Conditionally active polypeptides and methods of generating them

Abstract

This invention provides a method for preparing a conditionally active polypeptide from a parent polypeptide, comprising the steps of: evolving the DNA encoding the parent polypeptide by increasing the net charge of the parent polypeptide using one or more techniques selected from the following: increasing the total number of codons for charged amino acid residues in the DNA and decreasing the total number of codons for uncharged amino acid residues in the DNA to generate mutant DNA; expressing the mutant DNA to obtain a mutant polypeptide; and selecting the conditionally active polypeptide from the mutant polypeptide, wherein the conditionally active polypeptide exhibits reduced activity in a first analysis at a first value of the same condition compared to the same activity in a second analysis at a second value of the same condition. The conditionally active polypeptide, pharmaceutical compositions containing the same, nanoparticles thereof, and drug conjugates thereof are also provided, as well as their uses.

Description

Technical Field

[0001] This invention relates to the field of providing modified peptides with desired activity. Specifically, this invention relates to conditionally active peptides and methods for generating conditionally active peptides from parent peptides, wherein the conditionally active peptides exhibit stronger activity under one condition than under another. Prior Technology

[0002] Numerous studies describe methods for evolving various characteristics of proteins, especially enzymes or antibodies, to achieve activity or stability under different conditions. For example, enzymes can be evolved to be stable at higher temperatures. In cases where enzyme activity is improved at higher temperatures, most of the improvement can be attributed to the higher kinetic activity generally explained by the Q10 principle, which estimates that conversion doubles for every 10°C increase in temperature.

[0003] In addition, there are naturally occurring mutations that destabilize proteins under their normal operating conditions, thereby reducing their activity under normal conditions. For example, there are known temperature mutants that are active at lower temperatures, but generally have lower activity levels compared to the wild-type proteins from which they are derived.

[0004] It is desirable to generate peptides with conditional activity, such as peptides that are less active or virtually inactive under one condition and more active under another. It is also desirable to generate peptides that are activated or inactive in certain environments, or that become activated or inactive over time. Besides temperature, other conditions under which peptides can evolve or be modified for conditional activity include pH, osmotic pressure, osmotic weight molar concentration, oxidative stress, and electrolyte concentration. In addition to peptide activity, it is generally desirable to modify other properties during evolution, including chemical resistance and proteolytic resistance.

[0005] Several known mutant polypeptides possess modified properties, achieved through substitution of one or more amino acids. For example, U.S. Patent No. 8,318,469 discloses a mutant *Thermus brockianus* nucleic acid polymerase possessing one of two mutations: G43D and F665Y. Compared to polymerases without these mutations, the mutant polymerase exhibits reduced 5′–3′ exonuclease activity. 5′–3′ exonuclease activity is generally undesirable for DNA polymerases because this activity is required to digest nucleic acids with an unprotected 5′ end (including primers).

[0006] U.S. Patent No. 8,536,301 discloses a mutant peptide that binds with high affinity to cell surface receptors of fibronectin α5β1 or fibronectin αvβ3 and αvβ5 integrin. The mutant peptide is based on a molecular scaffold into which a subsequence containing the RGD integrin-binding motif is inserted. This subsequence is approximately 9-13 amino acids in length, with multiple amino acids flanked by the RGD motif. The molecular scaffold is preferably based on knotting agents, such as EETI, AgRP, and Agatoxin IVB, which have strictly defined three-dimensional conformations.

[0007] U.S. Patent No. 8,921,086 discloses a mutant DNA polymerase with improved tolerance to nucleotide analogs entering the active site region. The mutant polymerase may have amino acid substitutions at positions 375 and 512 relative to the 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] U.S. Patent No. 6,329,178 discloses a mutant DNA polymerase derived from a natural DNA polymerase by mutating one or more amino acids in its active site. The mutant DNA polymerase exhibits altered fidelity or altered enzyme activity compared to the natural DNA polymerase. The natural DNA polymerase has an active site comprising the following amino acid sequence motifs: AspTyrSerGlnIleGluLeuArg or LeuLeuVa1AlaLeuAspTyrSerGlnIleGluLeuArg. The mutant DNA polymerase has (a) substitution of two or more amino acids in the amino acid sequence motif, or (b) substitution of an amino acid other than Glu in the amino acid sequence motif.

[0009] These mutant proteins exhibit altered properties compared to the wild-type proteins from which they are derived. However, these altered properties of the mutant proteins do not change with varying conditions.

[0010] Evolving a parental peptide into one that is inactive or virtually inactive (less than 50%, 30%, or 10% activity, especially 1% activity) under one condition, while maintaining equivalent or better activity under another condition, may require one or more destabilizing mutations coexisting with one or more activity-enhancing mutations. There is a need in the industry to develop conditionally active peptides.

[0011] U.S. Patent No. 8,709,755 discloses a method for generating conditionally active antibodies, comprising the steps of: evolving DNA encoding a wild-type antibody to generate a mutant antibody; subjecting the mutant antibody and the wild-type antibody to analyses under a first condition and an analysis under a second condition; and selecting a conditionally active antibody from at least one mutant antibody, the conditionally active antibody exhibiting both of the following: (a) decreased binding activity to the antigen in the analysis under the first condition compared to the binding activity of the wild-type antibody in the same analysis under the first condition; and (b) increased binding activity to the antigen in the analysis under the second condition compared to the activity of the wild-type antibody in the same analysis under the second condition.

[0012] When used as therapeutic antibodies, conditionally active antibodies preferably exhibit reduced activity under normal physiological conditions and increased activity under abnormal conditions (e.g., the tumor microenvironment) where the therapeutic site may be present. Due to this preferential action, conditionally active antibodies can potentially cause less damage to normal tissues / organs where normal physiological conditions exist, thereby producing fewer side effects. This allows for longer treatment durations and / or the use of higher doses of conditionally active antibodies.

[0013] This invention provides a novel method for generating conditionally active peptides, which efficiently utilizes directed induction. This invention also provides novel conditionally active peptides generated by this method. Summary of the Invention

[0014] In one state, the present invention provides a conditionally active polypeptide that can be obtained by increasing the net charge of a parent polypeptide, wherein the conditionally active polypeptide exhibits reduced activity in a first analysis at a first value of the same conditions compared to the same activity in a second analysis at a second value of the same conditions. The charged amino acid residues may be selected from aspartic acid, glutamic acid, arginine, lysine, and, where appropriate, histidine.

[0015] In the foregoing embodiments, net charge can be increased by one or more techniques selected from the following: 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 the foregoing embodiments, conditions can be selected from pH, temperature, osmotic pressure, osmotic weight molar concentration, oxidative pressure, electrolyte concentration, protein concentration, and combinations of two or more of these conditions.

[0016] In the foregoing embodiments, the condition may be pH, and the conditionally active peptide may exhibit reduced activity at normal physiological pH and increased activity at abnormal pH that differs from normal physiological pH.

[0017] In the foregoing embodiments, conditional activity can be analyzed in the presence of at least one substance having a molecular weight less than 900 amu and a pKa that is at least 2 or 3 units away from the pH at which the activity is expected to increase. In the foregoing embodiments, the pKa of the substance can be between a first pH value and a second pH value.

[0018] In the foregoing embodiments, conditional activity can be analyzed in the presence of at least one substance selected from the following: histidine, histamine, hydrogen diphosphate, hydrogen sulfide, hydrogen triphosphate, citrate, acetate, lactate, disulfide, ammonium, bicarbonate, dihydrogen phosphate, and combinations thereof.

[0019] In the foregoing embodiments, the first and second analyses can be performed in the analytical solution in the absence of serum.

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

[0021] In the foregoing embodiments, one or more charged amino residues may be introduced into the active site of the parental polypeptide. In the foregoing embodiments, one or more charged amino residues may be introduced into a region outside the active site of the parental polypeptide. In 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 complementarity-determining region of the conditionally active antibody.

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

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

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

[0025] In the foregoing embodiments, the first value of the condition may be a value of normal physiological conditions, which is within the normal range of physiological conditions at the site where the conditionally active polypeptide is administered to the individual or at the tissue or organ at the site of action of the conditionally active polypeptide in the individual; and the second value of the condition may be a value of abnormal conditions, which deviates from the normal range of physiological conditions at the site where the conditionally active polypeptide is administered or at the tissue or organ at the site of action of the conditionally active polypeptide.

[0026] In the foregoing embodiments, the conditionally active polypeptide may be a protein or a protein fragment. In the foregoing embodiments, the conditionally active polypeptide may be an antibody, a single-chain antibody, or an antibody fragment, and its activity is related to antigen-binding activity; the conditionally active polypeptide may be the Fc region of an antibody; or the conditionally active polypeptide may be an enzyme with enzyme activity; or the conditionally active polypeptide may be a receptor, regulatory protein, soluble protein, interleukin, fragments of receptors and regulatory proteins, stress protein, fornix-related protein, neuronal protein, digestive tract protein, growth factor, mitochondrial protein, cytoplasmic protein, animal protein, structural protein, or plant protein.

[0027] In another embodiment, the present invention provides a pharmaceutical composition comprising an effective amount of the conditionally active polypeptide of any of the preceding embodiments and a pharmaceutically acceptable carrier.

[0028] In the foregoing embodiments, the pharmaceutical composition may further comprise at least one anticancer agent. In the foregoing embodiments, the at least one anticancer agent may be a chemotherapeutic agent, a radiation therapy agent, a hormone therapy agent, a toxin, or an immunotherapy agent. In the foregoing embodiments, the at least one anticancer agent may be a toxin selected from the following: taxane, maytansinoid, auristatin, calicheamicin, duocramycin, mmae, hypnotics, chlortetracycline, anthracyclines, CC-1065 analogs, docetaxel, autolysins, ricin, gelonin, Pseudomonas exotoxin, diphtheria toxin, RNase, and toxic radioisotopes. In the foregoing embodiments, the conditionally active polypeptide may be coupled to at least one anticancer agent, or at least four anticancer agents, or at least six anticancer agents.

[0029] In another embodiment, the present invention provides the use of the conditionally active polypeptides of the foregoing embodiments for the treatment of solid tumors, inflammatory joint or brain diseases or conditions, or for the removal of senescent cells from an individual.

[0030] In another embodiment, the present invention provides a method for treating solid tumors, inflammatory joints, or brain diseases or conditions, comprising the step of administering the conditionally active polypeptides of the foregoing embodiments.

[0031] In another embodiment, the present invention provides a nucleic acid molecule encoding the conditionally active polypeptides of the foregoing embodiments. In yet another embodiment, the present invention provides an expression vector comprising the nucleic acid molecule of the foregoing embodiments or a host cell comprising such an expression vector.

[0032] In another embodiment, the present invention provides a method for diagnosing the presence of cancer in an individual, comprising the steps of: (a) contacting the individual’s cells or tissues with the detectable labeled conditionally active polypeptides of the foregoing embodiments, wherein the conditionally active polypeptides bind to a target on a cancer cell; and (b) determining whether the conditionally active polypeptides bind to the individual’s cells or tissues.

[0033] In the foregoing embodiments, contacting cells or tissues with the conditionally active polypeptide can be performed in vitro. In the foregoing embodiments, contacting cells or tissues with the conditionally active polypeptide can be performed in vivo.

[0034] In another embodiment, the present invention provides a bispecific antibody comprising at least one binding domain, wherein the at least one binding domain contains at least one conditionally active polypeptide of the foregoing embodiments. In another embodiment, the present invention provides a chimeric protein comprising two different binding or catalytic domains, wherein one or both of such domains contain at least one conditionally active polypeptide of the foregoing embodiments. In yet another embodiment, the present invention provides an oncolytic virus comprising at least one conditionally active polypeptide of the foregoing embodiments.

[0035] In another embodiment, the present invention provides a method for preparing a conditionally active polypeptide from a parent polypeptide. In this method, one or more techniques selected from the following are used to evolve the DNA encoding the parent polypeptide to increase the net charge of the polypeptide: 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 combinations thereof, to produce mutant DNA. The mutant DNA is expressed to obtain a mutant polypeptide; and the conditionally active polypeptide is selected from the mutant polypeptide. The selected conditionally active polypeptide exhibits reduced activity in a first analysis at a first value of the same conditions compared to the same activity in a second analysis at a second value of the same conditions. The charged amino acid residues are selected from aspartic acid, glutamic acid, arginine, lysine, and histidine.

[0036] In the foregoing embodiments, the evolutionary step may employ site-directed mutagenesis, selected as appropriate from oligonucleotide-mediated mutagenesis, PCR mutagenesis, and cassette mutagenesis. In the foregoing embodiments, the evolutionary step may employ codon substitution or codon insertion.

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

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

[0039] In 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 the foregoing embodiments, the region outside the active site may be the antibody constant region or the antibody framework region.

[0041] In the foregoing embodiments, the conditionally active polypeptide may be reversibly inactivated at a first value of the condition.

[0042] In the foregoing embodiments, the conditions can be selected from pH, temperature, osmotic pressure, osmotic weight mole concentration, oxidative pressure, electrolyte concentration, and protein concentration.

[0043] In the foregoing embodiments, the first and second analyses can be performed in an analytical solution containing proteins found in blood. In the foregoing embodiments, the blood protein can be albumin.

[0044] In the foregoing embodiments, the first and second analyses can be performed in an analytical solution in the absence of serum.

[0045] In the foregoing embodiments, the value of the second condition may be a value of normal physiological conditions, which is within the normal range of physiological conditions at the site where the conditionally active polypeptide is administered to the individual or at the tissue or organ at the site of action of the conditionally active polypeptide in the individual; and the value of the first condition may be a value of abnormal conditions, which deviates from the normal range of physiological conditions at the site where the conditionally active polypeptide is administered or at the tissue or organ at the site of action of the conditionally active polypeptide.

[0046] In the foregoing embodiments, the conditions may be pH and the first and second analyses may be performed in an analytical medium comprising at least one substance having a molecular weight of less than 900 amu and a pKa that is at least 3 units away from a first value of pH.

[0047] In the foregoing embodiments, the conditions may be pH and the first and second analyses may be performed in an analytical medium comprising at least one substance having a molecular weight of less than 900 amu and a pKa between a second pH value and a first pH value.

[0048] In the foregoing embodiments, the first and second analyses may be performed in an analytical medium comprising substances selected from the following: histidine, histamine, adenosine diphosphate, adenosine triphosphate, citrate, acetate, lactate, hydrogen sulfide, disulfide, ammonium, bicarbonate, dihydrogen phosphate, and combinations thereof.

[0049] In the foregoing embodiments, the selection step may further include selecting conditionally active peptides based on properties selected from affinity, performance level, and humanization.

[0050] In the foregoing embodiments, the expression step may employ phage display or a eukaryotic cell-generated host. In the foregoing embodiments, the expression step may be performed in a eukaryotic cell-generated host, and the selected conditionally active peptide may be expressed in the same eukaryotic cell-generated host. Simple Explanation of the Diagram

[0051] Figure 1 shows the selectivity of the conditionally active antibody generated in Example 9 at pH 6.0 relative to pH 7.4. [ ] Figure 2 shows the binding activity of several conditionally active antibodies to antigens analyzed in different buffer solutions. [ ] Figure 3 shows the effect of changing the composition of Krebs buffer on the binding activity of conditionally active antibodies. [ ] Figure 4 shows that the binding activity of the three different conditionally active antibodies depends on the presence and concentration of bicarbonate at pH 7.4, as described in Example 12. [ ] Figure 5 is a simplified diagram showing the structure of a chimeric antigen receptor (CAR). [ ] Figure 6 is a simplified diagram showing the formation of salt bridges in deoxyheme, where three amino acid residues form two salt bridges, which stabilize the T quaternary structure of deoxyheme, resulting in a lower affinity for oxygen. [ ] Figure 7 shows the activity of conditionally active antibodies against ROR2 in different buffer solutions. [ ] Figure 8 shows the activity of conditionally active antibodies against Axl in different buffer solutions. [ ] Figures 9A-9E show the results of cell killing of A549 cells using wild-type anti-Axl antibody and conditionally active anti-Axl antibody at different antibody concentrations at pH 6.0 and pH 7.4. [ ] Figures 10A-10D show the binding affinity of conditionally active anti-Axl antibodies for human Axl and cynomolgus monkey Axl in different buffer solutions and at different pH values. [ ] Figures 11A-11H show the cell killing effects of conditionally active anti-Axl antibodies conjugated to chlortetracycline on different cell lines at different pH values. [ ] Figure 12 shows the cell killing effect of conditionally active anti-Axl antibodies conjugated to chlortetracycline on A549 cells at different pH values. [ ] Figure 13 shows the effect of chlortetracycline-conjugated conditionally active anti-Axl antibody treatment on tumor volume in xenograft mice. [ ] Figures 14A-14B show the presence of conditionally active anti-Axl antibodies conjugated with chlortetracycline in the blood of cynomolgus monkeys over time after injection of the anti-Axl antibody / chlortetracycline conjugate. [ ] Figure 15A shows the presence of aspartate aminotransferase (AST) in the blood of cynomolgus monkeys over time, from before injection of chlortetracycline-conjugated conditionally active anti-Axl antibody (before injection (D-3)) until 3 days after injection (D-3) after injection. [ ] Figure 15B shows the presence of alanine aspartate aminotransferase (ALT) in the blood of cynomolgus monkeys over time, from before injection of the conditionally active anti-Axl antibody conjugated with chlortetracycline (before injection (D-3)) until 3 days after injection (D-3) after injection. [ ] Figure 16 shows the lymphocyte count over time in the blood of cynomolgus monkeys after injection of chlortetracycline-conjugated conditionally active anti-Axl antibody. [ ] Figures 17A-17C show the effect of treatment with a conditionally active anti-Ror2 antibody conjugated with paclitaxel on tumor volume in xenograft mice, as described in Example 21. Implementation

[0052] [ ] [definition] [ ] To facilitate understanding of the examples provided herein, certain frequently used methods and / or terms will be defined herein. The following definitions are incorporated herein by reference from U.S. Patent No. 8,709,755 B2: "reagent," "polysemous base requirement," "amino acid," "amplification," "chimeric property," "homology," "comparison window," "conserved amino acid substitution," "corresponding to," "degradation-effective," "defined sequence frame," "defined sequence nucleus," "digestion," "directed ligation," "DNA shuffling," "drug" or "drug molecule," "effective amount," "epitope," "enzyme," "evolution" or "evolving," "fragment" or "derivative" or "analyte," "monoamine." "All range of amino acid substitutions", "gene", "genetic instability", "heterologous", "homologous" or "partially homologous", "industrial application", "identical" or "consistent", "conformity region", "isolated", "isolated nucleic acid", "ligand", "linker", "linker or spacer", "microenvironment", "molecular property to be evolved", "mutation", "N,N,G / T", "normal physiological conditions" or "wild-type action conditions", "nucleic acid molecule", "nucleic acid molecule", "nucleic acid sequence encoding ..." or "DNA coding sequence of ..." or "encoded Nucleotide sequence encoding…, nucleic acid encoding enzyme (protein) or DNA encoding enzyme (protein) or polynucleotide encoding enzyme (protein), specific nucleic acid molecule, assembly of working nucleic acid sample into nucleic acid library, nucleic acid library, "construct", "oligonucleotide" (or synonymous "oligonucleotide"), "homology", "operable linker", "parental polynucleotide set", "patient" or "individual", "physiological condition", "population", "proto-form (pro- "form", "pseudo-random", "quasi-repetitive unit", "random peptide library", "random peptide sequence", "receptor", "recombinant" enzyme, "synthetic" enzyme, "related polynucleotide", "reducing reassortment", "reference sequence", "repetition index (RI)", "restriction site", "optional polynucleotide", "sequence consistency", "similarity", "specific binding", "specific hybridization", "specific polynucleotide", "strict hybridization conditions", "substantially identical", "substantially pure enzyme", "substantially pure", "therapeutic", "variable segment" and "variant".

[0053] As used herein, the term "about" refers to the normal variation in the measured value, which would be expected by a skilled person performing the measurement with a certain degree of care, and is commensurate with the measurement objective and the precision of the measuring equipment used. Unless otherwise indicated, "about" means a variation of + / - 10% of the provided value.

[0054] As used herein, the term "activity" refers to any function a protein can perform, including catalytic reactions and binding to a partner. For enzymes, activity can be enzyme activity. For antibodies, activity can be the binding activity between the antibody and its antigen (i.e., binding activity). For receptors or ligands, activity can be the binding activity between the receptor and its ligand.

[0055] As used herein, the term "adoptive cell therapy" refers to the transfer of immune cells with anti-tumor activity to a cancer patient. Adoptive cell therapy involves identifying lymphocytes with anti-tumor activity in vitro, expanding these cells to a large number in vitro, and infusing them into a host with cancer. This invention is particularly interested in autologous adoptive cell therapy, i.e., adoptive cell therapy in which the immune cells with anti-tumor activity to be transferred to a cancer patient are derived from the same patient.

[0056] As used herein, the term "antibody" refers to the complete immunoglobulin molecule, as well as fragments of the immunoglobulin molecule capable of binding to antigenic epitopes, such as Fab, Fab', (Fab')2, Fv, and SCA fragments. These antibody fragments retain the selective binding ability of the antibodies from which they are derived to antigens (e.g., peptide antigens) and can be prepared using methods well-known in the art (e.g., see Harlow Lane, above), further elaborated below. Antibodies can be used to prepare antigens by immunoaffinity chromatography. Various other uses of these antibodies include diagnosing diseases (e.g., neoplasia) and / or staging diseases, and for therapeutic applications to treat diseases such as neoplasia, autoimmune diseases, AIDS, cardiovascular diseases, infections, and the like. Chimeric, human-like, humanized, or fully human antibodies are particularly useful for administration to human patients.

[0057] Fab fragments consist of monovalent antigen-binding fragments of antibody molecules and can be produced by digesting intact antibody molecules with papain to obtain fragments composed of a portion of the complete light chain and heavy chain.

[0058] The Fab' fragment of an antibody molecule can be obtained by treating the complete antibody molecule with pepsin and then reducing it to obtain a molecule composed of a portion of the complete light chain and heavy chain. Each antibody molecule treated in this way yields two Fab' fragments.

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

[0060] Fv fragments are defined as genetically engineered fragments containing variable regions of both the light and heavy strands that exhibit double-stranded characteristics.

[0061] Single-chain antibodies ("SCA" or scFv") are genetically engineered single-chain molecules containing variable regions of a light chain and a variable region of a heavy chain linked by suitable flexible polypeptide linkers, and may include additional amino acid sequences at the amino terminus and / or carboxyl terminus. For example, single-chain antibodies may include chalaza segments for linking to polynucleotides. Functional single-chain antibodies typically contain sufficient portions of the light chain variable region and the heavy chain variable region to retain the property of the full-length antibody to bind 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), enabling these cytotoxic effector cells to specifically bind to target cells carrying antigens and subsequently kill those target cells with cytotoxins. Ligand-specific, high-affinity IgG antibodies directed to the surface of target cells stimulate the cytotoxic cells and are necessary for this killing process. The lysis of target cells occurs extracellularly, requires direct cell-to-cell contact, and does not involve complement.

[0063] This assay can analyze the ability of any specific antibody to induce target cell lysis via ADCC. To evaluate ADCC activity, the antibody of interest is added to a combination of target cells displaying the target ligand and immune effector cells. These effector cells are activated by the antigen-antibody complex, leading to cell lysis of the target cells. Cell lysis is typically detected by the release of markers from the lysed cells (e.g., radioactive receptors, fluorescent dyes, or native intracellular proteins). Effector cells that can be used for these analyses include peripheral blood mononuclear cells (PBMCs) and natural killer (NK) cells. Specific examples of in vitro ADCC analysis are described in the following literature: Bruggemann et al., 1987, J. Exp. Med, Vol. 166, p. 1351; Wilkinson et al., 2001, J. Immunol. Methods, Vol. 258, p. 183; Patel et al., 1995, J. Immunol. Methods, Vol. 184, p. 29. Alternatively, the ADCC activity of the antibody of interest can be evaluated in vivo, for example in animal models, as revealed by Clynes et al., 1998, PNAS USA, Vol. 95, p. 652.

[0064] As used herein, "antigen" or "Ag" is defined as a molecule that can trigger an immune response. This immune response may involve antibody production or activation of specific immune-competent cells, or both. Those skilled in this art will understand that any macromolecule, including virtually all proteins or peptides, can be used as an antigen. It is evident that antigens can be generated, synthesized, or derived from biological samples. This biological sample may include, but is not limited to, tissue samples, tumor samples, cells, or biological fluids.

[0065] As used herein, "antisense RNA" refers to an RNA molecule capable of forming a double helix with another RNA molecule through complementarity or partial complementarity. Antisense RNA molecules can be complementary to the translated or untranslated regions of another RNA molecule. Antisense RNA does not need to be perfectly complementary to the other RNA molecule. Antisense RNA may or may not have the same length as the other RNA molecule; antisense RNA molecules may be longer or shorter than the other RNA molecule. If the other RNA molecule is mRNA, the binding of antisense RNA will prevent the mRNA from being fully or partially translated into a functional protein product.

[0066] The terms "biosimilar" or "follow-on biologic" are used in accordance with the working definition published by the U.S. Food and Drug Administration (FDA), which defines a biosimilar as a product that is "highly similar" to a reference product (but with minor differences in clinically inactive components). In practice, there may be no clinically significant differences in safety, purity, and efficacy between the reference product and the biosimilar (Public Health Service (PHS) Act §262). A biosimilar may also be a product that meets one or more of the guidelines adopted by the European Medicines Agency's Committee on Medicinal Products for Human Use (CHMP) on 30 May 2012 and published by the European Union in "Guideline on similar biological medicinal products containing monoclonal antibodies—non-clinical and clinical issues" (Document Reference No. EMA / CHMP / BMWP / 403543 / 2010). For example, "biosimilar antibody" typically refers to a subsequent form of an innovative antibody (reference antibody) manufactured by a different company. Differences between biosimilar antibodies and reference antibodies may include post-translational modifications, such as attaching other biochemical groups (e.g., phosphate esters, various lipids, and carbohydrates) to the antibody; post-translational proteolytic cleavage; alteration of amino acid chemistry (e.g., methionization); or various other mechanisms. Other post-translational modifications may be the result of process operations, such as saccharification by exposing the product to reducing sugars. In some cases, storage conditions may allow certain degradation pathways to occur, such as oxidation, deacetylation, or aggregation. All such product-related variants may be included in biosimilar antibodies.

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

[0068] As used herein, the term "chimeric antigen receptor" or "CAR" refers to an engineered receptor that specifically transplants an antigen onto cytotoxic cells (e.g., T cells, NK cells, and macrophages). The CAR of this invention may include at least one antigen-specific targeting region (ASTR), an extracellular space domain (ESD), a transmembrane domain (TM), one or more co-stimulatory 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 recognizes two different antigens or epitopes. After the ASTR specifically binds to the target antigen, the ISD activates intracellular signaling in the cytotoxic cell. For example, the ISD can redirect T cell specificity and cytotoxicity toward a selected target in a non-MHC-restricted manner, depending on the antigen-binding properties of the CAR. Non-MHC-restricted antigen recognition gives cytotoxic cells expressing the CAR the ability to recognize antigens independently of antigen processing, thereby bypassing the main mechanisms of tumor escape. Furthermore, when expressed in T cells, the CAR advantageously does not dimerize with the α and β chains of the endogenous T cell receptor (TCR).

[0069] As used herein, the term "complementarity-determining region" or "CDR" refers to the region in an antibody containing amino acid residues involved in antigen binding. The CDR exhibits the highest variability in amino acid alignment of antibody variable domains. Databases (e.g., the Kabat database) can be used for CDR identification, defined, for example, as containing 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, 5th Edition, U.S. Department of Health and Human Services, NIH Publication No. 91-3242). Alternatively, CDRs can be defined as residues derived from the "hypervariant ring" (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 carried out using the method described by Kabat et al. (above). Phrases such as "Kabat position," "Kabat residue," and "according to Kabat" in this document refer to the numbering system described by Kabat et al. for heavy or light chain variable domains. Using the Kabat numbering system, the actual linear amino acid sequence of the variable domain may contain fewer or additional amino acids corresponding to shortened or inserted segments of the variable domain frame (FR) or CDR. For example, the variable domain of the heavy chain may include an amino acid insertion after residue 52 of CDR H2 (residues 52a, 52b, and 52c according to Kabat) and an insertion residue after residue 82 of the heavy chain FR (e.g., residues 82a, 82b, and 82c according to Kabat). The Kabat number of residues in a given antibody can be determined by comparing the antibody sequence with homologous regions of a "standard" Kabat numbered sequence.

[0070] The term "conditionally active polypeptide" refers to a variant or mutant of a parent polypeptide that exhibits higher activity than the parent polypeptide under at least one condition and lower activity under a second condition, or a variant or mutant of a parent polypeptide wherein the activity of the variant or mutant polypeptide under the first condition is at least 1.3 times that under the second condition. This conditionally active polypeptide may exhibit activity at one or more selected sites in the body, and / or increased or decreased activity at another site in the body. For example, in one state, an evolved conditionally active polypeptide may be practically 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, intercytokines and their fragments, hormones and their fragments, ligands and their fragments, regulatory proteins and their fragments, and growth factors and their fragments. The conditionally active polypeptides described herein are preferably conditionally active biological polypeptides.

