiratory syncytial virus, and TNF-a.

[0184] Further examples of target antigens include surface proteins found in a specific or amplified manner on cancer cells, such as the IL-14 receptor, CD19, CD20 and CD40 of B-cell lymphomas, Lewis Y and CEA antigens of various cancers, Tag72 antigen of breast and colorectal cancers, EGF-R of lung cancer, folate binding protein and HER-2 protein, which are often amplified in human breast and ovarian cancers, or viral proteins, such as the gp120 and gp41 envelope proteins of HIV, envelope proteins from hepatitis B and C viruses, glycoprotein B and other envelope glycoproteins of human cytomegalovirus, and envelope proteins from oncoviruses such as Kaposi's sarcoma-associated herpesvirus. Other potential target antigens include CD4, whose ligand is the HIV gp120 envelope glycoprotein, and other viral receptors, such as ICAM, the receptor for human rhinovirus, and related receptor molecules for poliovirus.

[0185] In another embodiment, the CAR can target antigens associated with cancer therapeutic cells, such as NK cells, to activate these cancer therapeutic cells by acting as immune effector cells. An example of this is a CAR that targets the CD16A antigen to engage NK cells to fight CD30-expressing malignancies. The bispecific tetravalent AFM13 antibody is an example of an antibody that can deliver this effect. For further details of this type of embodiment, see, for example, Rothe, A., et al., "A phase 1 study of the bispecific anti-CD30 / CD16A antibody construct AFM13 in patients with relapsed or refractory Hodgkin lymphoma," Blood, 25 June 2015, Vol. 125, no. 26, pp. 4024-4031.

[0186] In some embodiments, the extracellular spacer domain and the transmembrane domain may be ubiquitination resistant, which can enhance CAR-T cell signaling and thus increase anti-tumor activity (Kunii et al., "Enhanced function of redirected human t cells expressing linker for activation of t cells that is resistant to ubiquitylation," Human Gene Therapy, vol. 24, pp. 27-37, 2013). Within this region, the extracellular spacer domain is on the outside of the CAR-T cell and therefore exposed to different conditions, potentially making it conditionally ubiquitination resistant.

[0187] Engineering masked conditionally active polypeptides The conditionally active polypeptides of the present invention, particularly the conditionally active antibodies, may have their conditional activity masked by a masking moiety and / or the activity of their conjugated agents masked. The masked activity may become available when the masking moiety is removed or cleaved from the conditionally active polypeptide. Suitable masking techniques are described, for example, in Desnoyers et al., "Tumor-Specific Activation of an EGFR-Targeting Probody Enhances Therapeutic Index," Sci.Transl.Med.5,207ra144,2013.

[0188] In some embodiments, the conditionally active antibody is linked to a masking moiety that masks the conditional activity and / or the activity of its conjugated drug. For example, when the conditionally active antibody is coupled to a masking moiety, such coupling or modification may cause a conformational change that reduces or inhibits the conditionally active antibody's ability to specifically bind to its antigen. When the conditionally active antibody reaches the target tissue or microenvironment, the masking moiety is cleaved by an enzyme present in the target tissue or microenvironment, thus releasing the masked activity. For example, the enzyme may be a protease that is generally active in the tumor microenvironment, which may cleave the masking moiety to release the conditionally active antibody that is active in the tumor tissue.

[0189] In some embodiments, the activity is masked to be less than about 50% of the original activity, or less than about 30% of the original activity, or less than about 10% of the original activity, or less than about 5% of the original activity, or less than about 2% of the original activity, or less than about 1% of the original activity, or less than about 0.1% of the original activity, or less than about 0.01% of the original activity. In some embodiments, for example, to ensure adequate time for delivery, the masking effect is designed to last at least 2, 4, 6, 8, 12, 28, 24, 30, 36, 48, 60, 72, 84, 96 hours, or 5, 10, 15, 30, 45, 60, 90, 120, 150, 180 days, or 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 months or more, as measured in vivo or in a target transfer in vitro immunoabsorption assay.

[0190] In certain embodiments, the masking moiety is structurally similar to the natural binding partner (antigen) of the conditionally active antibody. The masking moiety may be a modified version of the natural binding partner of the conditionally active antibody, which contains amino acid changes that at least slightly reduce the affinity and / or avidity of binding to the conditionally active antibody. In some embodiments, the masking moiety is free or substantially free of homology with the natural binding partner of the conditionally active antibody. In other embodiments, the masking moiety has no more than 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, or 80% sequence identity with the natural binding partner of the conditionally active antibody.

[0191] The masking moiety may be provided in a variety of different forms. In certain embodiments, the masking moiety may be a known binding partner of the conditionally active antibody, provided that the masking moiety binds to the conditionally active antibody with a lower affinity and / or avidity than the target protein targeted by the conditionally active antibody after cleavage of the masking moiety, to reduce interference of the masking moiety with the desired binding to the target. Thus, the masking moiety preferably masks the conditionally active antibody from target binding before cleavage of the masking moiety, but does not substantially or significantly interfere with or compete for binding of the active molecule to the target after cleavage of the masking moiety from the antibody. In specific embodiments, the conditionally active antibody and the masking moiety do not comprise the amino acid sequence of a naturally occurring binding partner pair, and thus at least one of the conditionally active antibody and the masking moiety does not have the amino acid sequence of a member of a naturally occurring binding partner.

[0192] Alternatively, the masking moiety may not specifically bind to the conditionally active antibody, but rather may prevent conditionally active antibody-target binding through non-specific interactions such as steric hindrance, etc. For example, the masking moiety may be located such that the structure or conformation of the antibody allows the masking moiety to mask the conditionally active antibody, e.g., through charge-based interactions, thereby preventing the target from reaching the conditionally active antibody.

[0193] In some embodiments, the masking moiety is coupled to the conditionally active antibody by a covalent bond. In another embodiment, the conditionally active antibody is prevented from binding to its target by attaching the masking moiety to the N-terminus of the conditionally active antibody. In yet another embodiment, the conditionally active antibody is coupled to the masking moiety by a cysteine-cysteine ​​disulfide bridge between the masking moiety and the conditionally active antibody.

[0194] In some embodiments, the conditionally active antibody is further coupled to a cleavable moiety (CM). The CM has the ability to be cleaved by an enzyme, or the CM has the ability to be reduced by a reducing agent, or the CM has the ability to be photolyzed. In one embodiment, the amino acid sequence of the CM can overlap or be included within the masking moiety. In another embodiment, the CM is between the conditionally active antibody and the masking moiety. It should be noted that all or a portion of the CM may facilitate masking of the conditionally active antibody prior to cleavage. Once the CM is cleaved, the conditionally active antibody becomes more active in binding to its antigen.

[0195] The CM may be a substrate for an enzyme that is co-present with the target antigen at the treatment site of the subject. Alternatively or in addition, the CM may have an intercysteine ​​disulfide bond that is cleavable as a result of the disulfide bond being reduced. The CM may also be a photolabile substrate that can be activated by a light source.

[0196] In the desired target tissue of the conditionally active antibody, the enzyme that cleaves the CM should be preferentially located, where the conditionally active antibody is more active under conditions (abnormal conditions) exhibited in the target tissue, such as diseased or tumor tissue. For example, there are known proteases whose levels are increased in a number of cancers, such as solid tumors. See, for example, La Rocca et al, (2004) British J. of Cancer 90(7):1414-1421. Non-limiting examples of such diseases include all types of cancer (breast cancer, lung cancer, colorectal cancer, prostate cancer, head and neck cancer, pancreatic cancer, etc.), rheumatoid arthritis, Crohn's disease, melanoma, SLE, cardiovascular injury, ischemia, etc. Thus, a suitable CM can be selected that contains a peptide substrate that can be cleaved by a protease present in tumor tissue, particularly a protease that is present at a higher level in tumor tissue as compared to non-cancerous tissue.

[0197] In some embodiments, the CM can be a substrate for an enzyme selected from legumain, plasmin, TMPRSS-3 / 4, MMP-9, MT1-MMP, cathepsin, caspase, human neutrophil elastase, β-secretase, uPA, and PSA. The enzyme that cleaves the CM is present at a relatively higher level in the target tissue at the treatment site (e.g., diseased tissue or tumor tissue; e.g., subject to therapeutic or diagnostic treatment) compared to the tissue at the non-treatment site (e.g., healthy tissue). Thus, in addition to the conditional activity of the antibody, which may be more active in the diseased or tumor tissue, the enzymes present in the diseased or tumor tissue can cleave the CM, which further enhances the activity of the conditionally active antibody, or the activity of the conjugated drug. The unmodified or uncleaved CM can allow for efficient inhibition or masking of the activity of the conditionally active antibody, which thus has low activity in normal tissue (normal physiological conditions). The dual mechanism for suppressing the activity of conditionally active antibodies in normal tissues (the conditionally active and masking moieties) allows the use of much higher dosages of the conditionally active antibodies without causing significant adverse effects.

[0198] In some embodiments, the CM can be a substrate for an enzyme selected from the enzymes listed in Table 1 below.

[0199] [Table 1]

[0200] Alternatively or in addition, the CM may contain a disulfide bond of a cysteine ​​pair that is therefore cleavable by reducing agents, such as cellular reducing agents that may be present in abundance in or around solid tumor tissues, including glutathione (GSH), thioredoxin, NADPH, flavins, ascorbate, etc.

[0201] In some embodiments, the conditionally active antibody contains both a CM and a masking moiety. The activity of the conditionally active antibody is unmasked when the CM is cleaved by an enzyme. In some embodiments, it may be desirable to insert one or more linkers, such as flexible linkers, between the antibody, the masking moiety and the CM to provide flexibility. For example, the masking moiety and / or the CM may not contain a sufficient number of residues (e.g., Gly, Ser, Asp, Asn, particularly Gly and Ser, particularly Gly) to provide the desired flexibility. Therefore, it may be beneficial to introduce one or more amino acids to provide a flexible linker. For example, the masked conditionally active antibody may have the following structure (where the following formula represents the amino acid sequence in either the N-terminal to C-terminal direction or the C-terminal to N-terminal direction): (MM)-L1-(CM)-(AB) (MM)-(CM)-L1-(AB) (MM)-L1-(CM)-L2-(AB) Cyclo[L1-(MM)-L2-(CM)-L3-(AB)] wherein MM is a masking moiety, AB is a conditionally active antibody; L1, L2, and L3 are each independently, and optionally present or absent, the same or different flexible linkers comprising at least one flexible amino acid (e.g., Gly); and cyclo, when present, the entire structure is in the form of a cyclic structure due to the presence of a disulfide bond between a pair of cysteines at or near both the N-terminus and C-terminus of the structure.

[0202] Linkers suitable for use in the present invention are generally those that provide flexibility to the masking moiety to facilitate inhibition of the activity of the conditionally active antibody. Such linkers are generally referred to as flexible linkers. Suitable linkers can be readily selected and may be of any suitable length, such as 1 amino acid (e.g., Gly) to 20 amino acids, 2 to 15 amino acids, 3 to 12 amino acids, for example, 4 to 10 amino acids, 5 to 9 amino acids, 6 to 8 amino acids, or 7 to 8 amino acids, and may be 1, 2, 3, 4, 5, 6, or 7 amino acids.

[0203] Exemplary flexible linkers include glycine polymers (G) n , glycine-serine polymers (e.g., (GS) n , (GSGGS) n and (GGGS) n where n is an integer of at least 1. Exemplary flexible linkers include glycine-alanine polymers, alanine-serine polymers, and other flexible linkers known in the art, including: Gly-Gly-Ser-Gly-Gly, Gly-Gly-Ser-Gly-Gly, Gly-Ser-Gly-Gly-Ser-Gly, Gly-Ser-Gly-Gly-Gly-Gly, Gly-Ser-Gly-Gly-Gly, Gly-Ser-Ser-Ser-Gly, and the like.

[0204] Some of the techniques used to mask the activity of conditionally active antibodies are described in WO2010081173A2.

[0205] In some embodiments, the conditionally active polypeptide is a conditionally active antibody that can be engineered using techniques described herein. Non-limiting examples of antibody engineering techniques include antibody conjugation, engineering multispecific antibodies, engineering bispecific conditionally active antibodies against immune effector cell surface antigens and target antigens, engineering the Fc region of an antibody.

[0206] Methods for conjugating conditionally active antibodies are described in WO 2015 / 175375. In one embodiment, the conditionally active antibody can be conjugated at the Fc region of the antibody. The conjugate molecules, compounds or agents described above can be conjugated to the Fc region as described in U.S. Pat. No. 8,362,210. For example, the Fc region can be conjugated to a cytokine or toxin that is delivered to the site where the conditionally active antibody exhibits preferential activity. Methods for conjugating a polypeptide to the Fc region of an antibody are known in the art. See, for example, U.S. Patent Nos. 5,336,603, 5,622,929, 5,359,046, 5,349,053, 5,447,851, 5,723,125, 5,783,181, 5,908,626, 5,844,095, and 5,112,946. , European Patent No. 307,434, European Patent No. 367,166, European Patent No. 394,827, International Publication No. WO 91 / 06570, International Publication No. WO 96 / 04388, International Publication No. WO 96 / 22024, International Publication No. WO 97 / 34631, and International Publication No. WO 99 / 04813, Ashkenazi See, e.g., Traunecker et al., Nature, vol. 331, pages 84-86, 1988; Zheng et al., J. Immunol., vol. 154, pages 5590-5600 1995; and ViI et al., Proc. Natl. Acad. Sci. USA, vol. 89, pages 11337-11341, 1992.

[0207] In one embodiment, the conditionally active antibody may be covalently attached to the conjugating agent via an intermediate linker having at least two reactive groups, one that reacts with the conditionally active antibody and one that reacts with the conjugating agent. The linker, which may include any compatible organic compound, may be selected such that reaction with the conditionally active antibody or the conjugating agent does not adversely affect the reactivity and / or selectivity of the conditionally active antibody. Furthermore, the addition of the linker to the conjugating agent must not destroy the activity of the conjugating agent.

[0208] Suitable linkers for oxidized conditionally active antibodies include those containing a group selected from primary amine, secondary amine, hydrazine, hydrazide, hydroxylamine, phenylhydrazine, semicarbazide and thiosemicarbazide groups. Suitable linkers for reduced conditionally active antibodies include those having a specific reactive group capable of reacting with a sulfhydryl group of a reduced conditionally active antibody. Such reactive groups include, but are not limited to, reactive haloalkyl groups (including, for example, haloacetyl groups), p-mercuric benzoate groups, and groups capable of Michael-type addition reactions (including, for example, maleimides and the types of groups described in Mitra and Lawton, J. Amer. Chem. Soc. Vol. 101, pages 3097-3110, 1979).

[0209] Methods for engineering multispecific conditionally active antibodies are described in WO 2015 / 175375. Conditionally active antibodies can be engineered to generate bispecific conditionally active antibodies against immune effector cell surface antigens and target antigens. The bispecific conditionally active antibodies of the present invention are capable of attracting immune effector cells to disease sites where the target antigen is present. Bispecific conditionally active antibodies are antibodies capable of specifically binding to two different antigens: immune effector cell surface antigens and target antigens. Bispecific antibodies can be full-length antibodies comprising two arms, one arm of which binds to the immune effector cell surface antigen and the other arm of which binds to the target antigen. Bispecific antibodies are antibodies that bind to a heavy chain variable domain (V H) and the light chain variable domain (V L In one embodiment, the antibody fragment may comprise at least two V(II) arms, one of which binds to an immune effector cell surface antigen and the other of which binds to a target antigen. H V L In other embodiments, the antibody fragment comprises at least two single variable domains (Vs) with one arm binding to an immune effector cell surface antigen and the other arm binding to a target antigen. H Or V L In some embodiments, the bispecific conditionally active antibody comprises two scFvs, one that binds to an immune effector cell surface antigen and the other that binds to a target antigen.

[0210] The attracting immune effector cells can attract immune effector cells to diseased cells or diseased tissues containing target antigens by binding activity to both the immune effector cells and the target antigen on the diseased cells or diseased tissues. The attracting immune effector cells will then attack the diseased cells or diseased tissues. The immune effector cells can suppress and even destroy the diseased cells or diseased tissues, thus promoting the cure of the disease. For example, the immune effector cells can destroy tumor cells or infected cells. The immune effector cells include natural killer cells (NK cells), macrophages, lymphokine-activated killer (LAK) cells, and T cells.

[0211] The bispecific conditionally active antibody has two binding activities, one each to the immune effector cell surface antigen and the target antigen. In one embodiment, both binding activities are conditional. That is, the binding activities of the bispecific conditionally active antibody to the immune effector cell surface antigen and the target antigen are lower than the binding activities of the parent antibody under normal physiological conditions and higher than the binding activities of the parent antibody under abnormal conditions. In one embodiment, only one of the two binding activities is conditional. That is, either the binding activity of the bispecific conditionally active antibody to the immune effector cell surface antigen or the binding activity of the bispecific conditionally active antibody to the target antigen is conditional. In this case, either the binding activity of the bispecific conditionally active antibody to the immune effector cell surface antigen or the binding activity of the bispecific conditionally active antibody to the target antigen is lower than the corresponding activity of the parent antibody under normal physiological conditions and higher than the corresponding activity of the parent antibody under abnormal conditions.

