Conditionally active polypeptide

Abstract

To provide a conditionally active polypeptide having higher activity and / or selectivity in a specific environment and / or under a specific condition.SOLUTION: A non-naturally occurring polypeptide or an isolated polypeptide wherein the ratio of the activity of the assay at a first pH in presence of at least one species having a molecular weight of less than 900a. m.u. and a pKa that is a maximum of 4pH unit away from the first pH to the activity of the assay at a second pH in presence of the same at least one species is at least 1.3. The species has a pKa between the first pH and the second pH and may be a small molecule.SELECTED DRAWING: None

Description

[Technical Field]

[0001] The present disclosure relates to the field of providing improved polypeptides with desired activities. Specifically, the present disclosure relates to methods for generating conditionally active polypeptides from parent polypeptides, which are more active under certain conditions in the presence of particular compounds or ionic species than under other conditions. [Background technology]

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

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

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

[0005] Many strategies for protein evolution have been described. For example, U.S. Patent Application Publication No. 2005 / 0100985 discloses a rapid and easy method for generating a set of mutant polynucleotides from a parent template polynucleotide by replacing each original codon position in the template polynucleotide with a codon encoding one of the 20 naturally occurring amino acids. This method, simply called saturation mutagenesis, can be used in combination with other mutagenesis methods, such as introducing two or more related polynucleotides into a suitable host cell to generate hybrid polynucleotides by recombination and reductive reassortment.

[0006] Giver et al., "Directed evolution of a thermostable esterase," Proc. Natl. Acad. Sci. USA, vol. 95, pp. 12809-12813 (1998), used in vitro evolution to investigate the relationship between stability and activity in mesophilic esterases. Six generations of random mutagenesis, recombination, and screening significantly stabilized Bacillus subtilis p-nitrobenzyl esterase (a T increase of >14°C) without compromising its catalytic activity at low temperatures. This study found that mutations that increased thermostability while maintaining low-temperature activity were extremely rare. Whether these two properties were inversely correlated or not correlated at all, improvements in one property, resulting from the accumulation of amino acid substitutions, were typically obtained at the expense of the other.

[0007] To evolve a parent polypeptide that is inactive or virtually inactive (less than 50%, 30%, or 10% activity, and especially 1% activity) under its normal operating conditions but maintains equivalent or better activity under abnormal conditions, it may be necessary for destabilizing mutations to coexist with activity-increasing mutations that do not counteract the destabilizing effect. It is expected that destabilizing mutations will reduce the activity of the polypeptide beyond the effect predicted by standard rules such as Q10, thus creating a conditionally active polypeptide, for example, by allowing the evolution of a polypeptide that is less active or inactive under its normal operating conditions but functions efficiently under abnormal conditions.

[0008] Thus, the conditionally active polypeptide has increased activity compared to the parent protein under abnormal conditions and decreased activity compared to the parent protein under normal physiological conditions. Therefore, when used as a therapeutic protein, the conditionally active polypeptide preferably acts where abnormal conditions exist, such as the tumor microenvironment. As a result of this preferential action, the conditionally active polypeptide may be less likely to harm normal tissues / organs where normal physiological conditions exist, and thus less likely to cause side effects. This allows the conditionally active polypeptide to be used for longer treatment periods or at higher doses, leading to greater efficacy of the therapy.

[0009] WO 2010 / 104821 and WO 2011 / 009058 disclose methods for evolving and screening for conditionally active proteins. Summary of the Invention [Problem to be solved by the invention]

[0010] There remains a need for conditionally active polypeptides that are more active and / or selective in particular environments and / or under particular conditions. [Means for solving the problem]

[0011] In one embodiment, the disclosure relates to a non-naturally occurring or isolated polypeptide having a ratio of activity assayed at a first pH in the presence of at least one species having a molecular weight of less than 900 amu and a pKa at most 0.5, 1, 2 or 4 units away from the first pH to activity assayed at a second pH in the presence of the same at least one species of at least 1.3.

[0012] In one embodiment, the disclosure relates to a non-naturally occurring or isolated polypeptide having a ratio of assay activity at a first pH in the presence of at least one species having a molecular weight of less than 900 amu to assay activity at a second pH in the presence of the same at least one species, and wherein the species has a pKa between the first pH and the second pH, of at least 1.3.

[0013] In one embodiment, the present disclosure provides a compound selected from the group consisting of histidine, histamine, hydrogenated adenosine diphosphate, hydrogenated adenosine triphosphate, citric acid ion , bicarbonate ion , acetic acid ion , lactic acid ion , disulfide ion , hydrogen sulfide, ammonium ion , dihydrogen phosphate ion and any combination thereof, wherein the ratio of activity in an assay at a first pH in the presence of a species selected from the group consisting of:

[0014] The polypeptide of any of the foregoing embodiments may have a ratio of activity assayed at a first pH to activity assayed at a second pH of at least 1.5, or at least 1.7, or at least 2.0, or at least 3.0, or at least 4.0, or at least 6.0, or at least 8.0, or at least 10.0, or at least 20.0, or at least 40.0, or at least 60.0, or at least 100.0. The polypeptide of any of the foregoing embodiments may be assayed at a first pH that is an acidic pH and a second pH that is alkaline or neutral. The second pH may be a normal physiological pH that is within the normal range of physiological conditions at the site of administration of the polypeptide to a subject or in a tissue or organ at the site of action of the polypeptide in a subject, and the first pH may be an abnormal pH that deviates from the normal range of physiological conditions at the site of administration of the polypeptide or in a tissue or organ at the site of action of the polypeptide.

[0015] The first pH may be in the range of 5.5 to 7.2, or in the range of 6.2 to 6.8. The second pH may be in the range of 7.2 to 7.6. The first pH may be about 6.0, and the second pH may be about 7.4.

[0016] The polypeptide of any of the foregoing embodiments may be a non-naturally occurring mutant polypeptide evolved from a parent polypeptide. The mutant polypeptide of any of the foregoing embodiments may be derived from a wild-type parent polypeptide, including a non-naturally occurring polypeptide. The mutant polypeptide of any of the foregoing embodiments may include at least one amino acid substitution compared to the parent polypeptide. The mutant polypeptide of any of the foregoing embodiments may have a higher proportion of charged amino acid residues than the parent polypeptide.

[0017] The polypeptide or variant polypeptide of any of the foregoing embodiments may be a protein or protein fragment. The polypeptide or variant polypeptide of any of the foregoing embodiments may be selected from an antibody, a single-chain antibody, and an antibody fragment, and the activity is antigen binding activity. The polypeptide or variant polypeptide may be an Fc region of an antibody. The polypeptide or variant polypeptide may be an enzyme, and the activity may be enzymatic activity. The polypeptide or variant polypeptide may be selected from a receptor, a regulatory protein, a soluble protein, a cytokine, and a fragment of a receptor, a regulatory protein, a soluble protein, or a cytokine.

[0018] The species of any of the foregoing embodiments may be hydrogen sulfide, bicarbonate, ion or disulfide ion The species of any of the foregoing embodiments may have a pKa greater than 6.2.

[0019] The polypeptide of any of the foregoing embodiments may have two functional domains, and the activity is the activity of one of the two functional domains. Both of the two functional domains may have a pH-dependent activity. The polypeptide may be a bispecific antibody.

[0020] In another embodiment, the polypeptide of any of the preceding embodiments may be used to treat solid tumors, inflamed joints, or brain diseases or disorders.

[0021] In another embodiment, the present disclosure relates to a method of treating a solid tumor, an inflamed joint, or a brain disease or disorder comprising administering the polypeptide of any of the preceding embodiments, which may be administered as part of a chimeric antigen receptor on a T cell comprising the polypeptide or linked to a nanoparticle, or as an antibody-drug conjugate comprising the polypeptide.

[0022] In another embodiment, the present disclosure relates to a chimeric antigen receptor for T cells comprising the polypeptide. In each of the foregoing embodiments, the polypeptide may be linked to a nanoparticle.

[0023] In another embodiment, the present disclosure relates to an antibody-drug conjugate comprising the subject polypeptide.

[0024] In another embodiment, the present disclosure provides a pharmaceutical composition comprising a conditionally active biological protein and a pharmaceutically acceptable carrier.

[0025] In another aspect, the present disclosure provides a method for producing a conditionally active polypeptide from a parent polypeptide, the method comprising: (i) evolving a parent polypeptide by mutating at least one region outside its active site, thereby generating one or more mutant polypeptides; (ii) subjecting one or more polypeptides and the parent polypeptide to a first assay under normal physiological conditions to measure the activity of the active site under normal physiological conditions, and a second assay under abnormal conditions to measure the activity of the active site under abnormal conditions, wherein the normal physiological conditions and the abnormal conditions are the same conditions but have different values; and (iii) selecting from the one or more mutant polypeptides a conditionally active polypeptide that exhibits both (a) a decreased activity compared to the same activity of the parent polypeptide in a first assay, and (b) an increased activity compared to the same activity of the parent polypeptide in a second assay. [Brief explanation of the drawings]

[0026] [Figure 1] 1 shows the conditionally active antibodies generated in Example 9 and their selectivity at pH 6.0 compared to pH 7.4. [Figure 2] 1 shows the antigen-binding activity of conditionally active antibodies assayed in various buffer solutions. [Figure 3]1 shows the effect of changing the composition of Krebs buffer on the binding activity of a conditionally active antibody. [Figure 4] As described in Example 12, the binding activity of three different conditionally active antibodies was dependent on the presence and concentration of bicarbonate at pH 7.4. [Figure 5] FIG. 1 shows the structure of a chimeric antigen receptor (CAR). [Figure 6] Diagram showing salt bridge formation in deoxyhemoglobin, where three amino acid residues form two salt bridges, which stabilize the T quaternary structure of deoxyhemoglobin, leading to a reduced affinity for oxygen. [Figure 7] 1 shows the activity of conditionally active antibodies against Ror2 in various buffer solutions. [Figure 8] 1 shows the activity of conditionally active antibodies against Axl in various buffer solutions. [Figure 9] 1 shows the activity of conditionally active antibodies against Axl discovered using an assay solution containing 10 mM disulfide ions. DETAILED DESCRIPTION OF THE INVENTION

[0027] definition To facilitate understanding of the examples provided herein, some frequently occurring methods and / or terms are defined here.The definitions of the following terms are incorporated by reference from U.S. Pat. No. 8,709,755 B2: "drug," "ambiguous base requirement," "amino acid," "amplification," "chimeric property," "cognate," "comparison window," "conservative amino acid substitution," "corresponding to," "degradation-effective," "defined sequence framework," "defined sequence kernel," "digestion," "directional ligation," "DNA shuffling," "drug" or "drug molecule," "effective amount," "epitope," "enzyme," "evolution" or "evolve," "fragment" or "derivative" or "analog," "global single amino acid substitution," "gene," "genetic instability," "heterologous," "homologous" or "homologous," "industrial application," "identical" or "identity," "identity range," "isolated," "isolated nucleic acid," "ligand," "ligation," "linker" or "spacer," "microenvironment," "molecular property to evolve," "mutation," "N,N,G / T," "normal physiological conditions" or "wild-type operating conditions," "nucleic acid molecule," "nucleic acid molecule," "nucleic acid sequence encoding" or "DNA coding sequence for" or "nucleotide sequence encoding," "enzyme (protein "nucleic acid encoding a protein" or "DNA encoding an enzyme (protein)" or "polynucleotide encoding an enzyme (protein)", "specific nucleic acid molecular species", "assembling a working nucleic acid sample into a nucleic acid library", "nucleic acid library", "construct", "oligonucleotide" (or synonymously "oligo"), "homologous", "operably linked", "parent polynucleotide set", "patient" or "subject", "physiological condition", "population", "proform", "pseudorandom", "quasi-repeated unit", "random peptide library", "random peptide sequence", "receptor", "recombinant" enzyme, "synthetic" enzyme, "related polynucleotide", "reduced reassortment", "reference sequence", "repeat index (RI)", "restriction site", "selectable polynucleotide", "sequence identity", "similarity", "specifically binds", "specific hybridization", "specific polynucleotide", "stringent hybridization conditions", "substantially identical", "substantially pure enzyme", "substantially pure", "treating", "variable segment", and "variant".

[0028] The term "about," as used herein in connection with a measured quantity, relates to the normal variation in the measured quantity that would be expected by one of ordinary skill in the art making the measurement and exercising a level of care appropriate to the purpose of the measurement and the precision of the measuring equipment being used. Unless otherwise specified, "about" relates to a + / - 10% variation of the given value.

[0029] The term "activity," as used herein, refers to any function a protein can perform, including catalyzing a reaction and binding to a partner. For an enzyme, the activity can be enzymatic activity. For an antibody, the activity can be binding activity between the antibody and its antigen (i.e., avidity). For a receptor or ligand, the activity can be binding activity between the receptor and its ligand.

[0030] The term "antibody," as used herein, refers to intact immunoglobulin molecules and fragments of immunoglobulin molecules, such as Fab, Fab', (Fab')2, Fv, and SCA fragments, that retain the ability to selectively bind to an epitope of an antigen. These antibody fragments, which retain some ability to selectively bind to the antigen (e.g., polypeptide antigen) of the antibody from which they were derived, can be produced using methods well known in the art (see, e.g., Harlow and Lane, supra), and are further described below. Antibodies can be used for the isolation of preparative quantities of antigens by immunoaffinity chromatography. Various other uses of such antibodies are in the diagnosis and / or staging of disease (e.g., neoplasia), as well as therapeutic applications for treating diseases such as neoplasia, autoimmune diseases, AIDS, cardiovascular diseases, and infectious diseases. Chimeric, human-like, humanized, or fully human antibodies are particularly useful for administration to human patients.