[0071] As used herein, the term "cytokines" refers to a broad class of biomolecules that exert or influence the cellular immune system. This definition is intended to include, but is not limited to, biomolecules that act locally or via blood circulation at locations other than their secretory sites 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 to 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 chemoattractant protein (MCP)-1, intracellular adhesion molecule (ICAM), macrophage community-stimulating factor (M-CSF), granulocyte-mass cytokine community-stimulating factor (G-CSF), and granulocyte-macrophage community-stimulating factor (GM-CSF).

[0072] As used herein, the term "electrolyte" is used to define a charged mineral in blood or other body fluids. For example, in a given state, normal physiological conditions and abnormal conditions may have different "electrolyte concentrations." Exemplary electrolytes include, but are not limited to, ionized calcium, sodium, potassium, magnesium, hydrochloride, citrate, lactate, bicarbonate, and phosphate.

[0073] The term "full-length antibody" refers to an antibody that contains an antigen-binding variable region (VH or VL) and a light chain constant domain (CL) and heavy chain constant domains CH1, CH2, and CH3. The constant domains can be native sequence constant domains (e.g., human native sequence constant domains) or variants of their amino acid sequences. Depending on the amino acid sequence of the constant domains in the heavy chain of a full-length antibody, it can be designated as a different "class". There are five main classes of full-length antibodies: IgA, IgD, IgE, IgG, and IgM, and several of these classes can be further divided into "subclasses" (isotypes), such as IgG1, IgG2, IgG3, IgG4, IgA, and IgA2. The heavy chain constant domains corresponding to different antibody classes are respectively called α, δ, ε, γ, and μ.

[0074] As used herein, the term "growth factor" refers to polypeptide molecules that enable 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 growth factor (ECGF), granulocyte-macrophage community-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), chondrocyte morphogenetic protein (CDMP), and platelet-derived growth factor (PDGF).

[0075] As used herein, the term "hormone" refers to substances typically identified as mediators, usually released by cells or glands in one part of an organism, acting as messengers to the rest of the organism. Examples of hormones include endocrine hormones released directly into the bloodstream and extracellular hormones (or ectohormones) secreted directly into ducts and flowing from those ducts into the bloodstream, or diffused from cells to the outside via a process called paracrine signaling. Vertebrate hormones can be classified into three chemical categories: peptide hormones, lipid and phospholipid-derived hormones, and monoamines. Peptide hormones consist of polypeptide chains. Examples of peptide hormones include insulin and growth hormone. Lipid and phospholipid-derived hormones are derived from lipids (e.g., linoleic acid and arachidonic acid) and phospholipids. The main category is steroid hormones derived from cholesterol and arachidic acids. Examples of steroid hormones are testosterone and cortisol. Monoamines are derived from aromatic amino acid decarboxylases, such as phenylalanine, tyrosine, and tryptophan. Examples of monoamines include thyroxine and adrenaline.

[0076] As used herein, the term "immunomodulator" refers to an agent whose action on the immune system results in an immediate or delayed enhancement or reduction of the activity of at least one pathway involved in an immune response. This response may be triggered naturally or artificially as part of the innate or adaptive immune system or both. Examples of immunomodulators include interleukins, stem cell growth factors, lymphotoxins (e.g., tumor necrosis factor (TNF)) and hematopoietic factors (e.g., interleukins (e.g., interleukin-1 (IL-1), IL-2, IL-3, IL-6, IL-10, IL-12, IL-18, and IL-21)), community-stimulating factors (e.g., granulocyte-macrophage-community-stimulating factor (G-CSF) and granulocyte-macrophage-community-stimulating factor (GM-CSF)), interferons (e.g., interferon-α, -β, and -γ), stem cell growth factors named "S1 factor," erythropoietin, and thrombopoietin. Examples of suitable immunomodulatory agents include IL-2, IL-6, IL-10, IL-12, IL-18, IL-21, interferon, TNF (e.g., TNF-α), and the like.

[0077] "Individual" or "subject" refers to mammals. Mammals include, but are 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] As used herein, the term "library" refers to a collection of proteins within a single pool. Libraries can be generated using recombinant DNA techniques. For example, a collection of cDNA or any other protein-coding DNA can be inserted into an expression vector to generate a protein library. Alternatively, a collection of cDNA or protein-coding DNA can be inserted into a phage genome to generate a phage display library of wild-type proteins. The cDNA collection can be generated from a selected cell population or tissue sample, for example, using the method described by Sambrook et al. (Molecular Cloning, Cold Spring Harbor Laboratory Press, 1989). CDNA collections from selected cell types can also be obtained from suppliers such as Stratagene®. Wild-type protein libraries, as used herein, are not collections of biological samples.

[0079] As used herein, the term "ligand" refers to a molecule that is recognized by a specific receptor and specifically binds to one or more binding sites of that receptor. Examples of ligands include, but are not limited to, agonists and antagonists of cell membrane receptors, toxins and venoms, viral epitopes, hormones, hormone receptors, peptides, enzymes, enzyme receptors, cofactors, drugs (e.g., opiates, steroids), lectins, sugars, polynucleotides, nucleic acids, oligosaccharides, proteins, and monoclonal antibodies. Typically, a ligand comprises two structural parts: a first part that participates in the binding of the ligand to its receptor; and a second part that does not participate in this binding.

[0080] As used herein, the term "receptor" refers to a molecule that has an affinity for a given ligand. Receptors can be natural or synthetic molecules. Receptors can be used in their unchanged state or as aggregates with other substances. Receptors can be directly or covalently or non-covalently attached to a binding member, either via a specific binding agent. Examples of receptors include, but are not limited to, antibodies (including monoclonal antibodies and antisera) that react with specific antigenic determinants (e.g., on viruses, cells, or other materials), cell membrane receptors, complex carbohydrate and glycoprotein receptors, enzyme and hormone receptors. The binding of a ligand to its receptor indicates that the ligand and its receptor molecules combine via specific molecular recognition to form a complex, which can be detected by a variety of ligand-receptor binding assays known to those skilled in the art.

[0081] As used herein, the terms "microRNA" or "miRNA" refer to untreated or treated RNA transcripts derived from miRNA genes. Untreated microRNA gene transcripts typically contain RNA transcripts of approximately 70-100 nucleotides in length. Transcribed microRNAs can be processed by digesting them with RNases (such as Dicer, Argonaut, or RNase III) into active RNA molecules of 19-25 nucleotides. These active RNA molecules of 19-25 nucleotides are also referred to as "treated" microRNA gene transcripts or "mature" microRNAs.

[0082] As used herein, the term "multispecific antibody" refers to an antibody that has binding specificity to at least two distinct epitopes. An example multispecific antibody may 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 antibody). This also includes engineered antibodies having two, three, or more (e.g., four) functional antigen-binding sites (e.g., see US 2002 / 0004587 A1). [ ]

[0083] As used herein, the term "mutagenic primer" refers to an oligonucleotide primer used in linear circular amplification reactions (such as polymerase chain reaction or PCR) where the primer does not precisely match the template DNA sequence. The mismatched nucleotide in the mutagenic primer is called the mutation site for the mutagenic primer. Therefore, during the amplification reaction, the mismatched nucleotide of the primer is incorporated into the amplification product, resulting in the synthesis of a mutagenic DNA strand containing the mutagenic primer. The mutagenic primer thus introduces a specific mutation into the template DNA sequence.

[0084] As used herein, the term "nanoparticle" refers to microparticles with a size in the nanometer (nm) range and a maximum linear dimension 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 between any two points on a nanoparticle measured as a straight line. The nanoparticles of this invention may be irregular, rectangular, spindle-shaped, rod-shaped, disc-shaped, disc-shaped, cylindrical, red blood cell-like, spherical, or substantially spherical, as long as their shape and size allow for interaction. Preferably, the nanoparticles of this invention are derived from biocompatible materials (polymers or lipids).

[0085] When applied to objects, the term "natural" as used herein refers to the fact that an object can be found in nature. For example, a polypeptide or polynucleotide sequence present in an organism (including viruses) that can be isolated from natural sources and has not been intentionally modified by humans in a laboratory is natural. A polypeptide excised from a larger polypeptide is not a natural polypeptide because the telomeres of the excised polypeptide differ in the form of the excised polypeptide from those in the larger natural polypeptide, as these telomeres will no longer bind to adjacent polypeptides. Generally, the term "natural" refers to objects present in non-pathological (unaffected) individuals, which may be typical for a species.

[0086] The "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, where applicable, histidine. Uncharged amino acid residues include the other 15 natural amino acids. The net charge of a polypeptide can be increased by 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] As used herein, “oxidative stress” refers to the imbalance between the generation of reactive oxygen free radicals (e.g., superoxide anions (O2''), nitric oxide, hydroxyl radicals (OH), and hydrogen peroxide (H2O2)) as byproducts of metabolic processes utilizing molecular oxygen (e.g., see Coyle et al., Science 262:689-695 (1993)) and the biological system’s ability to readily detoxify reactive intermediates or readily repair the resulting damage. Oxidative stress can refer to the state of animal cells or tissues, either in vitro or in vivo. Disruptions to the normal redox state within living cells can cause toxic effects by generating peroxides and free radicals that damage all cellular components, including proteins, lipids, and DNA.

[0088] In humans, oxidative stress is involved in a variety of diseases, such as atherosclerosis, Parkinson's disease, heart failure, myocardial infarction, Alzheimer's disease, X-chromosome fissile syndrome, and chronic fatigue syndrome. However, short-term oxidative stress can also play an important role in preventing aging by inducing mitochondrial activation. Reactive oxygen species (ROS) can be beneficial because they are used by the immune system to attack and kill pathogens. ROS are also involved in cell signaling, a process known as redox signaling.

[0089] In chemistry, oxidative stress elevates the cell's reduction potential (decreases its negativity) or reduces the reducing power of cellular redox couples (e.g., glutathione). The effects of oxidative stress depend on the magnitude of these changes, and cells can overcome small disturbances and return to their initial state. However, more severe oxidative stress can cause cell death, and even moderate oxidation can trigger apoptosis, while even stronger stress can cause necrosis.

[0090] As used herein, the terms "parental polypeptide" and "parental protein" refer to polypeptides or proteins that can be evolved using the methods of this invention to produce conditionally active polypeptides or proteins. Parental polypeptides or proteins can be wild-type or non-wild-type proteins. For example, therapeutic polypeptides or proteins, or mutant or variant polypeptides or proteins, can be used as parental polypeptides or proteins. Examples of parental polypeptides and proteins include antibodies, antibody fragments, enzymes, enzyme fragments, interleukins and their fragments, hormones and their fragments, ligands and their fragments, receptors and their fragments, regulatory proteins and their fragments, and growth factors and their fragments.

[0091] As used in this article, "pH-dependent" refers to peptides whose properties or activities differ at different pH values.

[0092] As used herein, the term "peptide" refers to a polymer in which monolithic amino acids are linked together by peptides or disulfide bonds. A peptide can be a full-length natural amino acid chain or fragment thereof, a mutant or variant thereof, such as a selected region of interest within an amino acid chain in terms of binding interactions. A peptide can also be a synthetic amino acid chain, or a combination of a natural amino acid chain or a fragment thereof with a synthetic amino acid chain. A fragment refers to an amino acid sequence that is part of a full-length protein and is typically between about 8 and about 500 amino acids in length, preferably about 8 to about 300 amino acids, more preferably about 8 to about 200 amino acids, and even more preferably about 10 to about 50 or 100 amino acids. Additionally, peptides may include amino acids that are not natural (e.g., β-alanine, phenylglycine, and high-arginine). Peptides may also include commonly encountered non-genetically encoded amino acids. Amino acids may be D- or L-optical isomers. D-isomers are preferred for specific cases, as further elaborated below. Additionally, other peptide mimics can be used, for example, in polypeptide linker sequences (see Spatola, 1983, Chemistry and Biochemistry of Amino Acids. Peptides and Proteins, edited by Weinstein, Marcel Dekker, New York, p. 267). Generally, the term "protein" is not intended to convey any significant difference from the term "peptide," except that it includes structures comprising two or more polypeptide chains held together by covalent or non-covalent bonds.

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

[0094] As used herein, "recombinant antibody" refers to an antibody expressed by a host cell containing a nucleic acid encoding an antibody (e.g., a chimeric, humanized, or human antibody or its antigen-binding fragment). Examples of "host cells" used to produce recombinant antibodies include: (1) mammalian cells, such as Chinese hamster ovary (CHO), COS, myeloma cells (including Y0 and NSO cells), young 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., tobacco (Nicotiana tabacum)); (4) yeast cells, such as those belonging to the genus Saccharomyces (e.g., brewer's yeast (Saccharomyces cerevisiae)) or the genus Aspergillus (e.g., Aspergillus niger); (5) bacterial cells, such as Escherichia coli cells or Bacillus subtilis cells, etc.

[0095] As used herein, "regulatory protein" 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; alters the interaction between another polypeptide or RNA molecule and other polypeptides, DNA or RNA molecules, or any other binding receptor; and / or alters the cellular location of another polypeptide or RNA molecule. Regulatory proteins that increase or decrease gene transcription rates are generally referred to as transcription factors that act on gene promoter or enhancer regions. 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 / α, cErb-A, myc, mad and max, p53, mdml, and other transcription factors described in Latchman, 1998, Eukaryotic Transcription Factors, 3rd edition, Academic Press: New York. Fusion protein derivatives of these or other transcription factors may also be used (wherein at least one of the fusion proteins provides a binding-specific DNA-binding motif fused to a small molecule regulator binding site).

[0096] As used herein, "site-directed mutagenesis" refers to a method in which a predetermined mutation is generated at a specific site in the parent polypeptide. A specific site is a site that is desired to be selected as needed for the study. A predetermined mutation may be the substitution of an amino acid residue at the specific site with a specific amino acid, the insertion of a specific amino acid residue at the specific site, or the deletion of a specific amino group at the specific site.

[0097] As used herein, “small interfering RNA” or “siRNA” refers to RNA or RNA-like molecules that can interact with and disrupt mRNA molecules that share sequence homology with the siRNA (Elbashir et al., Genes Dev, Vol. 15, pp. 188-200, 2001). It is thought that siRNA can be incorporated into a ribonucleoprotein complex called 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 these target mRNA molecules or inhibits their translation. Typical siRNAs are double-stranded nucleic acid molecules, each strand containing approximately 19 to approximately 28 nucleotides (i.e., approximately 19, 20, 21, 22, 23, 24, 25, 26, 27, or 28 nucleotides). siRNAs can also be single-stranded RNAs, but are less effective than double-stranded siRNAs. Single-stranded siRNAs are approximately 19 to approximately 49 nucleotides in length. Single-stranded siRNAs have a 5′ phosphate ester or are phosphorylated at the 5′ position in situ or in vivo. Single-stranded siRNAs can be synthesized chemically or through in vitro transcription, or expressed intrinsically from expression vectors or expression cassettes. The 5′ phosphate group can be added via kinases or may result from RNA nuclease cleavage.

[0098] The term "small molecule" refers to molecules or ions with a molecular weight typically less than 900 amu, or more preferably less than 500 amu, or more preferably less than 200 amu, or even more preferably less than 100 amu. In the analytical and environmental context of this invention, small molecules can typically exist as molecular mixtures and deprotonated ions of molecules, depending primarily on the pH of the analytical or environmental context.

[0099] As used herein, the term "therapeutic protein" refers to any protein and / or polypeptide that, when administered to mammals, elicits a biological or medical response in tissues, systems, animals, or humans, as sought by researchers or clinicians. Therapeutic proteins can elicit more than one biological or medical response. Examples of therapeutic proteins include antibodies, enzymes, hormones, cytokines, regulatory proteins, and fragments thereof.

[0100] As used herein, the term "therapeuticly effective dose" means any dose that, compared to a corresponding individual who has not received that dose, results in, but is not limited to, the healing, prevention, or improvement of disease, condition, or side effects, or a reduction in the rate of disease or condition progression. The scope of this term also includes doses that effectively enhance normal physiological function and doses that effectively induce physiological function in a patient or contribute to the therapeutic effect of another medication.

[0101] As used in this article, the term "tumor microenvironment" refers to the microenvironment within or around a solid tumor that supports tumor cell growth and metastasis. The tumor microenvironment includes the perivascular zone, immune cells, fibroblasts, other cells, soluble factors, signaling molecules, extracellular matrix, and mechanotransmitters. It can promote tumor transformation, support tumor growth and invasion, protect the tumor against the host's immune system, cultivate therapeutic resistance, and provide a favorable environment for dormant metastasis. Tumors and their surrounding microenvironment are closely related and constantly interact. Tumors can influence their microenvironment by releasing extracellular signals, promoting tumor angiogenesis, and inducing peripheral immune tolerance, while immune cells within the microenvironment can influence the growth and evolution of cancer cells. See Swarts et al., "Tumor Microenvironment Complexity: Emerging Roles in Cancer Therapy", Cancer Res, Vol. 72, pp. 2473-2480, 2012; Weber et al., "The tumor microenvironment", Surgical Oncology, Vol. 21, pp. 172-177, 2012; Blagosklonny, "Antiangiogenic therapy and tumor progression", Cancer Cell, Vol. 5, pp. 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 biological protein," or "wild-type biological protein" can refer to a protein that can be isolated from nature, will have the same level of activity as that found in nature, and will contain an amino acid sequence found in nature. The terms "parental molecule" and "target protein" also encompass wild-type proteins. A. Conditionally active polypeptides

[0103] This invention provides conditionally active peptides and methods for generating conditionally active peptides from parental peptides. The conditionally active peptide has an increased net charge compared to the parent peptide. This increase in net charge can be achieved by introducing one or more charged amino acid residues into the parent peptide, or by deleting one or more uncharged amino acid residues from the parent peptide, or both, thereby increasing the net charge of the parent peptide. The charged amino acid residues are selected from aspartic acid, glutamic acid, arginine, lysine, and histidine. In one state, the mutation can be a substitution of one or more uncharged amino acid residues in the parent peptide with one or more charged amino acid residues. In another state, the mutation can be an insertion of one or more additional charged amino acid residues into the parent peptide. Combinations of such substitutions and insertions can also be used. In yet another state, the mutation can be a deletion of one or more uncharged amino acid residues from the parent peptide.

[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 normally charged at normal physiological pH. Histidine can also be charged at normal physiological pH.

[0105] Conditionally active peptides with an increased number of charged amino acid residues exhibit both of the following: (a) decreased activity in the first analysis at a first value of conditions compared to the same activity of the parent peptide in the first analysis, and (b) increased activity in the second analysis at a second value of conditions compared to the same activity of the parent peptide in the second analysis. [ ]

[0106] In some embodiments, the conditionally active peptide exhibits reduced activity in a first analysis at a first value of conditions compared to the same activity in a second analysis at a second value of the same conditions. [ ]

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

[0108] In another state, the conditionally active polypeptide is derived from the light chain variable region (SEQ ID NOS: 11-13) of an antibody against the Ror2 protein, which is derived from the parental light chain variable region having SEQ ID NO: 10. In another state, the conditionally active polypeptide is derived from the heavy chain variable region (SEQ ID NOS: 15-18) of an antibody against the Ror2 protein, which is derived from the parental heavy chain variable region having SEQ ID NO: 14. In another state, the conditionally active antibody may contain both the light chain variable region selected from SEQ ID NOS: 11-13 and the heavy chain variable region selected from SEQ ID NOS: 15-18. [ ]

[0109] Conditional active antibodies against Axl or Ror2 are further illustrated in Examples 15-22 of the present invention. These conditionally active light chain variable regions and heavy chain variable regions include amino acid substitutions, wherein charged amino acid residues, particularly E and D, are introduced into the complementarity-determining regions of the parental light chain variable region and the parental heavy chain variable region. [ ]

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

[0111] In one state, the conditional active peptide exhibits pH-dependent activity in the presence of a substance with a pKa within 2 or 3 pH units of the pH at which the desired activity is desired. In another state, the invention relates to a conditional active peptide exhibiting pH-dependent activity in the presence of a substance with 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 yet another state, the invention relates to a conditional active peptide exhibiting pH-dependent activity in the presence of a substance selected from disulfides, bicarbonates, histidine, citrates, acetates, lactates, and mixtures thereof.

[0112] Substances present in analytical media that significantly influence the activity of conditionally active peptides are often those with at least two ionization states: an uncharged or lightly charged state, and a charged or heavily charged state. Therefore, the pKa of a substance affecting the activity of a conditionally active peptide can be useful in determining the extent to which that substance will affect the specific activity of the peptide and / or the degree of its influence at a specific pH.

[0113] pH-dependent conditionally active peptides exhibit higher activity at the first pH compared to different second pH values, with both activities measured in the presence of one or more of the substances listed above. To determine the pH dependence of a conditionally active peptide, the same activity of the peptide is analyzed at two different pH values ​​in the same analytical medium.

[0114] The ratio of activity at a first pH to the same activity at a second pH in the same analytical medium can be termed the selectivity of a pH-dependent conditionally active peptide. A pH-dependent conditionally active peptide may have 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 peptides typically exhibit different activities in the presence of different substances in the analytical medium. Substances possessing at least two ionization states (i.e., uncharged or lightly charged states and charged or heavily charged states) can dissociate to a greater extent at a specific pH, depending on the pKa value, thereby increasing the probability of interaction with charged amino acid residues present in the conditionally active peptide. This factor can be used to enhance the selectivity and / or pH-dependent activity of conditionally active peptides.

[0116] Not limited to theory, it is believed that as the pH of the analytical medium changes, the introduction of charged amino acid residues into the parent peptide alters the charge on the conditionally active peptide, thus changing its activity at different pH levels. For example, in the acidic pH range of the analytical medium, negatively charged amino acid residues (e.g., E and D) in the conditionally active peptide are uncharged or have a small charge, but in the neutral or alkaline pH range, they are negatively charged. It is thought that this change in charge on the conditionally active peptide allows it to interact differently with one of the substances discussed above at different pH values. In a given sample, the conditionally active peptide is more likely to interact with charged substances in the analytical medium at one pH, but less likely to interact with the same charged substances in the same analytical medium at another different pH. This is subsequently believed to affect the activity of the conditionally active peptide.

[0117] The charge properties of a conditionally active peptide can be a factor in determining suitable substances that affect its activity. In some embodiments, the conditionally active peptide may have more positively charged amino acid residues—lysine, arginine, and histidine—compared to its parent peptide. The conditionally active peptide can thus be selected to have a desired degree of interaction with specific substances present in an environment where the desired activity is desired, and / or a desired degree of interaction with specific substances present in an environment where the activity is desired to be reduced. Similarly, the conditionally active peptide may have more negatively charged amino acid residues—aspartate and glutamate—compared to its parent peptide.

[0118] The position of charged amino acid residues on pH-dependent conditionally active peptides can also affect their activity. For example, charged amino acid residues located near the binding site of the conditionally active peptide can be used to influence peptide activity.

[0119] In some embodiments, the interaction between charged environmental substances and conditionally active peptides can block or impede the activity of pH-dependent conditionally active peptides. For example, charged amino acids interacting with charged environmental substances can exhibit allotropic effects on the binding sites of conditionally active peptides.

[0120] In other embodiments, the interaction between charged environmental substances and conditionally active peptides can form salt bridges between different parts of the peptide (especially charged or polarized parts). It is known that the formation of salt bridges can stabilize peptide 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 the structure of proteins that typically undergo constant, minute structural changes known as "breathing" (Parak, "Proteins in action: the physics of structural fluctuations and conformational changes", Curr Opin Struct Biol., 13(5):552-557, 2003). Protein structural "breathing" is crucial for protein function and its binding to its partner, as structural fluctuations allow conditionally active proteins to effectively recognize and bind to their partner (Karplus et al., "Molecular dynamics and protein functions," PNAS, Vol. 102, pp. 6679-6685, 2015). By forming salt bridges, binding sites (especially binding pockets) on conditionally active peptides may become less accessible to their partner, possibly because the salt bridge directly blocks the partner from reaching the binding site. Even if the salt bridge is far from the binding site, allosteric effects can alter the conformation of the binding site to inhibit binding. Therefore, after the structure of a conditionally active peptide is stabilized (fixed) by a salt bridge, the peptide may become less active in binding to its partner, leading to reduced activity.

[0121] A known example of how peptides and their structures are stabilized by salt bridges is heme. Structural and chemical studies have revealed that at least two sets of chemical groups are responsible for the salt bridge: the amino terminus and side chain of histidine β146 and α122, with pKa values ​​close to pH 7. In deoxyheme, the terminal carboxyl group of β146 forms a salt bridge with a lysine residue in the α subunit of another αβ dimer. This interaction locks the side chain of histidine β146 at a position where it can participate in the salt bridge together with the negatively charged aspartic acid 94 in 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 does not form a salt bridge. However, as pH decreases, the side chain of histidine β146 becomes protonated, forming a salt bridge between histidine β146 and aspartic acid β94. This bridge stabilizes the quaternary structure of deoxyheme, leading to a greater tendency to release oxygen in metabolically active tissues (with lower pH). Heme exhibits pH-dependent oxygen-binding activity, where at low pH, oxygen-binding activity decreases due to the formation of the salt bridge. Conversely, at high pH, ​​oxygen-binding activity increases due to the absence of the salt bridge.

[0122] Similarly, small molecules such as bicarbonates can reduce the binding activity of conditionally active peptides to their conjugates by forming salt bridges within the peptide. For example, at pH values ​​below their pKa of 6.4, bicarbonates are protonated and thus uncharged. Uncharged bicarbonates cannot form salt bridges and therefore have little effect on the binding of conditionally active peptides to their conjugates. Therefore, at low pH, conditionally active peptides exhibit high binding activity to their conjugates. On the other hand, at high pH values ​​above the pKa of bicarbonates, bicarbonates ionize due to proton loss and become negatively charged. The negatively charged bicarbonates will form salt bridges between the positively charged or polarized portions of the conditionally active peptide to stabilize its structure. This will block or reduce the binding of the conditionally active peptide to its conjugate. Therefore, conditionally active peptides exhibit low activity at high pH. Conditionally active peptides therefore exhibit conditional activity in the presence of bicarbonate, with higher binding activity at low pH than at high pH.

[0123] When substances such as bicarbonate are absent in the analytical medium, conditionally active peptides may lose their conditional activity. This may be due to the lack of salt bridges on the conditionally active peptide to stabilize (fix) its structure. Therefore, at any pH, the partner peptide will have similar accessibility to the binding site on the conditionally active peptide, thus producing similar activity at the first and second pH.

[0124] In other embodiments, the interaction between small molecules or ions and conditionally active peptides can alter the peptide structure in a way that increases its activity. For example, structural changes can improve the binding affinity of conditionally active peptides by altering the position of the binding site required for binding affinity, steric hindrance, or binding energy. In such cases, it is desirable to selectively bind small molecules to the conditionally active peptide at the desired pH.

[0125] It should be understood that although salt bridges (ionic bonds) are the strongest and most common way in which compounds and ions influence the activity of conditionally active peptides, other interactions between these compounds and ions and conditionally active peptides can also help stabilize (fix) the structure of conditionally active peptides. 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 peptide is compared with its parental peptide to determine whether the conditionally active peptide has a higher proportion of negatively charged or positively charged amino acid residues. Subsequently, compounds with suitable charges at a second pH can be selected to influence the activity of the conditionally active peptide. For example, when the conditionally active peptide has a higher proportion of positively charged amino acid residues than the parental peptide, a suitable small molecule should generally be negatively charged at the second pH to interact with the conditionally active peptide. Conversely, when the conditionally active peptide has a higher proportion of negatively charged amino acid residues than the parental peptide, a suitable small molecule should generally be positively charged at the second pH to interact with the conditionally active peptide.

[0127] In other embodiments, the activity of the conditionally active peptide is controlled by the interaction between a small molecule or ion and the target peptide, which is the binding partner of the conditionally active peptide. In this case, the same principles described above also apply, but the target peptide generates the interaction between the small molecule or ion and the target peptide. The target peptide may be, for example, an antigen of a conditionally active antibody or a ligand of a conditionally active receptor.

[0128] Suitable small molecules can be any inorganic or organic molecule that transitions from an uncharged or less-charged state at a first pH to a charged or more-charged state at a second pH. Therefore, the pKa of a small molecule should generally be between the first and second pH values. For example, bicarbonate has a pKa of 6.4. Therefore, at higher pH values ​​(e.g., pH 7.4), the negatively charged bicarbonate will bind to the charged amino acid residues of the conditionally active peptide and reduce its activity. On the other hand, at lower pH values ​​(e.g., pH 6.0), the less-charged bicarbonate will not bind to the conditionally active peptide in the same amount, thus allowing for higher activity of the conditionally active peptide.

[0129] The pKa of disulfides is 7.05. Therefore, at higher pH levels (e.g., pH 7.4), more negatively charged disulfides will bind to positively charged amino acid residues in conditionally active peptides, reducing their activity. On the other hand, at lower pH levels (e.g., pH 6.2–6.8), less charged hydrogen sulfide / disulfide will not bind to conditionally active peptides to the same extent, thus allowing for higher activity of the conditionally active peptides.