[0212] The two arms of a bispecific conditionally active antibody (e.g., two V H V L The smallest antibody fragment that contains a complete antigen-binding site is a non-covalently associated heavy and light chain variable domain (VLCV). H and V L This configuration is achieved by forming a dimer of V and VD, with the three complementarity determining regions (CDRs) of each variable domain being V. H -V L The six CDRs correspond to those found in natural antibodies, which interact to define an antigen-binding site on the surface of the dimer. Collectively, the six CDRs confer antigen-binding specificity to the antibody. The frameworks (FRs) flanking the CDRs have a tertiary structure that is essentially conserved in natural immunoglobulins of species as diverse as human and mouse. These FRs serve to hold the CDRs in their proper orientation. The constant domains are not required for binding function, but are important in the V H -V LA single variable domain (or half an Fv containing only three CDRs specific for an antigen) has the ability to recognize and bind antigen, but usually with a lower affinity than the entire binding site (Painter et al., "Contributions of heavy and light chains of rabbit immunoglobulin G to antibody activity. I. Binding studies on isolated heavy and light chains," Biochemistry, vol. 11 pages 1327-1337, 1972). Therefore, the domains of the binding site of a bispecific conditionally active antibody are composed of the V domains of different immunoglobulins. H -V L , V H -V H Or V L -V L It may be structured as a pair of domains.

[0213] In some embodiments, the bispecific conditionally active antibody can be constructed by recombinant DNA techniques as a contiguous polypeptide chain, e.g., in a manner in which a nucleic acid molecule encoding the bispecific conditionally active antibody is expressed to construct a contiguous polypeptide chain (see, e.g., Mack et al., "A small bispecific antibody construct expressed as a functional single-chain molecule with high tumor cell cytotoxicity," Proc. Natl. Acad. Sci. USA, vol. 92, pages 7021-7025, 2005). H and V L The order of the domains is such that the antigen-binding site folds properly to form one binding site for the immune effector cell surface antigen and one binding site for the target antigen. H and V L As far as the domains are arranged, it is not critical to the invention.

[0214] Some of the techniques described herein for engineering multispecific conditionally active antibodies can be used to generate bispecific conditionally active antibodies against an immune effector cell surface antigen and a target antigen.

[0215] Bispecific antibodies may be constructed as single polypeptide chains, as described in WO 99 / 54440; Mack, J. Immunol. (1997), 158, 3965-3970; Mack, PNAS, (1995), 92, 7021-7025; Kufer, Cancer Immunol. Immunother., (1997), 45, 193-197; Loffler, Blood, (2000), 95, 6, 2098-2103; Bruhl, J. Immunol., (2001), 166, 2420-2426. A particularly preferred construction for bispecific antibodies is H and V L A polypeptide construct in which the domains are linked together by a linker domain. H and V L The order of the regions is not important. In one embodiment, the single polypeptide chain comprises H1 -Linker domain-V L1 -Linker domain-V H2 Linker domain-V L2 In another embodiment, the single polypeptide chain is configured as: L1 -Linker domain-V H1 -Linker domain-V L2 -Linker domain-V H2 In another embodiment, the single polypeptide chain is configured as: H1 -Linker domain-V H2 -Linker domain-V L1 -Linker domain-V L2 In another embodiment, the single polypeptide chain is configured as: H1 -Linker domain-V L2 -Linker domain-V L1 -Linker domain-V H2The single polypeptide chain can fold into two arms, each capable of binding to an immune effector cell surface antigen or a target antigen.

[0216] The linker domain of the bispecific conditionally active antibody is H and V L It is a peptide fragment of sufficient length to allow intermolecular association between the domains. The design of linkers suitable for this purpose is described in the prior art, for example in EP 623679B1, US 5,258,498, EP 573551B1 and US 5,525,491. The linker domain is preferably a hydrophilic flexible linker of 1 to 25 amino acids selected from glycine, serine and / or glycine / serine. In one embodiment, the linker domain is a 15 amino acid linker of the sequence (Gly4Ser)3.

[0217] The additional linker domain comprises an oligomerization domain that can promote the folding of two or several of the VH and VL domains into two arms, each capable of binding to an immune effector cell surface antigen or a target antigen. Examples of oligomerization domains include, but are not limited to, leucine zippers (e.g., jun-fos, GCN4, E / EBP, etc., Kostelny, J. Immunol. 148 (1992), 1547-1553; Zeng, Proc. Natl. Acad. Sci. 94 (1997), 3673-3678; Williams, Genes Dev. 5 (1991), 1553-1563; Suter, "Phage Display of Peptides and Proteins", Chapter 11, (1996), Academic Press), oligomerization domains derived from antibodies, such as the constant domains CH1 and CL (Mueller, FEBS Letters 422 (1998), 259-264) and / or tetramerization domains, such as GCN4-LI (Zerangue, Proc. Natl. Acad. Sci. 97 (2000), 3591-3595).

[0218] In some embodiments, the knobs-into-holes technique may be used to stabilize the folding of single polypeptide chain bispecific conditional antibodies. The knobs-into-holes technique has been described by Ridgway et al., ("'Knobs-into-holes' engineering of antibody CH3 domains for heavy chain heterodimerization," Protein Eng. 1996 July; 9(7):617-21). This method has been used to pack amino acid side chains between adjacent a-helices, where the side chains of residues in the a-helices are represented as knobs spaced apart on the surface of a cylinder alternating with holes into which the knobs of adjacent a-helices can fit (O'Shea et al., (1991) Science, 254, 539-544).

[0219] Immune effector cell surface antigens should be specific to one or a class of immune effector cells. Many immune effector cell surface antigens are known. Natural killer cells have surface antigens including CD56, CD8, CD16, KIR family receptors, NKp46, NKp30, CD244 (2B4), CD161, CD2, CD7, CD3, and killer cell immunoglobulin-like receptors (Angelis et al., “Expansion of CD56-negative, CD16-positive, KIR-expressing natural killer cells after T cell-depleted haploidentical hematopoietic stem cell transplantation,” Acta Haematol. 2011;126(1):13-20; Dalle et al., “Characterization of Cord Blood Natural Killer Cells: Implications for Transplantation and Neonatal Infections,” Pediatric Research (2005) 57,649-655; Agarwal et al., “Roles and Mechanism of Natural Killer Cells in Clinical and Experimental Transplantation,” Expert Rev Clin Immunol. 2008;4(1):79-91).

[0220] Macrophages have surface antigens including CD11b, F4 / 80, CD68, CSF1R, MAC2, CD11c, LY6G, LY6C, IL-4Rα, CD163, CD14, CD11b, F4 / 80 (mouse) / EMR1 (human), CD68 and MAC-1 / MAC-3, PECAM-1 (CD31), CD62, CD64, CD45, Ym1, CD206, CD45RO, 25F9, S100A8 / A9, and PM-2K (Murray et al., “Protective and pathogenic functions of macrophage subsets,” Nature Reviews Immunology, 11, 723-737; Taylor et al., “Macrophage receptors and immune recognition,” Annu Rev Immunol 2005; 23:901-44; Pilling, et al., “Identification of Markers that Distinguish Monocyte-Derived Fibrocytes from Monocytes,Macrophages,and Fibroblasts,”PLoS ONE 4(10):e7475.doi:10.1371 / journal.pone.0007475,2009).

[0221] Lymphokine-activated killer (LAK) cells have surface antigens including T3, T4, T11, T8, TII, Leu7, and Leu11 (Ferrini et al., "Surface markers of human lymphokine-activated killer cells and their precursors," Int J Cancer. 1987 Jan 15; 39(1): 18-24; Bagnasco et al., "Glycoproteic nature of surface molecules of effector cells with lymphokine-activated killer (LAK) activity," Int J Cancer. 1987 Jun 15; 39(6): 703-7; Kaufmann et al., "Interleukin 2 induces human acute lymphocytic leukemia cells to manifest lymphokine-activated-killer (LAK) cytotoxicity," The Journal of Immunology, August 1, 1987, vol. 139 no. 3 977-982).

[0222] T cells, particularly cytotoxic T cells, have surface antigens including CD2, CD3, CD4, CD5, CD6, CD8, CD28, T58, CD27, CD45, CD84, CD25, CD127, and CD196 (CCR6), CD197 (CCR7), CD62L, CD69, TCR, T10, T11, and CD45RO (Ledbetter et al., "Enhanced transmembrane signaling activity of monoclonal antibody heteroconjugates suggests molecular interactions between receptors on the T cell surface," Mol Immunol. 1989 Feb;26(2):137-45; Jondal et al., "SURFACE MARKERS ON HUMAN T AND B LYMPHOCYTES," JOURNAL OF EXPERIMENTAL MEDICINE, VOLUME 136, 1972, 207-215; Mingari et al., "Surface Markers on Human T and B Lymphocytes," Journal of Experimental Medicine, Vol. 136, 1972, 207-215). al., “Surface markers of human T lymphocytes,” Ric Clin Lab.1982 Jul-Sep;12(3):439-448).

[0223] The bispecific conditionally active antibody, after binding to the immune effector cell, is capable of delivering the immune effector cell to a cell or tissue where the target antigen is preferably present on the surface. When the bispecific conditionally active antibody (with immune effector cell) binds to the target antigen, the immune effector cell can attack the diseased cell or tissue. Immune effector cells such as natural killer cells, macrophages, LAK cells, T cells (cytotoxic), etc., are all capable of killing and / or destroying diseased cells or tissues, for example, destroying tumor tissues.

[0224] The diseased cells or tissues may be selected from cancer, inflammatory diseases, neuronal disorders, diabetes, cardiovascular diseases or infectious diseases. Examples of target antigens include antigens expressed by various immune cells, carcinomas, sarcomas, lymphomas, leukemias, germ cell tumors, blastomas, and cells associated with various hematological, autoimmune and / or inflammatory diseases.

[0225] Cancer-specific target antigens that can be targeted by bispecific conditionally active antibodies include 4-IBB, 5T4, adenocarcinoma antigen, alpha-fetoprotein, BAFF, B lymphoma cells, C242 antigen, CA-125, carbonic anhydrase 9 (CA-IX), C-MET, CCR4, CD152, CD19, CD20, CD200, CD22, CD221, CD23 (IgE receptor), CD28, CD30 (TNFRSF8), CD33, CD4, CD40, CD44v6, CD51, CD52, CD56, CD74, CD80, CEA, CNT0888, CTLA-4, DR5, EGFR, EpCAM, CD3, FAP, fibronectin extra domain B, folate receptor 1, GD2, GD3 ganglioside, glycoprotein 75, GPNMB, HER2 / neu, HGF, human scatter factor receptor kinase, IGF-1 receptor, IGF-I, IgGl, LI-CAM, IL-13, IL-6, insulin-like growth factor I receptor, integrin α5β1, integrin ανβ3, MORAb-009, MS4A1, MUC1, mucin CanAg, N-glycolylneuraminic acid, NPC-1C, PDGF-Ra, PDL192, phosphatidylserine, prostate cancer cells, RANKL, RON, ROR1, SCH 900105, SDC1, SLAMF7, TAG-72, tenascin-C, TGFβ2, TGF-β, TRAIL-R1, TRAIL-R2, tumor antigen CTAA16.88, VEGF-A, VEGFR-1, VEGFR2 or vimentin.

[0226] The types of cancers that may be treated with the genetically engineered cytotoxic cells or pharmaceutical compositions of the present invention include carcinomas, blastomas, and sarcomas, as well as certain leukemias or lymphoid malignancies, benign and malignant tumors, and malignant lesions, such as sarcomas, carcinomas, and melanomas. The cancer may be a non-solid tumor (e.g., a hematological tumor) or a solid tumor. Adult tumors / cancers and pediatric tumors / cancers are also included.

[0227] Hematological cancer is a cancer of the blood or bone marrow. Examples of hematological (or hematopoietic) cancer include leukemia, including acute leukemia (e.g., acute lymphocytic leukemia, acute myeloid leukemia, acute myelogenous leukemia, myeloblastic leukemia, promyelocytic leukemia, myelomonocytic leukemia, monocytic leukemia, and erythroleukemia), chronic leukemia (e.g., chronic myelocytic (granulocytic) leukemia, chronic myelogenous leukemia, and chronic lymphocytic leukemia), polycythemia vera, lymphoma, Hodgkin's disease, non-Hodgkin's lymphoma (indolent and aggressive), multiple myeloma, Waldenstrom's macroglobulinemia, heavy chain disease, myelodysplastic syndrome, hairy cell leukemia, and myelodysplasia.

[0228] A solid tumor is an abnormal mass of tissue that usually does not contain cysts or liquid areas. Solid tumors can be benign or malignant. Various types of solid tumors are named for the type of cells that form them (e.g., sarcoma, carcinoma, and lymphoma). Examples of solid tumors that can be treated include fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma, osteosarcoma, and other sarcomas, synovioma, mesothelioma, Ewing's sarcoma, leiomyosarcoma, rhabdomyosarcoma, colon cancer, lymphoid malignancies, pancreatic cancer, breast cancer, lung cancer, ovarian cancer, prostate cancer, hepatocellular carcinoma, squamous cell carcinoma, basal cell carcinoma, adenocarcinoma, sweat gland carcinoma, medullary thyroid carcinoma, papillary thyroid carcinoma, pheochromocytoma, sebaceous gland carcinoma, papillary carcinoma, papillary adenocarcinoma, medullary carcinoma, bronchogenic carcinoma, renal cell carcinoma, hepatoma, cholangiocarcinoma, choriocarcinoma, will. These include sarcomas and carcinomas, including mucosal tumors, cervical cancer, testicular tumors, seminomas, bladder cancer, melanomas, and CNS tumors (e.g., gliomas (e.g., brain stem gliomas and mixed gliomas), glioblastomas (also known as glioblastoma multiforme), astrocytomas, CNS lymphomas, germinomas, medulloblastomas, schwannomas, craniopharyngiomas, ependymomas, pinealomas, hemangioblastomas, acoustic neuromas, oligodendrogliomas, meningiomas, neuroblastomas, retinoblastomas, and brain metastases).

[0229] Antigens specific to inflammatory diseases that can be targeted by bispecific conditionally active antibodies include AOC3 (VAP-1), CAM-3001, CCL11 (eotaxin-1), CD125, CD147 (basigin), CD154 (CD40L), CD2, CD20, CD23 (IgE receptor), CD25 (IL-2 receptor chain), CD3, CD4, CD5, IFN-a, IFN-gamma, IgE, IgE Fc region, IL-1, IL-12, IL-23, IL-13, IL-17, IL-17A, IL-22, IL-4, IL-5, IL-5, IL-6, IL-6 receptor, integrin a4, integrin α4β7, LFA-1 (CD1 la), MEDI-528, myostatin, OX-40, rhuMAbβ7, sclerostin, SOST, TGFβ1, TNF-α, or VEGF-A.

[0230] Target antigens specific to neuronal disorders that may be targeted by the bispecific conditionally active antibodies of the present invention include one or more of beta amyloid or MABT5102A. Antigens specific to diabetes that may be targeted by the bispecific conditionally active antibodies of the present invention include one or more of L-Ιβ or CD3. Antigens specific to cardiovascular diseases that may be targeted by the bispecific conditionally active antibodies of the present invention include one or more of C5, cardiac myosin, CD41 (integrin α-lib), fibrin II, β chain, ITGB2 (CD18) and sphingosine-1-phosphate.

[0231] Infectious disease specific target antigens that may be targeted by the bispecific conditionally active antibodies of the invention include one or more of anthrax toxin, CCR5, CD4, clumping factor A, cytomegalovirus, cytomegalovirus glycoprotein B, endotoxin, Escherichia coli, hepatitis B surface antigen, hepatitis B virus, HIV-1, Hsp90, influenza A hemagglutinin, lipoteichoic acid, Pseudomonas aeruginosa, rabies virus glycoprotein, respiratory syncytial virus, and TNF-a.

[0232] Further examples of target antigens include surface proteins found specifically or amplified on cancer cells, such as the IL-14 receptor, CD19, CD20 and CD40 of B-cell lymphomas, Lewis Y and CEA antigens of various carcinomas, Tag72 antigen of breast and colorectal cancers, EGF-R of lung cancer, folate-binding protein and HER-2 protein, which are often amplified in human breast and ovarian cancers, or viral proteins, such as the gpl20 and gp41 envelope proteins of HIV, envelope proteins from Hepatitis B and C viruses, glycoprotein B and other envelope glycoproteins of human cytomegalovirus, and envelope proteins from oncoviruses such as Kaposi's sarcoma-associated herpesvirus. Other potential target antigens include CD4 (whose ligand is the HIV gpl20 envelope glycoprotein) and other viral receptors, such as ICAM, the receptor for human rhinovirus, and the related receptor molecule for poliovirus.

[0233] Human immunodeficiency virus (HIV) cannot enter human cells unless it first binds to two key molecules on the cell surface: CD4 and a co-receptor. The first co-receptor recognized is CCR5, and later in the viral life cycle, another chemokine receptor, CXCR4, becomes the co-receptor for HIV-1 (D'Souza, Nature Med. 2, 1293 (1996); Premack, Nature Med. 2, 1174; Fauci, Nature 384, 529 (1996)). The HIV-1 strains responsible for most cases of viral transmission through sexual contact are called M-tropic viruses. These HIV-1 strains (also known as non-syncytial-inducing (NSI) primary viruses) are able to replicate in primary CD4+ T cells and macrophages and use the chemokine receptors CCR5 (and less frequently CCR3) as their co-receptors. T-tropic viruses (sometimes called syncytium-inducing (SI) primary viruses) can also replicate in primary CD4+ T cells, but can also infect established CD4+ T cell lines in vitro, via the chemokine receptor CXCR4 (fusin). Many of these T-tropic strains can use CCR5 in addition to CXCR4, and some can enter macrophages via CCR5, at least under certain in vitro conditions (D'Souza, Nature Med. 2, 1293 (1996); Premack, Nature Med. 2, 1174; Fauci, Nature 384, 529 (1996)). M-tropic HIV-1 strains are responsible for approximately 90% of sexual transmission of HIV, so CCR5 is the major coreceptor for the virus in patients.