[0031] A Fab fragment consists of a monovalent antigen-binding fragment of an antibody molecule and can be produced by digestion of whole antibody molecules with the enzyme papain to produce a fragment consisting of an intact light chain and a portion of the heavy chain.

[0032] Fab' fragments of antibody molecules can be obtained by treating whole antibody molecules with pepsin, followed by reduction, to yield molecules consisting of an intact light chain and a portion of the heavy chain, resulting in two Fab' fragments per antibody molecule so treated.

[0033] A (Fab')2 fragment of an antibody can be obtained by treating whole antibody molecules with the enzyme pepsin without subsequent reduction. The (Fab')2 fragment is a dimer of two Fab' fragments held together by two disulfide bonds.

[0034] An Fv fragment is defined as a genetically engineered fragment containing the variable region of the light chain and the variable region of the heavy chain expressed as two chains.

[0035] Single-chain antibodies ("SCAs" or scFvs) are genetically engineered single-chain molecules comprising the variable region of a light chain and the variable region of a heavy chain joined by a suitable flexible polypeptide liner, and may contain additional amino acid sequences at the amino and / or carboxyl termini. For example, single-chain antibodies may include a tethering segment for linking to an encoding polynucleotide. A functional single-chain antibody generally comprises a sufficient portion of the variable region of the light chain and sufficient region of the variable region of the heavy chain so that it retains the properties of a full-length antibody for binding to a specific target molecule or epitope.

[0036] The term "antibody-dependent cell-mediated cytotoxicity" or "ADCC" refers to a form of cytotoxicity in which secreted immunoglobulins bind to Fc receptors (FcRs) present on certain cytotoxic cells (e.g., natural killer (NK) cells, neutrophils, and macrophages), thereby enabling these cytotoxic effector cells to specifically bind to antigen-bearing target cells and subsequently kill them with cytotoxins. Ligand-specific high-affinity IgG antibodies directed against the surface of target cells stimulate cytotoxic cells and are required for such killing. Lysis of target cells is extracellular, requires direct cell-to-cell contact, and does not involve complement.

[0037] The ability of any particular antibody to mediate target cell lysis by ADCC can be assayed. To assess ADCC activity, the antibody of interest is added to target cells presenting a target ligand in conjunction with immune effector cells; activation of the immune effector cells by the antigen-antibody complex can result in target cell lysis. Cytolysis is generally detected by the release of label (e.g., a radioactive substrate, a fluorescent dye, or a natural intracellular protein) from the lysed cells. Useful effector cells for such assays include peripheral blood mononuclear cells (PBMCs) and natural killer (NK) cells. Specific examples of in vitro ADCC assays are described in Bruggemann et al., 1987, J. Exp Med, vol. 166, p. 1351; Wilkinson et al., 2001, J. Immunol. Methods, vol. 258, p. 183; and Patel et al., 1995, J. Immunol. Methods, vol. 184, p. 29. Alternatively, or additionally, ADCC activity of an antibody of interest may be assessed in vivo, for example, in an animal model such as that disclosed in Clynes et al., 1998, PNAS USA, vol. 95, p. 652.

[0038] The term "antigen" or "Ag," as used herein, is defined as a molecule capable of eliciting an immune response. This immune response may include either or both antibody production or activation of specific immunologically competent cells. Those skilled in the art will understand that any macromolecule, including virtually any protein or peptide, can serve as an antigen. It is readily apparent that antigens may be generated, synthesized, or derived from biological samples. Such biological samples may include, but are not limited to, tissue samples, tumor samples, cells, or biological fluids.

[0039] The term "antisense RNA," as used herein, refers to an RNA molecule capable of forming a duplex with a second RNA molecule through complementarity or partial complementarity with the second RNA molecule. The antisense RNA molecule can be complementary to the translated or untranslated region of the second RNA molecule. The antisense RNA does not need to be perfectly complementary to the second RNA molecule. The antisense RNA may or may not be the same length as the second RNA molecule; the antisense RNA molecule may be either longer or shorter than the second RNA molecule. When the second RNA molecule is mRNA, binding of the antisense RNA will completely or partially prevent the mRNA from being translated into a functional protein product.

[0040] The terms "biosimilar" and "follow-on product" are used in a manner consistent 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 the reference product (although differing slightly in clinically inactive ingredients). In practice, there may be no clinically meaningful differences between the reference and biosimilar products in terms of safety, purity, and potency (Public Health Service Act (PHS) Article 262). Biosimilars may also meet one or more of the guidelines adopted by the European Medicines Agency's Committee for Medicinal Products for Human Use (CHMP) on May 30, 2012, and published by the European Union as "Guideline on similar biological medicinal products containing monoclonal antibodies - non-clinical and clinical issues" (document reference EMA / CHMP / BMWP / 403543 / 2010). For example, a "biosimilar antibody" refers to a generic version of a new antibody (reference antibody) typically produced by another company. Differences between a biosimilar antibody and a reference antibody can include post-translational modifications, such as those made by adding other biochemical groups to the antibody, such as phosphates, various lipids, and carbohydrates; by post-translational proteolytic cleavage; by changing the chemical nature of amino acids (e.g., formylation); or by many other mechanisms. Other post-translational modifications can be the result of manipulations in the manufacturing process—for example, glycation can occur when the formulation is exposed to reducing sugars. In some cases, storage conditions can be permissive for certain degradation pathways, such as oxidation, deamidation, or aggregation. All of these formulation variations can be included in a biosimilar antibody.

[0041] The terms "cancer" and "cancerous" refer to or describe the physiological condition in mammals that is typically characterized by unregulated cell growth / proliferation. A "tumor" contains one or more cancerous cells. Examples of cancer include, but are not limited to, carcinoma, lymphoma, blastoma, sarcoma, and leukemia or lymphoid malignancies. More particular examples of such cancers include squamous cell carcinoma (e.g., epithelial squamous cell carcinoma), lung cancer (including small cell lung cancer, non-small cell lung cancer ("NSCLC"), lung adenocarcinoma, and lung squamous cell carcinoma), peritoneal cancer, hepatocellular carcinoma, gastric cancer or stomach cancer (including gastrointestinal cancer), pancreatic cancer, glioblastoma, cervical cancer, ovarian cancer, liver cancer, bladder cancer, hepatoma, breast cancer, colon cancer, rectal cancer, colorectal cancer, endometrial cancer or uterine cancer, salivary gland cancer, kidney cancer or renal cancer, prostate cancer, vulvar cancer, thyroid cancer, liver cancer, anal cancer, penile cancer, and head and neck cancer.

[0042] The term "chimeric antigen receptor" or "CAR" or "CARs," as used herein, refers to an engineered receptor that grafts antigen specificity onto cytotoxic cells, such as T cells, NK cells, and macrophages. The CAR of the present invention may comprise at least one antigen-specific targeting region (ASTR), an extracellular spacer domain (ESD), a transmembrane domain (TM), one or more costimulatory domains (CSDs), and an intracellular signaling domain (ISD). In some embodiments, the ESD and / or CSD are optional. In one embodiment, the ASTR is bispecific and can recognize two different antigens or epitopes. After the ASTR specifically binds to the target antigen, the ISD activates intracellular signaling in the cytotoxic cell. For example, the ISD can redirect T cell specificity and cytotoxicity to a target of choice in an MHC-independent manner, depending on the antigen-binding properties of the CAR. MHC-independent antigen recognition allows CAR-expressing cytotoxic cells to recognize antigens independent of antigen processing, thereby circumventing a major tumor escape mechanism. Furthermore, when expressed in T cells, CARs advantageously do not dimerize with the endogenous T cell receptor (TCR) α and β chains.

[0043] The term "conditionally active polypeptide" refers to a variant or mutant of a parent polypeptide that is more active than the parent polypeptide under at least one condition and less active than the parent polypeptide under a second condition, or refers to a variant or mutant of a parent polypeptide where the variant or mutant polypeptide is at least 1.3-fold more active under a first condition than under a second condition. The conditionally active polypeptide may exhibit activity at one or more selected biological locations and / or may exhibit increased or decreased activity at other locations in the organism. For example, in one embodiment, an evolved conditionally active biological protein is effectively inactive at body temperature but active at lower temperatures. Conditionally active polypeptides include conditionally active proteins, protein fragments, antibodies, antibody fragments, enzymes, enzyme fragments, receptors and receptor fragments, cytokines and fragments thereof, hormones and fragments thereof, ligands and fragments thereof, regulatory proteins and fragments thereof, growth factors and fragments thereof, and proteins such as stress proteins, vault-associated proteins, neuronal proteins, gut proteins, growth factors, mitochondrial proteins, cytoplasmic proteins, animal proteins, structural proteins, plant proteins, and fragments of any of these proteins. Each of the conditionally active polypeptides described herein is preferably a conditionally active biological polypeptide.

[0044] The term "cytokine" or "cytokines," as used herein, refers to a general class of biomolecules that produce / affect cells of the immune system. This definition is intended to include, but is not limited to, biomolecules that act locally or at other locations distant from the site of secretion through the blood circulation to regulate or modulate an individual's immune response. Exemplary cytokines include, but are not limited to, interferon-α (IFN-α), interferon-β (IFN-β), and interferon-γ (IFN-γ), interleukins (e.g., IL-1 through IL-29, specifically IL-2, IL-5, IL-6, IL-7, IL-10, IL-12, IL-15, and IL-18), tumor necrosis factors (e.g., TNF-α and TNF-β), erythropoietin (EPO), MIP3a, monocyte chemoattractant protein (MCP)-1, intracellular adhesion molecules (ICAMs), macrophage colony-stimulating factor (M-CSF), granulocyte colony-stimulating factor (G-CSF), and granulocyte-macrophage colony-stimulating factor (GM-CSF).

[0045] As used herein, the term "electrolyte" is used to define a mineral in blood or other bodily fluids that carries an electrical charge. For example, in one aspect, the "electrolyte concentration" may be different between normal physiological conditions and abnormal conditions. Exemplary electrolytes include, but are not limited to, ionized calcium, sodium, potassium, magnesium, chloride, citrate, lactate, bicarbonate, and phosphate.

[0046] The term "full-length antibody" refers to an antibody that contains an antigen-binding variable region (V H or V L), and a light chain constant domain (CL) and heavy chain constant domains CH1, CH2, and CH3. The constant domains may be native sequence constant domains (e.g., human native sequence constant domains) or amino acid sequence variants thereof. Full-length antibodies can be assigned to different "classes" depending on the amino acid sequence of the constant domain of their heavy chain. There are five major classes of full-length antibodies: IgA, IgD, IgE, IgG, and IgM, some of which can be further divided into "subclasses" (isotypes), e.g., IgG1, IgG2, IgG3, IgG4, IgA, and IgA2. The heavy-chain constant domains that correspond to the different antibody classes are called α, δ, ε, γ, and μ, respectively.

[0047] The term "growth factor" as used herein refers to a polypeptide molecule capable of inducing 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 colony-stimulating factor (GM-CSF), bone morphogenetic protein (BMP), nerve growth factor (NGF), vascular endothelial growth factor (NEGF), fibroblast growth factor (FGF), insulin-like growth factor (IGF), cartilage-derived morphogenetic protein (CDMP), and platelet-derived growth factor (PDGF).

[0048] The term "hormone," as used herein, refers to a substance often identified as a mediator, typically released by cells or glands in one part of an organism to act as a messenger to another part of the organism. Exemplary hormones include endocrine hormones, which are released directly into the bloodstream, and exocrine hormones (or ectohormones), which are secreted directly into ducts from which they diffuse into the bloodstream or from cell to cell in a process known as paracrine signaling. Vertebrate hormones can be divided into three chemical classes: peptide hormones, lipid- and phospholipid-derived hormones, and monoamines. Peptide hormones consist of polypeptide chains. Examples of peptide hormones include insulin and growth hormone. Lipid- and phospholipid-derived hormones are derived from lipids and phospholipids, such as linoleic acid and arachidonic acid. A major class are steroid hormones, which are derived from cholesterol and eicosanoids. Examples of steroid hormones are testosterone and cortisol. Monoamines are derived from aromatic amino acids such as phenylalanine, tyrosine, and tryptophan by the action of aromatic amino acid decarboxylase enzymes. Examples of monoamines are thyroxine and adrenaline.

[0049] The term "immunomodulator," as used herein, refers to an agent whose effect on the immune system results in an immediate or delayed increase or decrease in the activity of at least one pathway involved in an immune response. Such responses can occur naturally as part of the innate or adaptive immune system, or both, or can be artificially induced. Examples of immunomodulators include cytokines, stem cell growth factors, lymphotoxins, such as tumor necrosis factor (TNF), and hematopoietic factors, such as interleukins (e.g., interleukin-1 (IL-1), IL-2, IL-3, IL-6, IL-10, IL-12, IL-18, and IL-21), colony-stimulating factors (e.g., granulocyte-colony stimulating factor (G-CSF) and granulocyte-macrophage colony-stimulating factor (GM-CSF)), interferons (e.g., interferon-α, -β, and -γ), stem cell growth factor known as "S1 factor," erythropoietin, and thrombopoietin. Examples of suitable immunomodulatory moieties include IL-2, IL-6, IL-10, IL-12, IL-18, IL-21, interferons, TNF (e.g., TNF-α), and the like.

[0050] An "individual" or "subject" is a mammal, including, but not limited to, domesticated animals (e.g., cows, sheep, cats, dogs, and horses), primates (e.g., humans and non-human primates such as monkeys), rabbits, and rodents (e.g., mice and rats).