[0130] Small molecules with a pKa between the first and second pH values ​​are preferred for use in this invention. Preferred substances are selected from disulfides, bicarbonates, histidine, histamine, citrates, acetates, and lactates. Each of these small molecules has a pKa between 6.2 and 7.0. Other small molecules, such as tris(hydroxymethyl)methylglycine (pKa 8.05) and dihydroxyethylglycine (pKa 8.26), can also be used. Other suitable small molecules can also be found in textbooks on the principles of this application, such as the CRC Handbook of Chemistry and Physics, 96th edition, CRC press, 2015; Chemical Properties Handbook, McGraw-Hill Education, 1998.

[0131] The concentration of small molecules in the analytical medium or environment is preferably at or close to the physiological concentration of the small molecules in an individual. For example, the physiological concentration of bicarbonate (in human serum) is in the range of 15 to 30 mM. Therefore, the concentration of bicarbonate in the analytical medium can be 10 mM to 40 mM, or 15 mM to 30 mM, or 20 mM to 25 mM, or about 20 mM. The physiological concentration of disulfide is also relatively low. The concentration of disulfide in the analytical medium can be 3 to 100 mM, or 5 to 80 mM, or 10 to 50 mM, or 10 to 30 mM.

[0132] In this invention, conditionally active peptides are selected and used at multiple concentrations, wherein the normal physiological concentration of a specific substance in the environment will have a significant effect on the activity of the conditionally active peptide within the pH range of interest. Therefore, in various therapeutic treatments, it is advantageous to have low activity of the conditionally active peptide at a pH of approximately 7.2-7.4 in blood or human serum, allowing for the delivery of therapeutic treatment via the bloodstream while minimizing or preventing activation of the conditionally active peptide. Therefore, for such treatments, it is advantageous to select small molecules with a pKa below pH 7.2-7.4 to ensure sufficient ionization of the small molecule at the bloodstream pH to have a significant effect on the activity of the conditionally active peptide. Simultaneously, the pKa of the small molecule should be at or above the pH at which the activity of the conditionally active peptide is desired, to ensure that the conditionally active peptide is activated by releasing the binding sites on the conditionally active peptide through protonation of the small molecule.

[0133] Small molecules preferably have low molecular weight and / or relatively small conformation to ensure maximum accessibility to pouches on target peptides or conditionally active peptides by minimizing steric hindrance. Therefore, the molecular weight of small molecules is typically less than 900 amu, or preferably less than 500 amu, or even more preferably less than 200 amu, or even more preferably less than 100 amu. For example, both disulfides and bicarbonates have low molecular weight and small structure, providing accessibility to pouches on target peptides or conditionally active peptides, as shown in Examples 13 and 14 below.

[0134] Small molecules can exist at substantially the same concentration in the analysis or environment, for example, bicarbonate at about 20 μM. In some embodiments, small molecules can exist at different concentrations in different environments, and therefore it is desirable to simulate this situation in the analysis. For example, the concentration of disulfides in the tumor microenvironment is higher than in human serum. Therefore, one analysis can simulate a tumor microenvironment with an acidic pH and a higher concentration of disulfides, while a second analysis can simulate human serum with a neutral or weakly alkaline pH and a lower concentration of disulfides. The acidic pH can be in the range of 6.0 to 6.8, while the neutral or weakly alkaline pH can be about 7.4. The higher concentration of disulfides in the first analysis can be 30 μM, while the lower concentration of disulfides in the second buffer can be 10 μM or lower, or 5 μM.

[0135] In some embodiments, the conditionally active peptide is pH-dependent in the presence of two or more different small molecules (e.g., a combination of bicarbonate and histidine).

[0136] In the absence of small molecules, conditionally active peptides can lose their pH dependence. Therefore, in the absence of small molecules, conditionally active peptides can exhibit similar activity between the first and second pH values.

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

[0138] Conditionally active peptides that exhibit higher activity at acidic pH and lower activity at alkaline or neutral pH can target tumor microenvironments with acidic pH, approximately 5.5 to 7.2, or approximately 6.2 to 6.8.

[0139] In other embodiments, the first pH at which the pH-dependent peptide activity is higher may be an alkaline pH, such as 7.6-7.9, for example in synovial fluid (see Jebens et al., "On the viscosity and pH of synovial fluid and pH of blood", Journal of Bone and Joint Surgery, Vol. 41 B, pp. 388-400, 1959). The second pH may be the blood pH of about 7.2-7.6, at which the conditionally active peptide activity is lower. These conditionally active peptides may be suitable for targeting joint diseases, such as joint inflammation.

[0140] In other embodiments, the conditionally active peptide may be designed to target the brain. A pH difference exists across the blood-brain barrier, with the brain side having a pH approximately 0.2 pH units lower than the blood side. Therefore, the first pH at which the conditionally active peptide exhibits higher activity is approximately 7.0 to 7.2 (brain pH), while the second pH may be approximately 7.4 (blood pH).

[0141] Conditionally active polypeptides can be enzymes, interleukins, receptors (especially cell receptors), regulatory polypeptides, soluble polypeptides, antibodies, or hormones.

[0142] Conditionally active polypeptides can be fragments of parental polypeptides. For example, conditionally active polypeptides can be antibody fragments, single-chain antibodies, enzyme fragments, receptor fragments, interleukin fragments, or hormone fragments. Antibody fragments can be Fc fragments of antibodies.

[0143] Fc fragments can be used as parental peptides to produce conditionally active Fc fragments that exhibit higher complement-binding activity at a first pH than at a second pH. The binding of Fc fragments to complement can provide antibody-dependent cell-mediated cytotoxicity. The first pH can be acidic, ranging from 5.5 to 7.2 or 6.2 to 6.8, as in the tumor microenvironment, while the second pH is in the range of 7.2–7.6. The first pH differs from the pH in lysosomes, which is typically around 4.0. Furthermore, in lysosome systems, Fc fragments are targeted to sites of degradation, just like any other peptide. Lysosomes lack complement and do not exhibit cell-mediated cytotoxicity induced via lysosomes.

[0144] Conditionally active peptides may possess two functional domains, at least one of which, preferably both, possess pH-dependent activity. These two functional domains may be simultaneously evolved and selected to identify two functional domains within the same mutant peptide. Alternatively, the two functional domains may be independently evolved and selected to individually identify pH-dependent activity. If the two functional domains are not in the same mutant peptide, they may be fused into a chimeric peptide with each functional domain possessing its own distinct activity.

[0145] Conditionally active peptides are reversibly or irreversibly inactive under a first condition or normal physiological condition, but become active under a second condition or abnormal condition to the same or equivalent degree as under the first condition or normal physiological condition. Such conditionally active peptides and methods for generating such peptides are described in U.S. Patent No. 8,709,755 B2. Conditionally active peptides are particularly valuable for the development of novel therapeutics that are active in the host for a short or limited time period. This conditionally active peptide is particularly valuable if the prolonged effect of a given dose is harmful to the host, but limited activity is required to implement the desired therapy. Examples of beneficial applications include local or systemic treatment at high doses, and localized treatment at high concentrations. Inactivation under the first condition or normal physiological condition can be determined by a combination of drug administration and the peptide inactivation rate. This condition-based inactivation is particularly important for enzyme therapeutics whose catalytic activity causes significant adverse effects over a relatively short period.

[0146] Conditionally active peptides are reversibly or irreversibly activated or inactive over time, or activated or inactive only in certain microenvironments in vivo (including in specific organs, such as the tumor microenvironment, synovial fluid, bladder, or kidney). In some cases, conditionally active peptides are antibodies or antibody fragments targeting one or more target proteins (antigens), as described herein.

[0147] The conditionally active polypeptide may be a separated polypeptide with pH-dependent activity, wherein, in the presence of a substance 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. In the absence of small molecules, the same activity is not pH-dependent. In some embodiments, the activity at the 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. The first pH may be an abnormal pH, in the range of about 5.5-7.2 or about 6.2-6.8, while the second pH may be a normal physiological pH, in the range of about 7.2-7.6.

[0148] For the selected conditionally active polypeptide, a nucleic acid (e.g., a DNA sequence) encoding the conditionally active polypeptide can be identified or synthesized. Nucleic acids can be used in recombinant technologies to generate conditionally active polypeptides in large quantities. For example, the nucleic acid can be inserted into an expression vector, wherein the expression vector has a promoter operatively linked to the nucleic acid to ensure a high degree of expression of the conditionally active polypeptide. The expression vector can be introduced into host cells, such as yeast, insect cells, or mammalian cells. Examples of host cells include 3T3 mouse fibroblasts; BHK21 Syrian hamster fibroblasts; MDCK, canine epithelial cells; HeLa human epithelial cells; PtK1 kangaroo epithelial cells; SP2 / 0 mouse plasma cells; and NSO mouse plasma cells; HEK 293 human embryonic kidney cells; COS monkey kidney cells; CHO, CHO-S Chinese hamster ovary cells; R1 mouse embryonic cells; E14.1 mouse embryonic cells; H1 human embryonic cells; H9 human embryonic cells; and PER C.6, human embryonic cells. B. Engineering of conditionally active peptides

[0149] Conditionally active peptides can be engineered using one or more of the protein engineering techniques described herein. Non-limiting examples of protein engineering techniques include conjugating conditionally active peptides to nucleic acids, conjugating conditionally active peptides to nanoparticles, engineering conditionally active peptides into chimeric antigen receptors, and engineering masked conditionally active peptides.

[0150] The conditionally active peptides of this invention can be coupled to nucleic acid molecules, such as DNA or RNA molecules, via linkers. Conditionally active peptides can facilitate the delivery of nucleic acid molecules to a target site in an individual, where the activity of the conditionally active peptide is higher than at other sites where that condition is not present. For example, a conditionally active peptide can be a conditionally active antibody, whose binding activity to its antigen is higher under conditions in the tumor microenvironment than under conditions at other sites (e.g., in human serum). This effect can be used to deliver nucleic acid molecules to the tumor microenvironment by coupling nucleic acid molecules to conditionally active peptides and delivering the conjugate to an individual.

[0151] In some embodiments, nucleic acid molecules can be agents that regulate gene expression at target sites. Abnormal gene expression is associated with a variety of diseases. Therefore, correcting abnormal gene expression can help control or even cure these diseases. For example, abnormal gene expression is characteristic of most cancer cells, and some genes are overexpressed in cancer cells, such as various oncogenes (e.g., epidermal growth factor receptor 2 (HER2) is overexpressed in breast cancer cells). Selectively inhibiting constitutively elevated expression of oncogenes provides an opportunity to suppress cancer cell proliferation.

[0152] Nucleic acid molecules that can suppress gene expression include antisense RNA, small interfering RNA (siRNA), microRNA, oligoDNA, and oligonucleotide mimics with an uncharged, achiral polyamide backbone linked to 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 RNAs are short RNA molecules that can bind to specific complementary regions of mRNA through base pairing, thereby inhibiting mRNA expression in a sequence-specific manner. Antisense RNAs can induce RNase H cleavage of mRNA at their binding sites, or they can physically block mRNA processing and translation or other steps in protein synthesis.

[0154] Small interfering RNAs (siRNAs) are typically short double-stranded RNA segments whose sequence is at least partially complementary to the mRNA sequence to be blocked from translation. siRNAs function through post-transcriptional mechanisms of gene silencing, using chromatin remodeling, inhibition of protein translation, or direct mRNA degradation. They are 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] Specifically, via RISC, siRNA can initiate an effective cascade of sequence-specific degradation of mRNAs homologous to siRNA (Fire et al., "Potent and specific genetic interference by doubles-stranded RNA in Caenorhabditis elegans", Nature 391:806-811, 1998). When siRNA is introduced into cells, it is treated by an RNase III enzyme called Dicer, which cleaves long siRNA into short 21-23 nucleotide double strands. These double strands have symmetrical 2-3 nucleotide 3' overhangs, 5' phosphate esters, 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). Therefore, effective siRNA only needs a small segment of adjacent complementary sequence to pair with mRNA to trigger siRNA-mediated silencing (Jackson and Linsley, "Noise amidst the silence: off-target effects of siRNA?" Trends Genet., 20:521-524, 2004). siRNA does not integrate into the genome, thus providing greater safety than plasmids or viral vectors.

[0156] MicroRNAs (miRNAs) are a class of naturally occurring non-coding small RNA molecules, 21-25 nucleotides in length. MicroRNAs are partially complementary to the mRNA molecules they act upon. The primary function of microRNAs is to reduce gene expression through translational repression, mRNA cleavage, and deadenylation. A central online repository of miRNA material, sequence data, annotations, and target predictions is called miRBase, hosted by the Sanger Institute in Great Britain. MicroRNA genes are transcribed by RNA polymerase II to produce primary miRNAs with a 5' cap and multiple A tails. In the nucleus, primary miRNAs are processed by a microprocessor complex to produce precursor miRNAs, which consists of the RNAe III enzyme Drosha and the double-stranded RNA Pasha / DGCR8. These precursor miRNAs are exported to the cytoplasm by the nuclear transport protein export protein (Exp5) and the Ran-GTP complex, where Ran GTPase binds to Exp5 to form a nuclear heterotrimer with the precursor miRNA. These precursor miRNAs are further processed by the RNAe III enzyme Dicer to produce mature microRNAs.

[0157] Another class of nucleic acid oligonucleotide mimics that can be delivered via conditionally active peptides comprises an uncharged, achiral polyamide backbone linked to nucleobases. Oligonucleotide mimics are commonly referred to as peptide nucleic acids (PNAs). More specifically, PNAs are DNA analogs in which N-(2-aminoethyl)glycine polyamide replaces the phosphate-ribose ring backbone, and methylene-carbonyl linkers connect native and non-native nucleobases to the central amine of N-(2-aminoethyl)glycine. Despite the significant changes in backbone structure, PNAs can bind specifically to DNA and mRNA according to the Watson-Crick base pairing principle.

[0158] PNA binds to complementary DNA / RNA with a higher affinity than native nucleic acids. This is due to the lack of negative charge on the backbone, thus reducing charge repulsion, and favorable geometry. The PNA-DNA / mRNA complex is extremely stable in biological fluids, leading to the inhibition of target gene transcription and translation through specific hybridization with DNA or mRNA. Typically, PNA is synthesized using well-known solid-phase peptide synthesis protocols. See 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., BioTechniques, 19, 472-480, 1995; Tomac et al., J. Am. Chem. Soc., 118, 5544-5552, 1996). Compared to DNA that is depurinated upon treatment with strong acids and hydrolyzed in alkaline hydroxides, the PNA system is completely acid-stable and sufficiently stable to weak bases.

[0159] OligoDNA, another type of nucleic acid, can be delivered via conditionally active peptides. OligoDNA consists of short, single-stranded segments of DNA that, upon entering the cytoplasm, selectively inhibit the expression of genes complementary to the oligoDNA sequence. For antisense applications, oligoDNA interacts with target mRNA or pre-mRNA to form a double strand and inhibits its translation or processing, thus suppressing protein biosynthesis. For antigen applications, oligoDNA must enter the nucleus, form a triple strand with double-stranded genomic DNA, and inhibit gene transcription, resulting in less mRNA and consequently less protein production.

[0160] Another type of nucleic acid that can be delivered via conditionally active peptides is the spherical nucleic acid (SNA, see Zhang, J Am Chem Soc., 134(40):16488-16491, 2012). SNAs comprise densely functionalized and highly oriented nucleic acids covalently attached to a metallic, semiconductive, or insulating inorganic or polymeric core material. They can also be coreless hollow structures, composed almost entirely of nucleic acid molecules. These spherical nucleic acids can bypass the individual's natural defenses against exogenous nucleic acids. Spherical nucleic acids utilize the unique properties derived from their densely packed, highly oriented nucleic acid shells to achieve nucleic acid protection and efficient delivery. These shells create regions of high local salt concentration, which, when combined with steric inhibition, reduce nuclease activity and prevent nucleic acid degradation by enzymes. In addition, these spherical nucleic acids recruit scavenging proteins from the natural extracellular environment to their surface, thus promoting endocytosis.

[0161] Once inside the cytoplasm, globular nucleic acids can inhibit the expression of target genes via antisense or siRNA pathways. Therefore, globular nucleic acids offer several advantages over viral vectors and many other synthetic systems, including low toxicity, low immunogenicity, resistance to enzymatic degradation, and more persistent gene knockdown. Conditionally active peptides, especially conditionally active antibodies, can deliver globular nucleic acids to target sites, such as diseased or inflamed tissues (e.g., tumors and inflamed joints).

[0162] Conditionally active peptides can also be coupled to nanoparticles via linkers to aid in the delivery of nanoparticles to the target site, where the conditional activity of the peptide is enhanced under the conditions at that target site. Nanoparticles are known mediators of toxins, radioactive agents, or other therapeutic agents, which can be encapsulated within nanoparticles.

[0163] The therapeutic agent encapsulated in nanoparticles can be a protein that can dedifferentiate tumor cells and potentially revert them back to normal cells (Friedmann-Morvinski and Verma, "Dedifferentiation and reprogramming: origins of cancer stem cells", EMBO Reports, 15(3):244-253, 2014). Nanoparticles can be linked to conditionally active antibodies to selectively deliver the linked nanoparticles and the encapsulated therapeutic agent to the environment where the conditionally active antibody has the highest activity.

[0164] Several types of nanoparticles with different configurations can be used in this invention. The nanoparticles can be derived from a variety of 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, when a polymer is exposed to bodily fluids (e.g., blood), it can be gradually absorbed and / or eliminated by enzymes in vivo. Methods for producing nanoparticles with different degradation rates are known to those skilled in the art, for example, see U.S. Patent Nos. 6,451,338, 6,168,804, and 6,258,378.

[0165] The exemplary nanoparticles of this invention include liposomes, polymer vesicles, and polymer particles. A lipid system refers to a completely enclosed bilayer of compartments typically composed of phospholipids. Liposomes can be prepared according to standard techniques known to those skilled in the art. One technique involves suspending a suitable lipid (e.g., phosphatidylcholine) in an aqueous medium, followed by ultrasonic treatment of the mixture. Another technique involves rapidly mixing the lipid in an ethanol-water solution, for example, by injecting the lipid into an agitated ethanol-water solution using a needle. In some embodiments, the liposomes may also additionally or alternatively contain other amphiphilic substances, such as sphingomyelin or lipids containing poly(ethylene glycol) (PEG).

[0166] Polymer vesicles comprise diblock or triblock copolymers modified to form a bilayer structure similar to liposomes. Depending on the length and composition of the block copolymers, polymer vesicles can be significantly more robust than liposomes. Furthermore, the ability to control the chemistry of each block in the block copolymer allows for tuning the composition of the polymer vesicles to suit desired applications. For example, the membrane thickness of the polymer vesicle, i.e., the thickness of the bilayer structure, can be controlled by altering the chain length of individual blocks in the block copolymer. Adjusting the glass transition temperature of each block in the copolymer affects flowability and thus the permeability of the polymer vesicle membrane. Even the release mechanism of the encapsulated reagent can be modified by altering the characteristics of the copolymer.

[0167] Polymer vesicles can be prepared by a method involving the following steps: (i) dissolving a block copolymer in an organic solvent, (ii) applying the resulting solution to a container surface, and then (iii) removing the solvent, leaving a copolymer film on the container wall. The film is then hydrated to form polymer vesicles. Alternatively, polymer vesicles can also be produced by dissolving the block copolymer in a solvent and then adding a weak solvent of one of the blocks in the copolymer.

[0168] Several techniques can be used to encapsulate therapeutic agents in polymer vesicles. For example, the therapeutic agent can be mixed in water and then used to rehydrate the copolymer film. Another example is by using osmotic pressure to force the therapeutic agent into the core of a pre-formed polymer vesicle, a method known as force loading. Another example is the use of dual emulsification technology, which can produce polymer vesicles with relatively monodispersity and high loading efficiency. Dual emulsification involves using microfluidics to produce a dual emulsion comprising water droplets surrounded by an organic solvent layer. The droplet-in-a-drop structures in these droplets are then dispersed in a continuous aqueous phase. The block copolymer is dissolved in the organic solvent and self-assembles into proto-polymer vesicles at the concentric interfaces of the dual emulsion. The final polymer vesicles are formed after the organic solvent has completely evaporated from the shell of the proto-polymer vesicle. This technique allows for precise control of polymer vesicle size. Furthermore, the ability to maintain complete separation of the internal and external fluids throughout the process allows for highly efficient encapsulation of the therapeutic agent.

[0169] Compared to the shell structure of liposomes and polymer vesicles, polymer particles refer to solid or porous particles. Methods for adhering therapeutic agents to the surface of polymer particle structures or integrating bioactive agents into polymer particle structures are known to those skilled in the art.

[0170] Polymers that can be used to prepare the nanoparticles of this invention include, but are not limited to, poly(N-acetylglucosamine) (chitin), chitosan, poly(3-hydroxyvalerate), poly(lactic acid lactide-co-glycolic acid), poly(3-hydroxybutyrate), poly(4-hydroxybutyrate), poly(3-hydroxybutyrate-co-3-hydroxyvalerate), polyorthoester, polyanhydride, poly(glycolic acid), poly(glycolic acid), poly(glycolic acid), poly(L-lactic acid), poly(L-lactic acid), poly(D,L-lactic acid), poly(D,L-lactic acid), poly(L-lactic acid lactide-co-D,L-lactic acid), poly(caprolactone), poly(L-lactic acid lactide-co-caprolactone), poly(D,L-lactic acid lactide-co-caprolactone), poly(glycolic acid-co-caprolactone), poly(glycolic acid-co-caprolactone), poly(glycolic acid-co-caprolactone), poly(glycolic acid-co-caprolactone), poly(glycolic acid-co-caprolactone), poly(glycolic acid-co-caprolactone), poly(glycolic acid-co-caprolactone), poly(glycolic acid-co-caprolactone), poly(glycolic acid-co-caprolactone), poly(ether-ester). (e.g., PEO / PLA), polyphosphazene, biomolecules (e.g., fibrin, fibrin glue, fibrinogen, cellulose, starch, collagen and hyaluronic acid, elastin and hyaluronic acid), polyurethane, polysiloxane, polyester, polyolefin, polyisobutylene and ethylene-α-olefin copolymers, acrylic polymers and copolymers other than polyacrylates, ethylene halide polymers and copolymers (e.g., polyvinyl chloride), polyvinyl ether (e.g., polyvinyl methyl ether), polyvinylidene halogen (e.g., polyvinyl chloride), polyacrylonitrile, polyvinyl ketone, polyvinyl aromatic hydrocarbons Aromatic hydrocarbons (e.g., polystyrene), polyvinyl esters (e.g., polyvinyl acetate), acrylonitrile-styrene copolymers, acrylonitrile butadiene styrene (ABS) resins, polyamides (e.g., Nylon 66 and polycaprolactam), polycarbonates (including tyrosine-based polycarbonates), polyoxymethylene, polyimide, polyether, polyurethane, 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, in addition to the selectivity derived from conditionally active peptides, nanoparticles can also provide tissue selectivity via coatings. For example, nanoparticles can be coated with electrostatically adsorbed poly(glutamate)-based peptide coatings to alter the external composition of the core particles. Compared to particles coated with disordered sequences containing RDG instead of RGD, negatively charged poly(glutamate)-based peptides containing arginine-glycine-aspartic acid (RGD) ligands can promote in vitro gene delivery to endothelial cells. These peptides consist of three components: a negatively charged poly(glutamate) extension, a polyglycine linker, and a charge-changing terminal sequence that can alter the biophysical properties and tissue selectivity of the particles. The coating and the particles themselves are biodegradable via 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] The mammalian immune system uses T cells to target substances or cells with foreign antigens. When encountering solid tumors, T cells often fail to respond effectively. Even when T cells reach the tumor site, they face resistance from immunosuppressive factors that allow cancer cells to escape the immune system. CAR-T technology uses genetic engineering to reprogram naturally occurring circulating T cells by inserting chimeric antigen receptors (CARs) into T cells to generate highly specific CAR-T cells. The CARs specifically bind to antigens on the surface of the target tissue, guiding the engineered CAR-T cells to the target tissue. Therefore, CAR-T cells can specifically target tumor cells, making them significantly more effective than naturally occurring circulating T cells. CAR-T cells can also be engineered to target other tissues, such as inflamed joints and brain tissue.

[0173] The CAR of this invention comprises at least one antigen-specific targeting region (ASTR), an extracellular space domain (ESD), a transmembrane domain (TM), one or more co-stimulatory domains (CSD), and an intracellular signal transduction domain (ISD), see Figure 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 the target antigen on a tumor or other targeted tissue, the ISD activates intracellular signal transduction in the CAR-T cells. For example, the ISD can redirect the specificity and responsiveness of CAR-T cells toward a selected target (e.g., tumor cells or other targeted cells) in a non-MHC-restricted manner, thereby utilizing the antigen-binding properties of the antibody. Non-MHC-restricted antigen recognition gives CAR-T cells the ability to recognize tumor cells and initiate antigen processing, thereby bypassing the main mechanisms by which tumors escape from the immune system's surveillance. In the embodiments, the ESD and / or CSD are optional. In another embodiment, ASTR has bispecificity, which allows it to bind specifically to two different antigens or epitopes.

[0174] The conditionally active peptides of this invention can be engineered into ASTRs or portions thereof, such that the CAR's binding activity to the target antigen is higher in a specific environment (e.g., the tumor microenvironment or synovial fluid) than in the blood or another part of the body in a different environment. These CARs can preferentially deliver T cells to disease sites, thereby significantly reducing the side effects of T cell attack on normal tissues. This allows for the use of higher doses of T cells to enhance therapeutic efficacy and improve individual tolerance to treatment.

[0175] These CARs are particularly valuable for developing novel therapeutic agents required for short or limited time periods in an individual. Examples of beneficial applications include high-dose systemic therapy and high-concentration localized therapy. See Maher, "Immunotherapy of Malignant Disease Using Chimeric Antigen Receptor Engrafted T Cells", ISRN Oncology, Vol. 2012, Article No. 278093, 2012.

[0176] ASTRs can contain conditionally active peptides, such as antibodies, especially single-chain antibodies or fragments thereof, that specifically bind to antigens on tumors or other targeted tissues. Some examples of peptides suitable for ASTRs include those linked to interleukins (which lead to recognition of cells with interleukin receptors), affinity molecules, ligand-binding domains from natural receptors, and soluble protein / peptide ligands of the receptor, for example, on tumor cells. In fact, almost any molecule that can bind to a given antigen with high affinity can be used in 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 the CAR, the ASTRs may be arranged in tandem and may be separated by linker peptides (Figure 5).

[0178] In another embodiment, the ASTR comprises a bivalent antibody. In the bivalent antibody, the scFv is generated using a linker peptide that is too short for the two variable regions to fold together, thereby driving scFv dimerization. Shorter linkers (one or two amino acids) result in the formation of a trimer, i.e., a so-called trivalent antibody or triple-chain antibody. Tetravalent antibodies can also be used in the ASTR.

[0179] The antigens targeted by CARs are present on the surface or inside cells of cells in tissues targeted for removal, such as tumors, glandular (e.g., prostate) hyperplasia, warts, and undesirable adipose tissue. While the ASTRs of CARs more effectively recognize and bind to surface antigens, CARs can also target intracellular antigens. In some embodiments, the target antigen is preferably specific to cancer, inflammatory diseases, neurological disorders, diabetes, cardiovascular diseases, or infectious diseases. Examples of target antigens include antigens of various immune cells, carcinomas, sarcomas, lymphomas, leukemias, germ cell tumors, blastomas, and antigens of cellular manifestations associated with various hematological disorders, autoimmune diseases, and / or inflammatory diseases.

[0180] ASTR can target one or more of the following cancer-specific antigens: 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 extradomain-B, folate receptor 1, GD2, GD3 ganglioside, glycoprotein 75, GPNMB, HER2 / neu, HGF, human discrete 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-hydroxyacetylneuraminic acid, NPC-1C, PDGF-Ra, PDL192, phosphatidylserine, prostate cancer cells, RANKL, RON, ROR1, SCH 900105, SDC1, SLAMF7, TAG-72, tendinin C, TGF β2, TGF-β, TRAIL-R1, TRAIL-R2, tumor antigen CTAA16.88, VEGF-A, VEGFR-1, VEGFR2, or vimentin.

[0181] ASTR can target antigens specific to inflammatory diseases, including one or more of the following: AOC3 (VAP-1), CAM-3001, CCL11 (eosin-1), CD125, CD147 (basigin), CD154 (CD40L), CD2, CD20, CD23 (IgE receptor), CD25 (IL-2 receptor chain), CD3, CD4, CD5, IFN-α, IFN-γ, 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, llamas, LFA-1 (CD11a), MEDI-528, myostatin, OX-40, rhuMAb β7, osteosclerosingin, SOST, TGF β1, TNF-α or VEGF-A.

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

[0183] The antigens specific to infectious diseases that can be targeted by the ASTR of this invention include one or more of the following: anthrax toxin, CCR5, CD4, agglutination 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 fusion virus, and TNF-α.