[0234] The number and identity of co-receptor molecules on target cells and the ability of HIV-1 strains to enter cells via different co-receptors appear to be important determinants of disease progression. High expression of CCR3 and CCR5 has also been observed in T and B cells in lymph nodes from Hodgkin's disease patients. Type I diabetes is considered to be a T cell-mediated autoimmune disease. Expression of CCR5 receptors in the pancreas has been associated with the progression of type I diabetes in relevant animal models (Cameron (2000) J. Immunol. 165, 1102-1110). In one embodiment, bispecific conditionally active antibodies bind to CCR5 of target antigens and can be used to inhibit HIV infection of host cells as well as slow the progression of other diseases.

[0235] Several antibodies that specifically bind to (human) CCR5 are known in the art, including MC-1 (Mack (1998) J. Exp. Med. 187, 1215-1224 or MC-5 (Blanpain (2002) Mol Biol Cell. 13:723-37; Segerer (1999) Kidney Int. 56:52-64; Kraft (2001) Biol. Chem. 14;276:34408-18). Thus, a bispecific conditionally active antibody may be, for example, a V-type antibody specific for CCR5, preferably human CCR5. L and V H domain (i.e., the second domain from Ig) and the V domain of an antibody specific for the CD3 antigen on T cells. H and V L It is preferred that the suffix code includes a domain.

[0236] In another embodiment, the present invention provides a bispecific conditionally active antibody against CD3 and CD19 on T cells as target antigens. CD19 has been found to be a very useful pharmaceutical target. CD19 is expressed in the entire B cell lineage from pro-B cells to mature B cells and uniformly on all lymphoma cells, but is absent from stem cells (Haagen, Clin Exp Immunol 90 (1992), 368-75; Uckun, Proc. Natl. Acad. Sci. USA 85 (1988), 8603-7). Combination therapy utilizing both antibodies against CD19 and additional immunomodulatory antibodies has been disclosed for the treatment of B cell malignancies (WO 02 / 04021; US ​​2002006404; US 2002028178) and autoimmune diseases (WO 02 / 22212; US 2002058029). WO 00 / 67795 discloses the use of antibodies against CD19 for the treatment of indolent and aggressive B-cell lymphomas, as well as acute and chronic lymphocytic leukemias. WO 02 / 80987 discloses the therapeutic use of immunotoxins based on antibodies against the antigen CD19 for the treatment of diseases such as B-cell non-Hodgkin's lymphoma, Hodgkin's lymphoma, or B-cell leukemia (e.g., B-cell acute lymphocytic leukemia (B-ALL), (e.g., hairy cell lymphoma), B-cell precursor acute lymphocytic leukemia (pre-B-ALL), B-cell chronic lymphocytic leukemia (B-CLL)).

[0237] In a further embodiment, the present invention provides a bispecific conditionally active antibody against CD3 and CD20 on T cells as target antigens. CD20 is one of the cell surface proteins present on B lymphocytes. The CD20 antigen is found on normal and malignant pre-B lymphocytes and mature B lymphocytes, e.g., in more than 90% of B cell non-Hodgkin's lymphomas (NHL). This antigen is absent from hematopoietic stem cells, activated B lymphocytes (plasma cells) and normal tissues. Several antibodies, mainly of murine origin, have been described: 1F5 (Press et al., 1987, Blood 69 / 2, 584-591), 2B8 / C2B8, 2H7, 1H4 (Liu et al., 1987, J Immunol 139, 3521-3526; Anderson et al., 1998, U.S. Pat. No. 5,736,137; Haisma et al., 1998, Blood 92, 184-190; Shan et al., 1999, J. Immunol. 162, 6589-6595).

[0238] CD20 has been described in immunotherapeutic strategies for the treatment of plasma cell malignancies using vaccination with DNA encoding scFv linked to carrier proteins (Treon et al., 2000, Semin Oncol 27(5), 598), and immunotherapeutic treatment with the CD20 antibody (IDEC-C2B8) has been shown to be effective in the treatment of non-Hodgkin's B-cell lymphoma.

[0239] In some embodiments, the bispecific conditionally active antibody is a single polypeptide chain encoded by a polynucleotide molecule. The polynucleotide can be, for example, DNA, cDNA, RNA, or synthetically produced DNA or RNA, or a recombinantly produced chimeric nucleic acid molecule comprising any of these polynucleotides, either alone or in combination. The polynucleotide can be part of a vector, such as an expression vector, including plasmids, cosmids, viruses, and bacteriophages, or any expression system conventionally used in genetic engineering. The vector can contain additional genes, such as marker genes, that allow the selection of the vector under suitable conditions in a suitable host cell.

[0240] In one embodiment, the polynucleotide is operably linked to an expression control sequence that allows expression in prokaryotic or eukaryotic cells. Expression vectors derived from viruses such as retroviruses, vaccinia viruses, adeno-associated viruses, herpes viruses, bovine papilloma viruses, etc., can be used to deliver polynucleotides or vectors into mammalian cells. The vectors containing the polynucleotides of the present invention can be transferred into host cells by well-known methods that vary depending on the type of cell host. For example, calcium chloride transfection is commonly used in prokaryotic cells, while calcium phosphate treatment or electroporation can be used in other cell hosts.

[0241] In another embodiment, the conditionally active polypeptide can be engineered to generate a bispecific conditionally active polypeptide. The method of engineering a bispecific conditionally active polypeptide is similar to the method of engineering a bispecific conditionally active antibody described in WO2015 / 175375. For example, a bispecific conditionally active polypeptide can have two active sites, each of which has a conditional activity, i.e., is less active than the wild-type site under normal physiological conditions and is more active than the wild-type site under abnormal conditions. These two conditionally active sites can be independently evolved and screened, and then the two active sites can be linked to the same bispecific conditionally active polypeptide with a linker. In one embodiment, the linker that can be used in a bispecific conditionally active antibody is suitable for generating a bispecific conditionally active polypeptide by linking two conditionally active sites of a conditionally active polypeptide.

[0242] Methods for engineering the Fc region of conditionally active antibodies are described in WO 2015 / 175375.

[0243] Methods for engineering conditionally active viral particles are described in WO 2015 / 175375.

[0244] In some embodiments, the conditionally active polypeptide may be inserted into a viral particle that is an oncolytic virus using the methods described in WO 2015 / 175375. An oncolytic virus is a virus that can kill tumor cells when it contacts them. The conditionally active polypeptide inserted into the oncolytic virus is more active in the tumor microenvironment but less active at other sites in the subject. For example, the conditionally active polypeptide is more active at the pH of the tumor microenvironment (e.g., pH 6.2-6.8) but less active at the pH present at other sites in the subject (e.g., pH 7.2-7.6). The conditionally active polypeptide inserted into the oncolytic virus will allow delivery of the oncolytic virus to the tumor. There, the oncolytic virus can target and kill tumor cells.

[0245] Targeted oncolytic viruses include adenovirus, herpes simplex virus type 1, vaccinia virus, parvovirus, reovirus, Newcastle disease virus, etc. Vaccinia virus is of particular interest.

[0246] In one embodiment, the oncolytic virus is selected from the group consisting of paramyxovirus, reovirus, herpesvirus, adenovirus and Semliki Forest virus. In a further embodiment, the paramyxovirus is selected from the group consisting of Newcastle Disease Virus (NDV), measles virus and mumps virus. In another embodiment, the NDV is from a strain selected from the group consisting of MTH68 / H, PV-701 and 73-T.

[0247] In another embodiment, the oncolytic virus is selected from herpesvirus, reovirus, E1B deleted adenovirus, vesicular stomatitis virus and poxvirus, which have the potential to not only destroy tumor cells but also to elicit an immune response by releasing antigens from the destroyed tumor cells.

[0248] Specific examples of oncolytic viruses include, but are not limited to, adenoviruses (e.g., Delta-24, Delta-24-RGD, ICOVIR-5, ICOVIR-7, Onyx-015, ColoAdl, H101, AD5 / 3-D24-GMCSF), reoviruses, herpes simplex viruses (HSV, OncoVEX GMCSF), Newcastle disease virus, measles virus, retroviruses (e.g., influenza virus), poxviruses (e.g., vaccinia virus, including Copenhagen, Western Reserve, and Wyeth strains), myxoma virus, rhabdoviruses (e.g., vesicular stomatitis virus (VSV)), picornaviruses (e.g., Seneca Valley virus, SW-001), coxsackieviruses, and parvoviruses.

[0249] In one aspect, the oncolytic virus is an adenovirus, including any member of its 57 human serotypes (HAdV-1 to 57). In one embodiment, the adenovirus is an Ad5 serotype. In another aspect, the adenovirus is a hybrid serotype that may or may not include an Ad5 component. Non-limiting examples of suitable adenoviruses include Δ-24, Δ-24-RGD, ICOVIR-5, ICOVIR-7, ONYX-015, ColoAdl, H101, and AD5 / 3-D24-GMCSF. ONYX-015 is a hybrid of viral serotypes Ad2 and Ad5 with deletions in the E1B-55K and E3B regions to improve cancer selectivity. HI01 is a modification of Onyx-015. ICOVIR-5 and ICOVIR-7 contain a deletion of the Rb binding site of ElA and a replacement of the ElA promoter with the E2F promoter. ColoAd1 is a chimeric Addl lp / Ad3 serotype. AD5 / 3-D24-GMCSF (CGTG-102) is a serotype 5 / 3 capsid-modified adenovirus encoding GM-CSF (the Ad5 capsid protein knob has been replaced with the knob domain from serotype 3).

[0250] In a particularly preferred embodiment, the oncolytic virus is a Delta-24 or Delta-24-RGD adenovirus. Delta-24 is described in US Patent Application Publication Nos. 2003 / 0138405 and 2006 / 0147420. Delta-24 adenovirus is derived from adenovirus type 5 (Ad-5) and contains a 24 base pair deletion in the CR2 portion of the ElA gene. Delta-24-RGD further contains an insertion of the RGD-4C sequence (which binds strongly to ανβ3 and ανβ5 integrins) in the HI loop of the fiber knob protein (Pasqualini R. et al., Nat Biotechnol, 15:542-546, 1997).

[0251] Oncolytic adenoviruses can also be further modified to improve the ability of oncolytic adenoviruses to treat cancer. Such modifications of oncolytic adenoviruses are described in Jiang et al. (Curr Gene Ther. 2009 Oct 9(5):422-427), see also US Patent Application No. 2006 / 0147420.

[0252] Oncolytic viruses carrying conditionally active polypeptides may be administered locally or systemically. For example, but not limited to, the oncolytic viruses can be administered intravascularly (intra-arterially or intravenously), intratumorally, intramuscularly, intradermally, intraperitoneally, subcutaneously, orally, parenterally, intranasally, intratracheally, transdermally, intraspinal, intraocularly, or intracranially.

[0253] The oncolytic virus may be administered in a single dose or multiple doses. The virus is administered in a concentration of at least 1×10 5 Plaque-forming units (PFU), at least 5 × 10 5 PFU, at least 1 × 10 6 PFU, at least 5 × 10 6 Or at least 5 x 10 6 PFU, 1 × 10 7 , at least 1 × 10 7 PFU, at least 1 × 10 8 Or at least 1 × 10 8 PFU, at least 1 × 10 8 PFU, at least 5 × 10 8 PFU, at least 1 × 10 9 Or at least 1 × 10 9 PFU, at least 5 × 10 9 Or at least 5 x 10 9 PFU, at least 1 × 10 10 PFU or at least 1 × 10 10 PFU, at least 5 × 10 10 Or at least 5 x 10 10 PFU, at least 1 × 10 11 PFU or at least 1 × 10 11 PFU, at least 1 × 10 12 PFU or at least 1 × 1013 For example, the oncolytic virus may be administered in a dose of about 10 PFU. 7 ~10 13 PFU, approx. 10 8 ~10 13 PFU, approx. 10 9 ~10 12 PFU or about 10 8 ~10 12 It may be administered in a dosage of PFU.

[0254] In certain embodiments, the cancer treated with the oncolytic virus includes any solid tumor, such as lung, ovarian, breast, cervical, pancreatic, gastric, colon, skin, laryngeal, bladder, prostate, etc. In one embodiment, the cancer is a cancer of the central nervous system. The cancer may be an astrocytic tumor (e.g., astrocytoma, anaplastic astrocytoma, glioblastoma, gliosarcoma, pilocytic astrocytoma, giant cell astrocytoma, pleomorphic xanthoastrocytoma), oligodendroglioma, ependymoma, oligoastrocytoma, spongiocytoma, astroblastoma, choroid plexus papilloma, choroid plexus carcinoma, gangliocytoma, ganglioglioma, neurocytoma, neuroepithelial tumor, neuroblastoma, pineal tumor (e.g., pineocytoma, pineoblastoma, or mixed pineocytoma / pineoblastoma), medulloepithelioma, medulloblastoma, neuroblastoma, or ganglioblastoma, retinoblastoma, ependymoblastoma, or other neuroepithelial tumor. The cancer may be a central nervous system neoplasm such as a sellar tumor (e.g., a pituitary adenoma, a pituitary carcinoma, or a craniopharyngioma), a hematopoietic tumor (e.g., a primary malignant lymphoma, a plasmacytoma, or a granulocytic sarcoma), a germ cell tumor (e.g., a germinoma, an embryonal carcinoma, a yolk sac tumor, a choriocarcinoma, a teratoma, or a mixed germ cell tumor), a meningioma, a mesenchymal tumor, a melanoma, or a cranial or spinal nerve tumor (a schwannoma or a neurofibroma). The cancer may be a low-grade glioma (e.g., an ependymoma, an astrocytoma, an oligodendroglioma, or a mixed glioma) or a high-grade (malignant) glioma (e.g., a glioblastoma multiforme). The cancer may be a primary or metastatic brain tumor.

[0255] C. Generation of Conditionally Active Polypeptides A method for generating a conditionally active polypeptide from a parent polypeptide includes evolving a DNA encoding the parent polypeptide to increase the net charge of the parent polypeptide using one or more techniques selected from increasing the total number of codons for charged amino acid residues in the DNA, decreasing the total number of codons for uncharged amino acid residues in the DNA, and combinations thereof to generate a mutant DNA, expressing the mutant DNA to generate a mutant polypeptide, and subjecting the mutant polypeptide and the parent polypeptide to a screening assay under a first condition, which may be a normal physiological condition, and a screening assay under a second condition, which may be an abnormal condition. The conditionally active polypeptide is selected from mutant polypeptides that (a) exhibit a decrease in activity in a first assay at a first value of a condition compared to the same activity of the parent polypeptide in the first assay, and (b) exhibit an increase in the same activity in a second assay at a second value of the same condition compared to the same activity of the parent polypeptide in the second assay. The decrease in activity of the conditionally active polypeptide under the first condition or normal physiological condition may be reversible or irreversible.

[0256] i. Parent Polypeptide The parent polypeptide may be a wild-type polypeptide, a mutant polypeptide derived from a wild-type polypeptide, such as a therapeutic polypeptide, a chimeric polypeptide derived from a different wild-type polypeptide, or even a synthetic polypeptide. The parent polypeptide may be selected from antibodies, enzymes, cytokines, regulatory proteins, hormones, receptors, ligands, biosimilars, immunomodulators, growth factors, stress proteins, vault-associated proteins, neuronal proteins, gut proteins, growth factors, mitochondrial proteins, cytoplasmic proteins, animal proteins, structural proteins, plant proteins, and fragments of these polypeptides.

[0257] In some embodiments, the parent polypeptide may be a fragment of a wild-type polypeptide, a fragment of a therapeutic polypeptide, or an antibody fragment. In some other embodiments, the parent polypeptide may be a polypeptide selected from mutant polypeptides generated by a mutagenesis process, provided that the polypeptide has desired properties, such as high binding activity, high expression level, humanization, etc. The selected polypeptide (in this case not the wild-type polypeptide) may be used as the parent polypeptide to evolve by the methods disclosed herein.

[0258] A description of suitable wild-type polypeptides as well as how they may be evolved and selected to produce conditionally active polypeptides is provided in US Pat. No. 8,709,755 B2.

[0259] In some embodiments, the parent polypeptide may be selected from a library of wild-type or mutant polypeptides, such as a bacteriophage display library. In such embodiments, a large number of candidate polypeptides are expressed in the bacteriophage library, particularly by surface display techniques. The candidate polypeptides from the library are screened for suitable parent polypeptides. A typical bacteriophage library may include bacteriophages that express thousands or even millions of candidate polypeptides in a bacterial host. In one embodiment, the bacteriophage library may include a plurality of bacteriophages.

[0260] To construct a bacteriophage library, typically a filamentous bacteriophage, such as the filamentous coliphage M13, is genetically modified by inserting an oligonucleotide encoding a candidate polypeptide into the coding sequence of one of the bacteriophage coat proteins. The bacteriophage coat protein is then expressed together with the candidate polypeptide, resulting in the display of the candidate polypeptide on the surface of the bacteriophage particle. The displayed candidate polypeptide can then be screened for suitable parent polypeptides.

[0261] One common technique for screening for suitable parent polypeptides is by immobilizing bacteriophage particles containing the desired candidate polypeptide on a support. The support can be a plastic plate coated with a "bait" that can bind to the desired candidate polypeptide. Unbound bacteriophage particles can be washed off the plate. Bacteriophage particles that are bound to the plate (along with the desired candidate) are eluted by washing, and the eluted bacteriophage particles are amplified with bacteria. The sequence encoding the candidate polypeptide of the selected bacteriophage particles can then be determined by sequencing. The relationship between the candidate polypeptide and the bait can be, for example, a ligand-receptor or antigen-antibody relationship.

[0262] Another common technique for screening for suitable parent polypeptides is by using enzymatic assays of individual bacteriophage clones for the desired enzymatic activity exhibited by the candidate polypeptides. Depending on the specific enzymatic activity, one skilled in the art can design a suitable assay to screen for parent polypeptides with the desired level of enzymatic activity.