[0051] The term "library," as used herein, refers to a collection of proteins as a single pool. Libraries can be created using recombinant DNA technology. For example, a collection of cDNAs or any other protein-encoding DNA can be inserted into an expression vector to create a protein library. A collection of cDNAs or protein-encoding DNA can also be inserted into a phage genome to create a bacteriophage display library of wild-type proteins. A collection of cDNAs can be generated from a specific cell population or tissue sample, such as by the method disclosed by Sambrook et al. (Molecular Cloning, Cold Spring Harbor Laboratory Press, 1989). cDNA collections from specific cell types are also commercially available from suppliers such as Stratagene®. A library of wild-type proteins as used herein is not a collection of biological samples.

[0052] The term "ligand," as used herein, refers to a molecule that is recognized by and specifically binds to a particular receptor at one or more binding sites. Examples of ligands include, but are not limited to, agonists and antagonists of cell membrane receptors, toxins and venoms, viral epitopes, hormones, hormone receptor peptides, enzymes, enzyme substrates, cofactors, drugs (e.g., opiates, steroids, etc.), lectins, saccharides, polynucleotides, nucleic acids, oligosaccharides, proteins, and monoclonal antibodies. Typically, a ligand comprises two structural moieties: a first moiety that is involved in binding of the ligand to its receptor and a second moiety that is not involved in such binding.

[0053] The term "receptor," as used herein, refers to a molecule that has affinity for a given ligand. Receptors can be naturally occurring or synthetic. Receptors may be used unmodified or as aggregates with other species. Receptors can be covalently or noncovalently bound to a binding member directly or via a specific binding substance. Examples of receptors include, but are not limited to, antibodies and antisera, including monoclonal antibodies reactive with specific antigenic determinants (such as those found on viruses, cells, or other materials), cell membrane receptors, glycoconjugates and glycoproteins, enzymes, and hormone receptors. Binding of a ligand to its receptor indicates the formation of a complex by combination of the ligand and its receptor molecule through specific molecular recognition, which can be detected by various ligand-receptor binding assays known to those skilled in the art.

[0054] The terms "microRNA" or "miRNA," as used herein, refer to unprocessed or processed RNA transcripts from miRNA genes. Unprocessed microRNA gene transcripts typically comprise RNA transcripts of approximately 70-100 nucleotides in length. Transcribed microRNAs can be processed by digestion with an RNase (e.g., Dicer, Argonaute, or RNase III) to yield active 19-25 nucleotide RNA molecules. These active 19-25 nucleotide RNA molecules are also referred to as "processed" microRNA gene transcripts or "mature" microRNAs.

[0055] The term "multispecific antibody," as used herein, refers to an antibody that has binding specificities for at least two different epitopes. Exemplary multispecific antibodies can bind to both the BBB-R and a brain antigen. Multispecific antibodies can be prepared as full-length antibodies or antibody fragments (e.g., F(ab')2 bispecific antibodies). Engineered antibodies containing two, three, or more (e.g., four) functional antigen-binding sites are also contemplated (see, e.g., U.S. Patent Application Publication No. 2002 / 0004587 A1).

[0056] The term "nanoparticle," as used herein, refers to a microscopic particle having a size measured in nanometers (nm) and a maximum linear dimension of less than about 1000 nm, or less than about 500 nm, or less than about 200 nm, or less than about 100 nm, or less than about 50 nm. As used herein, linear dimension refers to the distance measured in a straight line between any two points on the nanoparticle. The nanoparticles of the present invention may be irregular, oval, spindle-shaped, rod-shaped, disk-shaped, pancake-shaped, cylindrical, red blood cell-like, spherical, or nearly spherical, so long as their shape and size allow for binding interactions. The nanoparticles of the present invention are preferably made of biocompatible materials (polymers or lipids).

[0057] The term "naturally occurring," as used herein, refers to the fact that an object is found in nature. For example, a polypeptide or polynucleotide sequence present in an organism (including a virus) that can be isolated from a source in nature and that has not been intentionally modified by humans in a laboratory is naturally occurring. A polypeptide excised from a larger polypeptide is not a naturally occurring polypeptide because the end groups of the excised polypeptide will differ from those of the larger, naturally occurring polypeptide in the excised form because those end groups are no longer attached to adjacent polypeptides. Generally, the term naturally occurring refers to an entity as it exists in a non-diseased (unaffected) individual, such as may be typical for that species.

[0058] The terms "parent polypeptide" and "parent protein," as used herein, refer to a polypeptide or protein that can be evolved to generate a conditionally active polypeptide or protein using the methods of the present invention. A parent polypeptide protein can be a wild-type protein, including a protein that does not occur in nature. For example, a therapeutic polypeptide or protein or a mutant or variant polypeptide or protein can be used as a parent polypeptide or protein. Examples of parent polypeptides and proteins include antibodies, antibody fragments, enzymes, enzyme fragments, cytokines and fragments thereof, hormones and fragments thereof, ligands and fragments thereof, receptors and fragments thereof, regulatory proteins and fragments thereof, and growth factors and fragments thereof.

[0059] The term "pH-dependent" as used herein refers to a polypeptide that has different properties or activities at different pH values.

[0060] The term "polypeptide" as used herein refers to a polymer in which the monomers are amino acids and are linked together by peptide or disulfide bonds. A polypeptide can be a full-length naturally occurring amino acid chain or a fragment, mutant, or variant thereof, e.g., a selected region of the amino acid chain that is of interest in a binding interaction. A polypeptide can also be a synthetic amino acid chain, or a combination of a naturally occurring amino acid chain or a fragment thereof with a synthetic amino acid chain. A fragment refers to an amino acid sequence that is a portion of a full-length protein and is typically about 8 to about 500 amino acids in length, preferably about 8 to about 300 amino acids, more preferably about 8 to about 200 amino acids, and even more preferably about 10 to about 50 or 100 amino acids in length. In addition, amino acids other than naturally occurring amino acids, such as β-alanine, phenylglycine, and homoarginine, can be included in proteins. Commonly occurring, non-genetically encoded amino acids can also be included in polypeptides. Amino acids can be either D- or L-optical isomers. D-isomers are preferred for use in certain contexts, as discussed in more detail below. In addition, other peptidomimetics are also useful, for example, in linker sequences of polypeptides (see Spatola, 1983, Chemistry and Biochemistry of Amino Acids, Peptides and Proteins, Weinstein, ed., Marcel Dekker, New York, p. 267). In general, the term "protein" is not intended to convey any significant difference from the term "polypeptide," other than to encompass structures comprising two or several polypeptide chains held together by covalent or noncovalent bonds.

[0061] The term "recombinant antibody," as used herein, refers to an antibody (e.g., a chimeric, humanized, or human antibody or antigen-binding fragment thereof) expressed by a host cell containing nucleic acid encoding the antibody. Examples of "host cells" for producing recombinant antibodies include: (1) mammalian cells, such as Chinese hamster ovary (CHO), COS, myeloma cells (including Y0 and NS0 cells), baby hamster kidney (BHK), Hela, and Vero cells; (2) insect cells, such as sf9, sf21, and Tn5; (3) plant cells, such as plants belonging to the genus Nicotiana (e.g., Nicotiana tabacum); (4) yeast cells, such as those belonging to the genus Saccharomyces (e.g., Saccharomyces cerevisiae) or those belonging to the genus Aspergillus (e.g., Aspergillus niger); (5) bacterial cells, such as Escherichia coli cells or Bacillus subtilis cells.

[0062] The term "regulatory protein," as used herein, refers to any protein that increases or decreases the activity of another polypeptide or RNA molecule; increases or decreases the abundance of another polypeptide or RNA molecule; alters the interaction between another polypeptide or RNA molecule and other polypeptides, DNA or RNA molecules, or any other binding substrate; and / or alters the cellular location of another polypeptide or RNA molecule. When a regulatory protein increases or decreases the rate of transcription of a gene, it is often referred to as a transcription factor, which exerts its effect on the promoter or enhancer region of the gene. Examples of transcription factors include mammalian transcription factors such as NFkB, NF1, cyclic AMP-responsive 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 others as described in Latchman, 1998, Eukaryotic Transcription Factors, 3rd Ed., Academic Press: New York. Fusion protein derivatives of these or other transcription factors can also be used, in which at least the DNA-binding motif of the fusion protein, which provides the binding specificity, is fused to a small molecule regulatory factor binding site.

[0063] The term "small interfering RNA" or "siRNA," as used herein, refers to an RNA or RNA-like molecule 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 believed that siRNAs can be incorporated into a ribonucleoprotein complex known as the RNA-induced silencing complex (RISC). RISC uses the siRNA sequence to identify mRNA molecules that are at least partially complementary to the incorporated siRNA strand, and then cleaves or inhibits the translation of these target mRNA molecules. Typical siRNAs are double-stranded nucleic acid molecules, each strand of which is about 19 to about 28 nucleotides (i.e., about 19, 20, 21, 22, 23, 24, 25, 26, 27, or 28 nucleotides). siRNAs can also be single-stranded RNAs, although with lower efficiency compared to double-stranded siRNAs. Single-stranded siRNAs have a length of about 19 to about 49 nucleotides. Single-stranded siRNA has a 5' phosphate or is phosphorylated in situ or in vivo at the 5' position.Single-stranded siRNA can be synthesized chemically or by in vitro transcription and endogenously expressed from an expression vector or expression cassette.The 5' phosphate group can be added via kinase or can be the result of nuclease cleavage of RNA.

[0064] The term "small molecule" refers to a molecule or ion that typically has a molecular weight of 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 assays and environments of the present invention, small molecules may often exist as a mixture of the molecule and its deprotonated ion, depending primarily on the pH of the assay or environment.

[0065] The term "therapeutic protein," as used herein, refers to any protein and / or polypeptide that can be administered to a mammal to produce a biological or medical response in a tissue, system, animal, or human that is being sought, for example, by a researcher or clinician. A therapeutic protein may produce more than one biological or medical response. Examples of therapeutic proteins include antibodies, enzymes, hormones, cytokines, regulatory proteins, and fragments thereof.

[0066] The term "therapeutically effective amount," as used herein, means any amount that results in, but is not limited to, a cure, prevention, or amelioration of a disease, disorder, or side effect, or a reduction in the rate of progression of a disease or disorder, when compared to a corresponding subject who does not receive such amount. The term also includes within its scope an amount effective to enhance normal physiological function, as well as an amount effective to cause a patient to have physiological function that enhances or supplements the therapeutic effect of a second pharmaceutical agent.

[0067] The term "tumor microenvironment," as used herein, refers to the microenvironment within and surrounding a solid tumor that supports tumor cell growth and metastasis. The tumor microenvironment includes surrounding blood vessels, immune cells, fibroblasts, other cells, soluble factors, signaling molecules, extracellular matrix, and mechanical cues that can promote neoplastic transformation, support tumor growth and invasion, protect tumors from host immunity, develop therapeutic resistance, and provide a niche for harboring latent metastases. Tumors and their surrounding microenvironment are intimately associated and constantly interact. Tumors can influence their microenvironment by releasing extracellular signals, promoting tumor angiogenesis, and inducing peripheral immune tolerance, while immune cells in the microenvironment can influence the growth and evolution of cancerous cells. Swarts et al. “Tumor Microenvironment Complexity: Emerging Roles in Cancer Therapy,” Cancer Res, vol.,72, pages 2473-2480, 2012; Weber et al., “The tumor microenvironment,” Surgical Oncology, vol. 21, pages 172-177, 2012; Blagosklonny, “Antiangiogenic therapy and tumor progression,” Cancer Cell, vol. 5, pages 13-17, 2004; Siemann, “Tumor microenvironment,” Wiley, 2010; and Bagley, “The tumor microenvironment,” Springer, 2010.

[0068] As used herein, the term "wild-type" means that the polynucleotide does not contain any mutations. "Wild-type protein," "wild-type protein," "wild-type biological protein," or "wild-type biological protein" can refer to a protein that can be isolated from nature, that can be active at a level of activity found in nature, and that can contain an amino acid sequence found in nature. The terms "parent molecule" and "target protein" also encompass wild-type proteins.

[0069] Detailed Description A. pH-dependent conditionally active polypeptides In one aspect, the invention relates to conditionally active polypeptides that have pH-dependent activity in the presence of species having a pKa within 0.5, 1, 2, or 4 units of the pH at which activity is desired. In another aspect, the invention relates to conditionally active polypeptides that have pH-dependent activity in the presence of species having a pKa of about 4 to about 10, or about 4.5 to about 9.5, or about 5 to about 9, or about 5.5 to about 8, or about 6.0 to about 7.0. In another aspect, the invention relates to conditionally active polypeptides that have pH-dependent activity in the presence of species having a pKa of histidine, histamine, hydrogenated adenosine diphosphate, hydrogenated adenosine triphosphate, citric acid, or the like. ion , bicarbonate ion , acetic acid ion , lactic acid ion , disulfide ion , hydrogen sulfide, ammonium ion , dihydrogen phosphate ion and any combination thereof.

[0070] Species present in the assay medium that have a significant effect on the activity of a conditionally active polypeptide tend to be species that have at least two ionization states: an uncharged or low-charged state and a charged or high-charged state. As a result, the pKa of a species that affects the activity of a conditionally active polypeptide can play a role in determining the extent of the effect that species may have on the particular activity of the polypeptide and / or at a particular pH.

[0071] A pH-dependent conditionally active polypeptide is more active at a first pH than at a second, different pH (both activities measured in assays in the presence of one or more of the species listed above). To determine the pH dependence of a conditionally active polypeptide, the same activity of the polypeptide is assayed at two different pH values ​​in the same assay medium.

[0072] The ratio of activity at a first pH to the same activity at a second pH in the same assay medium can be referred to as the selectivity of the pH-dependent conditionally active polypeptide. A pH-dependent conditionally active polypeptide has a selectivity of at least about 1.3, or at least about 1.5, or at least about 1.7, or at least about 2.0, or at least about 3.0, or at least about 4.0, or at least about 6.0, or at least about 8.0, or at least about 10.0, or at least about 20.0, or at least about 40.0, or at least about 60.0, or at least about 100.0.