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

[0185] In another embodiment, the CAR can target and bind to cancer therapeutic cells (e.g., NK cells) to activate the antigens of the cancer therapeutic cells by acting as immune effector cells. One example is a CAR that targets the CD16A antigen to bind to NK cells for targeting CD30-expressing malignancies. Examples of bispecific, tetravalent AFM13 anti-antibody systems that can deliver antibodies with this effect are also provided. Further details of this type of embodiment can be found, for example, in the following literature: 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, June 25, 2015, Vol. 125, No. 26, pp. 4024-4031.

[0186] In some embodiments, the extracellular space domain and transmembrane domain may possess ubiquitination resistance, which can enhance CAR-T cell signaling and thereby increase antitumor 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 space domain is external to the CAR-T cell and is thus exposed to different conditions, potentially giving it conditional ubiquitination resistance. C. Engineering of masked conditionally active peptides

[0187] The conditional activity of conditionally active peptides, particularly conditionally active antibodies, can be masked, and / or the activity of their conjugates can be partially masked. The masked activity becomes available after the masked portion is removed from or cleaved from the conditionally active peptide. Suitable masking techniques are described, for example, in the following literature: Desnoyers et al., "Tumor-Specific Activation of an EGFR-Targeting Probody Enhances Therapeutic Index", Sci. Transl. Med. 5, 207ra144, 2013.

[0188] In some embodiments, a conditionally active antibody is linked to a masking portion, which masks the conditional activity and / or the activity of its conjugate. For example, when the conditionally active antibody is coupled to the masking portion, the coupling or modification may achieve a structural change that reduces or inhibits the ability of the conditionally active antibody to bind specifically to its antigen. Upon arrival at the target tissue or microenvironment, the masking portion is immediately cleaved by an enzyme present in the target tissue or microenvironment, thereby releasing the masked activity. For example, the enzyme may be a protease generally active in the tumor microenvironment, which can cleave the masking portion to release the conditionally active antibody active within the tumor tissue.

[0189] In some embodiments, the activity is masked to less than about 50%, or less than about 30%, or less than about 10%, or less than about 5%, or less than about 2%, or less than about 1%, or less than about 0.1%, or less than about 0.01% of the initial activity. In some embodiments, for example, to ensure sufficient delivery time, the masking effect is designed to last for 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 longer when measured in vivo or in an in vitro immunosorbent assay at the target displacement.

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

[0191] The masking portion can be provided in various forms. In some embodiments, the masking portion may be a known binding partner of the conditional active antibody, wherein the condition is that the masking portion binds to the conditional active antibody with an affinity and / or affinity lower than that of the target protein targeted by the conditional active antibody after the masking portion has cleaved, and also reduces interference with the masking portion that has the desired binding to the target. Therefore, the masking portion preferably masks the target binding of the conditional active antibody before the masking portion has cleaved, but does not substantially or significantly interfere with or compete with the binding of the binding active molecule to the target after the masking portion has cleaved the antibody. In a specific embodiment, the conditional active antibody and the masking portion do not contain the amino acid sequence of the natural binding partner pair, such that at least one of the conditional active antibody and the masking portion does not have the amino acid sequence of a member of the natural binding partner.

[0192] Alternatively, the masking portion may not specifically bind to the conditionally active antibody, but rather interfere with the binding of the conditionally active antibody to the target through non-specific interactions (e.g., steric hindrance). For example, the masking portion may be positioned such that the structure or configuration of the antibody allows the masking portion to mask the conditionally active antibody through, for example, charge-based interactions, thereby interfering with the target reaching the conditionally active antibody.

[0193] In some embodiments, the masking portion is covalently coupled to the conditional active antibody. In another embodiment, the conditional active antibody is prevented from binding to its target by binding the masking portion to the N-terminus of the conditional active antibody. In yet another embodiment, the conditional active antibody is prevented from coupling to the masking portion by a cysteine-cysteine ​​disulfide bridge between the masking portion and the conditional active antibody.

[0194] In some embodiments, the conditionally active antibody is further coupled to a cleavable moiety (CM). The CM can be cleaved by an enzyme, reduced by a reducing agent, or photolyzed. In one embodiment, the amino acid sequence of the CM may overlap with or be included within the masking moiety. In another embodiment, the CM is located between the conditionally active antibody and the masking moiety. It should be noted that all or part of the CM may promote masking of the conditionally active antibody prior to cleavage. Upon cleavage of the CM, the conditionally active antibody's activity to bind to its antigen becomes enhanced.

[0195] CM can be a receptor for an enzyme that co-localizes with the target antigen at an individual therapeutic site. Alternatively, CM can have cysteine-cysteine ​​disulfide bonds that can be cleaved by reduction of these disulfide bonds. CM can also be a photoinstantaneous receptor that can be activated by a light source.

[0196] Enzymes that cleave CMs should preferentially target tissues containing conditionally active antibodies, which are more active under conditions present in the target tissue (abnormal conditions), such as diseased or tumor tissues. For example, known proteases are present in increased amounts in various cancers (e.g., 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, lung, colorectal, prostate, head and neck, pancreas, etc.), rheumatoid arthritis, Crohn's disease, melanoma, SLE, cardiovascular injury, ischemia, etc. Therefore, suitable CMs can be selected that contain peptide receptors that can be cleaved by proteases present in tumor tissues, especially in tumor tissues at elevated levels compared to non-cancerous tissues.

[0197] In some embodiments, CM may be a receptor for an enzyme selected from: legumain, cytoplasm, TMPRSS-3 / 4, MMP-9, MTI-MMP, autolysin, caspase, human neutrophil elastase, β-secretase, uPA, and PSA. Compared to tissues at non-therapeutic sites (e.g., healthy tissues), enzymes that cleave CM are present in relatively high concentrations in target tissues at therapeutic sites (e.g., diseased or tumor tissues; for example, for therapeutic or diagnostic treatment). Therefore, in addition to the conditional activity of antibodies that may be higher at diseased or tumor tissues, enzymes present in diseased or tumor tissues can cleave CM, further enhancing the activity of conditionally active antibodies or conjugates. Unmodified or uncleaved CM can allow for effective inhibition or masking of the activity of conditionally active antibodies, resulting in lower activity of conditionally active antibodies in normal tissues (normal physiological conditions). The dual mechanism of inhibiting the activity of conditionally active antibodies in normal tissues (conditional activity and masking) allows for the use of significantly high doses of conditionally active antibodies without causing serious adverse effects.

[0198] In some embodiments, CM may be a substrate of an enzyme selected from the enzymes listed in Table 1 below. [surface] [1.] [Example enzyme] [ / ] [Protein] [ ] ADAM10 Caspase 8 Cell autolysin S MMP 8 ADAM12 Caspase 9 FAP MMP 9 ADAM 17 Caspase 10 Granulase B MMP-13 ADAMTS Caspase 11 Guanidinobenzoic acid enzyme (GB) MMP 14 ADAMTS5 Caspase 12 Serine transmembrane protease MT-SP1 BACE Caspase 13 Human neutrophil elastase (HNE) Enkephalinase caspase Caspase 14 Aspartame endopeptidase NS3 / 4A Caspase 1 Cell autolysins Protein lyase 2 Plasmin Caspase 2 Cell autolysin A Methyldopa (meprin) PSA Caspase 3 Cell autolysin B MMP 1 PSMA Caspase 4 Cell autolysin D MMP2 TACE Caspase 5 Cell autolysin E MMP 3 TMPRSS3 / 4 Caspase 6 Cell autolysin K MMP 7 uPA Caspase 7 MT1-MMP

[0199] Alternatively, CM may include the disulfide bond of cysteine, which can therefore be cleaved by reducing agents (such as cellular reducing agents), including glutathione (GSH), thioredoxin, NADPH, flavin, ascorbate, and the like, which can be present in large quantities in or around solid tumor tissues.

[0200] In some embodiments, the conditionally active antibody contains both a CM and a masking portion. The activity of the conditionally active antibody is exposed upon enzymatic cleavage of the CM. In some embodiments, it may be desirable to insert one or more linkers (e.g., flexible linkers) between the antibody, the masking portion, and the CM to provide flexibility. For example, the masking portion and / or the CM may not contain a sufficient number of residues (e.g., Gly, Ser, Asp, Asn, especially Gly and Ser, and specifically Gly) to provide the desired flexibility. Therefore, it may be advantageous to introduce one or more amino acids to provide flexible linkers. For example, a masked conditionally active antibody may have the following structure (where the following formula represents an amino acid sequence in 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) Ring [L1-(MM)-L2-(CM)-L3-(AB)] MM represents the masked portion and AB represents the conditionally active antibody; L1, L2 and L3 represent the same or different flexible linkers, each of which may or may not contain at least one flexible amino acid (e.g., Gly); and if a cyclic group is present, the entire structure is in a cyclic form due to the disulfide bond between the cysteine ​​pairs at or near the N-terminus and C-terminus of the structure.

[0201] The linkers suitable for use in this invention are generally linkers that provide flexibility to the shielding portion to facilitate the activity of antibodies that inhibit conditionally active antibodies. Such linkers are generally referred to as flexible linkers. Suitable linkers can be readily selected and can have any suitable length, for example, from 1 amino acid (e.g., Gly) to 20 amino acids, from 2 amino acids to 15 amino acids, from 3 amino acids to 12 amino acids, including from 4 amino acids to 10 amino acids, from 5 amino acids to 9 amino acids, from 6 amino acids to 8 amino acids, or from 7 amino acids to 8 amino acids, and can be 1, 2, 3, 4, 5, 6, or 7 amino acids.

[0202] Exemplary flexible connectors include glycine polymers (G)n, glycine-serine polymers (including, for example, (GS)n, (GSGGS)n, and (GGGS)n, where n is an integer of at least 1), glycine-alanine polymers, alanine-serine polymers, and other flexible connectors known in the art. Exemplary flexible connectors include, but are not limited to, Gly-Gly-Ser-Gly, Gly-Gly-Ser-Gly, Gly-Ser-Gly-Ser-Gly, Gly-Ser-Gly-Gly, Gly-Ser-Ser-Ser-Gly, and the like.

[0203] Some techniques for masking the activity of conditionally active antibodies are described in WO2010081173A2. [ ]

[0204] In some cases, conditionally active peptides are conditionally active antibodies, which can be engineered using the techniques described herein. Non-limiting examples of antibody engineering techniques include antibody conjugation, engineering of multispecific antibodies, engineering of bispecific conditionally active antibodies against immune effectors—cell surface antigens and target antigens—and engineering of the Fc region of antibodies.

[0205] Methods for conjugating conditionally active antibodies are described in WO 2015 / 175375. In one state, a conditionally active antibody may be conjugated to the Fc region of an antibody. The aforementioned conjugating molecules, compounds, or drugs may be conjugated to the Fc region, as described in U.S. Patent No. 8,362,210. For example, the Fc region may be conjugated to a cytokine or toxin intended for delivery to a site where the conditionally active antibody exhibits preferential activity. Methods for conjugating peptides to the Fc region of antibodies are known in the art. See, for example, U.S. Patents 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; EP 307,434; EP 367,166; EP 394,827; WO 91 / 06570, WO 96 / 04388, WO 96 / 22024, WO 97 / 34631, and WO 99 / 04813; Ashkenazi et al., Proc. Natl. Acad. Sci. USA. Volume 88, pp. 10535-10539, 1991; Traunecker et al., Nature, Volume 331, pp. 84-86, 1988; Zheng et al., J. Immunol., Volume 154, pp. 5590-5600, 1995; and ViI et al., Proc. Natl. Acad. Sci. USA, Volume 89, pp. 11337-11341, 1992.

[0206] In one state, a conditionally active antibody can be covalently attached to a coupling reagent via an intermediate linker having at least two reactive groups, one of which reacts with the conditionally active antibody and the other with the coupling reagent. The linker may include any compatible organic compound, which may be selected such that the reaction with the conditionally active antibody or the coupling reagent does not adversely affect the reactivity and / or selectivity of the conditionally active antibody. Furthermore, attachment of the linker to the coupling reagent may not destroy the activity of the coupling reagent.

[0207] Linkers suitable for oxidatively active antibodies include those containing groups selected from: primary amines, secondary amines, hydrazine, acetylhydrazine, hydroxylamine, phenylhydrazine, aminourea, and thiourea groups. Linkers suitable for reductively active antibodies include those having certain reactive groups that can react with the thiosulfate group in the reductively active antibody. These reactive groups include, but are not limited to: reactive haloalkyl groups (including, for example, acetyl haloalkyl), mercuric p-benzoate groups, and groups capable of Michael-type addition reactions (including, for example, maleimine and groups of the type described in Mitra and Lawton, J. Amer. Chem. Soc. Vol. 101, pp. 3097-3110, 1979).

[0208] Methods for engineering multispecific conditionally active antibodies have been described in WO 2015 / 175375. Conditionally active antibodies can be engineered to produce bispecific conditionally active antibodies against an immune effector—a cell surface antigen—and a target antigen. The bispecific conditionally active antibodies of this invention can attract immune effector cells to disease sites where the target antigen is present. The bispecific conditionally active antibody system can specifically bind to two different antigens: an immune effector—a cell surface antigen—and a target antigen. The bispecific antibody can be a full-length antibody comprising two arms, one arm binding to the immune effector—a cell surface antigen and the other arm binding to the target antigen. The bispecific antibody can be an antibody fragment comprising only a heavy chain variable domain (VH) and a light chain variable domain (VL). In one embodiment, the antibody fragment comprises at least two VHVL units: one binding to the immune effector—a cell surface antigen and the other arm binding to the target antigen. In another embodiment, the antibody fragment comprises at least two single variable domains (VH or VL): one binding to the immune effector—a cell surface antigen and the other arm binding to the target antigen. In some embodiments, a bispecific conditionally active antibody comprises two scFvs: one that binds to an immune effector—a cell surface antigen—and the other that binds to a target antigen.

[0209] The attraction of immune effector cells and their binding activity to target antigens on both immune effector cells and diseased cells or tissues allows immune effector cells to be drawn to diseased cells or tissues containing the target antigens. The attracted immune effector cells then attack the diseased cells or tissues, thereby aiding in the treatment of the disease, because immune effector cells can inhibit or even destroy diseased cells or tissues. For example, immune effector cells can destroy tumor cells or infected cells. Immune effector cells include natural killer cells (NK cells), macrophages, lymphotropic killer (LAK) cells, and T cells.

[0210] Bispecific conditional antibodies possess two binding activities, each relating to an immune effector—a cell surface antigen—and a target antigen. In one embodiment, both binding activities are conditional, meaning the binding activity of the bispecific conditional antibody to both the immune effector—cell surface antigen and target antigen is lower than that of the parent antibody under normal physiological conditions, but higher than that of the parent antibody under abnormal conditions. In another embodiment, only one of the two binding activities is conditional, meaning either the binding activity of the bispecific conditional antibody to the immune effector—cell surface antigen or the binding activity of the bispecific conditional antibody to the target antigen is conditional. In this case, either the binding activity of the bispecific conditional antibody to the immune effector—cell surface antigen or the binding activity of the bispecific conditional antibody to the target antigen is lower than the corresponding activity of the parent antibody under normal physiological conditions, but higher than the corresponding activity of the parent antibody under abnormal conditions.

[0211] The two arms of a bispecific conditionally active antibody (e.g., two VHVL units or two scFvs) can be conjugated using conventional methods. As is well known in the art, the smallest antibody fragment containing a complete antigen-binding site has a non-covalently associated dimer of a heavy chain variable domain and a light chain variable domain (VH and VL). This conformation corresponds to the conformation found in native antibodies, where three complementarity-determining regions (CDRs) of each variable domain interact to define the antigen-binding site on the surface of the VH-VL dimer. The six CDRs collectively confer antigen-binding specificity to the antibody. The framework (FR) flanking the CDRs has a substantially conserved tertiary structure in diverse species of native immunoglobulins, such as humans and mice. These FRs serve to maintain the CDRs in their proper orientation. Binding function does not require a constant domain, but it helps stabilize the VH-VL interaction. Even a single variable domain (or containing only half of the three CDRs specific to the antigen) can recognize and bind to the antigen, but its affinity is usually lower than that of the complete 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, pp. 1327-1337, 1972). Therefore, the binding site of a bispecific conditionally active antibody can be constructed as a VH-VL, VH-VH, or VL-VL domain pair of different immunoglobulins.

[0212] In some embodiments, the bispecific conditionally active antibody can be constructed as an adjacent polypeptide chain using recombinant DNA technology. For example, the construction method may be such that the nucleic acid molecule encoding the bispecific conditionally active antibody is expressed to construct an adjacent polypeptide chain (e.g., see 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, pp. 7021-7025, 2005). The order of the VH and VL domains within the polypeptide chain is irrelevant to this invention, as long as the arrangement of the VH and VL domains allows the antigen-binding site to fold appropriately to form a binding site for an immune effector—a cell surface antigen—and a binding site for a target antigen.

[0213] Some of the techniques described in this article for engineering multispecific conditional active antibodies can be used to generate bispecific conditional active antibodies against both immune effector cell surface antigens and target antigens.

[0214] Bispecific antibodies can be configured as a single polypeptide chain, as described in the following literature: 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. The preferred configuration for bispecific antibodies is a polypeptide construct in which the VH and VL regions are linked together by linker-domains. The order of the VH and VL regions in a single polypeptide chain is irrelevant. In one embodiment, the single polypeptide chain has a configuration of VH1-linker domain-VL1-linker domain-VH2-linker domain-VL2. In another embodiment, the single polypeptide chain has a configuration of VL1-linker domain-VH1-linker domain-VL2-linker domain-VH2. In yet another embodiment, the single polypeptide chain has a configuration of VH1-linker domain-VH2-linker domain-VL1-linker domain-VL2. In yet another embodiment, the single polypeptide chain has a configuration of VH1-linker domain-VL2-linker domain-VL1-linker domain-VH2. The single polypeptide chain can fold into two arms, each arm of which can bind to an immune effector cell surface antigen or a target antigen.

[0215] The linker domain in a bispecific conditionally active antibody is long enough to allow for intermolecular association between the VH and VL domains of the peptide fragment. Suitable linker designs for this purpose are described in the prior art, such as EP 623 679 B1, U.S. Patent No. 5,258,498, EP 573 551 B1, and U.S. Patent No. 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 linker of 15 amino acids of sequence (Gly4Ser)3.

[0216] Other linker domains include oligomeric domains. These oligomeric domains can facilitate the folding of two or more of their VH and VL domains into two arms, each of which can bind to immune effector cell surface antigens or target antigens. Non-limiting examples of oligomeric domains include leucine zippers (such as jun-fos, GCN4, E / EBP; 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), antibody-derived oligomeric domains (such as constant domains CH1 and CL) (Mueller, FEBS Letters 422 (1998), 259-264) and / or tetrameric domains (such as GCN4-LI) (Zerangue, Proc. Natl. Acad. Sci. 97 (2000)). 3591-3595).

[0217] In some embodiments, the knock-in-hole technique can be used to stabilize the folding of single-chain bispecific conditional antibodies. The knock-in-hole technique is described in Ridgway et al., "'Knobs-into-holes' engineering of antibody CH3 domains for heavy chain heterodimerization", Protein Eng. July 1996; 9(7):617-21. This method has been used to package amino acid side chains between adjacent α-helices, where the side chains of residues in the α-helix are represented as spacer knocks on the surface of a cylinder, alternating with holes, and the knocks adjacent to the α-helix can fit into these holes (O'Shea et al., (1991) Science, 254, 539-544).

[0218] Immune effectors—cell surface antigens—are specific to one or a class of immune effector cells. Surface antigens of various immune effector cells are known. Natural killer cells possess 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).

[0219] Macrophages possess 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).

[0220] Lymphokine-activated killer (LAK) cells possess surface antigens, including T3, T4, T11, T8, T1I, 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).

[0221] T cells, especially cytotoxic T cells, possess 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, Vol. 136, 1972, 207-215; Mingari et al., "Surface markers of human T cells...") lymphocytes", Ric Clin Lab. 1982 Jul-Sep;12(3):439-448).

[0222] Bispecific conditional active antibodies, upon binding to immune effector cells, can transport these cells to cells or tissues containing the target antigen (preferably on the surface). Once the bispecific conditional active antibody (and the immune effector cell) binds to the target antigen, the immune effector cell can attack the diseased cells or tissues. Immune effector cells (such as natural killer cells, macrophages, LAK cells, and T cells (cytotoxic)) can kill and / or destroy diseased cells or tissues, such as destroying tumor tissue.

[0223] Diseased cells or tissues can be selected from cancer, inflammatory diseases, neuronal disorders, diabetes, cardiovascular diseases, or infectious diseases. Examples of target antigens include antigens manifested as: various immune cells, carcinomas, sarcomas, lymphomas, leukemia, germ cell tumors, blastomas, and cells associated with various hematological diseases, autoimmune diseases, and / or inflammatory diseases.

[0224] Bispecific conditional active antibodies can target cancer-specific antigens including one or more of the following: 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, CNT0888, CTLA-4, DR5, EGFR, EpCAM, CD3, FAP, fibronectin extradomain-B, folate receptor 1, GD2, GD3 ganglioside, glycoprotein 75, GPNMB, HER2 / neu, HGF, human discrete 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-hydroxyacetylneuraminic acid, NPC-1C, PDGF-RA, PDL192, phosphatidylserine, prostate cancer cells, RANKL, RON, ROR1, SCH 900105, SDC1, SLAMF7, TAG-72, tendinin C, TGF β2, TGF-β, TRAIL-R1, TRAIL-R2, tumor antigen CTAA16.88, VEGF-A, VEGFR-1, VEGFR2, or vimentin.

[0225] The types of cancers for which the genetically engineered cytotoxic cells or pharmaceutical compositions of this invention are intended for treatment include carcinomas, blastomas, and sarcomas, as well as certain leukemias or lymphomas, benign and malignant tumors, and malignant diseases such as sarcomas, carcinomas, and melanomas. Cancers can be non-solid tumors (e.g., hematologic malignancies) or solid tumors. This also includes adult tumors / cancers and pediatric tumors / cancers.

[0226] Hematologic cancers are cancers of the blood or bone marrow. Examples of hematologic (or blood-borne) cancers include leukemia, including acute leukemia (e.g., acute lymphoblastic leukemia, acute myeloid leukemia, acute myeloid leukemia, and myeloblastic, promyelocytic, myelomonocytic, mononuclear, and erythrocytic leukemia), chronic leukemia (e.g., chronic myeloid (granulocytic) leukemia, chronic myeloid leukemia, and chronic lymphocytic leukemia); polycythemia vera, lymphoma, Hodgkin's disease, non-Hodgkin's lymphoma (indolent and advanced forms), multiple myeloma, Waldenstrom's macroglobulinemia, heavy chain disease, myelodyplasia syndrome, hairy cell leukemia, and myelodyplasia.

[0227] Solid tumors are abnormal tissue masses that typically do not contain cysts or fluid-filled areas. Solid tumors can be benign or malignant. Different types of solid tumors are named according to the type of cells that form them (e.g., sarcoma, carcinoma, and lymphoma). Examples of treatable solid tumors include sarcomas and carcinomas, including fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma, osteosarcoma and other sarcomas, synovoma, mesothelioma, Ewing's tumor, leiomyosarcoma, rhabdomyosarcoma, colon cancer, lymphoma, 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 carcinoma, papillary carcinoma, papillary adenocarcinoma, medullary carcinoma, bronchial carcinoma, renal cell carcinoma, hepatocellular carcinoma, cholangiocarcinoma, choriocarcinoma, and Wilms' tumor. Tumors, cervical cancer, testicular tumors, seminoma, bladder cancer, melanoma, and CNS tumors (such as gliomas (e.g., brainstem glioma and mixed glioma), glioblastoma (also known as multimorphic glioblastoma), astrocytoma, CNS lymphoma, germ cell tumor, medulloblastoma, schwannoma, craniopharyngioma, ependymoma, pineal tumor, hemangioblastoma, acoustic neuroma, oligodendroglioma, spinal cord meningioma, neuroblastoma, retinoblastoma, and brain metastases).

[0228] Bispecific conditional active antibodies can target inflammatory disease-specific antigens including one or more of the following: AOC3 (VAP-1), CAM-3001, CCL11 (eosinophil-1), CD125, CD147 (basal immunoglobulin), CD154 (CD40L), CD2, CD20, CD23 (IgE receptor), CD25 (IL-2 receptor chain), CD3, CD4, CD5, IFN-α, IFN-γ, 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, LFA-1 (CD11a), MEDI-528, myostatin, OX-40, rhuMAb β7, osteosclerosingin, SOST, TGF β1, TNF-α or VEGF-A.

[0229] The bispecific conditional active antibody of this invention can target antigens specific to target neuronal diseases, including one or more of the following: β-amyloid or MABT5102A. The bispecific conditional active antibody of this invention can target antigens specific to diabetes, including one or more of the following: L-1β or CD3. The bispecific conditional active antibody of this invention can target antigens specific to cardiovascular diseases, including one or more of the following: C5, cardiac myosin, CD41 (integrin α-lib), fibrin II, β-chain, ITGB2 (CD 18), and sphingosine-1-phosphate.

[0230] The bispecific conditional active antibody of this invention can target infectious disease-specific target antigens including one or more of the following: anthrax toxin, CCR5, CD4, agglutination 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 fusion virus, and TNF-α.

[0231] Other examples of target antigens include surface proteins found on cancer cells in a specific or amplified manner, such as the IL-14 receptor, CD19, CD20, and CD40 in B-cell lymphoma, Lewis Y and CEA antigens in various cancers, Tag72 antigen in breast and colorectal cancer, EGF-R in lung cancer, folate-binding proteins and HER-2 proteins commonly amplified in human breast and ovarian cancer, or viral proteins such as HIV gp120 and gp41 capsid proteins, capsid proteins from hepatitis B and C viruses, glycoprotein B and other capsid glycoproteins from human cytomegalovirus, and capsid proteins from oncogenic viruses (such as Kaposi's sarcoma-associated herpesvirus). Other potential target antigens include CD4, which contains the HIV gp120 capsid glycoprotein, and other viral receptors, such as ICAM (the human rhinovirus receptor) and related receptor molecules for poliovirus.

[0232] 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 initial co-receptor is CCR5, and later, during the virus's 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 strain that causes most transmission through sexual contact is called M-tonic viruses. These HIV-1 strains (also known as non-fusion-induced (NSI) primary viruses) can replicate in primary CD4+ T cells and macrophages, using the chemokine receptor CCR5 (and less frequently CCR3) as their co-receptor. T-tonic viruses (sometimes called fusion-induced (SI) primary viruses) can also replicate in primary CD4+ T cells, but can infect established CD4+ T cell lines in vitro via the chemokine receptor CXCR4 (fusionin). Many of these T-tonic strains can use CCR5 in addition to CXCR4, and some can enter macrophages via CCR5 under certain in vitro conditions (D'Souza, Nature Med. 2, 1293 (1996); Premack, Nature Med. 2, 1174; Fauci, Nature 384, 529 (1996)). Since M-tonic HIV-1 strains are involved in approximately 90% of HIV sexual transmission, CCR5 is the primary co-receptor for this virus in patients.

[0233] The number and properties of co-receptor molecules on target cells, and the ability of HIV-1 strains to enter cells via different co-receptors, appear to be key determinants of disease progression. High expression of CCR3 and CCR5 has also been observed in T cells and B cells derived from lymph nodes of patients with Hodgkin's disease. Type 1 diabetes is considered a T cell-mediated autoimmune disease. In relevant animal models, the expression of the CCR5 receptor in the pancreas has been associated with the progression of type 1 diabetes (Cameron (2000) J. Immunol. 165, 1102-1110). In one embodiment, a bispecific conditionally active antibody binds to CCR5 as a target antigen, which can be used to inhibit HIV infection in host cells and slow the progression of other diseases.

[0234] Several antibodies that specifically bind to (human) CCR5 are known in the art and include 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). Therefore, bispecific conditionally active antibodies preferably include, for example, the VL and VH domains (i.e., the Ig-derived second domain) of antibodies specific to CCR5, preferably human CCR5, and the VH and VL domains of antibodies specific to CD3 antigens on T cells.

[0235] In another embodiment, the present invention provides a bispecific conditionally active antibody against CD3 on T cells and CD19 as a target antigen. CD19 has been proven to be an extremely useful medical target. CD19 is expressed in all B cell lineages, from proto-B cells to mature B cells, and is consistently expressed in all lymphoma cells, and is absent in stem cells (Haagen, Clin Exp Immunol 90 (1992), 368-75; Uckun, Proc. Natl. Acad. Sci. USA 85 (1988), 8603-7). Combination therapy using an antibody against CD19 and another immunomodulatory antibody has been disclosed for the treatment of B-cell malignancies (WO 02 / 04021, US2002006404, US2002028178) and autoimmune diseases (WO 02 / 22212, US2002058029). WO 00 / 67795 discloses the use of antibodies against CD19 for the treatment of indolent and aggressive forms of B-cell lymphoma and acute and chronic forms of lymphocytic leukemia. WO 02 / 80987 discloses the therapeutic use of immunotoxins based on antibodies against the CD19 antigen 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 lymphoblastic leukemia (B-ALL), B-cell precursor acute lymphoblastic leukemia (pre-B-ALL), and B-cell chronic lymphocytic leukemia (B-CLL)).