[0263] In some embodiments, the bacteriophage library is provided as an array, with each bacteriophage clone occupying a specific location on the array. Such an array may be provided on a solid support, such as a membrane, an agar plate, or a microtiter plate, where in the case of a microtiter plate, each bacteriophage clone of the library is disposed or attached to a specific predefined location on the solid support. In the case of an agar plate, such a plate preferably contains a bacterial growth medium to support bacterial growth. When the array is provided on a membrane, such as a nitrocellulose or nylon membrane, the bacterial culture is added onto the membrane, and the membrane is immersed in a nutrient growth medium. In addition, the bacteriophage clones may also be provided on beads, in which case a single bacteriophage clone can be attached to a single bead. Alternatively, the bacteriophage clones may each be provided on the end of an optical fiber, where the fiber is used to optically transmit ultraviolet radiation from a light source.

[0264] A typical bacteriophage library contains 10 6 ~10 10 In some cases, the library may include four bacteriophages, each of which is distinguished by a coat protein carrying a different polypeptide (e.g., gp3 or gp8 for phage M13). The bacterial host for the bacteriophage library may be selected from bacterial genera including, for example, Salmonella, Staphylococcus, Streptococcus, Shigella, Listeria, Campylobacter, Klebsiella, Yersinia, Pseudomonas, and Escherichia.

[0265] The oligonucleotides encoding the candidate polypeptides can be a collection of cDNAs encoding wild-type polypeptides. Methods for synthesizing cDNA from biological samples capable of expressing suitable parent polypeptides are known. Any genetic information that exhibits biological activity through transcription can be collected as cDNA. When producing cDNA, it is essential to synthesize full-length cDNA. There are several methods that can be used to synthesize full-length cDNA. For example, suitable methods include a method using yeast or HeLa cell cap-binding protein to label the 5' cap site (I. Edery et al., "An Efficient Strategy To Isolate Full-length cDNAs Based on a mRNA Cap Retention Procedure (CAPture)", Mol. Cell. Biol., vol. 15, pages 3363-3371, 1995); and a method in which phosphates of incomplete cDNAs without a 5' cap are removed using alkaline phosphatase, and then the entire cDNA is treated with a tobacco mosaic virus decapping enzyme so that only full-length cDNAs have phosphates (K. Maruyama et al., "Oligo-capping: a simple method to replace the cap structure of eukaryotic mRNAs with oligoribonucleotides", Gene, vol. 138, pages 171-174, 1995 and S. Kato et al., "Construction of a human full-length cDNA bank", Gene, vol. 150, pages 3363-3371, 1995). 243-250, 1995).

[0266] In embodiments where the parent polypeptide is an antibody, a library of candidate antibodies may be generated using recombinant antibodies derived from the complete antibody repertoire of an organism. The genetic information representing the repertoire is assembled into a large collection of complete antibodies, which may be screened for suitable parent antibodies with the desired antigen-binding activity and / or one or more other functional properties. In some embodiments, B cells from an animal immunized with an antigen are isolated, such as an immunized human, mouse, or rabbit. mRNA is recovered from the isolated B cells, converted to cDNA, and then sequenced. The most frequent cDNA fragments encoding the light chain and the most frequent cDNA fragments encoding the heavy chain are assembled into an antibody. In one embodiment, the 100 most frequent cDNA fragments encoding the light chain and the 100 most frequent cDNA fragments encoding the heavy chain are assembled to generate the candidate antibody. In another embodiment, the most frequent cDNA fragments only encode the variable region of the heavy chain and the variable region of the light chain, and are assembled to generate an antibody fragment in which the antibody contains only the variable region and no constant region.

[0267] In some embodiments, a cDNA fragment encoding a variable region of an IgG heavy chain is assembled with the most frequent variable region of an IgK or IgK light chain, and the assembled antibody contains a heavy chain variable region from an IgG and a light chain variable region from an IgK or IgK.

[0268] The assembled antibody-encoding cDNA is then cloned and expressed, preferably in a plate-based format. The binding activity of the expressed antibody may be analyzed by bead-based ELISA analysis, and suitable parent antibodies may be selected based on the ELISA analysis. The assembled antibody-encoding cDNA may also be expressed in a bacteriophage display library, which may then be screened for one or more desirable parent antibodies by any one of the techniques disclosed herein.

[0269] In embodiments where the parent polypeptide is an antibody, the parent antibody preferably has at least one particular property that facilitates its evolution into a conditionally active antibody. In certain embodiments, the parent antibody may have similar binding activity and / or properties under both normal physiological conditions and abnormal conditions. In such embodiments, the parent antibody is selected based on having the most similar binding activity and / or the most similar combination of one or more properties under both normal physiological conditions and abnormal conditions. For example, if the normal physiological conditions and abnormal conditions are pH 7.4 and pH 6.0, respectively, the parent antibody with the most similar binding activity at pH 7.4 and 6.0 may be selected over an antibody with a less similar binding activity at pH 7.4 and 6.0.

[0270] ii. Evolution of the parent polypeptide In some embodiments, the DNA encoding the parent polypeptide is mutated using one or more site-directed mutagenesis techniques to introduce one or more charged amino acid residues into the parent polypeptide. In one embodiment, the charged amino acid residues can be introduced at any position in the parent polypeptide. In another embodiment, the charged amino acid residues are introduced into the active site of the parent polypeptide. The active site is a region in the parent polypeptide that is responsible for a particular property of the parent polypeptide. The property can be enzyme substrate specificity, enzyme product selectivity, protein secretion properties, ligand selectivity, ligand binding activity, protein stability, and antibody binding activity. Examples of active sites include the catalytic site of an enzyme, the binding site of a ligand or receptor, the complementarity determining region of an antibody, and the epitope of an antigen.

[0271] In one embodiment, charged amino acid residues are introduced into two or more complementarity determining regions of the parent antibody or parent antibody variable regions. In another embodiment, charged amino acid residues are introduced into two or more regions of the parent polypeptide involved in binding to a ligand, receptor or hormone. In yet another embodiment, charged amino acid residues are introduced into two or more regions of the parent polypeptide involved in catalytic activity, such as forming the catalytic pocket of an enzyme.

[0272] In another embodiment, the charged amino acid residues can be introduced into a region outside the active site of the parent polypeptide. For example, such a region can be adjacent to, around, or even distant from the active site. In one embodiment, the region can be a constant region of an antibody heavy or light chain. The region can also be a framework region of a variable region of an antibody heavy or light chain.

[0273] To introduce one or more charged amino acid residues into a parent polypeptide, the DNA encoding the parent polypeptide can be mutated, either by substituting a codon in the DNA with a codon for a charged amino acid or by inserting a codon for a charged amino acid into the DNA to produce a mutant DNA, which is expressed to make the mutant polypeptide. Substitution or insertion of a codon for a charged amino acid residue can be accomplished using one or more of the site-directed mutagenesis techniques described herein.

[0274] The codons for charged amino acid residues introduced into the DNA encoding the parent polypeptide are shown in Table 1.

[0275] [Table 2]

[0276] In one embodiment, the mutant polypeptide has fewer than 6 mutations, or fewer than 5 mutations, or fewer than 4 mutations, or fewer than 3 mutations, or fewer than 2 mutations compared to the parent polypeptide. These numbers of mutations may be sufficient to generate the desired conditionally active polypeptide.

[0277] In some embodiments, the DNA encoding the parent polypeptide is mutated using one or more site-directed mutagenesis techniques to delete one or more codons for uncharged amino acid residues. The deletion of one or more uncharged amino acid residues in the parent polypeptide increases the net charge of the parent polypeptide.

[0278] In another embodiment, the parent polypeptide can be mutated by deleting a region with no or only few charged amino acid residues to increase the net charge of the mutant polypeptide compared to the net charge of the parent polypeptide. For example, a full-length antibody can have a constant region with no or only few charged amino acid residues. Deleting the constant region of an antibody and combining two variable regions to form a single-chain antibody can also result in an increase in the net charge of the mutant single-chain antibody compared to the net charge of the parent antibody. In one example, the full-length antibody is an IgG antibody and is converted to a single-chain antibody with an increased net charge.

[0279] In some aspects, any site-directed mutagenesis technique may be used to mutate the DNA encoding the parent polypeptide, such as those described in U.S. Patent Nos. 6,391,548, 7,132,265, and 6,713,285. In some embodiments, site-directed mutagenesis techniques that may be utilized include nucleotide-mediated mutagenesis, PCR mutagenesis, and cassette mutagenesis of the DNA encoding the parent polypeptide.

[0280] Nucleotide-mediated mutagenesis is a suitable method for making substitutions or insertions of one or more codons in DNA encoding a parent polypeptide. This technique has been previously described in Sambrook et al, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor, 1989, second edition, chapter 15.51, "Oligonucleotide-mediated mutagenesis". Briefly, DNA is modified by hybridizing an oligonucleotide primer carrying the desired mutation to the DNA reduced to a single-stranded form. After hybridization, the entire second complementary strand of DNA is synthesized using DNA polymerase, which will incorporate the oligonucleotide primer and generate a product containing the desired mutation.

[0281] Generally, oligonucleotides at least 25 nucleotides in length are used. An optimal oligonucleotide will have 12-15 nucleotides on either side of the mutation that are perfectly complementary to the template. This ensures that the oligonucleotide will hybridize properly to single-stranded DNA. Oligonucleotides are readily synthesized using techniques known in the art, such as those described in Crea et al. (Proc. Natl. Acad. Sci. USA, 75:5765, 1978).

[0282] In one embodiment, the DNA encoding the parent polypeptide is in a circular DNA molecule, such as a plasmid or cosmid. An oligonucleotide is hybridized to the single-stranded circular DNA molecule under suitable hybridization conditions. A DNA polymerizing enzyme (usually the Klenow fragment of DNA polymerase I) is then added to synthesize the complementary strand of the single-stranded circular DNA molecule using the oligonucleotide as a primer for synthesis. This results in the formation of a heteroduplex molecule in which one strand of DNA contains the mutation and the other strand (the original strand) does not contain the mutation. This heteroduplex molecule is then transformed into a suitable host cell, usually a prokaryote such as E. coli JM101. After cell growth, bacterial colonies containing mutant DNA are plated on agarose plates and screened with an oligonucleotide primer radioactively labeled with 32-phosphate to identify them. The mutant DNA is then removed and placed into a suitable expression vector for protein production.

[0283] This procedure can be modified so that a homoduplex molecule is formed in which both strands of the plasmid contain the mutation. The modification is as follows: An oligonucleotide primer is annealed to the single-stranded circular DNA molecule described above. A mixture of the three deoxyribonucleotides, deoxyriboadenosine (dATP), deoxyriboguanosine (dGTP) and deoxyribothymidine (dTTP), is combined with a modified thio-deoxyribocytosine called dCTP-(aS). This mixture is added to the single-stranded circular DNA molecule-oligonucleotide hybridization complex. Addition of DNA polymerase to this mixture produces a strand of DNA identical to the single-stranded circular DNA molecule except for the mutation. In addition, this new strand of DNA will contain dCTP-(aS) instead of dCTP, which serves to protect it from restriction endonuclease digestion. After nicking the heteroduplex with an appropriate restriction enzyme, the heteroduplex single-stranded circular DNA molecule can be digested with ExoIII nuclease or another appropriate nuclease through the region containing the mutated site. The reaction is then stopped to generate a DNA molecule that is only partially single-stranded. A complete double-stranded DNA homoduplex is then formed using DNA polymerase in the presence of all four deoxyribonucleotide triphosphates, ATP, and DNA ligase. This homoduplex molecule is then transformed into a suitable host cell, such as E. coli JM101, as described above.

[0284] The introduction of two or more mutations (substitutions and / or insertions) into the DNA encoding the parent polypeptide can be accomplished by one of several methods. If the positions to be mutated are in close proximity in the DNA molecule, all of the desired mutations can be mutated simultaneously using one oligonucleotide primer. However, if the positions to be mutated are somewhat distant from each other (e.g., more than about 25 nucleotides apart), it is difficult to generate a single oligonucleotide primer for all of the desired mutations. Instead, one of two alternative methods can be utilized.

[0285] In one alternative, a separate oligonucleotide primer is used for each mutation. The oligonucleotide primers are then simultaneously annealed to the single-stranded circular DNA molecule, and the second complementary strand of DNA that is synthesized will contain all of the desired mutations from the separate oligonucleotide primers.

[0286] Another alternative involves two or more rounds of site-directed mutagenesis to generate the desired DNA with all mutations. The first round is described as a single mutation, where an oligonucleotide primer carrying the first desired mutation generates a heteroduplex DNA molecule. The second round of mutagenesis utilizes the heteroduplex DNA molecule generated in the first round, which already contains one mutation, as a template. A second oligonucleotide primer encoding an additional mutation is then annealed to this template to generate a second DNA molecule containing mutations from both the first and second rounds of mutagenesis. Optionally, the second DNA molecule can be used as a template in a third round of mutagenesis.

[0287] Another suitable site-directed mutagenesis method is PCR mutagenesis, which can be used to make insertion and / or substitution mutations in a parent polypeptide. As a cyclic amplification technique, PCR has been widely adopted in site-directed mutagenesis, using mutagenic primers to introduce desired mutations (Allemandou et al., J Biomed Biotechnol S, 202-207, (2003); An et al., Appl Microbiol Biotechnol S 68, 774-778, (2005); Jeltsch et al., Methods MoI Biol S 182, 85-94 (2002); Hall, et al. Protein Eng. 4: 601 (1991); Hemsley, et al. Nucleic Acids Research 17: 6545-6551 (1989); Ho, et al. Gene 77: 51-59 (1989); Hultman, et al. Nucleic Acids Research 18:5107-5112(1990), Jones,et al.Nature344:793-794(1990), Jones,et al.Biotechniques 12:528-533(1992),Landt,et al.Gene 96:125-128(1990),Nassal,et al.Nucleic Acids Research 18:3077-3078(1990), Nelson, et al.Analytical Biochemistry 180:147-151(1989), Vallette,et al.Nucleic Acids Research 17:723-733(1989), Watkins,et al.Biotechniques 15:700-704 (1993), Weiner, et al. Gene 126:35-41 (1993), and Yao, et al. PCR Methods and Applications 1:205-207 (1992).

[0288] Specifically, a small amount of DNA encoding the parent polypeptide is used as template DNA in PCR. The mutagenic primers differ slightly in sequence from the corresponding region of the template DNA so that the mutagenic primers generate relatively large amounts of specific DNA fragments with only the desired mutation at different positions from the template DNA. For the introduction of a mutation into the circular DNA, one of the primers is designed to overlap the position of the mutation and contain the mutation, while the sequence of the other primer can be identical to a stretch of sequence of the opposite strand of the circular DNA, but this sequence can be located at any position along the circular DNA. However, it is preferable that the sequence of the second primer is finally located within 200 nucleotides of the first one so that the entire amplified region of DNA to which the primers bind can be easily sequenced for confirmation of the mutation. As a result of PCR amplification using the primer pair, DNA fragments with different mutation positions of the mutagenic primers are obtained.

[0289] It is possible to simultaneously introduce mutations at separate positions by either using a second mutagenic primer or performing a second round of PCR with a different mutagenic primer, ligating the two resulting PCR fragments, and simultaneously introducing the three (or more) ligated parts into the vector.

[0290] In a specific example of PCR mutagenesis, template plasmid DNA (1 μg) is linearized by digestion with a restriction endonuclease that has a unique recognition site in the plasmid DNA outside the region to be amplified. 100 ng of this material is added to a PCR mixture containing PCR buffer. This mixture contains the four deoxynucleotide triphosphates included in the GeneAmp® kit (obtained from Perkin-Elmer Cetus, Norwalk, Conn. and Emeryville, Calif.) and 25 pmoles of each oligonucleotide primer for a final volume of 50 μl. The reaction mixture is overlaid with 35 μl mineral oil. The reaction is denatured at 100° C. for 5 min, placed briefly on ice, and then 1 μl Thermus aquaticus (Taq) DNA polymerase (5 units / μl, purchased from Perkin-Elmer Cetus, Norwalk, Conn. and Emeryville, Calif.) is added below the mineral oil layer. The reaction mixture is then inserted into a DNA thermal cycler (purchased from Perkin-Elmer Cetus) programmed as follows: 2 min at 55°C, 30 s at 72°C, followed by 19 cycles of 30 s at 94°C, 30 s at 55°C, and 30 s at 72°C. At the end of the process, the reaction vial is removed from the thermal cycler, the aqueous phase is transferred to a new vial, extracted with phenol / chloroform (50:50:vol), precipitated with ethanol, and the DNA is recovered by standard procedures. This material is then subjected to appropriate processing for insertion into a vector.

[0291] Cassette mutagenesis is another site-directed mutagenesis technique that can be used to introduce insertions and / or substitutions into DNA encoding a parent polypeptide. This technique is described in Wells et al. (Gene, 34:315, (1985)). There must be a unique restriction endonuclease site on each side of the mutation. If no such restriction sites exist, they can be generated to be introduced into the DNA at the appropriate locations using the nucleotide-mediated mutagenesis technique described above. The DNA encoding the parent polypeptide is cleaved at these restriction sites. Separate double-stranded DNA fragments with identical DNA sequences between the restriction sites but containing the desired mutations are synthesized using standard chemical synthesis procedures. This synthesized DNA fragment containing the mutation is called a cassette. This cassette is designed to have 3' and 5' ends compatible with the ends of the restriction endonuclease-treated DNA, so that it can be directly ligated to the restriction endonuclease-treated DNA. The ligated DNA now contains the desired mutation.

[0292] Using one or more site-directed mutagenesis techniques, the DNA encoding the parent polypeptide is mutated to introduce one or more codons for charged amino acid residues. The mutant DNA thus generated encodes a mutant polypeptide having a charged amino acid residue corresponding to the introduced codon. As a result, the mutant polypeptide may have an increased total number of charged amino acid residues compared to the parent polypeptide.