[0073] It has been observed that pH-dependent conditionally active polypeptides contain an increased number (or percentage) of charged amino acid residues compared to the amino acid residues of the parent polypeptide from which the conditionally active polypeptide is derived. There are three positively charged amino acid residues: lysine, arginine, and histidine; and two negatively charged amino acid residues: aspartic acid and glutamic acid. These charged amino acid residues are over-represented in the pH-dependent conditionally active polypeptide compared to the parent polypeptide from which the pH-dependent conditionally active polypeptide is derived. As a result, the pH-dependent conditionally active polypeptide has an increased number of charged amino acid residues, making it more likely to interact with charged species in the assay medium. This, in turn, affects the activity of the conditionally active polypeptide.

[0074] It has also been observed that pH-dependent conditionally active polypeptides typically have different activities in the presence of different species in the assay medium. Species that have at least two ionization states: an uncharged or low-charged state and a charged or high-charged state, may dissociate to a greater extent at a particular pH depending on their pKa values, thereby increasing the likelihood of interacting with charged amino acid residues present in the conditionally active polypeptide. This factor may be used to enhance the selectivity and / or pH-dependent activity of the conditionally active polypeptide.

[0075] The nature of the charge on a conditionally active polypeptide can be one factor used to determine suitable species for affecting the activity of the conditionally active polypeptide. In some embodiments, a conditionally active polypeptide can have more positively charged amino acid residues, such as lysine, arginine, and histidine, than the parent polypeptide. Thus, a conditionally active polypeptide can be selected to have a desired level of interaction with a particular species present in an environment in which activity is desired and / or a desired level of interaction with a particular species present in an environment in which reduced activity is desired. Similarly, a conditionally active polypeptide can have more negatively charged amino acid residues, such as aspartic acid and glutamic acid, than the parent polypeptide.

[0076] The location of charged amino acid residues on a pH-dependent conditionally active polypeptide can also affect activity. For example, the proximity of charged amino acid residues to the binding site of a conditionally active polypeptide can be used to affect the activity of the polypeptide.

[0077] In some embodiments, the interaction of a charged environmental species with a conditionally active polypeptide can block or interfere with the activity of a pH-dependent conditionally active polypeptide. For example, a charged amino acid that interacts with a charged environmental species can have an allosteric effect on the binding site of the conditionally active polypeptide.

[0078] In other embodiments, when charged environmental species interact with a conditionally active polypeptide, salt bridges may form between different moieties on the polypeptide, particularly between charged or polar moieties. Salt bridge formation is known to stabilize polypeptide structure (Donald, et al., "Salt Bridges: Geometrically Specific, Designable Interactions," Proteins, 79(3):898-915, 2011; Hendsch, et al., "Do salt bridges stabilize proteins? A continuum electrostatic analysis," Protein Science, 3:211-226, 1994). Salt bridges can stabilize or fix protein structures that undergo minor structural fluctuations, commonly referred to as "breathing" (Parak, "Proteins in action: the physics of structural fluctuations and conformational changes," Curr Opin Struct Biol., 13(5):552-557, 2003). The "breathing" of protein structure is important for protein function and its binding with its partners because this structural fluctuation allows the conditionally active protein to efficiently recognize and bind to its partner (Karplus, et al., "Molecular dynamics and protein functions," PNAS, vol. 102, pp. 6679-6685, 2015). The formation of a salt bridge can make the binding site, particularly the binding pocket, on the conditionally active polypeptide less accessible to the partner, possibly because the salt bridge can directly block the partner's access to the binding site. Even salt bridges far from the binding site can disrupt binding by altering the conformation of the binding site through allosteric effects. Therefore, after the salt bridge stabilizes (locks) the structure of the conditionally active polypeptide, the polypeptide may become less active in binding to its partner, resulting in reduced activity.

[0079] One well-known example of how polypeptides and their structures are stabilized by salt bridges is hemoglobin. Structural and chemical studies have revealed that salt bridges involve at least two pairs of chemical groups: the amino terminus and side chains of histidines β146 and α122 (which have pKa values ​​near pH 7). In deoxyhemoglobin, the terminal carboxylate group of β146 forms a salt bridge with a lysine residue in the α subunit of the other αβ dimer. This interaction fixes the side chain of histidine β146 in a position where it can participate in a salt bridge with the negatively charged aspartic acid 94 of the same chain, provided that the imidazole group of the histidine residue is protonated (Figure 6). At high pH, ​​the side chain of histidine β146 is unprotonated, and a salt bridge cannot be formed. However, as the pH decreases, the side chain of histidine β146 becomes protonated, forming a salt bridge between histidine β146 and aspartic acid β94, which stabilizes the quaternary structure of deoxyhemoglobin, thereby increasing its propensity for oxygen release in actively metabolizing tissues (lower pH). Hemoglobin exhibits a pH-dependent binding activity for oxygen, where at low pH, the binding activity for oxygen decreases due to the formation of a salt bridge. On the other hand, at high pH, ​​the binding activity for oxygen increases due to the absence of the salt bridge.

[0080] Similarly, small molecules such as bicarbonate can reduce the binding activity of a conditionally active polypeptide to its partner by forming salt bridges in the conditionally active polypeptide. For example, at pHs below its pKa of 6.4, bicarbonate is protonated and therefore uncharged. Uncharged bicarbonate cannot form salt bridges and therefore has little effect on the binding of a conditionally active polypeptide to its partner. Thus, a conditionally active polypeptide has high binding activity with its partner at low pH. On the other hand, at pHs above the pKa of bicarbonate, bicarbonate becomes ionized by losing a proton and therefore becomes negatively charged. Negatively charged bicarbonate forms salt bridges between positively charged or polar moieties on the conditionally active polypeptide, stabilizing the structure of the conditionally active polypeptide. This can block or reduce the binding of a conditionally active polypeptide to its partner. Therefore, a conditionally active polypeptide has low activity at high pH. Thus, a conditionally active polypeptide has conditionally active activity in the presence of bicarbonate, with higher binding activity at low pH compared to high pH.

[0081] In the absence of species such as bicarbonate in the assay medium, a conditionally active polypeptide may lose its conditional activity. This is likely due to the absence of salt bridges on the conditionally active polypeptide that stabilize (lock) the structure of the polypeptide. Thus, the partner will have equal access to the binding site on the conditionally active polypeptide at any pH, resulting in comparable activity at the first and second pHs.

[0082] In other embodiments, interaction between a small molecule or ion and a conditionally active polypeptide may alter the structure of the polypeptide in a way that increases its activity. For example, the structural change may improve the binding affinity of the conditionally active polypeptide by altering the position, steric hindrance, or binding energy of the binding site required for binding affinity. In such cases, it may be desirable to select a small molecule that binds to the conditionally active polypeptide at the pH where activity is desired.

[0083] Although salt bridges (ionic bonds) are the strongest and most common way in which compounds and ions affect the activity of conditionally active polypeptides, it should be understood that other interactions between such compounds and ions and the conditionally active polypeptide can also contribute to stabilizing (fixing) the structure of the conditionally active polypeptide. Such other interactions include hydrogen bonds, hydrophobic interactions, and van der Waals interactions.

[0084] In some embodiments, to select suitable compounds or ions, a conditionally active polypeptide is compared to the parent polypeptide from which it was evolved to determine whether the conditionally active polypeptide has a higher proportion of negatively or positively charged amino acid residues. Compounds with suitable charges at a second pH, respectively, can then be selected to affect the activity of the conditionally active polypeptide. For example, when a conditionally active polypeptide has a higher proportion of positively charged amino acid residues than the parent polypeptide, suitable small molecules typically must be negatively charged to interact with the conditionally active polypeptide at the second pH. On the other hand, when a conditionally active polypeptide has a higher proportion of negatively charged amino acid residues than the parent polypeptide, suitable small molecules typically must be positively charged to interact with the conditionally active polypeptide at the second pH.

[0085] In other embodiments, the activity of the conditionally active polypeptide is controlled by the interaction of a small molecule or ion with a target polypeptide that is a binding partner of the conditionally active polypeptide. In this case, the same principles discussed above are equally applicable, except that the goal is to create an interaction between the small molecule or ion and the target polypeptide. The target polypeptide can be, for example, an antigen for a conditionally active antibody or a ligand for a conditionally active receptor.

[0086] A suitable small molecule may be any inorganic or organic molecule that transitions from an uncharged or low-charged state at a first pH to a charged or high-charged state at a second pH. Therefore, the small molecule must typically have a pKa between the first and second pHs. For example, bicarbonate has a pKa of 6.4. Therefore, at higher pHs, such as pH 7.4, negatively charged bicarbonate may bind to charged amino acid residues in a conditionally active polypeptide, reducing its activity. On the other hand, at lower pHs, such as pH 6.0, low-charged bicarbonate may not bind to the conditionally active polypeptide in the same amount, potentially allowing for greater activity of the conditionally active polypeptide.

[0087] Disulfides have a pKa of 7.05. Therefore, at higher pHs, such as pH 7.4, more negatively charged disulfides may bind to positively charged amino acid residues of a conditionally active polypeptide, reducing its activity. On the other hand, at lower pHs, such as pH 6.2-6.8, less charged hydrogen sulfide / disulfides do not bind to the conditionally active polypeptide at the same level, thereby allowing for greater activity of the conditionally active polypeptide.

[0088] Small molecules having a pKa between the first and second pH ranges are preferred for use in the present invention. Preferred species are selected from disulfide, hydrogen sulfide, histidine, histamine, citrate, bicarbonate, acetate, and lactate. Each of these small molecules has a pKa between 6.2 and 7.0. Additionally, other small molecules such as tricine (pKa 8.05) and bicine (pKa 8.26) may also be used. For other suitable small molecules, reference may be made to textbooks using the principles of the present application, such as CRC Handbook of Chemistry and Physics, 96th Edition, by CRC Press, 2015; Chemical Properties Handbook, McGraw-Hill Education, 1998.

[0089] The concentration of the small molecule in the assay medium or environment is preferably at or near the physiological concentration of the small molecule in the subject. For example, the physiological concentration of bicarbonate (in human serum) is in the range of 15-30 mM. Thus, the concentration of bicarbonate in the assay medium may be 10 mM-40 mM, or 15 mM-30 mM, or 20 mM-25 mM, or about 20 mM. The physiological concentration of disulfide is also low. The concentration of disulfide in the assay medium may be 3-500 nM, or 5-200 nM, or 10-100 nM, or 10-50 nM.

[0090] In the present invention, a conditionally active polypeptide is selected and utilized at a concentration such that the normal physiological concentration of a particular species in the environment can have a significant effect on the activity of the conditionally active polypeptide in the desired pH range. Thus, for many therapeutic treatments, it may be advantageous for the conditionally active polypeptide to have low activity at approximately pH 7.2-7.4, the pH of blood or human serum, to enable delivery of the therapeutic treatment via the bloodstream while minimizing or preventing activation of the conditionally active polypeptide. Consequently, for such treatments, it may be advantageous to select a small molecule with a pKa below pH 7.2-7.4, ensuring a sufficient amount of ionized small molecule is available at bloodstream pH to have a significant effect on the activity of the conditionally active polypeptide. At the same time, the pKa of the small molecule must be above the pH at which activity of the conditionally active polypeptide is desired to ensure activation of the conditionally active polypeptide by protonating the small molecule and freeing up a binding site on the conditionally active polypeptide.

[0091] Small molecules preferably have a low molecular weight and / or a relatively compact conformation to ensure maximum access to small pockets on the target polypeptide or conditionally active polypeptide by minimizing steric hindrance. Thus, small molecules typically have a molecular weight of less than 900 a.mu, or more preferably less than 500 a.mu, or more preferably less than 200 a.mu, or even more preferably less than 100 a.mu. For example, hydrogen sulfide, disulfide, and bicarbonate all have low molecular weights and compact structures that allow them to access pockets on the target polypeptide or conditionally active polypeptide, as shown in Examples 13 and 14 below.

[0092] Small molecules can be present in the assay or environment at substantially the same concentration, e.g., about 20 μM for bicarbonate. In some embodiments, small molecules can be present at different concentrations in different environments, and it may be desirable to mimic this in the assay. For example, disulfide has a higher concentration in the tumor microenvironment than in human serum. Thus, one assay can mimic a tumor microenvironment with an acidic pH and higher disulfide concentrations, while a second assay can mimic human serum with a neutral or slightly basic pH and lower disulfide concentrations. The acidic pH can be in the range of 6.0 to 6.8, while the neutral or slightly basic pH can be about 7.4. The higher disulfide concentration in the first assay can be 30 μM, while the lower disulfide concentration in the second buffer can be 10 μM or less, or even 5 μM.

[0093] In some embodiments, the conditionally active polypeptide is pH dependent in the presence of two or more different small molecules, for example, a combination of bicarbonate and histidine.

[0094] In the absence of a small molecule, the conditionally active polypeptide may lose its pH dependence, such that in the absence of a small molecule, the conditionally active polypeptide may have similar activity between a first pH and a second pH in the absence of the small molecule.

[0095] In some embodiments, the first pH is an acidic pH, while the second pH is a basic or neutral pH. In other embodiments, the first pH is a basic pH, while the second pH is an acidic or neutral pH. For example, the first pH may be 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.

[0096] Conditionally active polypeptides that are more active at acidic pH and less active at basic or neutral pH can target tumor microenvironments that are acidic at pHs between about 5.5 and 7.2, or between about 6.2 and 6.8.