[0236] In another embodiment, the present invention provides a bispecific conditionally active antibody against CD3 on T cells and CD20 as a target antigen. CD20 is a cell surface protein found on B lymphocytes. The CD20 antigen is found in normal and malignant pre-B and mature B lymphocytes, including in more than 90% of B-cell non-Hodgkin's lymphomas (NHL). This antigen is absent in hematopoietic stem cells, activated B lymphocytes (plasma cells), and normal tissues. Several antibodies primarily derived from rodents have been described: 1F5 (Press et al., 1987, Blood 69 / 2, 584-591), 2B8 / C2B8, 2H7, and 1H4 (Liu et al., 1987, J Immunol. 139, 3521-3526; Anderson et al., 1998, US Patent No. 5,736,137; Haisma et al., 1998, Blood 92, 184-190; Shan et al., 1999, J. Immunol. 162, 6589-6595).

[0237] CD20 has been described in immunotherapy strategies for treating plasma cell malignancies using vaccination with DNA encoding scFv linked to a carrier protein (Treon et al., 2000, Semin. Oncol. 27(5), 598), and immunotherapy using CD20 antibodies (IDEC-C2B8) has been shown to be effective in treating non-Hodgkin's B-cell lymphoma.

[0238] In some embodiments, the bispecific conditional active antibody system encodes a single polypeptide chain by a polynucleotide molecule. The polynucleotide can be, for example, DNA, cDNA, RNA, or synthetically produced DNA or RNA, or a chimeric nucleic acid molecule resulting from the recombination of any of these polynucleotides, individually or in combination. The polynucleotide can be a vector, such as part of an expression vector including plasmids, granules, viruses, and bacteriophages, or any expression system conventionally used for genetic engineering. The vector can contain other genes, such as marker genes, which allow selection of the vector within a suitable host cell and under suitable conditions.

[0239] In one state, the polynucleotide is operatively linked to an expression control sequence, allowing expression in prokaryotic or eukaryotic cells. Expression vectors derived from viruses (e.g., retroviruses, vaccinia virus, adeno-associated virus, herpesvirus, or bovine papillomavirus) can be used to deliver the polynucleotide or vector into mammalian cells. Vectors containing the polynucleotides of this invention can be transferred into host cells using well-known methods, depending on the type of cell host. For example, calcium chloride transfection is generally used for prokaryotic cells, while calcium phosphate treatment or electroporation can be used for other cell hosts.

[0240] In another state, conditionally active peptides can be engineered to produce bispecific conditionally active peptides. The method for engineering bispecific conditionally active peptides is similar to the method for engineering bispecific conditionally active antibodies as described in WO 2015 / 175375. For example, a bispecific conditionally active peptide may have two active sites, each possessing conditional activity, i.e., lower activity than the wild-type site under normal physiological conditions and higher activity than the wild-type site under abnormal conditions. These two conditionally active sites can evolve and be screened independently, and then a linker is used to connect the two active sites to the same bispecific conditionally active peptide. In one state, the linker used in a bispecific conditionally active antibody is adapted to generate a bispecific conditionally active peptide by connecting the two conditionally active sites in the conditionally active peptide.

[0241] Methods for engineering the Fc region of conditionally active antibodies are described in WO 2015 / 175375. Methods for engineering conditionally active viral particles are also described in WO 2015 / 175375.

[0242] In some cases, a conditionally active peptide can be inserted into a viral particle, which is an oncolytic virus, using the method described in WO 2015 / 175375. Oncolytic viruses are viruses that kill tumor cells upon contact. The conditionally active peptide inserted into the oncolytic virus exhibits higher activity in the tumor microenvironment but lower activity at other sites within the individual. For example, the conditionally active peptide is more active at the pH of the tumor microenvironment (e.g., pH 6.2–6.8) but less active at the pH of other sites within the individual (e.g., pH 7.2–7.6). Inserting the conditionally active peptide into the oncolytic virus allows the oncolytic virus to be delivered to the tumor, where it can target and kill tumor cells.

[0243] Oncolytic viruses of concern include adenoviruses; herpes simplex virus-1; vaccinia virus; parvoviruses; Rio virus; Newcastle disease virus; and similar viruses. Vaccinia virus is of particular concern.

[0244] In one sample, the oncolytic viruses were selected from the following groups: paramyxovirus, Rio virus, herpesvirus, adenovirus, and Semliki Forest virus. In another sample, the paramyxoviruses were selected from the following groups: Newcastle disease virus (NDV), measles virus, and mumps virus. In yet another sample, the NDV strains were selected from the group consisting of MTH68 / H, PV-701, and 73-T.

[0245] In another variant, oncolytic viruses are selected from herpesviruses, Rioviruses, E1B-deficient adenoviruses, vesicular stomatitis viruses, and poxviruses. These oncolytic viruses have the potential to not only destroy tumor cells but also release antigens from the destroyed tumor cells, thereby triggering an immune response.

[0246] Specific examples of oncolytic viruses include, but are not limited to, adenoviruses (e.g., Δ-24, Δ-24-RGD, ICOVIR-5, ICOVIR-7, Onyx-015, ColoAdl, H101, AD5 / 3-D24-GMCSF), Rio virus, herpes simplex virus (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, rod-shaped virus (e.g., vesicular stomatitis virus (VSV)), microRNA virus (e.g., Seneca Valley virus; SW-001), Coxsackie virus, and small viruses.

[0247] In one embodiment, the oncolytic virus is an adenovirus, including any of its 57 human serotypes (HAdV-1 to 57). In one embodiment, the adenovirus is the Ad5 serotype. In other embodiments, the adenovirus is a heterozygous serotype, which may or may not contain the Ad5 component. Suitable non-limiting examples of adenoviruses include Δ-24, Δ-24-RGD, ICOVIR-5, ICOVIR-7, ONYX-015, ColoAd1, H101, and AD5 / 3-D24-GMCSF. ONYX-015 is a heterozygous form of viral serotypes Ad2 and Ad5, with deletions in the E1B-55K and E3B regions to enhance cancer selectivity. HI 01 is a modified form of Onyx-015. ICOVIR-5 and ICOVIR-7 contain a deletion of the E1A Rb-binding site and replace the E1A promoter with an E2F promoter. Colo Ad 1 is a chimeric Addl lp / Ad3 serotype. AD5 / 3-D24-GMCSF (CGTG-102) is an adenovirus encoding GM-CSF with a modified capsid of serotype 5 / 3 (the Ad5 capsid protein clasp is replaced by a clasp domain derived from serotype 3).

[0248] In a preferred embodiment, the oncolytic virus is Δ-24 or Δ-24-RGD adenovirus. Δ-24 is described in U.S. Patent Application Publication Nos. 2003 / 0138405 and 2006 / 0147420. Δ-24 adenovirus is derived from adenovirus type 5 (Ad-5) and contains a 24-base pair deletion in the CR2 portion of the E1A gene. Δ-24-RGD further comprises the insertion of the RGD-4C sequence (which strongly binds to ανβ3 and ανβ5 integrin) into the HI loop of fibroblastin (Pasqualini R. et al., Nat. Biotechnol., 15:542-546, 1997).

[0249] Oncolytic adenoviruses can also be further modified to improve their ability to treat cancer. Such modifications to oncolytic adenoviruses have been described in Jiang et al. (Curr. Gene Ther., 2009 Oct 9(5):422-427), see also U.S. Patent Application No. 2006 / 0147420.

[0250] Oncolytic viruses with conditionally active polypeptides can be administered locally or systemically. For example, but not limited to, oncolytic viruses can be administered via the following routes: intravascular (intra-arterial or intravenous), intratumoral, intramuscular, intradermal, intraperitoneal, subcutaneous, oral, non-intestinal, intranasal, intratracheal, percutaneous, intraspinal, ocular, or intracranial.

[0251] Oncolytic viruses can be administered in a single dose or multiple doses. The virus can be administered in the following doses: at least 1 × 10⁵ plaque-forming units (PFU), at least 5 × 10⁵ PFU, at least 1 × 10⁶ PFU, at least 5 × 10⁶ or at least 5 × 10⁶ PFU, 1 × 10⁷, at least 1 × 10⁷ PFU, at least 1 × 10⁸ or at least 1 × 10⁸ PFU, at least 1 × 10⁸ PFU, at least 5 × 10⁸ PFU, at least 1 × 10⁹ or at least 1 × 10⁹ PFU, at least 5 × 10⁹ or at least 5 × 10⁹ PFU, at least 1 × 10¹⁰ PFU or at least 1 × 10¹⁰ PFU, at least 5 × 10¹⁰ or at least 5 × 10¹⁰ PFU, at least 1 × 10¹¹ PFU or at least 1 × 10¹¹ PFU, at least 1 × 10¹² PFU or at least 1 × 10¹³ PFU. For example, oncolytic viruses can be administered at doses between approximately 10⁷ and 10¹³ PFU, between approximately 10⁸ and 10¹³ PFU, between approximately 10⁹ and 10¹² PFU, or between approximately 10⁸ and 10¹² PFU.

[0252] In some cases, the cancers to be treated with oncolytic viruses include any solid tumors, such as lung, ovarian, breast, cervical, pancreatic, stomach, colon, skin, larynx, bladder, and prostate cancer. In another case, the cancer is a cancer of the central nervous system. Cancer can be a neuroepithelial tumor, such as astrocytoma (e.g., astrocytoma, poorly differentiated astrocytoma, glioblastoma, gliosarcoma, piloblastic astrocytoma, giant cell astrocytoma, pleomorphic xanthoma-type astrocytoma), oligodendritic glioma, ependymoma, oligoastrocytoma, cavernous mitochondria, astroblastoma, choroid plexus papilloma, choroid plexus carcinoma, gangliocytoma, ganglioglioma, neurocytoma, neuroepithelial tumor, neuroblastoma, pineal region tumor (e.g., pineal cell tumor, pineoblastoma, or mixed pineal cell tumor / pineoblastoma), medullary epithelioma, medulloblastoma, neuroblastoma or ganglioneuroblastoma, retinoblastoma or ependymoblastoma. Cancer can be a tumor of the central nervous system, such as a sellar region tumor (e.g., pituitary adenoma, pituitary carcinoma, or craniopharyngioma), a hematologic malignancy (e.g., primary malignant lymphoma, plasmacytoma, or granulosa sarcoma), a germ cell tumor (e.g., germ cell tumor, embryonal carcinoma, yolk sac tumor, choriocarcinoma, teratoma, or mixed germ cell tumor), a meningioma, a stromal tumor, a melanoma, or a tumor of a cranial or spinal nerve (e.g., schwannoma or neurofibroma). Cancer can be a low-grade glioma (e.g., ependymoma, astrocytoma, oligodendroglioma, or mixed glioma) or a high-grade (malignant) glioma (e.g., glioblastoma multiforme). Cancer can be a primary or metastatic brain tumor. D. Production of conditionally active polypeptides

[0253] A method for generating conditionally active peptides from parental peptides comprises the following steps: evolving the DNA encoding the parental peptide using one or more techniques selected from the following to increase the net charge of the parental peptide: 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, thereby generating mutant DNA; expressing the mutant DNA to generate the mutant peptide; and subjecting the mutant peptide and the parental peptide to screening analysis under a first condition (which may be a normal physiological condition) and a second condition (which may be an abnormal condition). The conditionally active peptide is selected from mutant peptides exhibiting both: (a) a decrease in activity in the first analysis at a first value of the condition compared to the same activity of the parental peptide in the first analysis, and (b) an increase in the same activity in the second analysis at a second value of the same condition compared to the same activity of the parental peptide in the second analysis. The decrease in activity of the conditionally active peptide under the first condition or normal physiological condition may be reversible or irreversible. i. Parental polypeptide

[0254] Parental peptides can be wild-type peptides, mutant peptides derived from wild-type peptides (e.g., therapeutic peptides), chimeric peptides derived from different wild-type peptides, or even synthetic peptides. Parental peptides can be selected from antibodies, enzymes, interleukins, regulatory proteins, hormones, receptors, ligands, biosimilars, immunomodulators, growth factors, stress proteins, fornix-related proteins, neuronal proteins, digestive tract proteins, growth factors, mitochondrial proteins, cytoplasmic proteins, animal proteins, structural proteins, plant proteins, and fragments of such peptides.

[0255] In some embodiments, the parental 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 parental polypeptide may be a polypeptide selected from mutant polypeptides generated by mutagenesis, wherein the polypeptide is selected to have desired properties, such as high binding activity, high expression level, or humanization. The selected polypeptide (in this case, not a wild-type polypeptide) may be used as the parental polypeptide to be evolved in the methods disclosed herein.

[0256] The description of suitable wild-type peptides and the ways in which they can be evolved and selected to produce conditionally active peptides is set forth in U.S. Patent No. 8,709,755 B2.

[0257] In some embodiments, the parental peptide may be selected from a library of wild-type peptides or mutant peptides, such as a phage display library. In these embodiments, a large number of candidate peptides are displayed in the phage library, particularly using surface display technology. Candidate peptides from the library are screened for suitable parental peptides. A typical phage library may contain phages that express thousands or even millions of candidate peptides in bacterial hosts. In one embodiment, the phage library may include a plurality of phages.

[0258] To construct a phage library, filamentous phages (e.g., filamentous E. coli phage M13) are typically genetically modified by inserting an oligonucleotide encoding a candidate peptide into the coding sequence of a phage coat protein. The phage coat protein is then expressed along with the candidate peptide, displaying the candidate peptide on the surface of the phage particle. The displayed candidate peptide is then screened for suitable parent peptides.

[0259] A common technique for screening suitable parental peptides involves immobilizing phage particles carrying the desired candidate peptide onto a carrier. The carrier can be a plastic plate coated with a "bait" that binds to the desired candidate peptide. Unbound phage particles can be washed away from the plate. The phage particles bound to the plate (carrying the desired candidate peptide) are dissolved by washing, and the dissolved phage particles are amplified in bacteria. Sequencing can then be used to determine the sequence encoding the candidate peptide in the selected phage particles. The relationship between the candidate peptide and the bait can be, for example, a ligand-receptor or antigen-antibody relationship.

[0260] Another commonly used technique for screening suitable parental peptides is to analyze the enzyme activity exhibited by candidate peptides using enzyme analysis of individual phage lines. Depending on the specific enzyme activity, those familiar with this technique can design appropriate analyses to screen parental peptides with the desired level of enzyme activity.

[0261] In some embodiments, the phage library is provided as an array, such that each pure phage line occupies a specific position on the array. This array can be provided on a solid carrier, such as a membrane, agar plate, or microtiter plate, onto which each pure phage line of the library is placed or adhered at a specific predetermined position on the solid carrier. In the case of an agar plate, such plates preferably include a bacterial growth medium to support bacterial growth. When the array is provided on a membrane (e.g., nitrocellulose or nylon membrane), a bacterial culture is applied to the membrane and the membrane is immersed in a nutrient growth medium. Alternatively, pure phage lines can also be provided on beads, in which case a single pure phage line can be adhered to a single bead. Alternatively, pure phage lines can each be provided on the end of an optical fiber, in which case the fiber optic communication is used to transmit ultraviolet radiation from a light source.

[0262] A typical phage library can contain 10⁶ to 10¹⁰ phages, each distinguished by its coat protein carrying different candidate polypeptides (e.g., gp3 or gp8 in the case of phage M13). Bacterial hosts used for phage libraries can be selected from a variety of genera, including, for example, *Salmonella*, *Staphylococcus*, *Streptococcus*, *Shigella*, *Listeria*, *Campylobacter*, *Klebsiella*, *Yersinia*, *Pseudomonas*, and *Escherichia*.

[0263] Oligonucleotides encoding candidate polypeptides can be sets of cDNAs encoding wild-type polypeptides. Methods for synthesizing cDNA from biological samples are known, in which suitable parental polypeptides can be expressed. Any genetic information expressing physiological activity via transcripts can be collected as cDNA. Synthesizing full-length cDNA is essential for cDNA production. Several methods exist for synthesizing full-length cDNA. For example, suitable methods include labeling the 5' cap site using cap-binding proteins from yeast or HeLa cells (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, pp. 3363-3371, 1995); and methods involving removing phosphate esters from incomplete cDNA lacking a 5' cap using alkaline phosphatase, followed by treating the entire cDNA with a tobacco mosaic virus decapping enzyme, so that only the full-length cDNA has phosphate esters (K. Maruyama et al., "Oligo-capping: a simple method to replace the cap structure of eukaryotic mRNAs with oligoribonucleotides," Gene, Vol. 138, pp. 171-174, 1995; and S. Kato et al., "Construction of a human full-length cDNA bank," Gene, Volume 150, pp. 243-250, 1995.

[0264] In embodiments of parental polypeptide-based antibodies, the candidate antibody library can be generated using recombinant antibodies with a complete antibody profile derived from an organism. Assembling genetic information representing the antibody profile into a large ensemble of complete antibodies allows for screening of suitable parental antibodies with desired antigen-binding activity and / or one or more other functional characteristics. In some embodiments, B cells are isolated from animals immunized with an antigen (e.g., immunized humans, mice, or rabbits). mRNA from the isolated B cells is collected and converted to cDNA, which is then sequenced. The most common cDNA fragments encoding the light chain and the most common cDNA fragments encoding the heavy chain are assembled into antibodies. In one embodiment, 100 most common cDNA fragments encoding the light chain and 100 most common cDNA fragments encoding the heavy chain are assembled to generate candidate antibodies. In another embodiment, the most common cDNA fragments encoding only the variable regions of the heavy chain and the variable regions of the light chain are assembled to generate antibody fragments containing only the variable regions and no constant regions.

[0265] In some embodiments, a cDNA fragment encoding the variable region of the IgG heavy chain is assembled with the most common variable region of IgK or the IgK light chain. The assembled antibody contains the variable region of the IgG heavy chain and the variable region of the IgK light chain or IgK.

[0266] Subsequently, cDNA encoding the assembled antibody is preferably selected and expressed in a plate-based format. The binding activity of the expressed antibody can be analyzed using bead-based ELISA, and suitable parent antibodies can be selected based on this ELISA. The cDNA encoding the assembled antibody can also be expressed in a phage display library, and then screened for one or more desired parent antibodies using any of the techniques disclosed herein.

[0267] In embodiments of parental polypeptide-based antibodies, the parental antibody preferably possesses at least one specific characteristic that makes it easier to evolve the parental antibody into a conditionally active antibody. In some embodiments, the parental antibody may have similar binding activity and / or characteristics under both normal physiological and abnormal conditions. In these embodiments, the parental antibody system is selected based on the combination of the most similar binding activity and / or the most similar one or more characteristics under both normal physiological and abnormal conditions. For example, if the normal physiological and abnormal conditions are pH 7.4 and pH 6.0, respectively, then the parental antibody with the most similar binding activity at pH 7.4 and 6.0 can be selected compared to antibodies with less similar binding activity at pH 7.4 and 6.0. ii. Evolutionary parental peptides

[0268] In some states, one or more site-directed mutagenesis techniques are used to mutate the DNA encoding the parent polypeptide to introduce one or more charged amino acid residues into the parent polypeptide. In one state, the charged amino acid residues can be introduced at any position within the parent polypeptide. In another state, the charged amino acid residues are introduced into the active site of the parent polypeptide. The active site is a region within the parent polypeptide responsible for a specific property of the parent polypeptide. This property can be enzyme receptor specificity, enzyme product selectivity, protein secretion properties, ligand selectivity, ligand binding activity, protein stability, and antibody binding activity. Examples of active sites include enzyme catalytic sites, ligand or receptor binding sites, antibody complementarity-determining regions, and antigenic epitopes.

[0269] In one state, charged amino acid residues are introduced into two or more complementarity-determining regions of the parent antibody or its variable region. In another state, 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 state, charged amino acid residues are introduced into two or more regions of the parent polypeptide involved in catalytic activity (e.g., forming the catalytic pocket of an enzyme).

[0270] In another state, charged amino acid residues may be introduced into a region outside the active site of the parent polypeptide. For example, this region may be adjacent to, around, or even far from the active site. In one state, this region is a constant region of the antibody heavy or light chain. This region may also be a framework region of a variable region of the antibody heavy or light chain.

[0271] To introduce one or more charged amino acid residues into a parental polypeptide, the DNA encoding the parental polypeptide is mutated by either replacing a codon in the DNA with a codon of a charged amino acid or by inserting a codon of a charged amino acid into the DNA, thereby generating mutant DNA that can express the mutant polypeptide. The substitution or insertion of codons of charged amino acid residues can be accomplished using one or more site-directed mutagenesis techniques described herein.

[0272] The codons for the charged amino acid residues in the DNA encoding the parental polypeptide to be introduced are shown in Table 2. [surface] [2.] [Codons for charged amino acids] [ ] [Amino acids] [Three-letter symbol] [Single-letter symbols] [codon] Aspartic acid Asp D GAU, GAC glutamic acid Glu E GAA, GAG Arginine Arg R CGU, CGC, CGA, CGG, AGA, AGG lysine Lys K AAA, AAG histidine His H CAU, CAC

[0273] In a single-state sample, the mutant peptide has fewer than 6, 5, 4, 3, or 2 mutations compared to the parent peptide. Such a number of mutations is sufficient to produce the desired conditionally active peptide.

[0274] In some samples, one or more site-directed mutagenesis techniques are used to mutate the DNA encoding the parental polypeptide, resulting in the deletion of one or more codons of uncharged amino acid residues. The deletion of one or more uncharged amino acid residues in the parental polypeptide increases the net charge of the parental polypeptide.

[0275] In another approach, the parental polypeptide can be mutated by deleting regions containing little or no charged amino acid residues, thereby increasing the net charge of the mutant polypeptide relative to the parent polypeptide. For example, a full-length antibody may have a constant region containing little or no charged amino acid residues. Deleting this constant region and linking two antibody variable regions to form a single-chain antibody can also result in an increased net charge of the mutant single-chain antibody compared to the parent antibody. In one instance, a full-length antibody was converted into an IgG antibody and then into a single-chain antibody with an increased net charge.

[0276] In some samples, any site-directed mutagenesis technique can be used to mutate the DNA encoding the parental polypeptide, such as those described in U.S. Patents 6,391,548, 7,132,265, and 6,713,285. In some embodiments, the site-directed mutagenesis techniques that may be employed include oligonucleotide-mediated mutagenesis of the DNA encoding the parental polypeptide, PCR mutagenesis, and cassette mutagenesis.

[0277] Oligonucleotide-mediated mutagenesis is a suitable method for replacing or inserting one or more codons into DNA encoding a parental polypeptide. This technique has been previously described in Sambrook et al., *Molecular Cloning: A Laboratory Manual*, Cold Spring Harbor, 1989, 2nd edition, Chapter 15.51, "Oligonucleotide-mediated mutagenesis". In short, DNA is altered by hybridizing an oligonucleotide primer carrying the desired mutation with DNA that has been reduced to a single strand. After hybridization, the entire second complementary strand of the DNA is synthesized using DNA polymerase, which incorporates the oligonucleotide primer and produces a product containing the desired mutation.

[0278] Typically, oligonucleotides with a length of at least 25 nucleotides are used. Optimal oligonucleotides will have 12 to 15 nucleotides that are perfectly complementary to the template on either side of the mutation. This ensures that the oligonucleotide will hybridize correctly with single-stranded DNA. Oligonucleotides are easily synthesized using techniques known in the industry, such as those described in the following literature: Crea et al. (Proc. Natl. Acad. Sci. USA, 75: 5765, 1978).

[0279] In a single-stranded sample, DNA encoding the parental polypeptide is contained within a circular DNA molecule, such as a plastid or granule. Oligonucleotides are hybridized to the single-stranded circular DNA molecule under suitable hybridization conditions. DNA polymerase (typically a Klenow fragment of DNA polymerase I) is then added to synthesize the complementary strand of the single-stranded circular DNA molecule using the oligonucleotides as a synthetic primer. This forms a heteroduplex molecule, where one strand of the DNA is mutated and the other (the initial strand) is unmutated. This heteroduplex molecule is then transformed into a suitable host cell (typically a prokaryote, such as *E. coli* JM101). After cell growth, the cells are plated on agarose plates and screened using a 32-phosphate radiolabeled oligonucleotide primer to identify bacterial communities containing the mutated DNA. The mutated DNA is then removed and the cells are placed in a suitable expression vector for protein production.

[0280] This procedure can be modified to produce homologous duplex molecules in which both strands of the plastid contain mutations. The modification is as follows: Anneal the oligonucleotide primer to the single-stranded circular DNA molecule as described above. Combine a mixture of three deoxyribonucleotides (dATP, dGTP, and dTTP) with a modified thio-deoxycytosine called dCTP-(aS). Add this mixture to the single-stranded circular DNA molecule-oligonucleotide hybridization complex. After adding DNA polymerase to this mixture, a DNA strand identical to the single-stranded circular DNA molecule except for the mutation is produced. Additionally, this new DNA strand will contain dCTP-(aS) instead of dCTP, which prevents digestion by restriction endonucleases. After the heteroduplex is cleaved with an appropriate restriction enzyme, the single-stranded circular DNA molecule in the heteroduplex can be digested by ExoIII nuclease or another suitable nuclease, bypassing the region containing the desired mutagenic site. The reaction is then stopped to produce a partially single-stranded DNA molecule. 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 can then be transferred to a suitable host cell, such as *E. coli* JM101, as described above.

[0281] Introducing (inserting and / or substituting) two or more mutations into the DNA encoding the parental polypeptide can be accomplished in several ways. If the desired mutation sites are close together in the DNA molecule, they can be simultaneously mutated using a single oligonucleotide primer with all the desired mutations. However, if the desired mutation sites are some distance apart (e.g., more than about 25 nucleotides apart), it is more difficult to generate a single oligonucleotide primer with all the desired mutations. Therefore, one of two alternative methods can be used.

[0282] In an alternative approach, each mutation uses a separate oligonucleotide primer. The oligonucleotide primers are then simultaneously annealed to a single-stranded circular DNA molecule, and the second complementary strand of the synthesized DNA will contain all the desired mutations from the individual oligonucleotide primers.

[0283] Another alternative approach involves two or more rounds of site-directed mutagenesis to generate the desired DNA with all the desired mutations. The first round is used for a single mutation as described: a heteroduplex DNA molecule is generated using an oligonucleotide primer with the first desired mutation. The second round of mutagenesis uses the heteroduplex DNA molecule generated in the first round as a template, which already contains one mutation. A second oligonucleotide primer encoding another mutation is then annealed to this template to generate a second DNA molecule containing mutations from both the first and second rounds of mutagenesis. If desired, the second DNA molecule can be used as a template in a third round of mutagenesis, and so on.

[0284] Another suitable site-directed mutagenesis method is PCR mutagenesis, which can be used to perform insertion and / or substitution mutations in parental peptides. As a circular amplification technique, PCR has been widely used in site-directed mutagenesis, in which mutagenic primers are used to introduce desired mutations (see 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., Nature 344:793-794 (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)).

[0285] Specifically, a small amount of DNA encoding the parental polypeptide is used as the template DNA in PCR. The sequence of the mutagenic primer differs slightly from the corresponding region in the template DNA, used to generate a relatively large number of specific DNA fragments containing the desired mutation only at the location where the mutagenic primer differs from the template DNA. For introducing a mutation into circular DNA, one primer is designed to overlap with the mutation location and contain the mutation; the sequence of the other primer may be consistent with the sequence extension of the opposite strand of the circular DNA, but this sequence can be located anywhere along the circular DNA. However, the sequence of the second primer is preferably located within 200 nucleotides of the sequence of the first primer, so that the entire DNA amplification region bound to the final primer can be easily sequenced to confirm the mutation. PCR amplification using primer pairs yields DNA fragments with different mutation locations in the mutagenic primer.

[0286] Mutations at separate sites can be introduced simultaneously by performing a second round of PCR using a second mutagenic primer or a different mutagenic primer, and simultaneously ligating the two resulting PCR fragments into the vector in three (or more) parts.

[0287] In a specific example of PCR mutagenesis, template plasso DNA (1 μg) was linearized by digestion with a restriction endonuclease, which has a unique recognition site in the plasso DNA outside the region to be amplified. 100 ng of this material was added to a PCR mixture containing four deoxyribonucleotide triphosphates included in the GeneAmp® kit (from Perkin-Elmer Cetus, Norwalk, Conn. and Emeryville, Calif.) and 25 pmole of each oligonucleotide primer, for a final volume of 50 μl. The reaction mixture was covered with 35 μl of mineral oil. The reaction was denatured at 100°C for 5 minutes, briefly placed on ice, and then 1 μl of Thermus aquaticus (Taq) DNA polymerase (5 units / μl, from Perkin-Elmer Cetus, Norwalk, Conn. and Emeryville, Calif.) was added below the mineral oil layer. The reaction mixture was then inserted into a DNA thermal cycler (Perkin-Elmer Cetus) following the programmed sequence: 2 min. 55°C, 30 sec. 72°C, followed by up to 19 cycles: 30 sec. 94°C, 30 sec. 55°C, and 30 sec. 72°C. At the end of the procedure, the reaction vial was removed from the thermal cycler, and the aqueous phase was transferred to a new vial. Extraction was performed with phenol / chloroform (50:50:vol.), followed by ethanol precipitation, and DNA was recovered using a standard procedure. This material was then subjected to appropriate processing for insertion into a vector. [ ]

[0288] Cascade mutagenesis is another site-directed mutagenesis technique that can be used to introduce insertions and / or substitutions into DNA encoding parental polypeptides. This technique has been described by Wells et al. (Gene, 34:315, (1985)). A unique restriction endonuclease site must be present on each side of the mutation. If such restriction sites are absent, they can be generated by introducing them at the appropriate location in the DNA using the oligonucleotide-mediated mutagenesis technique described above. The DNA encoding the parental polypeptide is cleaved at these restriction sites. A single double-stranded DNA fragment with the same DNA sequence between the restriction sites but containing the desired mutation is synthesized using a standard chemical synthesis procedure. This synthetic DNA fragment containing the mutation is called a cassette. The cassette is designed with 3' and 5' ends compatible with the ends of restriction endonuclease-treated DNA, allowing it to be directly ligated to the restriction endonuclease-treated DNA. The ligated DNA now contains the desired mutation.