[0293] iii. Expression of mutant polypeptides Mutant DNA is generated using one or more site-directed mutagenesis techniques, and then the mutant DNA is expressed to produce the mutant polypeptide. A suitable method for expressing mutant DNA to produce mutant polypeptides is described in U.S. Patent No. 8,709,755 B2.

[0294] In one embodiment, mutant DNA can be expressed using peptide display methods for efficient screening of conditionally active polypeptides, as further described in PCT Publication Nos. WO 91 / 17271, WO 91 / 18980, WO 91 / 19818 and WO 93 / 08278.

[0295] WO 93 / 08278 describes a recombinant DNA method for displaying peptide ligands that involves the creation of a library of fusion proteins, each fusion protein consisting of a first polypeptide portion, typically containing a variable sequence, available for potential binding to a macromolecule, and a second polypeptide portion that binds to DNA, such as a DNA vector, encoding an individual fusion protein. When transformed host cells are cultured under conditions that allow expression of the fusion protein, the fusion protein binds to the DNA vector that encodes it. Upon lysis of the host cells, the fusion protein / vector DNA complexes can be screened against the immobilized macromolecule in the same way that bacteriophage particles are screened in phage-based display systems, and replication and sequencing of the DNA vector of selected fusion protein / vector DNA complexes provides a basis for identification of selected polypeptide sequences.

[0296] Peptide display techniques include in vitro and in vivo display methods for single-chain antibodies, such as nascent scFvs on polysomes or scFvs displayed on phage, and allow large-scale screening of scFv libraries with a wide range of diverse variable region sequences and binding specificities.

[0297] Other methods of expressing mutant polypeptides use cell-free enzymatic machinery to achieve in vitro synthesis of mutant polypeptides. Methods that rely on in vitro translation and generally involve stabilized polysome complexes are further described in PCT Publication Nos. WO 88 / 08453, WO 90 / 05785, WO 90 / 07003, WO 91 / 02076, WO 91 / 05058 and WO 92 / 02536.

[0298] Affinity enrichment methods allow for the screening of very large mutant polypeptide libraries and the selection of mutant DNAs encoding the desired polypeptides, where the mutant DNAs can be isolated and shuffled to combinatorially recombine the amino acid sequence of a given polypeptide (or a predetermined portion thereof) or single chain antibody (or only the VH, VL, or CDR portions thereof). Using such methods, mutant polypeptides or single chain antibodies with the desired binding affinity to a molecule can be identified, and the shuffling process can be exploited to rapidly converge on the desired high affinity mutant polypeptide or scFv.

[0299] iv. Screening for conditionally active polypeptides Methods for screening mutant polypeptides to select for conditionally active polypeptides are described in US Pat. No. 8,709,755 B2, which methods are incorporated herein by reference.

[0300] v. Assay conditions for screening and selecting conditionally active polypeptides The first and second conditions, or normal and abnormal conditions, of the assay used in the screening step may be performed using conditions selected from temperature, pH, osmolality, osmolality, oxidative stress, electrolyte concentration, protein concentration, and combinations of two or more such conditions. For example, the normal physiological condition of temperature may be normal human body temperature of 37.0°C, while the abnormal condition of temperature may be a temperature different from 37.0°C, for example, a temperature in a tumor microenvironment that may be 1-2°C higher than the normal physiological temperature. In another example, the normal and abnormal conditions may also be a normal physiological pH in the range of 7.2-7.8, or 7.2-7.6, and an abnormal pH present in the tumor microenvironment, such as in the range of 5.5-7.2, 6-7, or 6.2-6.8.

[0301] Both assays under the first and second conditions, or under normal and abnormal physiological conditions, may be performed in an assay medium. The assay medium may be a solution that may contain, for example, a buffer and other components. Common buffers that may 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 the assay may also be used. These buffers may be used to mimic the compositional properties or components of human or animal body fluids, such as plasma or lymph.

[0302] 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 ions, where bicarbonate is not part of the PBS buffer. Alternatively, bicarbonate ions are a component of the Krebs buffer.

[0303] 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, etc. 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.

[0304] 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.

[0305] 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, ferrous 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, 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.

[0306] Examples of normal physiological concentrations of some of the 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 about 80 μM, histidine ion in the range of 0.05-2.6 mM, histamine in the range of 0.3-1 μM, HAPT ion (hydrogen-added adenosine triphosphate) in the range of 1-20 μM, and HADP ion in the range of 1-20 μM.

[0307] In some embodiments, the ions present in the assay solutions in both the first and second conditions, or in the normal physiological and abnormal conditions, 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.

[0308] The organic compounds present in the assay solutions under both the first and second conditions, or under both normal and abnormal physiological conditions, may be selected from amino acids such as, for example, 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.

[0309] Examples of normal physiological concentrations of some of the amino acids include alanine at 3.97 ± 0.70 mg / dL, arginine at 2.34 ± 0.62 mg / dL, glutamic acid at 3.41 ± 1.39 mg / dL, glutamine at 5.78 ± 1.55 mg / dL, glycine at 1.77 ± 0.26 mg / dL, histidine at 1.42 ± 0.18 mg / dL, isoleucine at 1.60 ± 0.31 mg / dL, and riboflavin at 1.77 ± 0.26 mg / dL. These included leucine at 91±0.34 mg / dL, lysine at 2.95±0.42 mg / dL, methionine at 0.85±0.46 mg / dL, phenylalanine at 1.38±0.32 mg / dL, threonine at 2.02±6.45 mg / dL, tryptophan at 1.08±0.21 mg / dL, tyrosine at 1.48±0.37 mg / dL, and valine at 2.83±0.34 mg / dL.

[0310] The organic compounds present in the assay solutions under both the first and second conditions, or under both the normal and abnormal physiological conditions, may 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 creatine at 1.07±0.76 mg / dL, creatinine at 0.9-1.65 mg / dL, guanidinoacetic acid at 0.26±0.24 mg / dL, uric acid at 4.0±2.9 mg / dL, allantoin at 0.3-0.6 mg / dL, adenosine at 1.09±0.385 mg / dL, urea at 27.1±4.5 mg / dL, and choline at 0.3-1.5 mg / dL.

[0311] The organic compounds present in the assay solutions under both the first and second conditions, or under both normal physiological and abnormal conditions, may 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 acid, acetic acid, and volatile fatty acids. Examples of normal physiological concentrations of some of these organic acids include citric acid at 2.5±1.9 mg / dL, α-ketoglutaric acid at 0.8 mg / dL, succinic acid at 0.5 mg / dL, malic acid at 0.46±0.24 mg / dL, acetoacetic acid at 0.8-2.8 mg / dL, β-hydroxybutyric acid at 0.5±0.3 mg / dL, lactic acid at 8-17 mg / dL, pyruvic acid at 1.0±0.77 mg / dL, α-ketonic acid at 0.6-2.1 mg / dL, and volatile fatty acids at 1.8 mg / dL.

[0312] The organic compounds present in the assay solutions under both the first and second conditions, or under both normal and abnormal physiological conditions, may be selected from sugars (carbohydrates), such as glucose, pentoses, hexoses, xylose, ribose, mannose, and galactose, as well as disaccharides, including lactose, GlcNAcβ1-3Gal, Galα1-4Gal, Manα1-2Man, GalNAcβ1-3Gal, and O-, N-, C-, or S-glycosides. Examples of normal physiological concentrations of some of these sugars include 83±4 mg / dL glucose, 102±73 mg / dL polysaccharides (as hexoses), 77±63 mg / dL glucosamine, 0.4-1.4 mg / dL hexuronates (as glucuronic acid), and 2.55±0.37 mg / dL pentoses.

[0313] The organic compounds present in the assay solutions under both the first and second conditions, or under both normal and abnormal physiological conditions, may be selected from fats or derivatives thereof, 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).

[0314] The organic compounds present in the assay solutions under both the first and second conditions, or under both normal and abnormal physiological conditions, may be selected from proteins such as fibrinogen, antihemophilic globulins, immune gamma-globulins, immune euglobulins, isoagglutinins, beta-pseudoglobulins, glycoproteins, lipoproteins, 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.

[0315] In some embodiments, the assay solution may include blood proteins, such as those found in the subject's blood. A person skilled in the art can determine suitable blood proteins from a blood sample for a particular subject. For example, suitable human blood proteins may include albumin and hemoglobin.

[0316] The organic compounds present in the assay solutions under both the first and second conditions, or 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 between 0.019 and 0.036 mg / dL, vitamin E between 0.90 and 1.59 mg / dL, inositol between 0.42 and 0.76 mg / dL, folic acid between 0.00162 and 0.00195 mg / dL, and biotin between 0.00095 and 0.00166 mg / dL.

[0317] The concentration of an inorganic compound, ion, or organic molecule in the assay solution (both assays under first and second conditions, or under normal and abnormal physiological conditions) may be within the range of the normal physiological concentration of that inorganic compound, ion, or organic molecule in human or animal serum. However, concentrations outside the normal physiological range may also be used. For example, the normal range for magnesium ion in human serum is 1.7-2.2 mg / dL, and for calcium is 8.5-10.2 mg / dL. The magnesium ion concentration in the assay solution may be from about 0.17 mg / dL to about 11 mg / dL. The calcium ion concentration in the assay solution may 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.

[0318] The activity of the mutant polypeptide is measured using an assay under a first condition and a second condition, or under normal physiological conditions and abnormal conditions. During the assay, both the mutant polypeptide and its binding partner are present in the assay solution. The relationship between the mutant polypeptide and its binding partner can be, for example, antibody-antigen, ligand-receptor, enzyme-substrate, or hormone-receptor. For the mutant polypeptide to exhibit its activity, it must be able to contact and bind to its binding partner. The activity of the mutant polypeptide towards its binding partner is then exhibited and measured following binding between the mutant polypeptide and its binding partner.

[0319] In some embodiments, the ions used in the assay may function in forming bridges between the mutant polypeptide to be screened and its binding partner, especially those that contain charged amino acid residues. Thus, the ions may be capable of binding to both the mutant polypeptide and its binding partner through hydrogen bonds and / or ionic bonds. This may aid in the binding between the mutant polypeptide and its binding partner by allowing the ions to reach sites that may be difficult to reach by large molecules (mutant polypeptide or its binding partner). In some cases, the ions in the assay solution may increase the likelihood that the mutant polypeptide and its binding partner will bind to each other. Furthermore, the ions may additionally or alternatively aid in the binding between the mutant polypeptide and its binding partner by binding to the large molecule (mutant polypeptide or its binding partner). This binding may change the conformation of the large molecule to a particular conformation that facilitates binding to the binding partner and / or may hold the large molecule in such a conformation.

[0320] It has been observed that ions can assist the binding between the mutant polypeptide and its binding partner, possibly by forming ionic bonds between the mutant polypeptide and its binding partner. Thus, the 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.

[0321] It has been found that ions function to support binding between the mutant polypeptide and its binding partner at a pH close to the pKa of the ion. Such ions are preferably relatively small compared to the size of the mutant polypeptide.

[0322] 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 the candidate conditionally active polypeptide 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 at most 3 pH units away from the abnormal pH, at most 2 pH units away from the abnormal pH, at most 1 pH unit away from the abnormal pH, at most 0.8 pH units away from the abnormal pH, at most 0.6 pH units away from the abnormal pH, at most 0.5 pH units away from the abnormal pH, at most 0.4 pH units away from the abnormal pH, at most 0.3 pH units away from the abnormal pH, at most 0.2 pH units away from the abnormal pH, or at most 0.1 pH units away from the abnormal pH.

[0323] Exemplary pKa's of ions useful in the present invention (which may vary very slightly with 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, 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 + ).

[0324] In one embodiment, the conditionally active polypeptide is assayed and selected in the presence of disulfide. Disulfide has a pKa of 7.05. In some embodiments, different disulfide concentrations may be used for assays corresponding to normal and abnormal physiological conditions. Alternatively, the assay media for both normal and abnormal physiological conditions may have approximately the same disulfide concentration and may differ in some value for the specific condition, for example, the assay may be performed at a different pH. The disulfide concentration used in the assay may be between 1 mM and 100 mM. Preferably, the assay media has a disulfide concentration between 2 and 50 mM, or between 3 and 35 mM, or between 5 and 20 mM. Assays performed in the presence of disulfide are known.

[0325] In certain embodiments, once the pH of the abnormal condition (i.e., the abnormal pH) is known, ions suitable for increasing the number of 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 at most 3 pH units away from the abnormal pH, at most 2 pH units away from the abnormal pH, at most 1 pH unit away from the abnormal pH, at most 0.8 pH units away from the abnormal pH, at most 0.6 pH units away from the abnormal pH, at most 0.5 pH units away from the abnormal pH, at most 0.4 pH units away from the abnormal pH, at most 0.3 pH units away from the abnormal pH, at most 0.2 pH units away from the abnormal pH, or at most 0.1 pH units away from the abnormal pH.

[0326] As mentioned above, ions are most effective at supporting binding between mutant polypeptides and their binding partners at a pH at or near the pKa of the ion. For example, in an assay solution of pH 7.2-7.6, bicarbonate ions (which have a pKa of about 6.4) have been found to be less effective at supporting binding between mutant polypeptides and their binding partners. As the pH of the assay solution is lowered to 6.7 or even to about 6.0, bicarbonate ions become increasingly effective at supporting binding between mutant polypeptides and their binding partners. As a result, an assay at pH 6.0 may identify more hits compared to an assay at pH 7.2-7.6. Similarly, histidine is less effective at supporting binding between mutant polypeptides and their binding partners at pH 7.4. As the pH of the assay solution is lowered to 6.7 or even to about 6.0, histidine becomes increasingly effective at supporting binding between mutant polypeptides and their binding partners, and more hits may be identified, for example, at a pH range of about 6.2-6.4.

[0327] In the present invention, it has been unexpectedly found that when the pH of the assay solution under normal physiological conditions (i.e., normal physiological pH) is different from the pH of the assay solution under abnormal conditions (i.e., abnormal pH), ions having pKas ranging from about the midpoint between normal physiological pH and abnormal pH to about abnormal pH can significantly support the binding between the mutant polypeptide to be screened and its binding partner. As a result, the screening assay is much more efficient in finding more hit or candidate conditional polypeptides that are highly active under abnormal conditions.

[0328] In some embodiments, the pKa may even be at least one pH unit away from the abnormal pH. If the abnormal pH is an acidic pH, the pKa of the suitable ion may range from (abnormal pH-1) to the midpoint between the abnormal pH and normal physiological pH. If the abnormal pH is a basic pH, the pKa of the suitable ion may range from (abnormal pH+1) to the midpoint between the abnormal pH and normal physiological pH. The ions may be selected from those described in this application. However, many more ions not explicitly described in this application may also be used. It is understood that once the abnormal pH and normal physiological pH of the screening assay are selected, the skilled artisan may use the guidance of the present invention to select any ion with a suitable pKa to increase the efficiency of the screening in identifying more hits with high activity in abnormal conditions.

[0329] For example, if the abnormal pH for an 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) may be used for screening. Some ions with pKa in this range include ions 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 an 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) may be used for screening. Some ions with pKa 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 (6.46). Those skilled in the art can refer to numerous chemistry manuals and textbooks to identify known chemical compounds, including both inorganic and organic chemical compounds, that can be converted into ions with pKa in this range. Among chemical compounds with suitable pKa, those with smaller molecular weights may be preferred.

[0330] Thus, the present invention has unexpectedly found that the creation of the finally identified conditionally active polypeptide not only depends on the generation of the correct polypeptide mutant from the wild-type polypeptide, but also on the use of ions with suitable pKa in the assay solution. Since ions can facilitate the efficient selection of highly active mutants from a large library, the present invention believes that in addition to the generation of a large library of mutant polypeptides (e.g., by CPE and CPS), efforts should be made to find suitable ions (with appropriate pKa) to use in the assay solution. Furthermore, in the absence of suitable ions, the efficiency of screening may be low, decreasing the chance of finding highly active mutants. As a result, multiple screening rounds may be required to obtain the same number of highly active mutants without suitable ions.

[0331] The 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.

[0332] The concentration of this component in the assay solution (for both the first or normal physiological condition assay and the second or abnormal condition assay) 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 aid in binding between a mutant polypeptide and its binding partner, since it has been observed that a higher concentration of such ions may result in the formation of ionic bonds between the mutant polypeptide and its binding partner, in effect facilitating binding and increasing the likelihood of finding more hit or candidate conditionally active polypeptides.

[0333] In some embodiments, the concentration of ions in the assay solution may 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 about 15-30 mM. In one example, when the bicarbonate ion concentration in the assay solution was increased from 3 mM to 10 mM, 20 mM, 30 mM, 50 mM, and 100 mM, the number of hits in the assay increased with each increase in bicarbonate concentration. With this in mind, the assay solution may 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.

[0334] In another embodiment, the citrate concentration in the assay solution can be from about 30 μM to about 120 μM, or from about 40 μM to about 110 μM, or from about 50 μM to about 110 μM, or from about 60 μM to about 100 μM, or from about μM to about 90 μM, or about μM.

[0335] In one embodiment, the normal physiological conditions are normal physiological pH in the range of 7.2-7.6 and the abnormal conditions are abnormal pH in the range of 5.5-7.2, 6-7, or 6.2-6.8. The assay solution for assaying under normal physiological conditions has normal physiological pH and 50 mM bicarbonate ion. The assay solution for assaying under abnormal conditions has abnormal pH and 50 mM bicarbonate ion. Since the pKa of bicarbonate ion is about 6.4, bicarbonate ion can support binding between the mutant polypeptide and its binding partner at abnormal pH of pH pf 6.0-6.4, e.g. pH 6.0 or 6.2.