[0097] In other embodiments, the first pH at which the pH-dependent polypeptide is more active may be a basic pH, e.g., 7.6-7.9, such as that of synovial fluid (see Jebens et al., "On the viscosity and pH of synovial fluid and pH of blood," Journal of Bone and Joint Surgery, vol. 41 B, pp. 388-400, 1959). The second pH may be the pH of blood, about 7.2-7.6, at which the conditionally active polypeptide is less active. These conditionally active polypeptides may be suitable for targeting joint diseases, particularly joint inflammation.

[0098] In other embodiments, the conditionally active polypeptide can be designed to target the brain. There is a pH difference on either side of the blood-brain barrier, with the brain pH being approximately 0.2 pH units lower than the blood pH. Thus, the first pH in the brain, at which the conditionally active polypeptide is more active, can be approximately 7.0-7.2 (brain pH), while the second pH can be approximately 7.4 (blood pH).

[0099] The conditionally active polypeptide may be an enzyme, a cytokine, a receptor, particularly a cellular receptor, a regulatory polypeptide, a soluble polypeptide, an antibody, or a hormone.

[0100] The conditionally active polypeptide may be a fragment of the parent polypeptide. For example, the conditionally active polypeptide may be an antibody fragment, a single-chain antibody, an enzyme fragment, a receptor fragment, a cytokine fragment, or a hormone fragment. The antibody fragment may be an Fc fragment of an antibody.

[0101] An Fc fragment can be used as a parent polypeptide to generate a conditionally active Fc fragment whose complement binding activity at a first pH is higher than that at a second pH. Complement binding of the Fc fragment can be used to mediate antibody-dependent cell-mediated cytotoxicity. The first pH can be acidic, ranging from 5.5 to 7.2 or 6.2 to 6.8, such as the pH of the tumor microenvironment, while the second pH is in the range of 7.2 to 7.6. The first pH differs from the pH of lysosomes, which are typically about 4.0. Furthermore, lysosomes are a target for degradation of Fc fragments, just like any other polypeptide. Lysosomes lack complement, and cell-mediated cytotoxicity cannot occur via lysosomes.

[0102] A conditionally active polypeptide may have two functional domains, at least one of which, preferably both, may have a pH-dependent activity. These two functional domains may be simultaneously evolved and selected to identify both functional domains in the same mutant polypeptide. Alternatively, these two functional domains may be independently evolved and selected to identify the pH-dependent activities separately. If the two functional domains are not in the same mutant polypeptide, they may be fused together to form a chimeric polypeptide containing both of the separately identified functional domains.

[0103] In one embodiment, the conditionally active polypeptide exhibits increased activity at a first pH relative to the parent polypeptide and decreased activity at a second pH relative to the parent polypeptide, both in the presence of a factor such as a protein. The protein may be a protein present in blood, human serum, or a biological microenvironment, such as a tumor microenvironment, an inflammatory site, etc. One suitable protein may be albumin, particularly a mammalian albumin such as bovine albumin or human albumin.

[0104] In one embodiment, a protein such as albumin is present in an assay solution used to screen and select conditionally active polypeptides from mutant polypeptides generated by the evolution process, hi another embodiment, an assay solution containing a protein such as albumin is also used to test for activity of the selected conditionally active polypeptides under the same or different conditions.

[0105] B. Engineering Conditionally Active Polypeptides The conditionally active polypeptide may be engineered by one or more of the protein engineering techniques described herein, non-limiting examples of which include conjugating the conditionally active polypeptide to a nucleic acid, conjugating the conditionally active polypeptide to a nanoparticle, engineering the conditionally active polypeptide in a chimeric antigen receptor, and engineering a masked conditionally active polypeptide.

[0106] The conditionally active polypeptide of the present invention can be conjugated to a nucleic acid molecule, such as a DNA or RNA molecule, via a linker. This conditionally active polypeptide can help deliver the nucleic acid molecule to a target location in a subject that has a condition in which the conditionally active polypeptide is more active than other locations where the condition does not exist. For example, the conditionally active polypeptide can be a conditionally active antibody that has higher binding activity to its antigen under conditions in the tumor microenvironment than under conditions in other locations, such as human serum. This effect can be used to deliver a nucleic acid molecule to the tumor microenvironment by conjugating the nucleic acid molecule to the conditionally active polypeptide and administering the conjugate to a subject.

[0107] In some embodiments, the nucleic acid molecule may be an agent that regulates the expression of a gene at a target location. Abnormal gene expression is associated with many diseases. Therefore, correcting abnormal gene expression may contribute to the control or even cure of these diseases. For example, abnormal gene expression is a characteristic of the majority of cancer cells, and in cancer cells, the expression levels of some genes, such as many oncogenes, are elevated (e.g., epidermal growth factor receptor 2 (HER2) is overexpressed in breast cancer cells). Selective inhibition of the constitutively elevated expression of oncogenes provides an opportunity to inhibit the proliferation of cancer cells.

[0108] Nucleic acid molecules that can inhibit gene expression include antisense RNA, small interfering RNA (siRNA), microRNA, oligo-DNA, and oligonucleotide mimetics with uncharged achiral polyamide backbones linking the nucleobases (Pooga et al., Curr Cancer Drug Targets, 1(3):231-9, 2001; Pandey et al., Expert Opin Biol Ther., 9(8):975-89, 2009).

[0109] Antisense RNA is a short RNA molecule that can bind to a specific complementary region of mRNA through base pairing to inhibit mRNA expression in a sequence-specific manner. Antisense RNA can contain RNase H, which can cleave the mRNA at the site of binding to the antisense RNA or physically block translation or other steps in mRNA processing and protein synthesis.

[0110] Small interfering RNAs (siRNAs) are typically short double-stranded RNA segments that are at least partially complementary to the mRNA sequence whose translation is to be blocked. siRNAs function through post-transcriptional mechanisms of gene silencing using chromatin remodeling, inhibition of protein translation, or direct mRNA degradation, and 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; Bertrand et al., "Comparison of antisense oligonucleotides and siRNAs in cell culture and in vivo," Biochem Biophys Res Commun., 296(4):1000-1004, 2002).

[0111] In particular, through RISC, siRNAs can initiate a potent cascade of sequence-specific degradation of mRNAs that share homology with the siRNA (Fire et al., "Potent and specific genetic interference by double-stranded RNA in Caenorhabditis elegans," Nature 391:806-811, 1998). When siRNAs are introduced into cells, they are processed by an RNase III enzyme called Dicer, which cleaves long siRNAs into short 21- to 23-nucleotide duplexes with symmetric 2- to 3-nucleotide 3' overhangs and 5' phosphate and 3' hydroxyl groups (Tuschl et al., "Targeted mRNA degradation by double-stranded RNA in vitro," Genes Dev. 13:3191-3197, 1999; Hamilton and Baulcombe, "A species of small antisense RNA in posttranscriptional gene silencing in plants," Science 286:950-952, 1999). Thus, effective siRNAs require only small segments of contiguous complementary sequence to pair with mRNA to initiate siRNA-mediated silencing (Jackson and Linsley, "Noise amidst the silence: off-target effects of siRNAs?" Trends Genet., 20:521-524, 2004). siRNAs do not integrate into the genome and therefore offer increased safety compared to plasmid or viral vehicles.

[0112] MicroRNAs (miRNAs) are a class of naturally occurring small, non-coding RNA molecules, 21–25 nucleotides in length. They are partially complementary to the mRNA molecules they act on. The primary function of microRNAs is to downregulate gene expression through translational repression, mRNA cleavage, and deadenylation. A central online repository of miRNA species, sequence data, annotations, and target predictions, called miRBase, is maintained by the Sanger Institute in the UK. When a microRNA gene is transcribed by RNA polymerase II, a pri-miRNA with a 5' cap and poly(A) tail is produced. In the nucleus, the pri-miRNA is processed by the microprocessor complex, consisting of the RNase III enzyme Drosha and the double-stranded RNA Pasha / DGCR8, to generate pre-miRNAs. These pre-miRNAs are transported into the cytoplasm by the karyopherin exportin (Exp5) and Ran-GTP complex, where Ran GTPase binds to Exp5 to form a nuclear heterotrimer with the pre-miRNA. These pre-miRNAs are further processed by the RNase III enzyme Dicer to generate mature microRNAs.

[0113] Another class of nucleic acids that can be delivered by conditionally active polypeptides are oligonucleotide mimetics containing an uncharged, achiral polyamide backbone to which the nucleobases are linked. These oligonucleotide mimetics are often referred to as peptide nucleic acids (PNAs). More specifically, PNAs are DNA analogs in which N-(2-aminoethyl)glycine polyamides replace the phosphate-ribose ring backbone, and methylene carbonyl linkers connect natural and unnatural nucleobases to the central amine of the N-(2-aminoethyl)glycine. Despite the radical change in backbone structure, PNAs possess sequence-specific binding to DNA and mRNA, following Watson-Crick base-pairing rules.

[0114] PNAs bind to complementary DNA / RNA with higher affinity than natural nucleic acids, due in part to the lack of negative backbone charges, resulting in reduced charge-charge repulsion, as well as favorable geometric factors. PNA-DNA / mRNA complexes are highly stable in biological fluids, leading to the inhibition of target gene transcription and translation by specifically hybridizing with DNA or mRNA. PNAs are generally synthesized using well-known solid-phase peptide synthesis protocols. Kim et al.,J.Am.Chem.Soc.,115,6477-6481,1993;Hyrup et al.,J.Am.Chem.Soc.,116,7964-7970,1994;Egholm et al.,Nature,365,566-568,1993;Dueholm et al.,New J.Chem.,21,19-31,1997;Wittung et al.,J.Am.Chem.Soc.,118,7049-7054,1996;Leijon et al.,Biochemistry,33,9820-9825,1994,Orum et al. al.,BioTechniques,19,472-480,1995;Tomac et al. (See, e.g., J. Am. Chem. Soc., 118, 5544-5552, 1996.) In contrast to DNA, which is depurinated when treated with strong acid and hydrolyzed in alkaline hydroxide, PNA is completely acid-stable and quite stable to weak bases.

[0115] Another class of nucleic acids that can be delivered by conditionally active polypeptides is oligo-DNA. Oligo-DNA is a short, single-stranded segment of DNA that can selectively inhibit the expression of genes containing sequences complementary to the oligo-DNA upon entry into cellular plasma. For antisense applications, oligo-DNA interacts with target mRNA or pre-mRNA to form a duplex, inhibiting its translation or processing, resulting in inhibition of protein biosynthesis. For antigen applications, oligo-DNA must enter the cell nucleus and form a triplex with double-stranded genomic DNA, inhibiting gene transcription, which in turn leads to reduced mRNA production and reduced protein gene product production.

[0116] Another class of nucleic acids that can be delivered by conditionally active polypeptides is spherical nucleic acids (SNAs; see Zhang, J Am Chem Soc., 134(40):16488-16491, 2012). SNAs comprise densely functionalized and highly oriented nucleic acids covalently attached to the surface of metallic, semiconducting, or insulating inorganic or polymeric core materials. SNAs may also be coreless hollow structures composed almost entirely of nucleic acid molecules. Such spherical nucleic acids have the ability to evade a subject's natural defenses against exogenous nucleic acids. Spherical nucleic acids utilize unique properties resulting from their densely packed, highly oriented nucleic acid shell to protect and efficiently deliver nucleic acids. Such shells create regions of high local salt concentration, which, combined with steric hindrance, reduce nuclease activity and protect nucleic acids from enzymatic degradation. In addition, these spherical nucleic acids recruit scavenger proteins from the natural extracellular environment to their surface, thereby facilitating endocytosis.

[0117] After entering the cytoplasm, spherical nucleic acid can inhibit the expression of target genes by either antisense or siRNA pathway.As a result, compared with viral vectors and many other synthetic systems, spherical nucleic acid offers several advantages, including low toxicity, low immunogenicity, resistance to enzymatic degradation, and more sustained gene knockdown.Conditionally active polypeptides, particularly conditionally active antibodies, can deliver spherical nucleic acid to target locations, such as diseased tissues or inflamed tissues (e.g., tumors and inflamed joints).

[0118] Conditionally active polypeptides can also be conjugated to nanoparticles via linkers to aid in delivery of the nanoparticles to target locations where the conditional active polypeptide is more active. Nanoparticles are known vehicles for toxins, radiopharmaceuticals, or other therapeutic agents, which can be encapsulated in the nanoparticles.

[0119] The therapeutic agent encapsulated in the nanoparticles may be a protein that can dedifferentiate tumor cells, potentially reversing tumor cell differentiation and reverting tumor cells to normal cells (Friedmann-Morvinski and Verma, "Dedifferentiation and reprogramming: origins of cancer stem cells," EMBO Reports, 15(3):244-253, 2014). By linking the nanoparticles to a conditionally active antibody, the linked nanoparticles and the encapsulated therapeutic agent can be selectively delivered to the environment where the conditionally active antibody is most active.

[0120] Several types of nanoparticles with different configurations may be used in the present invention. Nanoparticles may be made of various biocompatible materials, including biostable polymers, biodegradable polymers, fullerenes, lipids, or combinations thereof. A biostable polymer refers to a polymer that does not degrade in vivo. A biodegradable polymer refers to a polymer that can be degraded after delivery to a patient. For example, if the polymer is exposed to bodily fluids such as blood, the polymer may be gradually absorbed and / or excreted by enzymes in the body. Methods for producing nanoparticles with various degradation rates are known to those skilled in the art; see, for example, U.S. Pat. Nos. 6,451,338, 6,168,804, and 6,258,378.

[0121] Exemplary nanoparticles of the present invention include liposomes, polymersomes, and polymer particles. Liposomes refer to compartments completely surrounded by a bilayer, typically composed of phospholipids. Liposomes can be prepared according to standard techniques known to those skilled in the art. One technique involves suspending a suitable lipid, such as phosphatidylcholine, in an aqueous medium, followed by sonication of the mixture. Another technique involves rapidly mixing a solution of lipid in ethanol-water, for example, by injecting the lipid with a needle into a stirred ethanol-water solution. In some embodiments, liposomes can also, in addition to or instead of, lipids containing other amphiphiles, such as sphingomyelin or poly(ethylene glycol) (PEG).