[0289] One or more site-directed mutagenesis techniques are used to mutate the DNA encoding a parental polypeptide to introduce codons for one or more charged amino acid residues. The resulting mutant DNA encodes a mutant polypeptide having charged amino acid residues corresponding to the introduced codons. Therefore, the mutant polypeptide may have an increased total number of charged amino acid residues compared to the parental polypeptide. iii. Expression of mutant peptides

[0290] After generating mutant DNA using one or more site-directed mutagenesis techniques, the mutant DNA is expressed to produce a mutant polypeptide. A suitable method for expressing mutant DNA to produce a mutant polypeptide is described in U.S. Patent No. 8,709,755 B2.

[0291] In a single-state sample, mutant DNA can be represented using peptide display methods for efficient screening of conditionally active peptides. These methods are further described in PCT Patent Publications Nos. WO 91 / 17271, WO 91 / 18980, WO 91 / 19818 and WO 93 / 08278.

[0292] WO 93 / 08278 describes a method for displaying recombinant DNA containing peptide ligands, comprising generating a library of fusion proteins, each consisting of a first polypeptide moiety containing a variable sequence, which can be used to efficiently bind to macromolecules, and a second polypeptide moiety binding to DNA (e.g., a DNA vector encoding the individual fusion protein). When transformed host cells are cultured under conditions that allow for the expression of the fusion protein, the fusion protein binds to the DNA vector encoding it. After host cell lysis, the fusion protein / vector DNA complex can be screened against immobilized macromolecules in the same manner as screening phage particles in a phage-based display system, wherein replication and sequencing of the DNA vector in the selected fusion protein / vector DNA complex serves as the basis for identifying the selected polypeptide sequence.

[0293] Peptide display methods include methods for displaying single-chain antibodies in vitro and in vivo, such as nascent scFvs on polyribosomes or scFvs displayed on bacteriophages, which enable large-scale screening of a wide variety of scFv libraries with variable region sequences and binding specificity.

[0294] Other methods for expressing mutant peptides utilize cell-free enzymatic mechanisms to achieve in vitro synthesis of the mutant peptides. These methods rely on in vitro translation and typically involve stabilized polyribosome complexes, as further elaborated in PCT patent publications WO 88 / 08453, WO 90 / 05785, WO 90 / 07003, WO 91 / 02076, WO 91 / 05058, and WO 92 / 02536.

[0295] Affinity enrichment methods allow for the screening of a large library of mutant peptides and the selection of mutant DNA encoding desired peptides. The mutant DNA can then be isolated and recombined to combinatorially reconstitute the amino acid sequences of the selected peptide (or a predetermined portion thereof) or single-chain antibody (or only its VH, VL, or CDR portions). Using these methods, mutant peptides or single-chain antibodies can be identified as having the desired binding affinity to molecules, and the recombining method can be used to rapidly converge to the desired high-affinity mutant peptide or scFv. iv. Screening of conditionally active peptides

[0296] Methods for screening mutant peptides for selection of conditionally active peptides have been described in U.S. Patent No. 8,709,755 B2, which are incorporated herein by reference. v. Analytical conditions for screening and selecting conditionally active peptides

[0297] The first and second conditions, or normal and abnormal conditions, used for analysis in the screening step can be implemented using conditions selected from: temperature, pH, osmotic pressure, osmolar concentration, oxidative pressure, electrolyte concentration, protein concentration, and combinations of two or more of these conditions. For example, the normal physiological condition for temperature can be the normal human body temperature of 37.0°C, while the abnormal temperature condition can be a temperature different from 37.0°C, such as the temperature in the tumor microenvironment, which can be 1-2°C higher than the normal physiological temperature. In another example, the normal physiological condition and the abnormal condition can also be a normal physiological pH in the range of 7.2-7.8 or 7.2-7.6 and an abnormal pH in the range of 5.5-7.2, 6-7, or 6.2-6.8, for example, present in the tumor microenvironment.

[0298] Analysis under both the first and second conditions, or under both normal and abnormal conditions, can be performed in an analytical medium. The analytical medium can be a solution containing, for example, buffers and other components. Common buffers that can be used in analytical media include citrate buffer (e.g., sodium citrate), phosphate buffer, bicarbonate buffer (e.g., Krebs buffer), phosphate-buffered saline (PBS) buffer, Hank's buffer, Tris buffer, HEPES buffer, etc. Other buffers known to those skilled in the art as suitable for these analyses may be used. These buffers can be used to mimic the characteristics or components of human or animal bodily fluids (e.g., plasma or lymph).

[0299] The analytical solution used in the methods of this invention may contain at least one component selected from: inorganic compounds, ions, and organic molecules, preferably components generally found in the body fluids of mammals (e.g., humans or animals). Examples of such components include nutrients and metabolites, as well as any other components found in body fluids. This invention covers components that may or may not be part of a buffer system. For example, the analytical solution may be a PBS buffer supplemented with bicarbonate ions, wherein the bicarbonate ions are not part of the PBS buffer. Alternatively, the bicarbonate ions may be a component of a Krebs buffer.

[0300] This component can exist at substantially the same concentration in two analytical solutions (for the first and second conditions), while the two analytical solutions differ in another state, such as pH, temperature, electrolyte concentration, or osmotic pressure. Therefore, this component is used as a constant rather than a difference between the first and second conditions, or between normal physiological conditions and abnormal conditions.

[0301] In some embodiments, the component is present in two analytical solutions at a concentration close to or the same as the normal physiological concentration of the component in mammals, particularly in humans.

[0302] The inorganic compounds or ions may be selected from one or more of the following: boric acid, calcium chloride, calcium nitrate, diammonium hydrogen phosphate, magnesium sulfate, ammonium dihydrogen phosphate, potassium dihydrogen phosphate, potassium chloride, potassium sulfate, copper sulfate, ferric sulfate, manganese sulfate, zinc sulfate, magnesium sulfate, calcium nitrate, chelates of calcium, copper, iron, manganese and zinc, ammonium molybdate, ammonium sulfate, calcium carbonate, magnesium phosphate, potassium bicarbonate, potassium nitrate, hydrochloric acid, carbon dioxide, sulfuric acid, phosphoric acid, carbonic acid, uric acid, hydrogen chloride, urea, phosphate ions, sulfate ions, chloride ions, magnesium ions, sodium ions, potassium ions, ammonium ions, iron ions, zinc ions and copper ions.

[0303] Examples of normal physiological concentrations of some inorganic compounds include: uric acid in the range of 2–7.0 mg / dL, calcium ions in the range of 8.2–11.6 mg / dL, chloride ions in the range of 355–381 mg / dL, iron ions in the range of 0.028–0.210 mg / dL, potassium ions in the range of 12.1–25.4 mg / dL, sodium ions in the range of 300–330 mg / dL, carbonic acid in the range of 15–30 mM, citrate ions in the range of approximately 80 μM, histidine ions in the range of 0.05–2.6 mM, histamine in the range of 0.3–1 μM, HAPT (hydrogenated adenosine triphosphate) ions in the range of 1–20 μM, and HADP ions in the range of 1–20 μM.

[0304] In some embodiments, the ions present in the analytical solution under the first and second conditions or under both normal physiological and abnormal conditions are selected from hydroxide ions, halide ions (chlorine, bromine, iodine), halide oxide ions, sulfate ions, magnesium ions, calcium ions, hydrogen sulfate ions, carbonate ions, bicarbonate ions, sulfonate ions, halide oxide ions, nitrate ions, nitrite ions, phosphate ions, hydrogen phosphate ions, dihydrogen phosphate ions, persulfate ions, monopersulfate ions, borate ions, ammonium ions; or organic ions, such as carboxylate ions, phenolic ions, sulfonate ions (organic sulfate ions, such as methyl sulfate ions), vanadate ions, tungstate ions, borate ions, organic borate ions, citrate ions, oxalate ions, acetate ions, pentaborate ions, histidine ions, and phenolic ions.

[0305] The organic compounds present in the analytical solution under the first and second conditions or under both normal physiological and abnormal conditions may be selected from, for example, amino acids, such as histidine, alanine, isoleucine, arginine, leucine, aspartic acid, lysine, aspartic acid, methionine, cysteine, phenylalanine, glutamic acid, threonine, glutamine, tryptophan, glycine, valine, pyrrolidine, proline, selenocysteine, serine, tyrosine, and mixtures thereof.

[0306] Examples of normal physiological concentrations of some amino acids include: alanine 3.97 ± 0.70 mg / dL, arginine 2.34 ± 0.62 mg / dL, glutamic acid 3.41 ± 1.39 mg / dL, glutamine 5.78 ± 1.55 mg / dL, glycine 1.77 ± 0.26 mg / dL, histidine 1.42 ± 0.18 mg / dL, isoleucine 1.60 ± 0.31 mg / dL, leucine 1.91 ± 0.34 mg / dL, lysine 2.95 ± 0.42 mg / dL, methionine 0.85 ± 0.46 mg / dL, phenylalanine 1.38 ± 0.32 mg / dL, threonine 2.02 ± 6.45 mg / dL, and 1.08 ± 0.21 mg / dL. Tryptophan at 1 mg / dL, Tyrosine at 1.48±0.37 mg / dL, and Valine at 2.83±0.34 mg / dL.

[0307] Organic compounds present in analytical solutions under conditions one and two, or under normal physiological conditions and abnormal conditions, may be selected from non-protein nitrogenous 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 1.07 ± 0.76 mg / dL, creatinine 0.9 to 1.65 mg / dL, guanidinoacetic acid 0.26 ± 0.24 mg / dL, uric acid 4.0 ± 2.9 mg / dL, allantoin 0.3 to 0.6 mg / dL, adenosine 1.09 ± 0.385 mg / dL, urea 27.1 ± 4.5 mg / dL, and choline 0.3 to 1.5 mg / dL.

[0308] Organic compounds present in analytical solutions under both the first and second conditions or under both normal and abnormal conditions may be selected from organic acids, such as citric acid, α-ketoglutarate, succinic acid, malic acid, fumaric acid, acetic acid, β-hydroxybutyric acid, lactic acid, pyruvic acid, α-keto acid, acetic acid, and volatile fatty acids. Examples of normal physiological concentrations of these organic acids include: citric acid 2.5 ± 1.9 mg / dL, α-ketoglutarate 0.8 mg / dL, succinic acid 0.5 mg / dL, malic acid 0.46 ± 0.24 mg / dL, acetic acid 0.8 to 2.8 mg / dL, β-hydroxybutyric acid 0.5 ± 0.3 mg / dL, lactic acid 8 to 17 mg / dL, pyruvate 1.0 ± 0.77 mg / dL, α-keto acids 0.6 to 2.1 mg / dL, and volatile fatty acids 1.8 mg / dL.

[0309] Organic compounds present in analytical solutions under both the first and second conditions, or under both normal and abnormal 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 such sugars include: 83 ± 4 mg / dL glucose, 102 ± 73 mg / dL polysaccharides (calculated as hexoses), 77 ± 63 mg / dL glucosamine, 0.4 to 1.4 mg / dL hexuronates (calculated as glucuronic acid), and 2.55 ± 0.37 mg / dL pentoses.

[0310] Organic compounds present in analytical solutions under both the first and second conditions, or under both normal and abnormal conditions, may be selected from fats or their derivatives, such as cholesterol, lecithin, cephalin, sphingomyelin, and bile acids. Examples of normal physiological concentrations of some of these compounds include: 40 to 70 mg / dL of free cholesterol, 100 to 200 mg / dL of lecithin, 0 to 30 mg / dL of cephalin, 10 to 30 mg / dL of sphingomyelin, and 0.02 to 0.3 mg / dL of bile acids (calculated as bile acids).

[0311] The organic compounds present in the analytical solution under the first and second conditions, or under both normal and abnormal conditions, may be selected from proteins, such as fibrinogen, antihemophilic globulin, immunogamma globulin, immunoeuglobulin, homolectins, β-pseuglobulins, 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.

[0312] In some cases, the analytical solution may include blood proteins, such as those found in an individual's blood. Those skilled in this technique can determine the appropriate blood proteins for a specific individual from a blood sample. For example, appropriate human blood proteins may include albumin and heme.

[0313] Organic compounds present in analytical solutions under both the first and second conditions, or under both normal and abnormal conditions, may be selected from vitamins, such as vitamin A, carotene, vitamin E, ascorbic acid, thiamine, inositol, folic acid, biotin, pantothenic acid, and riboflavin. Examples of normal physiological concentrations of these vitamins include: vitamin A 0.019 to 0.036 mg / dL, vitamin E 0.90 to 1.59 mg / dL, inositol 0.42 to 0.76 mg / dL, folic acid 0.00162 to 0.00195 mg / dL, and biotin 0.00095 to 0.00166 mg / dL.

[0314] The concentrations of inorganic compounds, ions, or organic molecules in the analytical solution (for both analyses under first and second conditions, or under normal and abnormal conditions) can be within the normal physiological concentration range of the inorganic compounds, ions, or organic molecules in human or animal serum. However, concentrations outside the normal physiological range may also be used. For example, the normal range for magnesium ions in human serum is 1.7–2.2 mg / dL, and for calcium it is 8.5–10.2 mg / dL. The concentration of magnesium ions in the analytical solution can be from about 0.17 mg / dL to about 11 mg / dL. The concentration of calcium ions in the analytical solution can be from about 0.85 mg / dL to about 51 mg / dL. Generally, the concentration of inorganic compounds, ions, or organic molecules in analytical solutions can be as low as 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, or 80% of the normal physiological concentration of inorganic compounds, ions, or organic molecules in human serum, or as high as 1.5 times, 2 times, 3 times, 4 times, 5 times, 7 times, 9 times, 10 times, or even 20 times the normal physiological concentration of inorganic compounds, ions, or organic molecules in human serum. Different components of the analytical solution can be used relative to their respective normal physiological concentrations.

[0315] The activity of mutant peptides is measured using analyses performed under first and second conditions, or under normal physiological and abnormal conditions. During the analysis, both the mutant peptide and its binding partner are present in the analytical solution. The relationship between the mutant peptide and its binding partner can be, for example, antibody-antigen, ligand-receptor, enzyme-receptor, or hormone-receptor. For the mutant peptide to exhibit its activity, it should be able to contact and bind to its binding partner. The activity of the mutant peptide against its binding partner is then observed and measured after binding between the mutant peptide and its binding partner.

[0316] In some embodiments, the ions used in the analysis can act to form bridges between the screened mutant peptide and its binding partner, particularly those comprising charged amino acid residues. The ions can thus bind to both the mutant peptide and its binding partner via hydrogen bonds and / or ionic bonds. This can facilitate the binding between the mutant peptide and its binding partner by allowing the ions to reach sites that are difficult for larger molecules (the mutant peptide or its binding partner) to reach. In some cases, the presence of ions in the analytical solution can increase the probability of the mutant peptide and its binding partner binding to each other. Furthermore, ions can additionally or alternatively facilitate the binding between the mutant peptide and its binding partner by binding to larger molecules (the mutant peptide or its binding partner). This binding can alter the conformation of the larger molecule and / or cause the larger molecule to retain a specific conformation conducive to binding with its binding partner.

[0317] It has been observed that ions may facilitate the binding of mutant peptides to their binding partners by forming ionic bonds. Therefore, screening is significantly more efficient and can identify more hits (candidate conditionally active peptides) compared to the same analysis without using ions. Suitable ions can be selected from magnesium ions, sulfate ions, bisulfate ions, carbonate ions, citrate ions, HAPT ions, HADP ions, bicarbonate ions, nitrate ions, nitrite ions, phosphate ions, hydrogen phosphate ions, dihydrogen phosphate ions, persulfate ions, monopersulfate ions, borate ions, lactate ions, citrate ions, histamine ions, histamine ions, and ammonium ions.

[0318] It has been found that ions function to facilitate the binding of mutant peptides to their binding partners at pH values ​​close to the ion's pKa. The plasma is preferably relatively small relative to the size of the mutant peptide.

[0319] In one embodiment, when the abnormal condition is a pH different from the normal physiological pH under normal physiological conditions, ions suitable for increasing the hit count of candidate conditionally active peptides can be selected from ions whose pKa is close to the abnormal pH to be tested in the analysis. For example, the ion pKa may be up to 3 pH units away from the abnormal pH, up to 2 pH units away from the abnormal pH, up to 1 pH unit away from the abnormal pH, up to 0.8 pH units away from the abnormal pH, up to 0.6 pH units away from the abnormal pH, up to 0.5 pH units away from the abnormal pH, up to 0.4 pH units away from the abnormal pH, up to 0.3 pH units away from the abnormal pH, up to 0.2 pH units away from the abnormal pH, or up to 0.1 pH units away from the abnormal pH.

[0320] Examples of pKa values ​​for ions that can be used in this invention (whose pKa may vary slightly at different temperatures) are as follows: ammonium ion pKa is about 9.24, dihydrogen phosphate pKa is about 7.2, acetic acid pKa is about 4.76, histidine pKa is about 6.04, bicarbonate ion pKa is about 6.4, citrate pKa is 6.4, lactate ion pKa is about 3.86, histamine pKa is about 6.9, HATP pKa is 6.95 (HATP3- ⇌ ATP4- + H+) and HADP pKa is 6.88 (HADP3- ⇌ ADP4- + H+).

[0321] In one embodiment, conditionally active peptides are analyzed and selected in the presence of disulfide. The pKa of the disulfide is 7.05. In some embodiments, different concentrations of disulfide may be used in the analysis to represent normal and abnormal physiological conditions. Alternatively, the analytical media for normal and abnormal conditions may have approximately equal disulfide concentrations and certain differences in the values ​​of specific conditions; for example, the analysis may be performed at different pH values. The concentration of disulfide to be used in the analysis may be from 1 mM to 100 mM. Preferably, the analytical media has a disulfide concentration of 2 to 50 mM, or 3 to 35 mM, or 5 to 20 mM. Analyses performed in the presence of disulfide are known.

[0322] In some embodiments, after the pH of the abnormal condition (i.e., the abnormal pH) is known, the ions suitable for increasing the hit of candidate conditionally active peptides can be selected from ions with a pKa at or close to the abnormal pH. For example, the pKa of the candidate ions can be as far away from the abnormal pH as 3 pH units, 2 pH units, 1 pH unit, 0.8 pH units, 0.6 pH units, 0.5 pH units, 0.4 pH units, 0.3 pH units, 0.2 pH units, or 0.1 pH units.

[0323] As described above, ions are most effective at ion pKa values ​​or close to the ion's binding pair at pH values ​​close to or near the pKa. For example, it has been found that bicarbonate ions (pKa of approximately 6.4) are not very effective at facilitating the binding of mutant peptides to their binding pairs in analytical solutions at pH 7.2–7.6. As the pH of the analytical solution decreases to 6.7 and further to approximately 6.0, bicarbonate ions gradually become more effective in facilitating the binding of mutant peptides to their binding pairs. Therefore, more hits can be identified in analyses at pH 6.0 compared to analyses at pH 7.2–7.6. Similarly, histidine is not very effective at facilitating the binding of mutant peptides to their binding pairs at pH 7.4. As the pH of the analytical solution decreases to 6.7 and further to approximately 6.0, histidine gradually becomes more effective in facilitating the binding of mutant peptides to their binding pairs, allowing for more hits to be identified in pH ranges such as approximately 6.2–6.4.

[0324] This invention surprisingly reveals that, under different pH conditions (i.e., normal physiological pH) and abnormal conditions (i.e., abnormal pH), ions with pKa values ​​ranging from approximately the midpoint between normal and abnormal pH to approximately abnormal pH significantly aid in the binding between the screened mutant peptide and its binding partner. Therefore, screening analysis is significantly more effective in identifying more hit or candidate conditional peptides exhibiting high activity under abnormal conditions.

[0325] In some embodiments, the pKa can even be at least 1 pH unit away from the abnormal pH. When the abnormal pH is acidic, the pKa of a suitable ion can be in the range of (abnormal pH - 1) to the midpoint between the abnormal pH and the normal physiological pH. When the abnormal pH is alkaline, the pKa of a suitable ion can be in the range of (abnormal pH + 1) to the midpoint between the abnormal pH and the normal physiological pH. Ions can be selected from those described in this application. However, a variety of other ions not explicitly described in this application may also be used. It should be understood that, after selecting the abnormal pH and normal physiological pH for screening analysis, those skilled in the art can use the guiding principles of this invention to select any ion with a suitable pKa that is suitable for increasing screening efficiency to identify more hits that are highly active under abnormal conditions.

[0326] For example, when the abnormal pH for an exemplary screening is 8.4 and the normal physiological pH is 7.4, any ion with a pKa in the range of approximately 7.9 (midpoint) to 9.4 (i.e., 8.4+1) can be used in the screening. Some ions with pKa in this range include those derived from: tris(hydroxymethyl)methylglycine (pKa 8.05), hydrazine (pKa 8.1), dihydroxyethylglycine (pKa 8.26), N-(2-hydroxyethyl)hexahydropyrazine-N′-(4-butyric acid) (pKa 8.3), N-[hydroxymethyl]methyl-3-aminopropanesulfonic acid (pKa 8.4), and taurine (pKa 9.06). For another example, when the abnormal pH for an exemplary screening is 6 and the normal physiological pH is 7.4, any ion with a pKa in the range of approximately 5 (i.e., 6-1) to 6.7 (midpoint) can be used in the screening. Some ions with pKa values ​​in this range include those derived from: malate (pKa 5.13), pyridine (pKa 5.23), hexahydropyrazine (pKa 5.33), dimethylarsethanoate (pKa 6.27), succinate (pKa 5.64), 2-(N-morpholino)ethanesulfonic acid (pKa 6.10), citrate (pKa 6.4), histidine (pKa 6.04), and bistris (6.46). Those skilled in this technique will be able to consult numerous chemical handbooks and textbooks to identify known chemical compounds, including both inorganic and organic compounds, that can be converted into ions with pKa values ​​falling within these ranges. Among chemical compounds with suitable pKa values, those with smaller molecular weights are generally preferred.

[0327] Therefore, this invention unexpectedly reveals that the generation of conditionally active peptides for final identification depends not only on the generation of the correct peptide mutant from the wild-type peptide, but also on the use of ions with suitable pKa in the analytical solution. This invention encompasses efforts to find suitable ions (with appropriate pKa) for the analytical solution, in addition to generating a large library of mutant peptides (e.g., via CPE and CPS), as these ions facilitate efficient selection of highly active mutants from the large library. Furthermore, it encompasses that in the absence of suitable ions, screening is less efficient and the probability of finding highly active mutants decreases. Therefore, in the absence of suitable ions, multiple rounds of screening may be required to obtain the same number of highly active mutants.

[0328] Ions in an analytical solution can be formed in situ from the components of the analytical solution or are directly included in the analytical solution. For example, CO2 from the air can be dissolved in the analytical solution to provide carbonate and bicarbonate ions. As another example, sodium dihydrogen phosphate can be added to the analytical solution to provide dihydrogen phosphate ions.

[0329] The concentration of this component in the analytical solution (for both analyses under first or normal physiological conditions and analyses under second or abnormal conditions) may be the same as or substantially the same as the concentration of the same component commonly found in the natural bodily fluids of mammals (e.g., humans). In other embodiments, particularly where the component is an ion that can act to facilitate binding between the mutant peptide and its binding partner, the component concentration may be higher, as it has been observed that higher concentrations of this ion can form ionic bonds with the mutant peptide and its binding partner, effectively promoting binding and increasing the probability of discovering more hit or candidate conditionally active peptides.

[0330] In some embodiments, particularly when concentrations exceeding normal physiological levels are used, the concentration of ions in the analytical solution can be positively correlated with the probability of finding more hits using the analysis. For example, human serum has a bicarbonate ion concentration of approximately 15-30 mM. In one instance, as the concentration of bicarbonate ions in the analytical solution increased from 3 mM to 10 mM, to 20 mM, to 30 mM, to 50 mM, and to 100 mM, the number of hits in the analysis also increased with each increase in bicarbonate concentration. Therefore, the analytical solution may use bicarbonate concentrations in the following ranges: 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.

[0331] In another embodiment, the concentration of citrate in the analytical solution may be about 30 μM to about 120 μM, or about 40 μM to about 110 μM, or about 50 μM to about 110 μM, or about 60 μM to about 100 μM, or about 70 μM to about 90 μM, or about 80 μM.

[0332] In one embodiment, normal physiological conditions are defined as a normal physiological pH range of 7.2–7.6, and abnormal conditions are defined as abnormal pH ranges of 5.5–7.2, 6–7, or 6.2–6.8. Analytical solutions used for analysis under normal physiological conditions have a normal physiological pH and 50 mM bicarbonate ions. Analytical solutions used for analysis under abnormal conditions have an abnormal pH and 50 mM bicarbonate ions. Because the pKa of bicarbonate ions is approximately 6.4, bicarbonate ions can facilitate the binding between mutant peptides and their binding partners at abnormal pH ranges of 6.0–6.4 (e.g., pH 6.0 or 6.2).

[0333] In another embodiment, normal physiological conditions are defined as a normal physiological pH range of 7.2-7.6, and abnormal conditions are defined as abnormal pH ranges of 5.5-7.2, 6-7, or 6.2-6.8. Analytical solutions used for analysis under normal physiological conditions have a normal physiological pH and 80 μM citrate ions. Analytical solutions used for analysis under abnormal conditions have an abnormal pH and 80 μM citrate ions. Since the pKa of citrate ions is 6.4, citrate ions can effectively facilitate the binding between mutant peptides and their binding partners in analytical solutions with abnormal pH conditions of 6.0-6.4. Therefore, more candidate conditionally active peptides with higher binding activity at pH 6.0-6.4 and lower activity at pH 7.2-7.8 can be identified. Other ions (including acetate, histidine, bicarbonate, HATP, and HADP) function in a similar manner, enabling analytical solutions containing these ions to effectively screen mutant peptides that exhibit high binding activity at pH values ​​approximately equal to the ion's pKa and low binding activity at pH values ​​different from the ion's pKa (e.g., normal physiological pH).

[0334] In another embodiment, normal physiological conditions are defined as a normal physiological temperature of 37°C, and abnormal conditions are defined as an abnormal temperature of 38-39°C (temperatures found in some tumor microenvironments). The analytical solution used for analysis under normal physiological conditions has a normal physiological temperature and 20 mM bicarbonate ions. The analytical solution used for analysis under abnormal conditions has an abnormal temperature and 20 mM bicarbonate ions.

[0335] In another embodiment, normal physiological conditions refer to specific electrolyte concentrations in normal human serum, while abnormal conditions refer to different abnormal concentrations of the same electrolyte, which may exist in different locations in animals or humans or may originate from pathological conditions in animals or humans that alter the normal physiological concentrations of electrolytes in human serum.

[0336] The binding between mutant peptides and / or their binding partners can also be influenced in a variety of other ways. Typically, this influence is applied by including one or more additional components in the analytical solution. These additional components can be designed to interact with the mutant peptide, the binding partner, or both. Furthermore, these additional components can influence binding using combinations of two or more interactions and combinations of two or more types of interactions.

[0337] In one embodiment, the binding interaction of interest lies between the antibody and the antigen. In this embodiment, one or more additional components may be included in the analytical solution to influence the antibody, the antigen, or both. In this way, the desired binding interaction can be enhanced.

[0338] In addition to ions that can form ionic bonds with the mutant polypeptide and / or its binding partner to facilitate binding between the mutant polypeptide and its binding partner, the present invention also includes other components that can be used to facilitate binding between the mutant polypeptide and its binding partner. In one embodiment, molecules capable of forming hydrogen bonds with the mutant polypeptide and / or its binding partner are used. In another embodiment, molecules capable of hydrophobic interactions with the mutant polypeptide and / or its binding partner are used. In yet another embodiment, molecules capable of interacting with the mutant polypeptide and / or its binding partner, van der Waals, are included.

[0339] As used in this article, "hydrogen bond" refers to a relatively weak non-covalent interaction between a hydrogen atom covalently bonded to an electronegative atom (e.g., carbon, nitrogen, oxygen, sulfur, chlorine, or fluorine (hydrogen bond donor)) and a non-shared electron pair of an electron donor atom (e.g., nitrogen, oxygen, sulfur, chlorine, or fluorine (hydrogen bond acceptor)).