[0336] In yet another embodiment, the normal physiological condition is a normal physiological pH in the range of 7.2-7.6, and the abnormal condition is an abnormal pH in the range of 5.5-7.2, 6-7, or 6.2-6.8. The assay solution for the assay under normal physiological conditions has a normal physiological pH and 80 μM citrate ion. The assay solution for the assay under abnormal conditions has an abnormal pH and 80 μM citrate ion. Since the citrate ion has a pKa of 6.4, the citrate ion can effectively support the binding between the mutant polypeptide and the binding partner in the assay solution under abnormal conditions of pH 6.0-6.4. Therefore, more candidate conditionally active polypeptides having higher binding activity under conditions of pH 6.0-6.4 and lower activity under pH conditions of 7.2-7.8 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 mutant polypeptides that have higher binding activity at a pH around the ion's pKa and lower binding activity at a pH different from the ion's pKa (e.g., normal physiological pH).

[0337] In yet another embodiment, the normal physiological conditions are normal physiological temperature of 37° C. and the abnormal conditions are abnormal temperature of 38-39° C. (temperatures in some tumor microenvironments). The assay solution for assaying under normal physiological conditions has normal physiological temperature and 20 mM bicarbonate. The assay solution for assaying under abnormal conditions has abnormal temperature and 20 mM bicarbonate.

[0338] 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 at a different location in the animal or human, or that may be caused by an animal or human condition that alters the normal physiological electrolyte concentration in human serum.

[0339] The binding between a mutant polypeptide and / or its binding partner can also be influenced in a number of other ways. Typically, this can be influenced by including one or more additional components in the assay solution. These additional components can be designed to interact with either the mutant polypeptide, the binding partner, or both. In addition, these additional components can affect binding using a combination of two or more interactions as well as a combination of two or more types of interactions.

[0340] 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.

[0341] In addition to ions that may form ionic bonds with the mutant polypeptide and / or its binding partner to aid in binding between the mutant polypeptide and its binding partner, the present invention also includes other components that may be utilized to aid in binding between the mutant polypeptide and its binding partner. In one embodiment, molecules that may form hydrogen bonds with the mutant polypeptide and / or its binding partner may be utilized. In another embodiment, molecules capable of hydrophobic interactions with the mutant polypeptide and / or its binding partner may be used. In yet another embodiment, molecules capable of van der Waals interactions with the mutant polypeptide and / or its binding partner are contemplated.

[0342] As used herein, the term "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 an unshared pair of electrons of an electron-donating atom such as nitrogen, oxygen, sulfur, chlorine, or fluorine (hydrogen bond acceptor).

[0343] Moieties capable of forming hydrogen bonds with the mutant polypeptide and / or its binding partner include organic molecules with polar bonds as well as inorganic molecules. The mutant polypeptide and / or binding partner of the mutant polypeptide typically contain 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).

[0344] These amino acids can function as both hydrogen donors and hydrogen acceptors. For example, the oxygen atom of an -OH group, such as found in Ser, Thr, and Tyr; the oxygen atom of a -C=O group, such as found in Glu and Asp; the sulfur atom of an -SH group or -SC-, such as found in Cys and Met; the sulfur atom of an -NH3 group, such as found in Lys and Arg; + Nitrogen atoms of -NH- groups, such as those found in Trp, His, and Arg, can all function as hydrogen acceptors. Also, groups in this list that contain hydrogen atoms (e.g., -OH, -SH, NH3 + and -NH-) can also function as hydrogen donors.

[0345] In some embodiments, the backbone of the mutant 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 act as hydrogen acceptors, while the hydrogen atoms may participate in hydrogen bonds.

[0346] Inorganic compounds having at least one polar bond involving hydrogen or oxygen atoms that can be used for hydrogen bonding include, for example, H2O, NH3, H2O2, hydrazine, carbonates, sulfates, and phosphates. Organic compounds such as alcohols, phenols, thiols, aliphatic amines, amides, epoxides, carboxylic acids, ketones, aldehydes, ethers, esters, organic chlorides, and organic fluorines. Compounds capable of forming hydrogen bonds are well known in the chemical literature, for example, those discussed in "The Nature of the Chemical Bond," Linus Pauling, Cornell University Press, 1940, pages 284-334.

[0347] In some embodiments, alcohols may 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-butylmercaptan, pentanethiols, hexanethiol, thiophenol, dimercaptosuccinic acid, 2-mercaptoethanol, and 2-mercaptoindole. Suitable amines include methylamine, ethylamine, propylamine, isopropylamine, aniline, dimethylamine and methylethylamine, trimethylamine, aziridine, piperidine, N-methylpiperidine, benzidine, cyclohexylamine, ethylenediamine, hexamethylenediamine, o-, m-, and p-toluidine and N-phenylpiperidine.Suitable amides include ethanamide, N,N-dimethylacetamide, N,N-dimethylformamide, N,N-dimethylmethoxyacetamide, and N-methyl-Np-cyanoethylformamide.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, salicylaldehyde, 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 may be used in the present invention include dimethyl ether, methyl ethyl ether, diethyl ether, methyl propyl ether, and dimethoxyethane. Ethers may also be cyclic, such as ethylene oxide, tetrahydrofuran, and dioxane.

[0348] The organic chlorides include chloroform, pentachloroethane, dichloromethane, trichloromethane, carbon tetrachloride, tetrachloromethane, tetrachloroethane, pentachloroethane, trichloroethylene, tetrachloroethylene, and ethylene dichloride.The organic fluorines can include fluoromethane, difluoromethane, trifluoromethane, trifluoroethane, tetrafluoroethane, pentafluoroethane, difluoropropane, trifluoropropane, tetrafluoropropane, pentafluoropropane, hexafluoropropane, and heptafluoropropane.

[0349] Hydrogen bonds can be classified as strong, medium, or weak, depending 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.

[0350] In some embodiments, the components used in the present invention can form strong hydrogen bonds with mutant polypeptides and / or their binding partners. These components tend to have atoms with high electronegativity. The atoms known to have the highest electronegativity are, in order, F>O>Cl>N. Thus, 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.

[0351] In another embodiment, moieties capable of hydrophobic interaction with the mutant polypeptide and / or its binding partner are utilized. Such moieties include organic compounds with hydrophobic groups.

[0352] As used herein, the term "hydrophobic interaction" refers to a reversible attractive interaction between a hydrophobic compound or a hydrophobic region of a compound and another hydrophobic compound or a hydrophobic region of another compound. This type of interaction is described in "Hydrophobic Interactions," A. Ben-Nairn (1980), Plenum Press, New York.

[0353] Hydrophobic materials, due to their non-polar nature, experience the repulsive force of water molecules. When relatively non-polar molecules or groups in an aqueous solution associate with other non-polar molecules rather than with water, it is called "hydrophobic interaction".

[0354] The mutant polypeptides and their binding partners typically contain amino acids with hydrophobic interaction capacity. These amino acids can typically be characterized by having at least one side chain with a non-polar group with hydrophobic interaction capacity. 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).

[0355] Components capable of hydrophobic interaction with mutant polypeptides and / or their binding partners include organic compounds that are hydrophobic molecules or molecules containing at least one hydrophobic moiety. In some embodiments, these hydrophobic components 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 1-30 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.

[0356] 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 of hydrophobic moieties in the molecule involved in the hydrophobic interaction and / or the nature of the "hydrophobicity". For example, a hydrophobic moiety (which in its original form may comprise a hydrocarbon chain) can be modified to increase its hydrophobicity (its ability to increase the strength of the hydrophobic interaction in which it participates) by adding a hydrophobic side chain to one of the carbons of its carbon backbone. In a preferred embodiment of the present invention, this can include, for example, the addition of various steroid compounds and / or their derivatives, such as sterol-based compounds, more particularly various polycyclic compounds, including cholesterol. In general, the side chains can be linear, aromatic, aliphatic, cyclic, polycyclic, or any other various types of hydrophobic side chains as contemplated by those skilled in the art.

[0357] The types of moieties capable of van der Waals interactions with the mutant polypeptide and / or its binding partner are usually, but not necessarily, compounds that have 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, or molecules as a result of dipole-dipole interactions and / or quantum dynamics.

[0358] Van der Waals interactions in the present invention are attractive forces between mutant polypeptides or binding partners and components. 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).

[0359] Second, the presence of a molecule that is a permanent dipole can temporarily distort the electronic charges of other adjacent polar or non-polar molecules, thereby inducing further polarization. An additional attractive force arises 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 molecule involved is not a permanent dipole (e.g., the organic liquid benzene), there is still an attractive force between the molecules due to the two instantly induced dipoles in the molecule. This is the interaction between two instantly induced dipoles and can be referred to as a London dispersion force.

[0360] There are many amino acids in the mutant polypeptides and / or binding partners that have van der Waals interaction capabilities. 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), tryptophan (Trp). These amino acids may also have side chains with non-polar groups, including alanine (Ala), isoleucine (Ile), leucine (Leu), phenylalanine (Phe), valine (Val), proline (Pro), and glycine (Gly).

[0361] Components capable of van der Waals interactions with mutant polypeptides and / or their binding partners include polar or non-polar inorganic compounds that are 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 those that are polar such that they have the ability to interact dipole-dipole. For example, AlF3 has a polar Al-F bond and is soluble in water (about 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 about 1 g / 100 ml at 20°C.

[0362] 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.

[0363] Hydrogen bonding, hydrophobic and van der Waals components can be utilized to affect binding between a mutant polypeptide and its binding partner in a number of ways. In one embodiment, bridges can be formed between a mutant polypeptide and its binding partner by hydrogen bonding, hydrophobic interactions and / or van der Waals interactions. Such bridges can bring the mutant polypeptide and the binding partner into close proximity to one another to facilitate binding and / or place the mutant polypeptide and / or the binding partner in a position relative to one another to facilitate binding.

[0364] In another embodiment, hydrogen bonds and / or hydrophobic interactions may increase the likelihood that a mutant polypeptide will bind to its binding partner, for example, 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 may be used alone or in combination to cause the mutant polypeptide and binding partner to assemble closer to one another or to position the mutant polypeptide and binding partner in a manner that promotes binding, for example, by drawing the binding sites closer to one another or by positioning the non-binding portions of the molecule further away from one another, thereby positioning the binding sites closer to one another.

[0365] In yet another embodiment, hydrogen bonding and / or hydrophobic interactions may affect the conformation of the mutant polypeptide and / or its binding partner, resulting in a conformation that is more conducive to binding between the mutant polypeptide and its binding partner. Specifically, binding or interaction with one or more of the amino acids of the mutant polypeptide and / or binding partner may result in one or more conformational changes in the mutant polypeptide or binding partner that are favorable to the mutant polypeptide / binding partner binding reaction.

[0366] The present invention performs two pairs of assays, one that seeks a decrease in the activity of the mutant polypeptide in an assay under normal physiological conditions compared to the parent polypeptide from which it was derived in normal physiological conditions, and a second assay that seeks an increase in the activity of the mutant polypeptide in an assay under abnormal conditions compared to the parent polypeptide from which it was derived in abnormal conditions. In some cases, the parent polypeptide from which the mutant polypeptide is derived is a wild-type polypeptide. In other cases, the parent polypeptide may itself be a mutant polypeptide prepared using one or more of the mutation techniques described elsewhere herein.

[0367] The conditions used in the assay pairs of the invention may 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 may be used at substantially the same concentration in both pairs of assays. In such cases, the component is typically present for the purpose of simulating a particular environment in a human or animal, such as serum, a tumor microenvironment, a synovial fluid environment, a neurological environment, or any other environment that may be encountered at the point of administration, through which an administered treatment may pass, or at the point of treatment. An important aspect of the selection of one or more components that mimic these environments is that it may improve the results of the selection process performed with the assay pair. For example, simulating a particular environment allows the evaluation of different effects of particular components of that environment on mutant polypeptides during the selection process. A particular environmental component may, for example, alter or bind to the mutant polypeptide, inhibit the activity of the mutant polypeptide, inactivate the mutant polypeptide, etc.

[0368] In some embodiments, one or more components of the assay solution are preferably small compounds such as disulfide, bicarbonate, histidine, histamine, citrate, lactate, and acetate, etc. In one embodiment, the small molecule components are preferably present in the assay solution at a concentration of about 100 μm to about 100 mM, or more preferably about 0.5 to about 50 mM, or about 1 to about 10 mM.

[0369] The concentration of a component in the assay solution 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. 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.

[0370] 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 that is assayed, since it is the concentration of the components that is the condition that distinguishes between the assay solution for the assay under normal physiological conditions and the assay solution for the assay under abnormal conditions. Thus, the conditionally active polypeptides produced by this embodiment of the method of the invention may be selected for an activity that is at least partially dependent on the concentration of the components.

[0371] 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.

[0372] 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 the abnormal conditions present at a particular location in the body.

[0373] In another example, glucose may be absent in the abnormal condition assay solution to mimic the absence of glucose as may be found in a tumor microenvironment, while in the pair of normal physiological condition assay solutions glucose may be set at a level that mimics plasma glucose concentrations. Using this feature, a conditionally active polypeptide may be preferentially delivered to a location or environment that is inactive or minimally active during transport, and activated upon reaching an environment where a concentration of a component in the abnormal condition assay solution is present.

[0374] For example, the tumor microenvironment typically has both low glucose and high lactate concentrations compared to human serum. The normal physiological concentration of glucose ranges from about 2.5 mM to about 10 mM in serum. On the other hand, in the tumor microenvironment, the glucose concentration is typically very low, ranging from 0.05 mM to 0.5 mM. In one embodiment, the assay solution for assaying under normal physiological conditions has a glucose concentration ranging from about 2.5 mM to about 10 mM, and the assay solution for assaying under abnormal conditions has a glucose concentration ranging from about 0.05 mM to about 0.5 mM. The conditionally active polypeptide thus produced has higher activity in a low glucose environment (in the tumor microenvironment) than in a high glucose environment (in normal tissue or blood). The conditionally active polypeptide is functional in the tumor microenvironment, but may have low activity while passing through the bloodstream.

[0375] Normal physiological concentrations of lactate in serum range from about 1 mM to about 2 mM. On the other hand, in tumor microenvironments, lactate concentrations are typically in the range of 10 mM to 20 mM. In one embodiment, assay solutions for assays under normal physiological conditions have lactate concentrations in the range of about 1 mM to about 2 mM, and assay solutions for assays under abnormal conditions have lactate concentrations in the range of about 10 mM to about 20 mM. The conditionally active polypeptide thus produced has higher activity in a high lactate environment (in the tumor microenvironment) than in a low lactate environment (in normal tissue or blood). Thus, the conditionally active polypeptide is functional in the tumor microenvironment, but may have low activity while passing through the bloodstream.

[0376] Similarly, it is known that lactate concentrations are higher than normal (abnormal) in muscle pain. Thus, when searching for mutant polypeptides that may be active in a muscle pain environment, the abnormal condition assay pair can be performed in the presence of higher concentrations of lactate to mimic the muscle pain environment, while the normal physiological condition assay pair can be performed at lower concentrations of lactate or in the absence of lactate. In this way, mutant polypeptides can be selected that have improved activity with increasing lactate concentrations in a muscle pain environment. Such conditionally active polypeptides can be useful, for example, as anti-inflammatory agents.

[0377] 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 conditionally active polypeptides. For example, returning to the tumor microenvironment, the abnormal condition assay pair may be performed in an assay medium that includes both high lactate and low glucose concentrations, while the corresponding normal physiological condition assay pair may be performed in an assay medium that includes both relatively low lactate and relatively high glucose concentrations.

[0378] The present invention contemplates that each component 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.

[0379] 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.

[0380] 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. The 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 concentration of about 85000 μM in the blood; inositol has a concentration of about 21700 μM in the blood; glycine has a concentration of about 220-400 μM in the blood; and alanine has a concentration of about 220-300 μM in the blood.

[0381] Thus, these metabolites, including at least N-acetylaspartic acid, inositol, glycine, and alanine, may be used in different concentrations in the assay solution 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 with a concentration of 85000 μM N-acetylaspartic acid may be used in the assay pair under normal physiological conditions, and an assay solution with a concentration of 350 μM N-acetylaspartic acid may be used in the assay pair under abnormal conditions.

[0382] Mayers et al., “Elevated circulating branched chain amino acids are an early event in pancreatic adenocarcinoma development,” Nature Medicine, vol. 20, pp. 1193-1198, 2014, investigated the concentrations of various different metabolites, including branched chain amino acids, in the pre-diagnostic plasma of pancreatic patients. They found that pancreatic tumor patients have several metabolites that are 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. also found that pancreatic cancer patients have a significant elevation of branched chain amino acids in their plasma compared to normal subjects. Branched chain amino acids that are present at elevated concentrations include isoleucine, leucine, and valine (Table 1 in Mayers et al.). There are other metabolites shown in Figure 1 in Mayers that are present at significantly different concentrations in the plasma of pancreatic cancer patients compared to normal healthy humans. These metabolites include at least acetylglycine, glycine, phenylalanine, tyrosine, 2-aminoadipic acid, taurodeoxycholate / taurochenodeoxycholate, aconitate, isocitrate, lactate, a-glycerophosphate, and urate. Therefore, based on the knowledge 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.

[0383] Thus, in one embodiment, one or more of these metabolites may be used in an assay solution under normal physiological conditions in amounts that approximate the concentrations of these metabolites in plasma of a healthy individual (i.e., the normal physiological concentrations of those metabolites). For example, the known normal physiological concentrations in 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. An assay solution under normal physiological conditions may have normal physiological concentrations of one or more of these branched amino acids within these ranges. An assay solution under abnormal conditions may have a concentration of the same branched amino acid that is about 5 times, or about 10 times, or about 20 times, or about 50 times, or about 70 times, or about 100 times, or about 150 times, or about 200 times, or about 500 times higher than the normal physiological concentration of the corresponding branched amino acid in a healthy individual. This may reflect that the higher concentrations of these branched amino acids found in plasma by Mayers et al. arise from the tumor microenvironment and are diluted in the bloodstream, and therefore, based on the findings of Mayers et al., one would expect the concentrations of these branched 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 real environment, thereby selecting the most active variants for that particular environment.