[0122] Polymersomes contain diblock or triblock copolymers that can be modified to form bilayer structures similar to liposomes. Depending on the length and composition of the block copolymer, polymersomes can be substantially more robust than liposomes. In addition, because the chemistry of each block of the block copolymer can be controlled, the composition of the polymersome can be tailored to suit the desired application. For example, the membrane thickness of the polymersome, i.e., the thickness of the bilayer structure, can be controlled by varying the chain length of the individual blocks in the block copolymer. Tuning the glass transition temperature of each block of the copolymer can affect the fluidity and therefore permeability of the polymersome membrane. Varying the copolymer properties can even modify the release mechanism of the encapsulated drug.

[0123] Polymersomes can be prepared by a process that involves (i) dissolving a block copolymer in an organic solvent, (ii) applying the resulting solution to the vessel surface, and then (iii) removing the solvent, thereby leaving a thin film of the copolymer on the vessel wall. The thin film is then hydrated to form polymersomes. Alternatively, polymersomes can also be created by dissolving a block copolymer in a solvent and then adding a weak solvent for one of the blocks of the copolymer.

[0124] Therapeutic agents can be encapsulated in polymersomes using several techniques. For example, the therapeutic agent can be mixed into water and then used to rehydrate the copolymer thin film. Another example is by osmotically forcing the therapeutic agent into the core of a preformed polymersome, a process known as forced loading. Another example is by using the double emulsion method, which can produce polymersomes with relatively monodispersity and high loading efficiency. The double emulsion method involves using microfluidic technology to generate double emulsions containing water droplets surrounded by an organic solvent layer. This droplet-in-a-drop structure is then dispersed in a continuous aqueous phase. The block copolymer dissolves in the organic solvent and self-assembles at the concentric interface of the double emulsion to form protopolymersomes. The final polymersome is formed after complete evaporation of the organic solvent from the protopolymersome shell. This technique allows for fine control of the polymersome size. Additionally, the ability to keep the inner fluid completely separated from the outer fluid throughout the process allows for highly efficient encapsulation of therapeutic agents.

[0125] Polymer particles refer to solid or porous particles, in contrast to the shell structures of liposomes and polymersomes. Methods for attaching therapeutic agents to the surface of polymer particle structures or incorporating bioactive agents therein are known to those skilled in the art.

[0126] Polymers that can be used to prepare the nanoparticles of the present invention include, but are not limited to, poly(N-acetylglucosamine) (chitin), chitosan, poly(3-hydroxyvalerate), poly(lactide-co-glycolide), poly(3-hydroxybutyrate), poly(4-hydroxybutyrate), poly(3-hydroxybutyrate-co-3-hydroxyvalerate), polyorthoesters, polyanhydrides, poly(glycolic acid), poly(glycolide), poly(L-lactic acid), poly(L-lactide), poly(D,L-lactic acid), poly(D,L-lactide). , poly(L-lactide-co-D,L-lactide), poly(caprolactone), poly(L-lactide-co-caprolactone), poly(D,L-lactide-co-caprolactone), poly(glycolide-co-caprolactone), poly(trimethylene carbonate), polyesteramides, poly(glycolic acid-co-trimethylene carbonate), co-poly(ether esters) (e.g., PEO / PLA), polyphosphazenes, biomolecules (fibrin, fibrin glue, fibrinogen, cellulose, starch, collagen, and hyaluron) acid, elastin and hyaluronic acid, etc.), polyurethanes, silicones, polyesters, polyolefins, polyisobutylene and ethylene-alpha-olefin copolymers, acrylic polymers and copolymers other than polyacrylates, halogenated vinyl polymers and copolymers (such as polyvinyl chloride), polyvinyl ethers (such as polyvinyl methyl ether), halogenated polyvinylidenes (such as polyvinylidene chloride), polyacrylonitrile, polyvinyl ketones, polyvinyl aromatics (such as polystyrene), polyvinyl esters (poly vinyl acetate, acrylonitrile-styrene copolymer, acrylonitrile butadiene styrene (ABS) resin, polyamides (such as nylon 66 and polycaprolactam), polycarbonates including tyrosine-based polycarbonates, polyoxymethylenes, polyimides, polyethers, polyurethanes, rayon, rayon triacetate, cellulose, cellulose acetate, cellulose butyrate, cellulose acetate butyrate, cellophane, cellulose nitrate, cellulose propionate, cellulose ethers, and carboxymethyl cellulose.

[0127] In some embodiments, nanoparticles can also provide tissue selectivity through coating, in addition to the selectivity derived from the conditionally active polypeptide. For example, nanoparticles can be coated with an electrostatically adsorbed poly(glutamic acid)-based peptide coating to alter the outer composition of the core particle. A negatively charged polyglutamic acid-based peptide containing arginine-glycine-aspartic acid (RGD) ligand can increase in vitro gene delivery to endothelial cells compared to coated particles with a scrambled sequence containing RGD instead of RGD. This peptide consists of three components: a stretch of poly(glutamic acid) that provides a negative charge, a polyglycine linker, and terminal sequences with variable charge that may alter the particle's biophysical properties and tissue selectivity. This coating, as well as the particle itself, are biodegradable through their amide and ester bonds, respectively. See Harris et al. ("Tissue-Specific Gene Delivery via Nanoparticle Coating," Biomaterials, vol. 31, pp. 998-1006, 2010).

[0128] T cells are used by the mammalian immune system to fight substances or cells that carry foreign antigens. When T cells encounter solid tumors, they are often unable to mount an effective response. Even if T cells reach the tumor site, they face a barrage of immunosuppressive factors that allow cancer cells to escape the immune system. CAR-T technology involves reprogramming naturally occurring circulating T cells by inserting a chimeric antigen receptor (CAR) into the T cell using genetic engineering methods to create highly specific CAR-T cells. The CAR specifically binds to antigens on the surface of the target tissue, thereby directing the engineered CAR-T cells to the target tissue. Therefore, CAR-T cells can specifically target tumor cells, making them far more effective than naturally occurring circulating T cells. CAR-T cells can also be engineered to target other target tissues, such as inflamed joints and brain tissue.

[0129] The CARs of the present invention comprise at least one antigen-specific targeting region (ASTR), an extracellular spacer domain (ESD), a transmembrane domain (TM), one or more costimulatory domains (CSDs), and an intracellular signaling 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 a target antigen on a tumor or other target tissue, the ISD activates intracellular signaling in the CAR-T cell. For example, the ISD utilizes the antigen-binding properties of antibodies to redirect CAR-T cell specificity and reactivity to a target of choice (e.g., tumor cells or other target cells) in an MHC-independent manner. This MHC-independent antigen recognition allows CAR-T cells to recognize tumor cells and initiate antigen processing, thus circumventing a major mechanism of tumor escape from immune system surveillance. In certain embodiments, the ESD and / or CSD are optional. In another embodiment, the ASTR is bispecific and is therefore capable of specifically binding to two different antigens or epitopes.

[0130] The conditionally active polypeptides of the present invention may be engineered as ASTRs or portions thereof, such that the CAR is more active in binding to a target antigen in a specific environment, such as a tumor microenvironment or synovial fluid, than in blood or another body site where a different environment exists. Such CARs can preferentially deliver T cells to the disease site, thereby dramatically reducing the side effects caused by T cells attacking normal tissues. This allows for the use of higher doses of T cells, increasing therapeutic efficacy and improving the subject's tolerance of the treatment.

[0131] These CARs are particularly useful for developing novel therapeutics that require a short or limited time in the subject's body. Beneficial applications include systemic treatments at high doses as well as local treatments at high concentrations. See Maher, "Immunotherapy of Malignant Disease Using Chimeric Antigen Receptor Engrafted T Cells," ISRN Oncology, vol. 2012, article ID 278093, 2012.

[0132] ASTRs can include conditionally active polypeptides, such as antibodies, particularly single-chain antibodies, or fragments thereof, that specifically bind to antigens on tumors or other target tissues. Some examples of polypeptides suitable for ASTRs include linked cytokines (which provide for recognition of cells bearing cytokine receptors), affibodies, ligand-binding domains from naturally occurring receptors, and soluble protein / peptide ligands for receptors, for example, on tumor cells. Virtually any molecule capable of binding with high affinity to a given antigen can be used as an ASTR.

[0133] In some embodiments, the CAR of the present invention comprises at least two ASTRs that target at least two different antigens or two epitopes on the same antigen. In one embodiment, the CAR comprises three or more ASTRs that target at least three or more different antigens or epitopes. When multiple ASTRs are present in a CAR, the ASTRs may be arranged in tandem and separated by a linker peptide (Figure 5).

[0134] In yet another embodiment, the ASTR comprises a diabody, in which the two variable regions are too short to fold together and so an scFv is created with a linker peptide that drives dimerization of the scFv. Even shorter linkers (1 or 2 amino acids) lead to the formation of trimers, also known as triabodies or tribodies. Tetrabodies can also be used in ASTRs.

[0135] Antigens targeted by CARs are present on the surface or inside cells of tissues targeted for removal, such as tumors, glandular (e.g., prostate) hyperplasia, warts, and unwanted adipose tissue. Surface antigens are more efficiently recognized and bound by the ASTR of CARs, but intracellular antigens can also be targeted by CARs. In some embodiments, the target antigen is preferably specific to cancer, inflammatory disease, neuronal disorder, diabetes, cardiovascular disease, or infectious disease. Examples of target antigens include antigens expressed by various immune cells, carcinomas, sarcomas, lymphomas, leukemias, germ cell tumors, blastomas, and cells associated with various hematological, autoimmune, and / or inflammatory diseases.

[0136] Cancer-specific antigens that can be targeted by ASTR include 4-IBB, 5T4, adenocarcinoma antigen, alpha-fetoprotein, BAFF, B lymphoma cells, C242 antigen, CA-125, carbonic anhydrase 9 (CA-IX), C-MET, CCR4, CD152, CD19, CD20, CD200, CD22, CD221, CD23 (IgE receptor), CD28, CD30 (TNFRSF8), CD33, CD4, CD40, and CD44. v6, CD51, CD52, CD56, CD74, CD80, CEA, CTLA-4, DR5, EGFR, EpCAM, CD3, FAP, fibronectin extra domain-B, folate receptor 1, GD2, GD3 ganglioside, glycoprotein 75, GPNMB, HER2 / neu, HGF, human scatter factor receptor kinase, IGF-1 receptor, IGF-I, IgG1, LI-CAM, IL-13, IL-6, insulin-like growth factor I receptor, integrin α5β1, integrin ανβ3, MORAb-009, MS4A1, MUC1, mucin CanAg, N-glycolylneuraminic acid, NPC-1C, PDGF-Rα, PDL192, phosphatidylserine, prostate cancer cells, RANKL, RON, ROR1, SCH 900105, SDC1, SLAMF7, TAG-72, tenascin C, TGFβ2, TGF-β, TRAIL-R1, TRAIL-R2, tumor antigen CTAA16.88, VEGF-A, VEGFR-1, VEGFR2 or vimentin.

[0137] Antigens specific to inflammatory diseases that can be targeted by ASTR include AOC3 (VAP-1), CAM-3001, CCL11 (eotaxin-1), CD125, CD147 (basigin), CD154 (CD40L), CD2, CD20, CD23 (IgE receptor), CD25 (IL-2 receptor α 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 α4, integrin α4β7, Lama glama, LFA-1 (CD11a), MEDI-528, myostatin, OX-40, and rhuMAb. β7, scleroscin, SOST, TGFβ1, TNF-α or VEGF-A.

[0138] Antigens specific to neuronal disorders that can be targeted by ASTRs of the present invention include one or more of beta amyloid or MABT5102A. Antigens specific to diabetes that can be targeted by ASTRs of the present invention include one or more of L-Iβ or CD3. Antigens specific to cardiovascular diseases that can be targeted by ASTRs of the present invention include one or more of C5, cardiac myosin, CD41 (integrin α-lib), fibulin II, beta chain, ITGB2 (CD18), and sphingosine-1-phosphate.

[0139] Antigens specific to infectious diseases that can be targeted by the ASTRs of the invention include one or more of anthrax toxin, CCR5, CD4, clumping factor A, cytomegalovirus, cytomegalovirus glycoprotein B, endotoxin, Escherichia coli, hepatitis B surface antigen, hepatitis B virus, HIV-1, Hsp90, influenza A hemagglutinin, lipoteichoic acid, Pseudomonas aeruginosa, rabies virus glycoprotein, respiratory syncytial virus, and TNF-α.

[0140] Further examples of target antigens include surface proteins found in specific or amplified form on cancer cells, such as the IL-14 receptor, CD19, CD20, and CD40 in B-cell lymphomas, Lewis Y and CEA antigens in various cancers, Tag72 antigen in breast and colorectal cancers, EGF-R in lung cancer, folate-binding protein and HER-2 protein, which are often amplified in human breast and ovarian cancers, or viral proteins, such as the gp120 and gp41 envelope proteins of HIV, envelope proteins from hepatitis B and C viruses, glycoprotein B and other envelope glycoproteins of human cytomegalovirus, and envelope proteins from oncoviruses such as Kaposi's sarcoma-associated herpesvirus. Other potential target antigens include CD4, whose ligand is the HIV gp120 envelope glycoprotein, and other viral receptors, such as ICAM, the receptor for human rhinovirus, and related receptor molecules for poliovirus.