[0340] Components capable of forming hydrogen bonds with mutant peptides and / or their binding partners include organic molecules and inorganic molecules with polar bonds. Mutant peptides and / or their binding partners 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 glutamic acid (Gln), glutamic acid (Glu), arginine (Arg), aspartic acid (Asn), aspartic acid (Asp), lysine (Lys), histidine (His), serine (Ser), threonine (Thr), tyrosine (Tyr), cysteine ​​(Cys), methionine (Met), and tryptophan (Trp).

[0341] These amino acids can be used as both hydrogen donors and hydrogen acceptors. For example, the oxygen atom in the -OH group found in Ser, Thr, and Tyr; the oxygen atom in the -C=O group found in Glu and Asp; the sulfur atom in the -SH group or -SC- group found in Cys and Met; the nitrogen atom in the -NH3+ group found in Lys and Arg; and the nitrogen atom in the -NH- group found in Trp, His, and Arg can all be used as hydrogen acceptors. Similarly, groups that include hydrogen atoms in this list (e.g., -OH, -SH, NH3+, and -NH-) can be used as hydrogen donors.

[0342] 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-, such as in a peptide bond. The oxygen and nitrogen atoms in this structure can act as hydrogen acceptors, while the hydrogen atoms can participate in hydrogen bonding.

[0343] Inorganic compounds having at least one polar bond consisting of a hydrogen or oxygen atom that can be used for hydrogen bonding may include, for example, H₂O, NH₃, H₂O₂, hydrazine, carbonates, sulfates, and phosphates. Organic compounds include, for example, alcohols; phenols; thiols; aliphatic, amine, acetylamine; epoxides, carboxylic acids; ketones, aldehydes, ethers, esters, organochlorides, and organofluorines. Compounds capable of forming hydrogen bonds are well known in the chemical literature, for example, as discussed in, for example, "The Nature of the Chemical Bond," Linus Pauling, Cornell University Press, 1940, pp. 284–334.

[0344] In some embodiments, alcohols may include methanol, ethanol, propanol, isopropanol, butanol, pentanol, 1-hexanol, 2-octanol, 1-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-cresol, m-cresol, and p-cresol, thymol, α-naphthol and β-naphthol, gallol, guaiacol, and phloroglucinol. Suitable thiols include methanethiol, ethanethiol, 1-propanethiol, 2-propanethiol, butanethiol, tributylthiol, pentanethiol, hexanethiol, thiophene, dimercaptosuccinic acid, 2-mercaptoethanol, and 2-mercaptoindole. Suitable amines include methylamine, ethylamine, propylamine, isopropylamine, aniline, dimethylamine and methylethylamine, trimethylamine, aziridine, hexahydropyridine, N-methylhexahydropyridine, benzidine, cyclohexylamine, ethylenediamine, hexamethylenediamine, o-toluidine, m-toluidine and p-toluidine and N-phenylhexahydropyridine. Suitable acetamides include acetamide, N,N-dimethylacetamide, N,N-dimethylmethoxyacetamide, N,N-dimethylmethoxyacetamide and N-methyl-N-p-cyanoethylmethoxyacetamide. Epoxides may include ethylene oxide, propylene oxide, tert-butyl hydroperoxide, styrene oxide, glycidyl epoxide, cyclohexene oxide, di-tert-butyl peroxide, cumene hydroperoxide or ethylbenzene hydroperoxide, isobutylene oxide and 1,2-epoxyoctane. Carboxylic acids may 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-methylbenzoic acid, m-methylbenzoic acid, p-methylbenzoic acid, nicotinic acid, isonicotinic acid, and p-aminobenzoic acid. Ketones may include acetone, 3-propanone, butanone, pentanone, methyl ethyl ketone, diisobutyl ketone, ethyl butyl ketone, methyl isobutyl ketone, methyl tributyl ketone, cyclohexanone, acetone, methyl ethyl ketone, methyl propyl ketone, methyl butyl ketone, methyl pentanone, methyl hexyl ketone, diethyl ketone, ethyl butyl ketone, dipropyl ketone, diisobutyl ketone, diacetone alcohol, phorone, isophorone, cyclohexanone, methyl cyclohexanone, and acetophenone. Aldehydes may include formaldehyde, acetaldehyde, propionaldehyde, butyraldehyde, benzaldehyde, cinnamaldehyde, isobutyraldehyde, pentanal, octanal, benzaldehyde, cinnamaldehyde, cyclohexanone, salinomyl, and furfural. Esters include ethyl acetate, methyl acetate, ethyl formate, butyl acetate, ethyl lactate, ethyl butyrate, propyl acetate, ethyl formate, propyl formate, butyl formate, pentyl formate, methyl acetate, ethyl acetate, propyl acetate, butyl acetate, pentyl acetate, methyl isoamyl acetate, methoxybutyl acetate, hexyl acetate, cyclohexyl acetate, benzyl acetate, methyl propionate, ethyl propionate, butyl propionate, pentyl propionate, methyl butyrate, ethyl butyrate, butyl butyrate, pentyl butyrate, methyl acetate, and ethyl acetate. Ethers that can be used in this invention include dimethyl ether, methyl ethyl ether, diethyl ether, methyl propyl ether, and dimethoxyethane. Ethers can be cyclic, such as ethylene oxide, tetrahydrofuran, and dioxane.

[0345] Organochlorinated compounds include chloroform, pentachloroethane, dichloromethane, trichloromethane, carbon tetrachloride, tetrachloromethane, tetrachloroethane, pentachloroethane, trichloroethylene, tetrachloroethylene, and dichloroethylene. Organofluorinated compounds may include fluoromethane, difluoromethane, trifluoromethane, trifluoroethane, tetrafluoroethane, pentafluoroethane, difluoropropane, trifluoropropane, tetrafluoropropane, pentafluoropropane, hexafluoropropane, and heptafluoropropane.

[0346] Hydrogen bonds can be classified according to bond strength: strong, moderate, or weak hydrogen bonds (Jeffrey & George A.; An introduction to hydrogen bonding, Oxford University Press, 1997). Strong hydrogen bonds have a donor-acceptor distance of 2.2–2.5 Å and an energy range of 14–40 kcal / mol. Moderate hydrogen bonds have a donor-acceptor distance of 2.5–3.2 Å and an energy range of 4–15 kcal / mol. Weak hydrogen bonds have a donor-acceptor distance of 3.2–4.0 Å and an energy range of < 4 kcal / mol. Some examples of hydrogen bonds with multiple energy levels are: FH…:F (38.6 kcal / mol), OH…:N (6.9 kcal / mol), OH…:O (5.0 kcal / mol), NH…:N (3.1 kcal / mol), and NH…:O (1.9 kcal / mol). For further information, see the following references: Perrin et al., “Strong” hydrogen bonds in chemistry and biology, Annual Review of Physical Chemistry, Vol. 48, pp. 511-544, 1997; Guthrie, “Short strong hydrogen bonds: can they explain enzymic catalysis?” Chemistry & Biology, March 1996, 3:163-170.

[0347] In some embodiments, the components used in this invention can form strong hydrogen bonds with mutant polypeptides and / or their binding partners. These components often have atoms with strong anionic charge. The known sequence of atoms with the strongest anionic charge is F > O > Cl > N, in this order. Therefore, the present invention preferably uses organic compounds including fluorine, hydroxyl, or carbonyl groups for hydrogen bond formation. In one embodiment, an organofluorine compound can be used in this invention to form strong hydrogen bonds.

[0348] In another embodiment, a component capable of hydrophobically interacting with the mutant polypeptide and / or its binding partner is employed. Such components include organic compounds having hydrophobic groups.

[0349] As used herein, "hydrophobic interaction" refers to a reversible attractive interaction between a hydrophobic compound or a hydrophobic region of a compound and another hydrophobic compound or another hydrophobic region. This type of interaction has been described in "Hydrophobic Interactions" by A. Ben-Nairn (1980), Plenum Press, New York.

[0350] Hydrophobic materials are repelled by water molecules due to their nonpolar properties. When relatively nonpolar molecules or groups in an aqueous solution associate with other nonpolar molecules rather than with water, it is called a "hydrophobic interaction".

[0351] Mutant peptides and their binding partners typically comprise amino acids capable of hydrophobic interactions. These amino acids are typically characterized by having at least one side chain with a nonpolar group capable of hydrophobic interactions. 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).

[0352] Components capable of hydrophobic interactions 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, such hydrophobic components may be hydrocarbons selected from: aromatic hydrocarbons, substituted aromatic hydrocarbons, polyaromatic hydrocarbons, aromatic or non-aromatic heterocycles, cycloalkanes, alkanes, alkenes, and alkynes. Hydrophobic groups may include aromatic groups, alkyl groups, cycloalkyl groups, alkenyl groups, and alkyne groups. As used herein, the terms "alkyl," "alkenyl," and "alkynyl" refer to unsaturated aliphatic groups having 1 to 30 carbon atoms, including straight-chain alkenyl / alkynyl, branched alkenyl / alkynyl, cycloalkenyl (alicyclic) groups, alkyl-substituted cycloalkyl groups, and cycloalkyl-substituted alkenyl / alkynyl groups. These hydrocarbon moieties may also be substituted at one or more carbon atoms.

[0353] It is understood that the strength of hydrophobic interactions is based on the available amount of "hydrophobic" components that can interact with each other. Therefore, hydrophobic interactions can be modulated, for example, by increasing the amount and / or "hydrophobic" properties of the hydrophobic portion involved in the interaction within the molecule. For example, the hydrophobic portion in its initial form may include a hydrocarbon chain, which can be modified by attaching a hydrophobic side chain to a carbon atom of its carbon backbone to increase its hydrophobicity (increase the ability to strengthen the hydrophobic interaction involving that portion). In a preferred embodiment of the invention, this may include the addition of various polycyclic compounds, including, for example, various steroid compounds and / or their derivatives, such as steroidal compounds, more specifically cholesterol. Generally, the side chain can be linear, aromatic, aliphatic, cyclic, polycyclic, or any other type of hydrophobic side chain as contemplated by those skilled in the art.

[0354] The types of components that can interact with mutant polypeptides and / or their binding partners, van der Waals, are typically, but not always, compounds with polar portions. As used herein, "van der Waals interaction" refers to the attraction between atoms, parts, molecules, and surfaces, which arises from the correlation in dipole-dipole interactions and / or wave polarizations of neighboring atoms, parts, or molecules due to quantum dynamics.

[0355] The Van De Waal interaction of this invention refers to the attractive force between the mutant polypeptide or binding partner and the component. The Van De Waal interaction can arise from three sources. First, some molecules / parts, although electrically neutral, can still be permanent electric dipoles. Due to the fixed distortion in the electron charge distribution in the structure of some molecules / parts, one side of the molecule / part is always slightly positive and the opposite side is slightly negative. The tendency of these permanent dipoles to align with each other produces a net attractive force. This is the interaction between two permanent dipoles (Keesom force).

[0356] Second, the presence of a molecule with a permanent dipole can temporarily distort the electronic charge in other neighboring polar or nonpolar molecules, thereby inducing further polarization. Another attractive force originates from the interaction between a permanent dipole and an adjacent induced dipole. This interaction between a permanent dipole and its corresponding induced dipole can be called the Debye force. Third, even if the molecules involved are not permanent dipoles (e.g., the organic liquid benzene), there is an attractive force between molecules with two transient induced dipoles. This interaction between two transient induced dipoles can be called the London dispersion force.

[0357] The mutant peptide and / or its binding partner contain multiple amino acids capable of van der Waals interactions. These amino acids may have polar side chains, including glutamic acid (Gln), aspartic acid (Asn), histidine (His), serine (Ser), threonine (Thr), tyrosine (Tyr), cysteine ​​(Cys), methionine (Met), and tryptophan (Trp). These amino acids may also have side chains with nonpolar groups, including alanine (Ala), isoleucine (Ile), leucine (Leu), phenylalanine (Phe), valine (Val), proline (Pro), and glycine (Gly).

[0358] Components capable of interacting with mutant peptides and / or their binding partners, van der Waals, include polar or nonpolar inorganic compounds soluble in analytical solutions. The analytical solutions are typically aqueous solutions, and therefore these polar or nonpolar inorganic compounds are preferably soluble in water. Preferred materials for van der Waals interactions are polar materials, enabling dipole-dipole interactions. For example, AlF3 has a polar Al-F bond and is soluble in water (approximately 0.67 g / 100 ml water at 20°C). HgCl2 has a polar Hg-Cl bond and is soluble in water at 20°C at 7.4 g / 100 ml. PrCl2 has a polar Pr-Cl bond and is soluble in water at 20°C at approximately 1 g / 100 ml.

[0359] Suitable polar compounds for Nenvandewar interactions include alcohols, thiols, ketones, amines, amides, esters, ethers, and aldehydes. Suitable examples of these compounds have been described above with respect to hydrogen bonding. Suitable nonpolar compounds for Nenvandewar interactions include aromatic hydrocarbons, substituted aromatic hydrocarbons, polyaromatic hydrocarbons, aromatic or nonaromatic heterocycles, cycloalkanes, alkanes, alkenes, and alkynes.

[0360] Hydrogen-bonding components, hydrophobic components, and van der Waals components can be used to influence the binding of mutant peptides to their binding partners in a variety of ways. In one embodiment, hydrogen bonding, hydrophobic interactions, and / or van der Waals interactions can form a bridge between the mutant peptide and its binding partner. This bridge allows the mutant peptide and its binding partner to approach each other more closely, thereby promoting the binding and / or localization of the mutant peptide and / or its binding partner relative to each other in a binding-promoting manner.

[0361] In another embodiment, hydrogen bonding and / or hydrophobic interactions can increase the probability of a mutant polypeptide binding to its binding partner by, for example, causing the polypeptide and its binding partner to aggregate or associate with each other in a manner that increases the binding probability. Therefore, one or more of these interactions can be used alone or in combination to bring the binding sites closer together or to further distance the non-binding portions of the molecule from each other, thereby bringing the binding sites closer together, aggregating the mutant polypeptide and its binding partner more closely, or arranging the mutant polypeptide and its binding partner in a manner that promotes binding.

[0362] In another embodiment, hydrogen bonding and / or hydrophobic interactions can influence the conformation of the mutant peptide and / or its binding partner to provide a conformation more conducive to the binding of the mutant peptide to its binding partner. Specifically, binding to or interacting with one or more amino acids in the mutant peptide and / or its binding partner can cause one or more conformational changes in the mutant peptide or its binding partner that are favorable to the mutant peptide / binding partner binding reaction.

[0363] This invention employs two pairs of analyses. One analysis seeks to reduce the activity of the mutant peptide compared to the parent peptide derived under normal physiological conditions, and the second analysis seeks to increase the activity of the mutant peptide compared to the parent peptide derived under abnormal conditions. In some cases, the parent peptide from which the mutant peptide is derived is a wild-type peptide. In other cases, the parent peptide itself may be a mutant peptide, prepared using one or more mutation techniques described elsewhere herein.

[0364] The conditions used in the analytical pairs of this invention can be selected from temperature, pH, osmotic pressure, osmotic weight molar concentration, oxidative pressure, electrolyte concentration, and the concentration of any other component in the analytical solution or medium. Therefore, a specific component of the analytical medium can be used at substantially the same concentration in both analytical pairs. In this case, the component is typically present to simulate a specific environment in humans or animals, such as serum, tumor microenvironment, synovial fluid environment, neural environment, or any other environment encountered at the time of administration, experienced during administration, or encountered during treatment. An important aspect of selecting one or more components to simulate such environments is that it can improve the results of the selection process performed using the analytical pairs. For example, simulating a specific environment allows for the evaluation of various effects of a specific component of that environment on mutant peptides during the selection process. Components of the specific environment can, for example, alter or bind to the mutant peptide, inhibit the activity of the mutant peptide, or render the mutant peptide inactive.

[0365] In some embodiments, one or more components of the analytical solution are preferably small compounds, such as disulfides, bicarbonates, histidine, histamine, citrates, lactates, and acetates. In one embodiment, the small molecule component is preferably present in the analytical 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.

[0366] The concentration of a component in the analytical solution may be the same as or substantially the same as the concentration of the same component typically found in the natural body fluids of mammals (e.g., humans). This may be referred to as the normal physiological concentration of the component in the body fluid. In other embodiments, the concentration of a particular component in the analytical solution may be less than or greater than the concentration of the same component typically found in the natural body fluids of mammals (e.g., humans).

[0367] In another embodiment, the components can be present in significantly different concentrations in each pair of analyses. In this case, the presence, absence, or concentration of the component becomes a condition of analysis, since the component concentration is a distinguishing factor between analytical solutions analyzed under normal physiological conditions and analytical solutions analyzed under abnormal conditions. Therefore, conditionally active peptides produced by the embodiments of the method of the present invention can be selected for activity selection, at least partially depending on the component concentration.

[0368] In some embodiments, a component may be present in one pair of analytical solutions but completely absent in another pair. For example, the lactate concentration in the analytical solution under abnormal conditions may be set to a value simulating the lactate concentration in a tumor microenvironment. Lactate may be absent in the analytical solution pair under normal physiological conditions.

[0369] In one embodiment, normal physiological condition refers to a first lactate concentration under normal physiological conditions, and abnormal condition refers to a second lactate concentration under abnormal conditions present at a specific location in the body.

[0370] In another example, glucose may be absent in analytical solutions under anomalous conditions to simulate the absence of glucose found in the tumor microenvironment, while glucose may be set to a concentration simulating blood glucose levels in analytical solutions under normal physiological conditions. This feature can be used to preferentially deliver conditionally active peptides to a location or environment in a manner that is inactive or has minimal activity during transport, and to activate the conditionally active peptides upon arrival at an environment where the concentration of the component in the analytical solution under anomalous conditions is present.

[0371] For example, the tumor microenvironment usually has both lower glucose concentrations and higher lactate concentrations compared with human serum. The normal physiological concentration of glucose in serum is in the range of about 2.5 mM to about 10 mM. On the other hand, glucose concentrations in the tumor microenvironment are usually extremely low, in the range of 0.05 mM to 0.5 mM. In one embodiment, the glucose concentration of the analytical solution analyzed under normal physiological conditions is in the range of about 2.5 mM to about 10 mM; The resulting conditionally active polypeptides were more active in low glucose environments (in the tumor microenvironment) than in higher glucose environments (in normal tissues or blood). This conditionally active polypeptide will function in the tumor microenvironment but has low activity in blood flow transport.

[0372] The normal physiological concentration of lactate in serum was in the range of about 1 mM to about 2 mM. On the other hand, the concentration of lactate salts in the tumor microenvironment is usually in the range of 10 mM to 20 mM. In one embodiment, the lactate concentration of the analytical solution analyzed under normal physiological conditions is in the range of about 1 mM to about 2 mM; The resulting conditionally active polypeptides were more active in high lactate concentration environments (in the tumor microenvironment) than in lower lactate environments (in normal tissues or blood). This conditionally active polypeptide would thus function in the tumor microenvironment but with low activity in blood flow transport.

[0373] Similarly, muscle soreness is known to have a higher (abnormal) lactate concentration compared to normal. Therefore, in the search for mutant polypeptides that will be active in the muscle soreness environment, the analytical pairs under abnormal conditions can be implemented in the presence of higher concentrations of lactate to simulate the muscle soreness environment, while the analytical pairs under normal physiological conditions can be implemented at lower concentrations of lactate or in the absence of lactate. In this way, increased lactate concentrations targeting potentiating activity in muscle soreness environments can be used to select mutant polypeptides. This conditionally active polypeptide can, for example, be used as an anti-inflammatory agent.

[0374] In another embodiment, two or more components may be used in two pairs of analytical solutions. In this type of analysis, the characteristics of the two types of analyses described above can be used to select conditionally active peptides. Alternatively, two or more components may be used to increase the selectivity of conditionally active peptides. For example, returning to the tumor microenvironment, analyses under abnormal conditions can be performed in analytical media with both high lactate and low glucose concentrations, while corresponding analyses under normal physiological conditions can be performed in analytical media with both relatively low lactate and relatively high glucose concentrations.

[0375] This invention covers conditionally active polypeptides, each component selected from inorganic compounds, ions, and organic molecules, which can be used alone or in combination to select for higher activity at one component concentration than at different concentrations of the same component.

[0376] Analysis based on the different concentrations of one or more metabolites that are distinguishing conditions between normal environments (normal physiological conditions) and abnormal environments (abnormal conditions) is particularly suitable for selecting conditionally active peptides that are more active in the tumor microenvironment than in plasma, because the tumor microenvironment usually has a large number of metabolites with different concentrations compared to the same metabolites in plasma.

[0377] Kinoshita et al. ("Absolute Concentrations of Metabolites in Human Brain Tumors Using In Vitro Proton Magnetic Resonance Spectroscopy", NMR IN BIOMEDICINE, Vol. 10, pp. 2-12, 1997) compared metabolites in normal brain and brain tumors. This group found that the concentration of N-acetylaspartate in normal brain was 5000-6000 μM, but in glioblastoma it was only 300-400 μM, in astrocytoma it was 1500-2000 μM, and in poorly differentiated astrocytoma it was 600-1500 μM. Furthermore, the concentration of inositol in normal brain is 1500-2000 μM, but in glioblastoma it is 2500-4000 μM, in astrocytoma it is 2700-4500 μM, and in poorly differentiated astrocytoma it is 3800-5800 μM. The concentration of phosphatidylethanolamine in normal brain is 900-1200 μM, but in glioblastoma it is 2000-2800 μM, in astrocytoma it is 1170-1370 μM, and in poorly differentiated astrocytoma it is 1500-2500 μM. The concentration of glycine in the normal brain is 600-1100 μM, but it is 4500-5500 μM in glioblastoma, 750-1100 μM in astrocytoma, and 1900-3500 μM in poorly differentiated astrocytoma. The concentration of alanine in the normal brain is 700-1150 μM, but it is 2900-3600 μM in glioblastoma, 800-1200 μM in astrocytoma, and 300-700 μM in poorly differentiated astrocytoma. These metabolites can also have different concentrations in the blood. For example, the concentration of N-acetylaspartate in the blood is about 85,000 μM; the concentration of inositol in the blood is about 21,700 μM; the concentration of glycine in the blood is about 220-400 μM; and the concentration of alanine in the blood is about 220-300 μM.

[0378] Therefore, these metabolites (including at least N-acetylaspartate, inositol, glycine, and alanine) can be used in analytical solutions at different concentrations to select conditionally active peptides that are active in brain tumors but inactive in blood or normal brain tissue. For example, an analytical solution of N-acetylaspartate at a concentration of 85,000 μM can be used for analytical pairs under normal physiological conditions, and an analytical solution of N-acetylaspartate at a concentration of 350 μM can be used for analytical pairs under abnormal conditions to select conditionally active peptides that are active in the tumor microenvironment of glioblastoma but inactive or at least have low activity in blood or normal brain tissue.

[0379] 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) studied the concentrations of various metabolites, including branched-chain amino acids, in the pre-diagnostic plasma of pancreatic cancer patients. They found that several metabolites were present in the bloodstream of pancreatic cancer patients at concentrations different from those in the blood of individuals without pancreatic cancer. Mayers et al. also found that pancreatic cancer patients had significantly elevated levels of branched-chain amino acids in their plasma compared to healthy individuals. These elevated concentrations included isoleucine, leucine, and valine (Table 1, Mayers et al.). Figure 1 by Mayers shows other metabolites present in the plasma of pancreatic cancer patients at concentrations significantly different from those in healthy individuals. These metabolites include at least acetylglycine, glycine, phenylalanine, tyrosine, 2-aminoadipic acid ester, taurideoxycholate / tauridechenodeoxycholate, aconitate, isocitrate, lactate, α-glycerophosphate, and urate. Therefore, based on the finding that certain metabolites exist at different concentrations in the plasma of pancreatic cancer patients and healthy individuals, it can be predicted that the tumor microenvironment of pancreatic cancer will also have concentrations of these metabolites different from those present in the pancreatic microenvironment of healthy patients.

[0380] Therefore, in one embodiment, one or more of these metabolites can be used in an analytical solution under normal physiological conditions at concentrations close to those of the metabolites in the plasma of a healthy individual (i.e., normal physiological concentrations of the metabolites). For example, known normal physiological concentrations in the plasma of a healthy individual are about 1.60 ± 0.31 mg / dL isoleucine, about 1.91 ± 0.34 mg / dL leucine, and about 2.83 ± 0.34 mg / dL valine. The analytical solution under normal physiological conditions can have normal physiological concentrations of one or more of these branched-chain amino acids within these ranges. The analytical solution under abnormal conditions can have the same branched-chain amino acid at concentrations about 5 times, about 10 times, about 20 times, about 50 times, about 70 times, about 100 times, about 150 times, about 200 times, or about 500 times higher than the normal physiological concentrations of the corresponding branched-chain amino acids in a healthy individual. This reflects the fact that the pancreatic tumor microenvironment is expected to have significantly elevated concentrations of these branched-chain amino acids, as observed by Mayers et al., because the higher concentrations of these branched-chain amino acids found in plasma by Mayers et al. originated from the tumor microenvironment and were diluted in the bloodstream. Similarly, even when the concentrations of specific metabolites in cancer patients are significantly lower than in normal individuals, analysis under abnormal conditions can reflect the concentrations of other metabolites in the blood of pancreatic cancer patients. In this way, mutants that simulate the actual environment can be screened, thereby ensuring the selection of the most active mutants in that specific environment.

[0381] In some other embodiments, the analytical solution under normal physiological conditions may contain one or more branched-chain amino acids at concentrations that mimic those found in the plasma of pancreatic cancer patients, thus simulating the actual plasma environment of such patients. In these embodiments, the analytical solution under abnormal conditions may contain the same branched-chain amino acids at concentrations approximately 2, 3, 4, 5, 7, 8, 10, 15, 20, or 50 times higher than those found in the plasma of pancreatic cancer patients, reflecting the fact that these higher concentrations originate from the tumor microenvironment and that the concentrations in the bloodstream represent dilution of the actual concentrations in the tumor microenvironment. Similarly, other metabolites may also be present at different concentrations in the analytical solutions under normal and abnormal conditions, reflecting the expected actual differences in data collected from the bloodstream. In some cases, a deficiency of specific metabolites can be observed in the bloodstream of pancreatic cancer patients. In such cases, the concentrations measured in the bloodstream are suitable for analysis under normal physiological conditions, and even lower concentrations are suitable for analysis under abnormal conditions, suggesting that the metabolite may be being consumed in the tumor microenvironment. Conditionally active peptides selected using these analytical solutions will exhibit higher activity in the pancreatic cancer microenvironment than in the plasma of pancreatic cancer patients.

[0382] In some embodiments, whole plasma from pancreatic cancer patients can be used in this invention. For example, in one embodiment, a simulation of one or more components of pancreatic cancer patient plasma can be used in analytical solutions for one or both of the following analyses under normal physiological conditions and abnormal conditions. In an exemplary embodiment, the analytical solution under normal physiological conditions has a pH in the range of 7.2-7.6 and is supplemented with 30 wt.% pancreatic cancer patient plasma, and the analytical solution under abnormal conditions has a pH in the range of 6.2-6.8 and is supplemented with 30 wt.% pancreatic cancer patient plasma. In this embodiment, the presence of pancreatic cancer patient plasma achieves both of the following: (1) ensuring that the conditionally active peptide is not activated in blood at pH 7.2-7.6, and (2) also ensuring that even in the presence of this metabolite composition found in the blood of pancreatic cancer patients, the conditionally active peptide can be activated by the pH of the tumor microenvironment at 5.5-7.2, 6-7, or 6.2-6.8. This will adjust the treatment of pancreatic cancer patients.

[0383] In another exemplary embodiment, the analytical solution under normal physiological conditions has a pH in the range of 7.2-7.6 and is supplemented with 30 wt.% plasma from a pancreatic cancer patient, while the analytical solution under abnormal conditions has a pH in the range of 5.5-7.2 or 6.2-6.8 and is not supplemented with any plasma from a pancreatic cancer patient.

[0384] The same components selected from inorganic compounds, ions, and organic molecules can be used in each of the several types of analyses discussed above. For example, in the case of lactate, lactate can be used at substantially the same concentration in analytical solutions for both normal and abnormal conditions. Normal and abnormal conditions will then differ in one or more other states, such as temperature, pH, and the concentration of another component. In various embodiments, lactate can serve as a distinguishing factor between normal and abnormal conditions, reflecting the fact that lactate concentrations are higher in abnormal tumor microenvironments than in normal physiological conditions (non-tumor microenvironments).

[0385] In some embodiments, two or more components are added to two analytical solutions under normal physiological conditions and abnormal conditions at substantially the same concentration. For example, citrate and bovine serum albumin (BSA) are both added to the analytical solutions. In both analytical solutions, the citrate concentration may be about 80 μM and the BSA concentration may be about 10-20%. More specifically, the analytical solution for the analytical pair under normal physiological conditions may have a pH in the range of 7.2-7.6, and the citrate concentration is about 80 μM and the BSA concentration is about 10-20%. The analytical solution for the analytical pair under abnormal conditions may have a pH in the range of 6.2-6.8, and the citrate concentration is about 80 μM and the BSA concentration is about 10-20%.