[0384] In some other embodiments, the assay solution for normal physiological conditions may contain one or more branched amino acids at concentrations that mimic the concentrations in the plasma of patients with pancreatic cancer to mimic the actual plasma environment of these patients. In such embodiments, the assay solution for abnormal conditions may have concentrations of the same branched amino acids that are 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 concentrations of the corresponding branched amino acids in the plasma of patients with pancreatic cancer to reflect the fact that these higher concentrations occur in the tumor microenvironment and that the concentrations in the bloodstream represent a dilution of the actual concentrations in the tumor microenvironment. Similarly, other metabolites may have different concentrations in the assay solutions for normal physiological conditions and abnormal conditions, reflecting the actual differences that would be expected from the 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 may be used to assay for normal physiological conditions, and a lower concentration may be used to assay for abnormal conditions to take into account the expectation that the metabolite would be consumed in the tumor microenvironment. Conditionally active polypeptides selected using such assay solutions may be more active in the pancreatic cancer microenvironment compared to in the plasma of pancreatic cancer patients.

[0385] In some embodiments, whole plasma of a pancreatic cancer patient may be used in the present invention. For example, in one embodiment, one or more components of plasma of a pancreatic cancer patient may be simulated in the assay solution for one or both of the assays under normal physiological conditions and abnormal conditions. In an exemplary embodiment, the assay solution under normal physiological conditions has a pH in the range of 7.2-7.6 and is spiked with 30 wt.% plasma of a pancreatic cancer patient, and the assay solution under abnormal conditions has a pH in the range of 6.2-6.8 and is spiked with 30 wt.% plasma of a pancreatic cancer patient. In this embodiment, the plasma of the pancreatic cancer patient is present both to (1) ensure that the conditionally active polypeptide is not activated in blood at pH 7.2-7.6, and (2) also ensure that the conditionally active polypeptide can be activated by pH 5.5-7.2, 6-7, or 6.2-6.8 in the tumor microenvironment, even in the presence of this metabolic product composition found in the blood of a pancreatic cancer patient. This allows the treatment to be tailored to the pancreatic cancer patient.

[0386] In another exemplary embodiment, the assay solution under normal physiological conditions has a pH in the range of 7.2 to 7.6 and is supplemented with 30 wt.% 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 supplemented with any plasma from a pancreatic cancer patient.

[0387] In each of the several types of assays discussed above, the same components selected from inorganic compounds, ions, and organic molecules may be used. For example, in the case of lactate, lactate may be used at substantially the same concentration in both normal and abnormal conditions of the pair of assay solutions. The normal and abnormal 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 that distinguish between normal and abnormal conditions, thereby reflecting that lactate is at a higher concentration in the abnormal tumor microenvironment compared to the normal physiological condition (non-tumor microenvironment).

[0388] In some embodiments, two or more components are added to both the normal and abnormal conditions assay solutions at substantially the same concentration. 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%.

[0389] In one embodiment, serum may be added to both the normal and abnormal conditions assay solutions at substantially the same concentration. Since serum has a large number of inorganic compounds, ions, and organic molecules (including polypeptides), the assay solution may have a plurality and a large number of components selected from inorganic compounds, ions, and organic molecules that are present at substantially the same concentration between the two assay solutions. The assay solution may have 5-30 vol.%, or 7-25 vol.%, or 10-20 vol.%, or 10-15 vol.% serum. In some other embodiments, both the normal and abnormal conditions assay solutions 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.

[0390] Assay solutions for normal and abnormal physiological conditions may have different pHs, which may be adjusted using CO2 and O2 levels in the buffer using bicarbonate.

[0391] In some other embodiments, at least one of the 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 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 by 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.

[0392] In some embodiments, the assay solution may be designed to select for conditionally active polypeptides whose activity depends on two or more conditions. In one exemplary embodiment, the conditionally active polypeptide may have activity that is both pH and lactate dependent. The assay solution for selecting such conditionally active polypeptides may be an assay solution with a pH of 7.2-7.6 and a lactate concentration in the range of 8-15 mg / dL for normal physiological conditions. The assay solution for abnormal conditions may be an assay solution with a pH of 6.2-6.8 and a lactate concentration in the range of 30-50 mg / dL. Optionally, the assay solutions for both normal and abnormal physiological conditions may also contain ions that aid in binding between the mutant polypeptide and its binding partner, thus increasing the number of hits for candidate biologically active polypeptides.

[0393] In yet another exemplary embodiment, the conditionally active polypeptide may have activity that is dependent on pH, glucose, and lactate. The assay solution for selecting such conditionally active polypeptides may be an assay solution with a pH of 7.2-7.6, a glucose concentration in the range of 2.5-10 mM, and a lactate concentration in the range of 8-15 mg / dL for normal physiological conditions. The assay solution for abnormal conditions may be an assay solution with a pH of 6.2-6.8, a glucose concentration in the range of 0.05-0.5 mM, and a lactate concentration in the range of 30-50 mg / dL. Optionally, the assay solutions for both normal and abnormal physiological conditions may also contain ions that support binding between the mutant polypeptide and its binding partner, thus increasing the number of candidate biologically active polypeptides that bind to the binding partner at pH 6.2-6.8. A conditionally active polypeptide selected using such an assay solution is more active in an environment of pH 6.2-6.8, a glucose concentration of 0.05-0.5 mM, and a lactate concentration of 30-50 mg / dL than in an environment of pH 7.2-7.6, a glucose concentration of 2.5-10 mM, and a lactate concentration of 8-15 mg / dL.

[0394] The two or more components selected from inorganic compounds, ions, and organic molecules are for creating an abnormal condition assay solution that mimics the environment of the location / site (i.e., the target site) to which the selected conditionally active polypeptide is to be delivered. In some embodiments, at least three components in the target site environment may be added to the assay solution, or at least four components in the target site environment may be added to the assay solution, or at least five components in the target site environment may be added to the assay solution, or at least six components in the target site environment may be added to the assay solution.

[0395] In one embodiment, bodily fluid collected from a target site (where the conditionally active polypeptide may be more active) may be directly used as an assay solution for the abnormal condition assay. 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 abnormal condition assay pair to select the conditionally active polypeptide. By using the collected synovial fluid, optionally diluted, as an assay solution for the abnormal condition assay and an assay solution that mimics human plasma for the normal physiological condition assay, 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.

[0396] The development of a conditionally active polypeptide with activity dependent on multiple conditions will result in improved selectivity of the conditionally active polypeptide to target sites in the subject's body. Ideally, the conditionally active polypeptide is not active, or at least is significantly less active, in other locations where only some of the conditions are present. In one embodiment, a conditionally active polypeptide that is active at pH 6.2-6.8, glucose concentration 0.05-0.5 mM, and lactate concentration 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, and therefore are 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. However, it may not have the alternative assay conditions. Thus, the conditionally active polypeptide is not active, or at least is less active, in post-exercise muscle.

[0397] In some embodiments, a step may 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 may 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 may then be tested individually under each of these three conditions, and in environments that include pairs of the three conditions, to confirm that the conditionally active polypeptide is not active or has low activity under these test conditions or environments.

[0398] In some embodiments, certain components of serum are intentionally minimized or excluded from the assay medium. For example, when screening for antibodies, components of serum that bind or adsorb the antibodies can be minimized or excluded in 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 such that components that can potentially bind to the variants in the assay are minimized or excluded, the number of non-functional variants that may be falsely 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 mutant 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 mutant polypeptides that bind to components in human serum. Other similar substitutions can also be made in the detailed examples to achieve the same goal.

[0399] In some embodiments, the assay conditions mimic the environment near the cell membrane, such as inside the cell membrane, on the cell membrane, or outside the cell membrane, or the joint environment. Some factors that may affect the binding activity during screening in the cell membrane environment include receptor expression, internalization, antibody drug conjugate (ADC) potency, etc.

[0400] The format of the assay can be any suitable assay known to those skilled in the art. Examples include ELISA, enzyme activity assays, in vitro (such as organ) real tissue screening, tissue slides, whole animals, cell lines and the use of 3D systems. For example, suitable cell-based assays are described in WO2013 / 040445, tissue-based assays are described in US7,993,271, whole animal-based screening methods are described in US2010 / 0263599, and 3D system-based screening methods are described in US2011 / 0143960.

[0401] In some embodiments, the evolution process may generate mutant polypeptides that may simultaneously possess other desired properties in addition to the conditionally active properties discussed above. Suitable other desired 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 desired properties. Selection processes may be used to select for both the conditional activity and one or more other desired properties of the mutant polypeptide.

[0402] In some embodiments, the present invention generates conditionally active polypeptides. The selected conditionally active polypeptides can be further mutated, for example in a second evolution step, using one of the mutagenesis techniques disclosed herein, thereby improving another property of the selected conditionally active polypeptide, such as binding activity, expression, humanization, etc. After this second evolution step, mutant polypeptides can be screened for both conditional activity and improved properties.

[0403] In some embodiments, after evolving the parent polypeptide to generate mutant polypeptides, a first conditionally active polypeptide is selected that exhibits both (a) a decrease in the first activity compared to the parent polypeptide in an assay under normal physiological conditions, and (b) an increase in the first activity compared to the parent polypeptide in an assay under abnormal conditions. The first conditionally active polypeptide can then be further subjected to one or more additional evolution, expression and selection steps to select at least a second conditionally active polypeptide that exhibits both (1) (a) a decrease in the second activity compared to the parent polypeptide in an assay under normal physiological conditions, and (b) an increase in the second activity compared to the parent polypeptide in an assay under abnormal conditions, or (2) 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. It should be noted that the second conditionally active polypeptide can have both a higher first activity and a higher second activity under abnormal conditions compared to the parent polypeptide, and a lower first activity and a lower second activity under normal physiological conditions compared to the parent polypeptide.

[0404] In some embodiments, the expression step is carried out in a eukaryotic production host. The host may be 3T3 mouse fibroblast cells; BHK21 Syrian hamster fibroblast cells; MDCK, canine epithelial cells; Hela human epithelial cells; PtK1 rat kangaroo epithelial cells; SP2 / 0 mouse plasma cells; and NS0 mouse plasma cells; HEK293 human embryonic kidney cells; COS monkey kidney cells; CHO, CHO-S Chinese hamster ovary cells; R1 mouse embryo cells; E14.1 mouse embryo cells; H1 human embryo cells; H9 human embryo cells; Per C.6, human embryo cells; S. cerevisiae yeast cells and pichia yeast cells. The selected conditionally active polypeptide may then be produced in large quantities in the same eukaryotic production host. By using the same eukaryotic production host for both the expression step and the final production, higher expression levels of the conditionally active polypeptide may be achieved.

[0405] In certain embodiments, the present invention aims to generate conditionally active polypeptides that have a greater ratio of activity under abnormal conditions (or a second condition) to activity under normal physiological conditions (or a first condition) (e.g., greater selectivity between abnormal and normal physiological conditions). The ratio of activity under abnormal conditions (or second conditions) to activity under normal physiological conditions (or first 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.

[0406] 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 the tumor site (in the tumor microenvironment) and is significantly less active or inactive at non-tumor sites (normal physiological conditions).

[0407] In one embodiment, the conditionally active polypeptide is an antibody intended to be conjugated with another agent, such as those disclosed elsewhere herein. The conditionally active antibody may have a higher ratio of activity under abnormal conditions to activity under normal physiological conditions. For example, the conditionally active antibody conjugated with 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, since it is desirable to concentrate such conjugated agent at the disease or treatment site (where the abnormal condition exists).

[0408] D. Creation of Conditionally Active Polypeptides Selected conditionally active polypeptides with reversible or irreversible activity may be generated for therapeutic, diagnostic, research and related purposes and / or may be subjected to one or more additional evolution and selection cycles.

[0409] The conditionally active polypeptide may be produced using a polypeptide-expressing cellular production host or organism. To make the production process more efficient, the DNA encoding the conditionally active polypeptide may be subjected to codon optimization for the cellular production host or organism. Codon optimization has been described previously, for example, Narum et al., "Codon optimization of gene fragments encoding Plasmodium falciparum merzoite proteins enhances DNA vaccine protein expression and immunogenicity in mice". Infect. Immun. 2001 December, 69(12):7250-3 describes codon optimization in mouse systems. Outchkourov et al., "Optimization of the expression of Equistatin in Pichia pastoris, protein expression and purification", Protein Expr. Purif. 2002 February;24(1):18-24 describes codon optimization in yeast systems. Feng et al., "High level expression and mutagenesis of recombinant human phosphatidylcholine transfer protein using a synthetic gene: evidence for a C-terminal membrane binding domain," Biochemistry 2000 Dec.19,39(50):15399-409, describes codon optimization in E. coli.Humphreys et al., "High-level periplasmic expression in Escherichia coli using a eukaryotic signal peptide: importance of codon usage at the 5'end of the coding sequence", Protein Expr. Purif. 2000 Nov. 20(2):252-64, describes how codon usage affects protein secretion in E. coli.

[0410] The cell production host may be a mammalian system selected from one of the group consisting of CHO, HEK293, IM9, DS-I, THP-I, Hep G2, COS, NIH 3T3, C33a, A549, A375, SK-MEL-28, DU 145, PC-3, HCT 116, Mia PACA-2, ACHN, Jurkat, MM1, Ovcar 3, HT 1080, Panc-1, U266, 769P, BT-474, Caco-2, HCC 1954, MDA-MB-468, LnCAP, NRK-49F, and SP2 / 0 cell lines; and mouse splenocytes and rabbit PBMCs. In one embodiment, the mammalian system is selected from a CHO or HEK293 cell line. In a specific aspect, the mammalian system is a CHO-S cell line. In another embodiment, the mammalian system is a HEK293 cell line.

[0411] In some embodiments, the cellular production host is a yeast cell line, such as an S. cerevisiae yeast cell or a Pichia yeast cell. In some embodiments, the cellular production host is a prokaryotic cell, such as E. coli (Owens RJ and Young RJ, J. Immunol. Meth., vol. 168, p. 149, 1994; Johnson S and Bird RE, Methods Enzymol., vol. 203, p. 88, 1991). Conditionally active polypeptides can also be made in plants (Firek et al. Plant MoI. Biol., vol. 23, p. 861, 1993).

[0412] Conditionally active polypeptides can also be produced by synthetic methods using chemical methods well known in the art. For example, see Caruthers, "New chemical methods for synthesizing polynucleotides," Nucleic Acids Res. Symp. Ser. 215-223, 1980; Horn, "Synthesis of oligonucleotides on cellulose. Part II: design and synthetic strategy to the synthesis of 22 oligodeoxynucleotides coding for Gastric Inhibitory Polypeptide (GIP)" 1 ", Nucleic Acids Res. Symp. Ser. 225-232, 1980; Banga, A. K., Therapeutic Peptides and Proteins, Formulation, Processing and Delivery Systems Technomic Publishing Co., Lancaster, Pa, 1995. For example, peptide synthesis can be carried out using a variety of solid-phase techniques (see, e.g., Roberge "A strategy for a convergent synthesis of N-linked glycopeptides on a solid support", Science 269:202, 1995; Merrifield "Concept and early development of solid-phase peptide synthesis", Methods Enzymol. 289:3-13, 1997), or automated synthesis may be accomplished using, for example, an ABI 43 IA peptide synthesizer (Perkin Elmer) following instructions provided by the manufacturer.

[0413] Solid-phase chemical peptide synthesis methods have been known in the art since the early 1960s (Merrifield, R.B., "Solid-phase synthesis I. The synthesis of a tetrapeptide", J. Am. Chem. Soc, 85:2149-2154, 1963) (see also Stewart, J.M. and Young, J.D., Solid Phase Peptide Synthesis, 2nd Ed., Pierce Chemical Co., Rockford, 111., pp.11-12)) and are now used in commercially available laboratory peptide design and synthesis kits (Cambridge Research Biochemicals). Such commercially available laboratory kits generally utilize the teachings of HM Geysen et al., "Use of peptide synthesis to probe viral antigens for epitopes to a resolution of a single amino acid," Proc. Natl. Acad. Sci., USA, 81:3998, 1984, and provide for the synthesis of peptides at the tips of a large number of "rods" or "pins," all attached to a single plate. In using such a system, the plate of rods or pins is inverted and inserted into a second plate in corresponding wells or reservoirs, which contains a solution for attaching or fixing the appropriate amino acids to the tips of the pins or rods. By repeating this process step, i.e., inverting and inserting the tips of the rods and pins into the appropriate solutions, amino acids are built up into the desired peptide. In addition, there are many available FMOC peptide synthesis systems available. For example, assembly of polypeptides or fragments can be performed on a solid support using an Applied Biosystems, Inc. Model 431 A™ automated peptide synthesizer. Such equipment provides ready access to the peptides of the present disclosure, either by direct synthesis or by synthesis of a series of fragments that can be coupled using other known techniques.

[0414] The conditionally active polypeptides can also be glycosylated. Glycosylation can be added chemically or post-translationally by the cellular biosynthetic machinery, which incorporates the use of known glycosylation motifs, which may be native to the sequence or may be added as peptides or to the nucleic acid coding sequence. Glycosylation can be O-linked or N-linked.

[0415] The conditionally active polypeptide includes any form of "mimetic" and "peptidomimetic". The terms "mimetic" and "peptidomimetic" refer to a synthetic chemical compound having substantially the same structural and / or functional properties as the polypeptides of the present disclosure. The mimetics may be composed entirely of synthetic non-natural amino acid analogs, or are chimeric molecules of partly natural peptide amino acids and partly non-natural amino acid analogs. The mimetics can also incorporate any amount of natural amino acid conservative substitutions, so long as the substitutions do not also substantially alter the mimetic's structure and / or activity. As with the polypeptides of the present disclosure that are conservative variants, it will be determined by routine experimentation whether a mimetic is within the scope of the present disclosure, i.e., its structure and / or function are not substantially altered.