[0141] In another embodiment, the CAR can target an antigen associated with cancer-treating cells, such as NK cells, to activate those cancer-treating cells by acting as immune effector cells. One example of this is a CAR that targets the CD16A antigen to engage NK cells to combat CD30-expressing malignancies. The bispecific tetravalent AFM13 antibody is an example of an antibody that can achieve this effect. For further details of this type of embodiment, see, for example, Rothe, A., et al., "A phase 1 study of the bispecific anti-CD30 / CD16A antibody construct AFM13 in patients with relapsed or refractory Hodgkin lymphoma," Blood, 25 June 2015, Vol. 125, no. 26, pp. 4024-4031.

[0142] In some embodiments, the extracellular spacer domain and transmembrane domain may be ubiquitination-resistant, which can enhance CAR-T cell signaling and thus increase anti-tumor activity (Kunii et al., "Enhanced function of redirected human t cells expressing linker for activation of t cells that is resistant to ubiquitylation," Human Gene Therapy, vol. 24, pp. 27-37, 2013). Within this region, the extracellular spacer domain is on the outside of the CAR-T cell and therefore exposed to different conditions, potentially allowing for conditional ubiquitination resistance.

[0143] C. Engineering Masked Conditionally Active Polypeptides The conditionally active polypeptides of the present invention, particularly conditionally active antibodies, may have their conditional activity masked by a masking moiety, and / or the activity of their conjugated drugs may be masked. The masked activity may become available when the masking moiety is removed or cleaved from the conditionally active polypeptide. Suitable masking techniques are described, for example, in Desnoyers et al., "Tumor-Specific Activation of an EGFR-Targeting Probody Enhances Therapeutic Index," Sci. Transl. Med. 5, 207ra144, 2013.

[0144] In some embodiments, the conditionally active antibody is linked to a masking moiety that masks the conditional activity and / or the activity of its conjugated drug. For example, when a conditionally active antibody is coupled to a masking moiety, such coupling or modification may result in a conformational change that reduces or inhibits the ability of the conditionally active antibody to specifically bind to its antigen. When the conditionally active antibody reaches the target tissue or microenvironment, the masking moiety is cleaved by an enzyme present in the target tissue or microenvironment, thereby releasing the masked activity. For example, the enzyme may be a protease that is generally active in the tumor microenvironment, which may cleave the masking moiety to release the conditionally active antibody that is active in the tumor tissue.

[0145] In some embodiments, the activity is masked to less than about 50% of the original activity, or less than about 30% of the original activity, or less than about 10% of the original activity, or less than about 5% of the original activity, or less than about 2% of the original activity, or less than about 1% of the original activity, or less than about 0.1% of the original activity, or less than about 0.01% of the original activity. In some embodiments, for example, to allow adequate time for delivery, the masking effect is designed to last at least 2, 4, 6, 8, 12, 28, 24, 30, 36, 48, 60, 72, 84, 96 hours, or 5, 10, 15, 30, 45, 60, 90, 120, 150, 180 days, or 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 months or more as measured in vivo or in a target transfer in vitro immunoabsorption assay.

[0146] In certain embodiments, the masking moiety is structurally similar to the natural binding partner (antigen) of the conditionally activated antibody. The masking moiety may be a modified version of the natural binding partner of the conditionally activated antibody that contains amino acid changes that at least slightly reduce the affinity and / or avidity of binding to the conditionally activated antibody. In some embodiments, the masking moiety contains no or substantially no homology to the natural binding partner of the conditionally activated antibody. In other embodiments, the masking moiety shares no more than 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, or 80% sequence identity with the natural binding partner of the conditionally activated antibody.

[0147] The masking moiety can be provided in a variety of different forms. In certain embodiments, the masking moiety can be a known binding partner of the conditionally activated antibody, provided that the masking moiety binds to the conditionally activated antibody with lower affinity and / or avidity than the target protein targeted by the conditionally activated antibody after cleavage of the masking moiety, thereby reducing interference of the masking moiety with the desired target. Thus, the masking moiety preferably masks the conditionally activated antibody from target binding before cleavage of the masking moiety, but does not substantially or significantly interfere with or compete for binding of the activated molecule to the target after cleavage of the masking moiety from the antibody. In specific embodiments, the conditionally activated antibody and the masking moiety do not comprise the amino acid sequence of a naturally occurring binding partner pair, such that at least one of the conditionally activated antibody and the masking moiety does not have the amino acid sequence of a member of a naturally occurring binding partner.

[0148] Alternatively, the masking moiety may not specifically bind to the conditionally active antibody, but rather may prevent conditionally active antibody-target binding through non-specific interactions, such as steric hindrance. For example, the masking moiety may be located such that the structure or conformation of the antibody allows the masking moiety to mask the conditionally active antibody, e.g., through charge-based interactions, thereby preventing the target from reaching the conditionally active antibody.

[0149] In some embodiments, the masking moiety is coupled to the conditionally active antibody by a covalent bond. In another embodiment, the conditionally active antibody is prevented from binding to its target by attaching a masking moiety to the N-terminus of the conditionally active antibody. In yet another embodiment, the conditionally active antibody is coupled to the masking moiety by a cysteine-cysteine ​​disulfide bridge between the masking moiety and the conditionally active antibody.

[0150] In some embodiments, the conditionally active antibody is further coupled to a cleavable moiety (CM). The CM has the ability to be cleaved by an enzyme, or the CM has the ability to be reduced by a reducing agent, or the CM has the ability to be photolyzed. In one embodiment, the amino acid sequence of the CM overlaps with or can 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 can facilitate masking of the conditionally active antibody before cleavage. Once the CM is cleaved, the conditionally active antibody becomes more active in binding to its antigen.

[0151] The CM may be a substrate for an enzyme that coexists with the target antigen at the treatment site in the subject. Alternatively, or in addition, the CM may have a cleavable intercysteine ​​disulfide bond as a result of disulfide bond reduction. The CM may also be a photolabile substrate that can be activated by a light source.

[0152] The desired target tissue of the conditionally active antibody should preferentially contain an enzyme that cleaves the CM, where the conditionally active antibody is more active under conditions (abnormal conditions) present in the target tissue, such as diseased or tumor tissue. For example, there are known proteases whose levels are increased in several cancers, such as solid tumors. See, e.g., La Rocca et al., (2004) British J. of Cancer 90(7):1414-1421. Non-limiting examples of such diseases include all types of cancer (breast cancer, lung cancer, colorectal cancer, prostate cancer, head and neck cancer, pancreatic cancer, etc.), rheumatoid arthritis, Crohn's disease, melanoma, SLE, cardiovascular injury, ischemia, etc. Therefore, a suitable CM can be selected that contains a peptide substrate that can be cleaved by a protease present in tumor tissue, particularly a protease that is present at higher levels in tumor tissue compared to non-cancerous tissue.

[0153] In some embodiments, CM can be a substrate for an enzyme selected from legumain, plasmin, TMPRSS-3 / 4, MMP-9, MT1-MMP, cathepsin, caspase, human neutrophil elastase, β-secretase, uPA, and PSA. Enzymes that cleave CM are present at relatively higher levels in target tissues at the treatment site (e.g., diseased or tumor tissues; e.g., those targeted for therapeutic or diagnostic treatment) compared to tissues at non-treatment sites (e.g., healthy tissues). Thus, in addition to the conditional activity of the antibody, which may be more active in diseased or tumor tissues, enzymes present in diseased or tumor tissues can cleave CM, further enhancing the activity of the conditionally active antibody or the activity of the conjugated drug. Unmodified or uncleaved CM can enable efficient inhibition or masking of the activity of the conditionally active antibody, which therefore has low activity in normal tissues (normal physiological conditions). The dual mechanisms (conditional activity and masking moieties) that inhibit the activity of conditionally active antibodies in normal tissues allow for the use of much higher dosages of conditionally active antibodies without causing significant adverse effects.

[0154] In some embodiments, the CM can be a substrate for an enzyme selected from the enzymes listed in Table 1 below.

[0155] [Table 1]

[0156] Alternatively or additionally, the CM may contain a disulfide bond of a cysteine ​​pair, which is therefore cleavable by reducing agents such as cellular reducing agents that may be present in abundance in or around solid tumor tissues, including glutathione (GSH), thioredoxin, NADPH, flavins, ascorbate, etc.

[0157] In some embodiments, the conditionally active antibody contains both a CM and a masking moiety. The activity of the conditionally active antibody is unmasked when the CM is cleaved by an enzyme. In some embodiments, it may be desirable to insert one or more linkers, such as flexible linkers, between the antibody, the masking moiety, and the CM to confer flexibility. For example, the masking moiety and / or the CM may not contain a sufficient number of residues (e.g., Gly, Ser, Asp, Asn, particularly Gly and Ser, particularly Gly) to confer the desired flexibility. Therefore, it may be beneficial to introduce one or more amino acids to provide a flexible linker. For example, a masked conditionally active antibody may have the following structure (where the following formula represents the amino acid sequence in either the N-terminal to C-terminal direction or the C-terminal to N-terminal direction): (MM)-L1-(CM)-(AB) (MM)-(CM)-L1-(AB) (MM)-L1-(CM)-L2-(AB) Cyclo[L1-(MM)-L2-(CM)-L3-(AB)] wherein MM is a masking moiety, AB is a conditionally active antibody; L1, L2, and L3 each independently represent, and optionally are present or absent, the same or different flexible linkers comprising at least one flexible amino acid (e.g., Gly); and cyclo, when present, the entire structure is in the form of a cyclic structure due to the presence of disulfide bonds between cysteine ​​pairs at or near both the N-terminus and C-terminus of the structure.

[0158] Linkers suitable for use in the present invention generally confer flexibility to the masking moiety, facilitating inhibition of the activity of a conditionally active antibody. Such linkers are generally referred to as flexible linkers. Suitable linkers can be readily selected and may be of any suitable length, such as 1 amino acid (e.g., Gly) to 20 amino acids, 2 to 15 amino acids, 3 to 12 amino acids, e.g., 4 to 10 amino acids, 5 to 9 amino acids, 6 to 8 amino acids, or 7 to 8 amino acids, and may be 1, 2, 3, 4, 5, 6, or 7 amino acids.

[0159] Exemplary flexible linkers include glycine polymers (G) n , glycine-serine polymers (e.g., (GS) n , (GSGGS) n and (GGGS) n where n is an integer of at least 1. Exemplary flexible linkers include glycine-alanine polymers, alanine-serine polymers, and other flexible linkers known in the art, including: Gly-Gly-Ser-Gly, Gly-Gly-Ser-Gly-Gly, Gly-Ser-Gly-Ser-Gly, Gly-Ser-Gly-Gly, Gly-Ser-Gly-Gly, Gly-Ser-Gly-Gly, Gly-Ser-Ser-Gly, and the like.

[0160] Some of the techniques used to mask the activity of conditionally active antibodies are described in WO2010081173A2.

[0161] The present disclosure provides a method for preparing a conditionally active polypeptide from a parent polypeptide, such as a wild-type polypeptide or a therapeutic polypeptide. The method includes evolving DNA encoding the parent polypeptide using one or more evolutionary techniques to create a mutant DNA; expressing the mutant DNA to obtain a mutant polypeptide; subjecting the mutant polypeptide and the parent polypeptide to an assay under a first condition and an assay under a second condition; and selecting from the mutant polypeptides a conditionally active polypeptide that exhibits both (a) a decrease in activity in the assay under the first condition compared to the parent polypeptide, and (b) an increase in activity in the assay under the second condition compared to the parent polypeptide. The assay under the first condition and the assay under the second condition are performed in an assay solution containing at least one component selected from inorganic compounds, ions, and organic molecules. In some embodiments, the first condition is a normal physiological condition, and the second condition is an abnormal condition.

[0162] Conditionally active polypeptides are reversibly or irreversibly inactivated under a first condition or normal physiological conditions, but remain active under a second condition or abnormal condition at the same or equivalent level as under the first condition or normal physiological conditions. These conditionally active biological polypeptides and methods for producing them are described in U.S. Patent No. 8,709,755 B2. Conditionally active biological polypeptides are particularly valuable for the development of novel therapeutic agents that are active in a host for only a short or limited period of time. This is particularly valuable for the extended action of administered therapeutic agents, where limited activity is necessary to effect the desired treatment, even though they may be harmful to the host. Examples of useful applications include local or systemic treatment at high doses, as well as local treatment at high concentrations. Inactivation under a first condition or normal physiological conditions may be determined by the administration combination and the rate of inactivation of the polypeptide. This condition-based inactivation is particularly important in enzyme therapy, where catalytic activity can cause substantial negative effects in a relatively short period of time.

[0163] The present disclosure also relates to methods for engineering or evolving parent polypeptides to generate conditionally active polypeptides that are reversibly or irreversibly activated or inactivated over time, or that are activated or inactivated only when present in certain microenvironments of the body, including specific organs of the body (such as the tumor microenvironment, synovial fluid, bladder, or kidney). In some embodiments, the conditionally active polypeptides are antibodies or antibody fragments directed against one or more target proteins (antigens) as described herein.

[0164] The conditionally active polypeptide can be an isolated polypeptide having a pH-dependent activity, wherein the activity at a first pH is dependent on the activity of histidine, histamine, adenosine diphosphate, adenosine triphosphate, citrate, or the like. ion , bicarbonate ion , acetic acid ion , lactic acid ion , disulfide ion , hydrogen sulfide, ammonium ion , dihydrogen phosphate ion and any combination thereof, the activity at a first pH is at least about 1.3-fold greater than the activity at a second pH. The same activity is not pH-dependent in the absence of the small molecule. In some embodiments, the activity at a first pH is 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-fold greater than the same activity at a second pH. The first pH may be an abnormal pH in the range of about 5.5 to 7.2, or about 6.2 to 6.8, while the second pH may be a normal physiological pH in the range of about 7.2 to 7.6.