[0386] In one embodiment, serum may be added to both the normal physiological condition and abnormal condition analytical solutions at substantially the same concentration. Because serum contains a large number of inorganic compounds, ions, and organic molecules (including polypeptides), the analytical solutions will contain a variety of components selected from inorganic compounds, ions, and organic molecules, present at substantially the same concentration in both analytical solutions. The analytical solutions may contain 5 to 30 vol.%, or 7 to 25 vol.%, or 10 to 20 vol.%, or 10 to 15 vol.% serum. In some other embodiments, both the normal physiological condition and abnormal condition analytical solutions do not contain serum. The serum may be human serum, bovine serum, or serum from any other mammal. In some other embodiments, the analytical solutions do not contain serum.

[0387] Analytical solutions under normal physiological conditions and abnormal conditions may have different pH values. The pH of these analytical solutions can be adjusted by using bicarbonate in the buffer solution to control the CO2 and O2 content.

[0388] In some other embodiments, at least one of two or more components is added to analytical solutions under normal physiological conditions and abnormal conditions at different concentrations. For example, both lactate and bovine serum albumin (BSA) are added to the analytical solutions. The lactate concentration may differ between the analytical solutions under normal physiological conditions and abnormal conditions, while the BSA concentration may be the same in both analytical solutions. The lactate concentration in the analytical solution under abnormal conditions may be in the range of 30 to 50 mg / dL, and the concentration in the analytical solution under normal physiological conditions may be in the range of 8 to 15 mg / dL. On the other hand, the BSA concentration is the same in both analytical solutions, for example, about 10 to 20%. Thus, conditionally active peptides selected using such analytical solutions exhibit higher activity at high lactate concentrations of 30 to 50 mg / dL than at low lactate concentrations of 8 to 15 mg / dL in the presence of BSA.

[0389] In some embodiments, the analytical solution may be designed to select conditionally active peptides whose activity depends on two or more conditions. In one exemplary embodiment, the activity of a conditionally active peptide may depend on both pH and lactate. The analytical solution used to select such a conditionally active peptide may be an analytical solution under normal physiological conditions, with a pH of 7.2-7.6 and a lactate concentration in the range of 8 to 15 mg / dL. An analytical solution under abnormal conditions may have a pH of 6.2-6.8 and a lactate concentration in the range of 30 to 50 mg / dL. Depending on the circumstances, both the analytical solutions under normal physiological conditions and those under abnormal conditions may also contain ions to facilitate the binding between the mutant peptide and its binding partner, thereby increasing the hit rate of candidate bioactive peptides.

[0390] In another exemplary embodiment, the activity of the conditionally active peptide may depend on pH, glucose, and lactate. The analytical solution used to select this conditionally active peptide may be an analytical solution under normal physiological conditions, with a pH of 7.2-7.6, a glucose concentration in the range of 2.5-10 mM, and a lactate concentration in the range of 8-15 mg / dL. An analytical solution under abnormal conditions may have 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. Depending on the circumstances, both the normal and abnormal condition analytical solutions may also contain ions to facilitate the binding between the mutant peptide and its binding partner, thereby increasing the number of candidate bioactive peptides that bind to the binding partner at pH 6.2-6.8. The conditionally active peptides selected using these analytical solutions exhibited higher activity at pH 6.2–6.8, 0.05–0.5 mM glucose concentration, and 30–50 mg / dL lactate concentration than at pH 7.2–7.6, 2.5–10 mM glucose concentration, and 8–15 mg / dL lactate concentration.

[0391] Two or more components selected from inorganic compounds, ions, and organic molecules are used to prepare an analytical solution that simulates anomalous conditions in the environment at the site / location (i.e., the target site) where the selected conditionally active polypeptide is to be delivered. In some embodiments, at least three components present in the environment of the target site may be added to the analytical solution, or at least four components present in the environment of the target site may be added to the analytical solution, or at least five components present in the environment of the target site may be added to the analytical solution, or at least six components present in the environment of the target site may be added to the analytical solution.

[0392] In one embodiment, fluid recovered from the target site (where the activity of the conditionally active peptide will be stronger) can be used directly as an analytical solution for analysis under abnormal conditions. For example, synovial fluid can be recovered from an individual, preferably from an individual with joint disease requiring treatment. The recovered synovial fluid, diluted as needed, can be used as an analytical solution for selecting the conditionally active peptide in analysis under abnormal conditions. By using the recovered synovial fluid, diluted as needed, as an analytical solution for analysis under abnormal conditions and for analysis using simulated human plasma under normal physiological conditions, the selected conditionally active peptide (e.g., TNF-α) will have higher activity in the joint than in other locations or organs. For example, an individual with an inflamed joint (e.g., arthritis) may be treated with TNF-α. However, TNF-α often has serious side effects that damage other tissues and organs. Conditionally active TNF-α, which is more active in synovial fluid but inactive or less active in the blood, delivers the activity of TNF-α to the joint while reducing or potentially eliminating the side effects of TNF-α on the rest of the body.

[0393] The development of conditionally active peptides with activity dependent on multiple conditions will improve the selectivity of conditionally active peptides for target sites in an individual. Ideally, conditionally active peptides are inactive or at least have significantly low activity at other sites where only some of these conditions are present. In one embodiment, a conditionally active peptide active at pH 6.2–6.8, 0.05–0.5 mM glucose concentration, and 30–50 mg / dL lactate concentration can be specifically delivered to the tumor microenvironment because these conditions are present in the tumor microenvironment. Other tissues or organs may contain one or two of these conditions, but not all three, and therefore the conditionally active peptide is not fully activated in other tissues or organs. For example, exercised muscle may have a low pH in the range of 6.2–6.8. However, it may not have the other analytical condition. Therefore, the conditionally active peptide is inactive or at least has low activity in exercised muscle.

[0394] In some embodiments, multiple steps may be performed to confirm that the activity of a conditionally active peptide is indeed dependent on the conditions used to select the conditionally active peptide. For example, the conditionally active peptide is selected based on three conditions: pH 6.2–6.8, a glucose concentration of 0.05–0.5 mM, and a lactate concentration of 30–50 mg / dL. The selected conditionally active peptide can then be tested individually under each of these three conditions and in an environment having paired conditions of these three conditions to confirm that the conditionally active peptide is inactive or has low activity under these test conditions or in such environments.

[0395] In some embodiments, certain components of serum are intentionally minimized or omitted from the analytical medium. For example, when screening antibodies, components in serum that bind to or adsorb antibodies may be minimized or omitted from the analytical medium. These bound antibodies can produce false positives, including bound mutant antibodies that are not conditionally active but bind only to components present in serum under various conditions. Therefore, by carefully selecting analytical components to minimize or omit components that may bind to mutants in the analysis, the number of non-functional mutants that might be inadvertently identified as positive for conditionally active mutants due to binding to components other than the desired binding pair in the analysis can be reduced. For example, in some embodiments screening mutant peptides that tend to bind to components in human serum, BSA may be used in the analytical solution to reduce or eliminate the possibility of false positives caused by mutant peptides that bind to components in human serum. Similar substitutions may be made in certain situations to achieve the same goal.

[0396] In some embodiments, the analytical conditions simulate the environment near the cell membrane (e.g., inside the cell membrane, at the cell membrane, or outside the cell membrane) or the environment within a joint. When screening in a cell membrane environment, several factors can affect binding activity, including receptor expression, internalization, antibody-drug complex (ADC) efficacy, etc.

[0397] The analytical methods can be any suitable analysis known to a person skilled in this technique. Examples include ELISA, enzyme activity analysis, in vitro screening of real tissues (organs, etc.), tissue slides, whole animals, cell lines, and the use of 3D systems. For example, suitable cell-based analyses are described in WO 2013 / 040445, tissue-based analyses in US 7,993,271, whole animal-based screening methods in US 2010 / 0263599, and 3D system-based screening methods in US 2011 / 0143960.

[0398] In some embodiments, the evolutionary step may produce mutant peptides that possess other desired properties in addition to the conditional activity characteristics discussed above. Suitable other desired properties that can be evolved may include binding activity, expression, humanization, etc. Therefore, the present invention can be used to produce conditionally active peptides with at least one or more of these other desired properties also modified. The selection step may be used to select both the conditional activity and one or more other desired properties of the mutant peptide.

[0399] In some embodiments, the present invention generates conditionally active peptides. The selected conditionally active peptide can be further mutated using, for example, a mutagenesis technique disclosed herein in a second evolution step to improve another property of the selected conditionally active peptide, such as binding activity, expression, humanization, etc. After the second evolution step, mutant peptides can be screened for both conditional activity and the improved property.

[0400] In some embodiments, after evolving the parent peptide to generate a mutant peptide, a first conditionally active peptide is selected that exhibits both of the following: (a) a decreased first activity in analysis under normal physiological conditions compared to the parent peptide, and (b) an increased first activity in analysis under abnormal conditions compared to the parent peptide. The first conditionally active peptide may then undergo one or more additional evolution, expression, and selection steps to select at least a second conditionally active peptide that (1) exhibits both of the following: (a) a decreased second activity in analysis under normal physiological conditions compared to the parent peptide, and (b) an increased second activity in analysis under abnormal conditions compared to the parent peptide, or (2) a larger ratio between the first activity under abnormal conditions and the first activity under normal physiological conditions compared to the first conditionally active peptide and / or the parent peptide. Note that both the first and second activities of the second conditionally active peptide may be higher under abnormal conditions compared to the parent peptide, and both the first and second activities may be lower under normal physiological conditions compared to the parent peptide.

[0401] In some cases, the expression steps are performed in a eukaryotic cell-generating host. The host can be 3T3 mouse fibroblast cells; BHK21 Syrian hamster fibroblast cells; MDCK canine epithelial cells; HeLa human epithelial cells; PtK1 kangaroo epithelial cells; SP2 / 0 mouse plasma cells; and NSO mouse plasma cells; HEK 293 human embryonic kidney cells; COS monkey kidney cells; CHO and CHO-S Chinese hamster ovary cells; R1 mouse embryonic cells; E14.1 mouse embryonic cells; H1 human embryonic cells; H9 human embryonic cells; PER C.6 human embryonic cells; brewer's yeast cells; and Pichia pastoris cells. The selected conditionally active peptide can then be produced in large quantities in the same eukaryotic cell-generating host. By using the same eukaryotic cell-generating host in both expression steps and for the final production, a higher degree of expression of the conditionally active peptide can be achieved.

[0402] In some embodiments, the present invention aims to produce conditionally active peptides that exhibit a higher 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 or selectivity of the activity under abnormal conditions (or the second condition) to the activity under normal physiological conditions (or the first condition) may be at least about 2:1, or at least about 3:1, or at least about 4:1, or at least about 5:1, or at least about 6:1, or at least about 7:1, or at least about 8:1, or at least about 9:1, or at least about 10:1, or at least about 11:1, or at least about 12:1, or at least about 13:1, or at least about 14:1, or at least about 15:1, or at least about 16:1, or at least about 17:1, or at least about 18:1, or at least about 19:1, or at least about 20:1, or at least about 30:1, or at least about 40:1, or at least about 50:1, or at least about 60:1, or at least about 70:1, or at least about 80:1, or at least about 90:1, or at least about 100:1.

[0403] In one embodiment, the conditionally active peptide is an antibody whose activity under abnormal conditions is in a ratio to its 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 peptide is used to target tumor sites, wherein the conditionally active peptide is active at the tumor site (in the tumor microenvironment) and has significantly lower or no activity at non-tumor sites (normal physiological conditions).

[0404] In one embodiment, the conditionally active polypeptide is an antibody intended to be conjugated to another agent (such as those disclosed elsewhere herein). The ratio of the activity of the conditionally active antibody under abnormal conditions to its activity under normal physiological conditions can be high. For example, the ratio of the activity of the conditionally active antibody to its activity under normal physiological conditions under abnormal conditions to that under abnormal conditions may be 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 can be particularly important when the conjugating agent is, for example, toxic or radioactive, as the conjugating agent can be expected to be concentrated at the disease or treatment site (where abnormal conditions exist). E. Production of conditionally active polypeptides

[0405] It can produce selected conditionally active peptides with reversible or irreversible activity for therapeutic, diagnostic, research and related purposes, and / or it can undergo one or more additional cycles of evolution and selection.

[0406] Conditionally active peptides can be produced by peptide expression cells to generate a host or organism. To make the production method more efficient, the DNA encoding the conditionally active peptide can undergo codon optimization for cell-to-host or organism production. Codon optimization has been previously described, 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. Dec. 2001, 69(12):7250-3, which describes codon optimization in the mouse system; Outchkourov et al., "Optimization of the expression of Equistatin in Pichia pastoris, protein expression and purification", Protein Expr. Purif. Feb. 2002; 24(1): 18-24, which describes codon optimization in the yeast system; Feng et al., "High level expression and mutagenesis of recombinant human phosphatidylcholine transfer protein using a synthetic gene: evidence for a C-terminal membrane binding domain", Biochemistry Dec. 19, 2000, 39(50): 15399-409, which describes codon optimization in Escherichia coli; Humphreys et al., "High-level periplasmic expression in Escherichia coli using a eukaryotic signal peptide: importance of codon usage at the 5' end of the coding sequence", Protein Expr. Purif. November 2000, 20(2):252-64, which describes how codon usage affects protein secretion in Escherichia coli.

[0407] The cell-generating host may be a mammalian system selected from one of the following groups: 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, MMI, 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 spleen cells and rabbit PBMCs. In one embodiment, the mammalian system is selected from the CHO or HEK293 cell line. In one particular embodiment, the mammalian system is the CHO-S cell line. In another embodiment, the mammalian system is the HEK293 cell line.

[0408] In some embodiments, the cells generate host yeast cell systems, such as brewer's yeast cells or Pichia pastoris cells. In some embodiments, the cells generate host prokaryotic cells, such as Escherichia 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 peptides can also be produced in plants (Firek et al., Plant Mol. Biol., Vol. 23, p. 861, 1993).

[0409] Conditionally active peptides can also be produced using well-known chemical methods. 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)", Nucleic Acids Res. Symp. Ser. 225-232, 1980; Banga, AK, 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 (e.g., see 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), and automated synthesis can be achieved, for example, using an ABI 43 IA peptide synthesizer (Perkin Elmer) according to the manufacturer's instructions.

[0410] Solid-phase chemical peptide synthesis methods have been known in the industry since the early 1960s (Merrifield, RB, "Solid-phase synthesis I. The synthesis of a tetrapeptide", J. Am. Chem. Soc, 85:2149-2154, 1963) (see also Stewart, JM and Young, JD, Solid Phase Peptide Synthesis, 2nd ed., Pierce Chemical Co., Rockford, 111, pp. 11-12)) and have recently been used in commercially available laboratory peptide design and synthesis kits (Cambridge Research Biochemicals). These commercially available laboratory kits typically 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 synthetic peptides at the tips of multiple "rods" or "pins", each attached to a single plate. In using this system, the plate of rods or pins is inverted and inserted into a second plate containing a solution for attaching or anchoring the appropriate amino acid to the tip of the rod or pin. By repeating this process—inverting the tips of the rods and pins and inserting them into the appropriate solution—the amino acid is constructed into the desired peptide. Alternatively, various readily available FMOC peptide synthesis systems can be used. For example, the assembly of peptides or fragments can be performed on a solid support using the Applied Biosystems, Inc. Model 431 A™ Automated Peptide Synthesizer. This device makes it easy to obtain the peptides of the present invention by direct synthesis or by synthesizing a series of fragments that can be coupled using other known techniques.

[0411] Conditionally active peptides can also be glycosylated. Glycosylation can be added post-translationally chemically or through cellular biosynthetic mechanisms, which involve the use of known glycosylation motifs, which may be native to the sequence, added as a peptide, or added to a nucleic acid coding sequence. Glycosylation can be O-linked or N-linked.

[0412] Conditionally active polypeptides include all forms of "mimics" and "peptide mimics". The terms "mimic" and "peptide mimic" refer to synthetic chemical compounds that have substantially the same structural and / or functional characteristics as the polypeptides of the present invention. Mimics may consist entirely of synthetic non-natural analogs of amino acids, or be chimeric molecules of some natural peptide amino acids and some non-natural analogs of amino acids. Mimics may also incorporate any amount of conservative substitution of natural amino acids, as long as such substitutions do not significantly alter the structure and / or activity of the mimic. For conserved variants of the polypeptides of the present invention, routine experiments will determine whether the mimic is within the scope of the present invention, i.e., whether its structure and / or function are not significantly altered.

[0413] The polypeptide mimicry compositions of the present invention may contain any combination of non-natural structural components. In alternative forms, the mimicry compositions of the present invention comprise one or all of the following three structural groups: a) residue-linking groups other than natural amide bonds ("peptide bonds"); b) non-natural residues replacing natural amino acid residues; or c) residues that induce secondary structure mimicry, i.e., residues that induce or stabilize secondary structures (e.g., β-turns, γ-turns, β-folds, α-helical configurations, and the like). For example, the polypeptide of the present invention may be characterized as a mimicry when all or some residues are chemically linked in a manner other than natural peptide bonds. Individual peptide mimicry residues may be linked by peptide bonds, other chemical bonds, or coupling methods (e.g., glutaraldehyde, N-hydroxysuccinimide, bifunctional maleimide, N,N'-dicyclohexylcarbodiimide (DCC), or N,N'-diisopropylcarbodiimide (DIC)). Alternative linking groups that can be used for traditional amide bonds (“peptide bonds”) include, for example, ketomethylene (e.g., -(C=O)CH2-, for -(C=O)-NH-), aminomethylene (CH2-NH), ethyl, olefin (CH=CH), ether (CH2-O), thioether (CH2-S), tetrazolium (CN4--), thiazole, resamide, thioamide, or ester (e.g., see Spatola (1983), Chemistry and Biochemistry of Amino Acids, Peptides and Proteins, Vol. 7, pp. 267-357, “Peptide Backbone Modifications”, Marcell Dekker, NY).

[0414] The polypeptides of this invention can also be characterized as mimics by containing all or some non-natural residues in place of natural amino acid residues. Non-natural residues have been well described in scientific and patent literature; a few exemplary non-natural compositions and guidelines that can be used as mimics of natural amino acid residues are described below. Aromatic amino acid mimics can be produced by substitution with, for example, the following: D- or L-naphthylalanine; D- or L-phenylglycine; D- or L-2-thienylalanine; D- or L-1-, 2-, 3-, or 4-pyreneylalanine; D- or L-3-thienylalanine; D- or L-(2-pyridyl)-alanine; D- or L-(3-pyridyl)-alanine; D- or L-(2-pyrazinyl)-alanine; D- or L-(4-isopropyl)- Phenylated glycine; D-(trifluoromethyl)-phenylglycine; D-(trifluoromethyl)-phenylalanine; D-p-fluoro-phenylalanine; D- or L-p-biphenylalanine; D- or L-p-methoxy-biphenylalanine; D- or L-2-indole(alkyl)alanine; and D- or L-alkylamines, wherein the alkyl group may be substituted or unsubstituted methyl, ethyl, propyl, hexyl, butyl, pentyl, isopropyl, isobutyl, diisobutyl, isopentyl; or non-acidic amino acids. Aromatic rings of non-natural amino acids include, for example, thiazolyl, phenylthio, pyrazolyl, benzimidazolyl, naphthyl, furanyl, pyrroleyl, and pyridyl aromatic rings.

[0415] Analogs of acidic amino acids can be generated by substitutions such as: noncarboxylic acid amino acids while maintaining a negative charge; (phosphono)alanine; sulfated threonine. Carboxyl side groups (e.g., aspartic acid or glutamine) can also be selectively modified by reaction with carbodiimides (R'~N—C--N--R') (e.g., 1-cyclohexyl-3(2-morpholino-(4-ethyl)carbodiimide or 1-ethyl-3(4-azacation-4,4-dimethylpentyl)carbodiimide). Aspartic acid or glutamine can also be converted to aspartic acid and glutamine residues by reaction with ammonium ions. Analogs of basic amino acids can be generated by substitutions such as: (except for ionized amino acids) (Except for arginine and guanine, citrulline, or (guanidino)-acetic acid or (guanidino)alkyl-acetic acid, wherein the alkyl group is defined above. Aspartic acid or glutamine can be substituted with nitrile derivatives (e.g., containing a CN- portion replacing COOH). The aspartic acid amide and glutamine amide residues can be deamined in response to the aspartic acid amide or glutamine amide residue. Arginine residue mimics can be obtained by preferably being in basic conditions. Under certain conditions, spermidine residues can be generated by reacting the spermidine residue with one or more conventional reagents, such as phenylglyoxal, 2,3-butanedione, 1,2-cyclohexanedione, or ninhydrin. Tyrosine residue mimics can be generated by reacting the spermidine residue with, for example, aromatic diazo compounds or tetranitromethane. N-acetylimazole and tetranitromethane can be used to form O-acetylityliidine and 3-nitro derivatives, respectively. Cysteine ​​residue mimics can be generated by reacting the cysteine ​​residue with, for example, α-haloacetates (e.g., 2-chloroacetic acid or chloroacetamide) and the corresponding amine to give carboxymethyl or carboxyacetylmethyl derivatives. Cysteine ​​residue mimics can also be generated by reacting the cysteine ​​residue with, for example, bromo-trifluoroacetone, α-bromo-β-(5-)-( Imidazolyl propionic acid; chloroacetyl phosphate, N-alkylmaleimine, 3-nitro-2-pyridyl disulfide; methyl-2-pyridyl disulfide; p-chloromercuryl benzoate; 2-chloromercuryl-4-nitrophenol; or chloro-7-nitrobenzo[-]oxa-1,3-diazole. Lysine mimics can be generated by reacting the lysine group with, for example, succinic acid or other carboxylic anhydrides (and the amino-terminal residue can be modified). Lysine and other mimics containing α-amino residues can also be generated by reacting with imine esters (e.g., methyl pyridinylimine, pyridoxal phosphate). Methionine analogs can be produced by reactions with pyridoxal, chlorine borohydride, trinitrobenzenesulfonic acid, O-methylisourea, 2,4-pentanedione, and by transaminase-catalyzed reactions with glyoxylates. Methionine analogs can be produced by reactions with, for example, methionine sulfoxide. Proline analogs include, for example, hexahydronicotinic acid, thiazolyl carboxylic acid, 3- or 4-hydroxyproline, dehydroproline, 3- or 4-methylproline, or 3,3-dimethylproline. Histidine residue analogs can be produced by reacting histidine with, for example, diethyl pyrocarbonate or p-bromobenzoylmethyl bromide.Other analogues include those produced by, for example, the following: hydroxylation of proline and lysine; phosphorylation of the hydroxyl groups of serine or threonine residues; methylation of the α-amino groups of lysine, arginine, and histidine; acetylation of the N-terminal amine; methylation of the main-chain amide residues or substitution with an N-methylamino acid; or acetylation of the C-terminal carboxyl group.

[0416] The residues of the polypeptides of the present invention (e.g., amino acids) can also be replaced by amino acids of opposite chirality (or peptide mimic residues). Therefore, any amino acid that is naturally present in the L-configuration (which may also be called R or S, depending on the structure of the chemical entity) can be replaced by an amino acid or peptide mimic with the same chemical structure type but with opposite chirality (called D-amino acid, but may also be called R- or S-form).

[0417] This invention also provides methods for modifying conditionally active peptides by natural methods (e.g., post-translational processing (e.g., phosphorylation, acetylation, etc.)) or by chemical modification techniques. Modification can be performed at any position in the peptide, including the peptide backbone, amino acid side chains, and amino or carboxyl terminals. It should be understood that the same type of modification, or variations thereof, can exist at several sites on a given peptide. A given peptide can also have multiple types of modifications. Modifications include acetylation, acetylation, polyethylene glycolation, ADP-ribosylation, acetylation, covalent attachment of flavin, covalent attachment of heme moieties, covalent attachment of nucleotides or nucleotide derivatives, covalent attachment of lipids or lipid derivatives, covalent attachment of phosphatidylinositol, cross-linking cyclization, disulfide bond formation, demethylation, formation of covalent cross-links, formation of cysteine, formation of pyroglutamic acid, methionization, γ-carboxylation, glycosylation, GPI anchoring, hydroxylation, iodination, methylation, cardamomylation, oxidation, polyethylene glycolation, proteolytic treatment, phosphorylation, isopreneation, racemization, selenoylation, sulfation, and transfer RNA-mediated addition of amino acids to proteins (e.g., arginylation). For example, see Creighton, TE, as—Structure and Molecular Properties, 2nd ed., WH Freeman and Company, New York (1993); Posttranslational Covalent Modification of Proteins, BC Johnson (ed.), Academic Press, New York, pp. 1-12 (1983). F. Pharmaceutical Compositions

[0418] The selected conditionally active peptides or products engineered from conditionally active peptides can be used in pharmaceutical compositions. In one e...

Claims

1. A method for preparing a conditionally active antibody or antibody fragment from a parent antibody, a single-chain antibody, or an antibody fragment, the method comprising the steps of: (i) derivatizing DNA encoding the parent antibody, single-chain antibody, or antigen-binding antibody fragment to increase the number of aspartic acid residues, glutamic acid residues, or both of the parent antibody, single-chain antibody, or antibody fragment to generate mutant DNA; (ii) expressing the mutant DNA to obtain mutant antibodies or antibody fragments; and (iii) selecting the conditionally active antibody or antibody fragment from the mutant antibodies or antibody fragments, the conditionally active antibody or antigen-binding antibody fragment reversibly exhibiting reduced antigen binding in a first analysis within a first pH range of 6.0 compared to antigen binding in a second analysis at a second pH of 7.4; wherein the activity pair at pH 6.0 is reduced in pH range. The ratio of identical activities under 7.4 is at least 1.3:1, and the first and second analyses are performed in an analytical medium containing a buffer and at least one substance selected from the following: histidine, histamine, hydrogenated adenosine diphosphate (HADP), hydrogenated adenosine triphosphate (HATP), citrate, acetate, lactate, sulfide / disulfide and bicarbonate.

2. The method of claim 1, wherein the derivation step employs mutagenesis selected from the group consisting of: oligonucleotide-mediated mutagenesis, PCR mutagenesis, and site-directed mutagenesis using cassette mutagenesis.

3. The method of claim 1, wherein the derivation step comprises introducing a codon of one or more acidic amino acids into the DNA encoding the parent antibody, single-chain antibody, or antibody fragment.

4. The method of request item 1, wherein the derivation step comprises codon substitution, codon insertion, or both.

5. The method of claim 3, wherein the one or more codons are introduced into the region of the DNA encoding the parent antibody, single-chain antibody or antibody fragment that encodes the active site of the parent antibody, single-chain antibody or antibody fragment.

6. The method of claim 3, wherein the one or more codons are introduced into a region of the DNA encoding the parental antibody, single-chain antibody, or antibody fragment, the region being outside the active site region encoding the parental antibody, single-chain antibody, or antibody fragment.

7. The method of claim 1, wherein the derivatization step comprises derivatizing the DNA to introduce a codon of one or more acidic amino acid residues into a region of the DNA encoding the complementarity-determining region of the parent antibody, single-chain antibody, or antibody fragment.

8. The method of claim 1, wherein the first and second analyses are performed in an analytical solution that further contains proteins found in blood.

9. The method of claim 8, wherein the protein found in the blood is albumin.

10. The method of claim 1, wherein the first and second analyses are performed in an analytical solution in the absence of serum.

11. The method of claim 1, wherein the first and second analyses comprise sulfides / disulfides or bicarbonates.

12. The method of claim 1, wherein the first and second analyses comprise at least one substance selected from those having a pKa between a first pH 7.4 and a second pH 6.0: histidine, citrate, histamine, HADP and HATP.

13. The method of claim 1, wherein the buffer used for the first and second analyses is selected from: citrate buffer, phosphate buffer, bicarbonate buffer or Hank's buffer.

14. The method of claim 1, wherein the first and second analyses are performed in an analytical medium comprising a group of substances selected from the group consisting of hydrogen sulfide, disulfides, bicarbonates and any combination thereof.

15. The method of claim 1, wherein the disulfide is present at a concentration of 2 to 50 mM, or the bicarbonate is present at a concentration of 3 to 200 mM.

16. The method of claim 1, wherein the selection step further comprises selecting the conditionally active antibody or antibody fragment based on a group of characteristics consisting of: affinity, expression level, humanization, or a combination thereof.

17. The method of claim 1, wherein the presentation step employs phage display or eukaryotic cell-generated host.

18. The method of claim 17, wherein the expression step is performed in a eukaryotic cell-generating host and the selected conditionally active antibody or antibody fragment is expressed in the same eukaryotic cell-generating host.

19. The method of claim 1, wherein the ratio of activity at pH 6.0 to the same activity at pH 7.4 is at least 1.7:

1.

20. The method of claim 1, wherein the ratio of activity at pH 6.0 to the same activity at pH 7.4 is at least 3.

0.

21. The method of claim 1, wherein the ratio of activity at pH 6.0 to the same activity at pH 7.4 is up to 4.

0.

22. The method of claim 1, wherein the ratio of activity at pH 6.0 to the same activity at pH 7.4 is at least 8.

0.

23. The method of claim 1, wherein the analysis is selected from enzyme-linked immunosorbent assay (ELISA) or fluorescence-activated cell sorting (FACS).

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