[0416] The polypeptide mimetic compositions of the present disclosure may contain any combination of non-natural structural components. In alternative embodiments, the mimetic compositions of the present disclosure include one or all of the following three structural groups: a) residue linking groups other than natural amide bonds ("peptide bonds") linkages; b) non-natural residues in place of naturally occurring amino acid residues; or c) residues that induce secondary structure mimicry, i.e., induce or stabilize secondary structures, such as β-turns, γ-turns, β-sheets, α-helical conformations, etc. For example, a polypeptide of the present disclosure can be characterized as a mimetic when all or some of its residues are joined together by chemical means other than natural peptide bonds. Individual peptidomimetic residues can be joined together by peptide bonds, other chemical bonds or coupling means, such as glutaraldehyde, N-hydroxysuccinimide esters, bifunctional maleimides, N,N'-dicyclohexylcarbodiimide (DCC) or N,N'-diisopropylcarbodiimide (DIC), etc. Linking groups that can be used in place of the traditional amide bond ("peptide bond") linkage include, for example, ketomethylene (e.g., -C(C=O)CH2- as opposed to -(C=O)-NH-), aminomethylene (CH2-NH), ethylene, olefin (CH=CH), ether (CHO), thioether (CH2S), tetrazole (CN4--), thiazole, retroamide, thioamide, or ester (see, e.g., Spatola (1983), Chemistry and Biochemistry of Amino Acids, Peptides and Proteins, Vol. 7, pp 267-357, "Peptide Backbone Modifications," Marcell Dekker, NY).

[0417] The polypeptides of the present disclosure can also be characterized as mimetics by containing all or some non-naturally occurring residues in place of naturally occurring amino acid residues. Non-naturally occurring residues are well described in the scientific and patent literature; some exemplary non-natural compositions and guidelines useful as mimetics of natural amino acid residues are set forth below. Mimetics of aromatic amino acids include, for example, D- or L-naphylalanine; D- or L-phenylglycine; D- or L-2 thieneylalanine; D- or L-1,-2, 3-, or 4-pyreneylalanine; D- or L-3 thieneylalanine; D- or L-(2-pyridinyl)-alanine; D- or L-(3-pyridinyl)-alanine; D- or L-(2-pyrazinyl)-alanine; D- or L-(4-isopropyl)-phenylglycine; D-(trifluoromethyl ... D-(methyl)-phenylglycine; D-(trifluoromethyl)-phenylalanine; Dp-fluoro-phenylalanine; D- or Lp-biphenylphenylalanine; D- or Lp-methoxy-biphenylphenylalanine; D- or L-2-indole(alkyl)alanine; and D- or L-alkylanines, where alkyl may be substituted or unsubstituted methyl, ethyl, propyl, hexyl, butyl, pentyl, isopropyl, isobutyl, sec-isotyl, iso-pentyl, or non-acidic amino acids. Aromatic rings of unnatural amino acids include, for example, thiazolyl, thiophenyl, pyrazolyl, benzimidazolyl, naphthyl, furanyl, pyrrol...

Claims

1. A conditionally active polypeptide obtained by increasing the net charge of a parent polypeptide, wherein the conditionally active polypeptide exhibits a decrease in activity in a first assay at a first value of a condition compared to the same activity in a second assay at a second value of the same condition.

2. The conditionally active polypeptide of claim 1 , wherein the condition is pH.

3. The conditionally active polypeptide of claim 1 , wherein the first assay and the second assay are performed in an assay solution in the absence of serum.

4. 2. The conditionally active polypeptide of claim 1, wherein the net charge is increased by introducing one or more charged amino acid residues into the parent polypeptide through amino acid residue substitution, amino acid insertion, or a combination thereof.

5. 3. The conditionally active polypeptide of claim 2, wherein the charged amino acid residue is selected from aspartic acid, glutamic acid, arginine, histidine and lysine.

6. 6. The conditionally active polypeptide of claim 5, wherein the charged amino acid residue is selected from aspartic acid, glutamic acid, arginine and lysine.

7. The conditionally active polypeptide of claim 5 , wherein the one or more charged amino residues are introduced into the active site of the parent polypeptide.

8. The conditionally active polypeptide of claim 5 , wherein the one or more charged amino residues are introduced into a region outside the active site of the parent polypeptide.

9. 9. The conditionally active polypeptide of claim 8, wherein the region outside the active site is a constant region of an antibody or a framework region of the antibody.

10. 10. The conditionally active polypeptide of claim 4, wherein the one or more charged amino acid residues are less than 5 charged amino acid residues, or less than 4 charged amino acid residues, or less than 3 charged amino acid residues, or less than 2 charged amino acid residues.

11. The conditionally active polypeptide of any one of claims 4 to 10, wherein the one or more charged amino acid residues are selected from aspartic acid and glutamic acid.

12. The conditionally active polypeptide according to any one of claims 4 to 11, wherein the conditionally active polypeptide is a conditionally active antibody, and the one or more charged amino acid residues are introduced into at least one of the complementarity determining regions of the conditionally active antibody.

13. The conditionally active polypeptide of any one of claims 1 to 12, wherein the conditionally active polypeptide is reversibly inactivated at the first value of the condition.

14. The conditionally active polypeptide of any one of claims 1 to 13, wherein the condition is selected from pH, temperature, osmolality, osmolality, oxidative stress, electrolyte concentration.

15. The conditionally active polypeptide according to any one of claims 1 to 14, wherein the condition is pH.

16. 16. The conditionally active polypeptide of claim 15, wherein the first and second assays are performed in the presence of at least one species having a molecular weight of less than 900 a.m.u. and a pKa that is at most 3 units away from the second value of the pH.

17. 16. The conditionally active polypeptide of claim 15, wherein the first and second assays are performed at the same temperature and in the presence of at least one species having a molecular weight of less than 900 a.m.u. and a pKa between the first value of the pH and the second value of the pH.

18. 13. The conditionally active polypeptide of claim 12, wherein the first and second assays are performed in the presence of a species selected from histidine, histamine, adenosine diphosphate, hydrogen sulfide, adenosine triphosphate, citrate, acetate, lactate, bicarbonate, disulfide, ammonium, dihydrogen phosphate, and combinations thereof.

19. 19. The conditionally active polypeptide of any one of claims 1 to 18, wherein the ratio of increased activity in an assay at a second value of the condition to decreased activity in an assay at a first value of the condition 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, or at least 20.0, or at least 40.0, or at least 60.0, or at least 100.

0.

20. 20. The conditionally active polypeptide according to any one of claims 1 to 19, wherein the first value of the condition is a value of a normal physiological condition that is within the normal range of physiological conditions at the administration site of the conditionally active polypeptide to a subject or physiological conditions in a tissue or organ at the site of action of the conditionally active polypeptide in a subject, and the second value of the condition is a value of an abnormal condition that deviates from the normal range of physiological conditions at the administration site of the conditionally active polypeptide or physiological conditions in a tissue or organ at the site of action of the conditionally active polypeptide.

21. The conditionally active polypeptide according to any one of claims 1 to 20, wherein the conditionally active polypeptide is a protein or a protein fragment.

22. The conditionally active polypeptide according to any one of claims 1 to 21, wherein the conditionally active polypeptide is selected from an antibody, a single-chain antibody, and an antibody fragment, and the activity is binding activity to an antigen.

23. The conditionally active polypeptide according to any one of claims 1 to 22, wherein the conditionally active polypeptide is an Fc region of an antibody.

24. 22. The conditionally active polypeptide according to any one of claims 1 to 21, wherein the conditionally active polypeptide is an enzyme and the activity is an enzymatic activity.

25. 22. The conditionally active polypeptide of any one of claims 1 to 21, wherein the conditionally active polypeptide is selected from receptors, regulatory proteins, soluble proteins, cytokines and fragments of receptors, regulatory proteins, stress proteins, vault-associated proteins, neuronal proteins, gut proteins, growth factors, mitochondrial proteins, cytoplasmic proteins, animal proteins, structural proteins and plant proteins.

26. A pharmaceutical composition comprising an effective amount of the conditionally active polypeptide of any one of claims 1 to 25 and a pharma- ceutically acceptable carrier.

27. Use of a conditionally active polypeptide according to any one of claims 1 to 25 for the treatment of a solid tumor, an inflamed joint or a brain disease or disorder.

28. Use of a conditionally active polypeptide according to any one of claims 1 to 25 for removing senescent cells from a subject.

29. 26. A method for treating a solid tumor, an inflamed joint, or a brain disease or disorder, comprising the step of administering a conditionally active polypeptide according to any one of claims 1 to 25.

30. 30. The method of claim 29, wherein the conditionally active polypeptide is administered as part of a chimeric antigen receptor comprising the conditionally active polypeptide, and the chimeric antigen receptor is introduced into a T cell or a NK cell.

31. 31. The method of claim 30, wherein the T cells or NK cells are used as part of an adoptive cell therapy.

32. 30. The method of claim 29, wherein the conditionally active polypeptide is attached to a nanoparticle.

33. 30. The method of claim 29, wherein the conditionally active polypeptide is administered as an antibody-drug conjugate comprising the conditionally active polypeptide.

34. A nucleic acid molecule encoding the conditionally active polypeptide according to any one of claims 1 to 25.

35. 35. An expression vector comprising the nucleic acid molecule of claim 34.

36. A host cell comprising the expression vector of claim 35.

37. 27. The pharmaceutical composition of claim 26, further comprising at least one additional anti-cancer agent.

38. 38. The pharmaceutical composition of claim 37, wherein the at least one additional anti-cancer agent is a chemotherapeutic agent, a radiotherapeutic agent, a hormonal therapeutic agent, a toxin, or an immunotherapeutic agent.

39. 38. The pharmaceutical composition of claim 37, wherein the at least one additional anti-cancer agent is a toxin selected from a taxane, a maytansinoid, an auristatin, a calicheamicin, a duocarmycin, an mmae, a hypnotic agent, a duomycin, an anthracycline, a CC-1065 analog, docetaxel, a cathepsin, a ricin, gelonin, a Pseudomonas exotoxin, a diphtheria toxin, an RNase, and a toxic radioisotope.

40. 38. The pharmaceutical composition of claim 37, wherein the conditionally active polypeptide is conjugated to the at least one additional anti-cancer agent, or at least four additional anti-cancer agents, or at least six additional anti-cancer agents.

41. 1. A method of diagnosing the presence of cancer in a subject, comprising: (a) contacting a cell or tissue of the subject with a conditionally active polypeptide according to any one of claims 1 to 25, wherein the conditionally active polypeptide binds to a target on a cancer cell, and the conditionally active polypeptide is detectably labeled; (b) determining whether said conditionally active polypeptide binds to said cell or tissue of said subject. The method includes:

42. 42. The method of claim 41, wherein said contacting of said cell or tissue with said conditionally active polypeptide occurs in vitro.

43. 42. The method of claim 41, wherein said contacting of said cell or tissue with said conditionally active polypeptide occurs within said subject.

44. A bispecific antibody comprising at least one binding domain containing a conditionally active polypeptide according to any one of claims 1 to 25.

45. A chimeric protein containing two different binding or catalytic domains, one or both of which comprises a conditionally active polypeptide according to any one of claims 1 to 25.

46. An oncolytic virus comprising a conditionally active polypeptide according to any one of claims 1 to 25.

47. 26. The conditionally active polypeptide of any one of claims 1 to 25, wherein the conditionally active polypeptide is conjugated to a nucleic acid.

48. 48. The conditionally active polypeptide of claim 47, wherein the nucleic acid is non-covalently or covalently conjugated and the nucleic acid is a nucleic acid that includes at least one non-naturally occurring nucleotide.

49. 19. The conditionally active polypeptide of any one of claims 16 to 18, wherein the at least one species interacts with the polypeptide through hydrophilic, hydrophobic or covalent interactions.

50. 19. The conditionally active polypeptide of any one of claims 16 to 18, comprising two or more species.

51. The method according to any one of claims 29 to 33, wherein two or more conditionally active polypeptides according to any one of claims 1 to 25 are used, either simultaneously or sequentially.

52. The method according to any one of claims 29 to 33, wherein the conditionally active polypeptide according to any one of claims 1 to 25 has immunomodulatory activity and is used in combination therapy.

53. 53. The method of claim 52, wherein the combination therapy is in combination with a prodrug.

54. The conditionally active polypeptide of any one of claims 1 to 25, wherein the conditionally active polypeptide has prodrug activity.

55. 1. A method for preparing a conditionally active polypeptide from a parent polypeptide, comprising: i. evolving a DNA encoding said parent polypeptide to increase the net charge of said parent polypeptide using one or more techniques selected from increasing the total number of codons for charged amino acid residues in said DNA, decreasing the total number of codons for uncharged amino acid residues in said DNA, and combinations thereof, resulting in a mutant DNA; ii. expressing the mutant DNA to obtain a mutant polypeptide; iii. selecting from said mutant polypeptides said conditionally active polypeptides which exhibit a decrease in activity in a first assay at a first value of a condition compared to the same activity in a second assay at a second value of the same condition, wherein said charged amino acid residues are selected from aspartic acid, glutamic acid, arginine, lysine and histidine; The method includes:

56. 56. The method of claim 55, wherein the evolving step utilizes site-directed mutagenesis.

57. 57. The method of claim 56, wherein the site-directed mutagenesis is selected from oligonucleotide-mediated mutagenesis, PCR mutagenesis, and cassette mutagenesis.

58. 58. The method of any one of claims 55 to 57, wherein the evolving step comprises introducing one or more codons for a charged amino acid residue into the DNA.

59. 59. The method of claim 58, wherein the one or more codons for a charged amino acid residue are less than five codons, or less than four codons, or less than three codons, or less than two codons.

60. 60. The method of any one of claims 55 to 59, wherein the evolving step comprises codon substitution, codon deletion or codon insertion.

61. 62. The method of any one of claims 55 to 61, wherein the charged amino acid residues are selected from aspartic acid and glutamic acid.

62. 62. The method of any one of claims 58 to 61, wherein the one or more codons are introduced into a region of the DNA encoding an active site of the parent polypeptide.

63. 63. The method of claim 62, wherein the parent polypeptide is a parent antibody and the active site is a complementarity determining region of the parent antibody.

64. 62. The method of any one of claims 58 to 61, wherein the one or more charged amino residues are introduced into a region outside the active site of the parent polypeptide.

65. 65. The method of claim 64, wherein the region outside the active site is a constant region of an antibody or a framework region of the antibody.

66. 66. The method of any one of claims 55 to 65, wherein the conditionally active polypeptide is reversibly inactivated under the first value of the condition.

67. 67. The method of any one of claims 55 to 66, wherein the conditions are selected from pH, temperature, osmolality, osmolality, oxidative stress, electrolyte concentration and protein concentration.

68. 68. The method of any one of claims 55 to 67, wherein the first and second assays are carried out in an assay solution comprising proteins found in blood.

69. 69. The method of claim 68, wherein the blood protein is albumin.

70. 68. The method of any one of claims 55 to 67, wherein the first and second assays are carried out in an assay solution in the absence of serum.

71. The method of any one of claims 55 to 70, wherein the value of the second condition is a value of a normal physiological condition that is within the normal range of physiological conditions at the site of administration of the conditionally active polypeptide to a subject or physiological conditions in a tissue or organ prior to the site of action of the conditionally active polypeptide in a subject, and the value of the first condition is a value of an abnormal condition that deviates from the normal range of physiological conditions at the site of administration of the conditionally active polypeptide or physiological conditions in a tissue or organ prior to the site of action of the conditionally active polypeptide.

72. 68. The method of claim 67, wherein the condition is pH and the first and second assays are performed in an assay medium containing at least one species having a molecular weight less than 900 a.m.u. and a pKa at most 3 units away from the first value of pH.

73. 68. The method of claim 67, wherein said condition is pH and said first and second assays are performed in an assay medium comprising at least one species having a molecular weight less than 900 a.m.u. and a pKa between said second value of pH and said first value of pH.

74. 74. The method of any one of claims 55 to 73, wherein the first and second assays are carried out in an assay medium comprising a species selected from histidine, histamine, adenosine diphosphate, adenosine triphosphate, citrate, acetate, lactate, hydrogen sulfide, disulfide, ammonium, bicarbonate, dihydrogen phosphate, and combinations thereof.

75. The method of any one of claims 55 to 74, wherein the parent polypeptide is selected from an antibody, a single chain antibody and an antibody fragment, and the activity is antigen-binding activity.

76. The method of any one of claims 55 to 74, wherein the parent polypeptide is an Fc region of an antibody.

77. 75. The method of any one of claims 55 to 74, wherein the parent polypeptide is an enzyme and the activity is an enzymatic activity.

78. 75. The method of any one of claims 55 to 74, wherein the parent polypeptide is selected from receptors, regulatory proteins, soluble proteins, cytokines and fragments of receptors, regulatory proteins, stress proteins, vault-associated proteins, neuronal proteins, gut proteins, growth factors, mitochondrial proteins, cytoplasmic proteins, animal proteins, structural proteins and plant proteins.

79. 75. The method of any one of claims 55 to 74, wherein the selecting step further comprises selecting the conditionally active polypeptide based on a property selected from affinity, expression level, and humanization.

80. 75. The method of any one of claims 55 to 74, wherein the expression step utilises a phage display or eukaryotic production host.

81. 81. The method of claim 80, wherein said expressing step is carried out in said eukaryotic production host and said selected conditionally active polypeptide is expressed in the same eukaryotic production host.

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