[0165] D. Parent Polypeptide The parent polypeptide may be a wild-type polypeptide, including a non-naturally occurring polypeptide, a mutant polypeptide derived from the wild-type polypeptide, such as a therapeutic polypeptide, a chimeric polypeptide derived from a different wild-type polypeptide, or even a synthetic polypeptide. The parent polypeptide may be selected from an antibody, an enzyme, a cytokine, a regulatory protein, a hormone, a receptor, a ligand, a biosimilar, an immunomodulator, a growth factor, and fragments of these polypeptides.

[0166] A description of suitable wild-type polypeptides and how they can be evolved and selected to produce conditionally active polypeptides is provided in US Pat. No. 8,709,755 B2.

[0167] In some embodiments, the parent polypeptide may be selected from a library of wild-type or mutant polypeptides, such as a bacteriophage display library. In such embodiments, a large number of candidate polypeptides are expressed in the bacteriophage library, particularly by surface display techniques. The candidate polypeptides from the library are screened for suitable parent polypeptides. A typical bacteriophage library may contain thousands or even millions of bacteriophages expressing candidate polypeptides in a bacterial host. In one embodiment, the bacteriophage library may contain a plurality of bacteriophages.

[0168] To construct a bacteriophage library, typically, a filamentous bacteriophage, such as the filamentous coliphage M13, is genetically modified by inserting an oligonucleotide encoding a candidate polypeptide into the coding sequence of one of the bacteriophage coat proteins. The bacteriophage coat protein is then expressed along with the candidate polypeptide, resulting in the display of the candidate polypeptide on the surface of the bacteriophage particle. The displayed candidate polypeptide can then be screened for suitable parent polypeptides.

[0169] One common technique for screening for suitable parent polypeptides is by immobilizing bacteriophage particles containing the desired candidate polypeptide on a support. The support can be a plastic plate coated with a "bait" capable of binding to the desired candidate polypeptide. Unbound bacteriophage particles can be washed off the plate. Bacteriophage particles bound to the plate (along with the desired candidate) are eluted by washing, and the eluted bacteriophage particles are amplified with bacteria. The sequences encoding the candidate polypeptide of selected bacteriophage particles can then be determined by sequencing. The relationship between the candidate polypeptide and the bait can be, for example, a ligand-receptor or antigen-antibody relationship.

[0170] Another common technique for screening for suitable parent polypeptides is by using enzymatic assays of individual bacteriophage clones for the desired enzymatic activity exhibited by the candidate polypeptide. Depending on the specific enzymatic activity, one skilled in the art can design an appropriate assay to screen for parent polypeptides with the desired level of enzymatic activity.

[0171] In some embodiments, a bacteriophage library is provided as an array, with each bacteriophage clone occupying a specific location on the array. Such an array may be provided on a solid support, such as a membrane, agar plate, or microtiter plate, where each bacteriophage clone in the library is disposed or attached to a specific, predetermined location on the solid support. In the case of an agar plate, the plate preferably contains a bacterial growth medium to support bacterial growth. When the array is provided on a membrane, such as a nitrocellulose or nylon membrane, the bacterial culture is added to the membrane, and the membrane is immersed in a nutrient growth medium. In addition, bacteriophage clones may also be provided on beads, in which case a single bacteriophage clone can be attached to a single bead. Alternatively, the bacteriophage clones may each be provided on the end of an optical fiber, where the fiber is used to optically transmit ultraviolet radiation from a light source.

[0172] A typical bacteriophage library contains 10 6 ~10 10 The library may include multiple bacteriophages, each distinguished by a coat protein carrying a different polypeptide (e.g., gp3 or gp8 in the case of phage M13). Bacterial hosts for bacteriophage libraries may be selected from bacterial genera including, for example, Salmonella, Staphylococcus, Streptococcus, Shigella, Listeria, Campylobacter, Klebsiella, Yersinia, Pseudomonas, and Escherichia.

[0173] Oligonucleotides encoding candidate polypeptides can be a collection of cDNAs encoding wild-type polypeptides. Methods for synthesizing cDNA from biological samples capable of expressing suitable parent polypeptides are known. Any genetic information that exhibits physiological activity through transcription can be collected as cDNA. When producing cDNA, it is essential to synthesize full-length cDNA. There are several methods that can be used to synthesize full-length cDNA. For example, suitable methods include a method using yeast or HeLa cell cap-binding protein to label the 5' cap site (I. Edery et al., "An Efficient Strategy To Isolate Full-length cDNAs Based on a mRNA Cap Retention Procedure (CAPture)", Mol. Cell. Biol., vol. 15, pp. 3363-3371, 1995); and a method in which phosphates from incomplete cDNAs that do not have a 5' cap are removed using alkaline phosphatase, and then the entire cDNA is treated with a tobacco mosaic virus decapping enzyme, so that only full-length cDNAs have phosphates (K. Maruyama et al., "Oligo-capping: a simple method to replace the cap structure of eukaryotic mRNAs with oligoribonucleotides", Gene, vol. 138, pp. 171-174, 1995 and S. Kato et al., "Construction of a human full-length cDNA bank", Gene, vol. 150, pp. 171-174, 1995). 243-250, 1995).

[0174] In embodiments in which the parent polypeptide is an antibody, a library of candidate antibodies can be generated using recombinant antibodies derived from an organism's complete antibody repertoire. Genetic information representing the repertoire can be assembled into a large collection of complete antibodies, which can then be screened for suitable parent antibodies with desired antigen-binding activity and / or one or more other functional properties. In some embodiments, B cells from an animal immunized with an antigen are isolated, such as an immunized human, mouse, or rabbit. mRNA is recovered from the isolated B cells, converted to cDNA, and then sequenced. The most frequently occurring cDNA fragments encoding the light chain and the most frequently occurring cDNA fragments encoding the heavy chain are assembled to form an antibody. In one embodiment, the 100 most frequently occurring cDNA fragments encoding the light chain and the 100 most frequently occurring cDNA fragments encoding the heavy chain are assembled to generate candidate antibodies. In another embodiment, the most frequently occurring cDNA fragments only encode the variable regions of the heavy and light chains and are assembled to generate antibody fragments containing only the variable regions and no constant regions.

[0175] In some embodiments, a cDNA fragment encoding the variable region of an IgG heavy chain is assembled with the most frequently occurring variable region of an IgK or IgK light chain, and the assembled antibody contains a heavy chain variable region derived from an IgG and a light chain variable region derived from an IgK or IgK.

[0176] The cDNAs encoding the assembled antibodies are then cloned and expressed, preferably in a plate-based format. The binding activity of the expressed antibodies may be analyzed by bead-based ELISA assay, and suitable parent antibodies may be selected based on the ELISA assay. The cDNAs encoding the assembled antibodies may also be expressed in a bacteriophage display library, which may then be screened for one or more desirable parent antibodies by any one of the techniques disclosed herein.

[0177] In embodiments where the parent polypeptide is an antibody, the parent antibody preferably has at least one particular property that facilitates its evolution into a conditionally active antibody. In certain embodiments, the parent antibody may have similar binding activity and / or properties under both normal and abnormal physiological conditions. In such embodiments, the parent antibody is selected based on having the most similar binding activity and / or the most similar combination of one or more properties under both normal and abnormal physiological conditions. For example, if the normal and abnormal physiological conditions are pH 7.4 and pH 6.0, respectively, the parent antibody with the most similar binding activity at pH 7.4 and pH 6.0 may be selected over an antibody with a less similar binding activity at pH 7.4 and pH 6.0.

[0178] E. Identification of Conditionally Active Polypeptides After selection of a parent polypeptide, the DNA encoding the parent polypeptide can be evolved using suitable mutagenesis techniques to generate mutant DNAs that can then be expressed to generate mutant proteins that are screened to identify conditionally active polypeptides. In some embodiments, evolution can be minimal, e.g., only a few mutations are introduced into the parent polypeptide to generate mutant polypeptides with the desired conditional activity. For example, introducing fewer than about 20 changes, and sometimes fewer than about 18 changes at each site by comprehensive positional evolution (CPE), can be sufficient to generate a suitable conditionally active polypeptide. For comprehensive positional synthesis (CPS), a combination of fewer than about 6 upmutations, or fewer than about 5 upmutations, or fewer than about 4 upmutations, or fewer than about 3 upmutations, or fewer than about 2 upmutations in the parent polypeptide can be sufficient to generate the desired conditionally active polypeptide.

[0179] In some embodiments, if the library of candidate polypeptides (e.g., a bacteriophage library and / or a recombinant antibody library) is sufficiently large, the evolution and expression steps may not be necessary. Such large-scale libraries may contain candidate polypeptides with conditionally active properties (i.e., having both low activity in an assay under normal physiological conditions and high activity in an assay under abnormal conditions, both compared to a reference polypeptide, or having lower activity in an assay under normal physiological conditions compared to an assay under abnormal conditions). In these embodiments, the candidate polypeptides in the library are subjected to a selection step to identify conditionally active polypeptides that have lower activity in an assay under normal physiological conditions compared to the same polypeptide in an assay under abnormal conditions. In one embodiment, the candidate polypeptides in the library are subjected to separate assays under normal physiological conditions and abnormal conditions, along with a reference polypeptide. Conditionally active polypeptides selected from the library are those that exhibit lower activity under normal physiological conditions and higher activity of the same polypeptide under abnormal conditions, both compared to the reference polypeptide. In this embodiment, the library is large enough that candidate polypeptides with conditionally active properties already exist in the library. It is not necessary to evolve parent polypeptides to discover conditionally active polypeptides.

[0180] In some embodiments, the reference polypeptide may not be conditionally active in that it has similar or identical activity under both normal and abnormal physiological conditions. The reference polypeptide may be a polypeptide homologous to the candidate polypeptide in the library, such as a homologous enzyme, cytokine, regulatory protein, antibody, hormone, or functional peptide. The reference polypeptide may also be a homologous tissue plasminogen activator, streptokinase, urokinase, renin, hyaluronidase, calcitonin gene-related peptide (CGRP), substance P (SP), neuropeptide Y (NPY), vasoactive intestinal peptide (VTP), vasopressin, or angiostatin. For example, if the library contains a large number of candidate antibodies against a certain antigen, the reference polypeptide may be an antibody against the same antigen that has the same or similar binding activity to that antigen under both normal and abnormal physiological conditions.

[0181] Thus, in one embodiment, candidate polypeptides in a library are individually assayed under normal physiological conditions and under abnormal conditions along with a reference polypeptide, and conditionally active polypeptides are selected from the library that exhibit both (a) decreased activity under normal physiological conditions relative to the reference polypeptide, and (b) increased activity under abnormal conditions relative to the reference polypeptide.

[0182] F. Methods for Producing Conditionally Active Polypeptides The DNA encoding the parent polypeptide is evolved using one or more mutagenesis techniques to create mutant DNA; the mutant DNA is expressed to produce mutant polypeptides; and the mutant polypeptides are subjected to a screening assay under a first condition, which may be a normal physiological condition, and a second condition, which may be an abnormal condition. A conditionally active polypeptide is selected from mutant polypeptides that exhibit both (a) a decrease in activity in the assay under the first condition compared to the parent polypeptide, and (b) an increase in activity in the assay under the second condition compared to the parent polypeptide. The decrease in activity of the conditionally active polypeptide under the first condition or normal physiological condition may be reversible or irreversible.

[0183] In some embodiments, the polypeptide to be evolved may be a fragment of a wild-type polypeptide, a fragment of a therapeutic polypeptide, or an antibody fragment. In some other embodiments, the parent polypeptide may be a polypeptide selected from mutant polypeptides generated by a mutagenesis process, where the polypeptide is selected for having desired properties such as high binding activity, high expression level, or humanization. The selected polypeptide may be used as the parent polypeptide to be evolved in the methods disclosed herein.

[0184] Methods for generating mutant DNA from DNA encoding a parent polypeptide are described in US Pat. No. 8,709,755 B2.

[0185] Creation of mutant DNA by evolving DNA encoding a parent polypeptide can be accomplished using point mutations (substitutions, insertions, and / or deletions) or mutations of large segments within the DNA. In some embodiments, the evolution process does not alter the active site of the parent polypeptide, but instead only alters one or more of the regions surrounding the active site and / or one or more regions distant from the active site.

[0186] In one embodiment, the evolution process involves converting a parent full-length antibody into a single-chain antibody. In this case, the active site, i.e., the variable region, particularly the CDRs, may not have any mutations compared to the parent antibody, but the context in which the active site resides has been changed by the removal of the constant region. In one example, the parent full-length antibody is an IgG antibody and the mutant antibody is a single-chain antibody derived therefrom.

[0187] In some embodiments, single-chain antibodies are bispecific antibodies with two arms that each bind to a different epitope. Mutations in one arm can affect the activity of the other arm. Thus, the evolution process can involve mutating only one arm of a parent polypeptide that is a bispecific antibody. In one example, the length of one arm can be evolved by shortening the arm through deletion or lengthening the arm through insertion. Alternatively, the evolution process can evolve both arms of a bispecific antibody in the same evolution step or in sequential evolution steps, optionally with screening after each step.

[0188] In yet another embodiment, the parent polypeptide is an antibody or antibody fragment. The evolution process can involve mutation of the Fc region. Mutations in the Fc region can be substitutions, insertions, and / or deletions. The Fc region can be shortened by deleting a fragment of the Fc region, or lengthened by inserting a fragment into the Fc region.

[0189] In yet another aspect, the parent polypeptide comprises multiple complementarity determining regions separated by framework regions. Such a parent polypeptide may be, for example, an antibody, light or heavy chain variable region. In certain embodiments, the evolution process may involve mutating framework regions alone or a combination of complementarity determining regions and framework regions. Evolution of the framework regions and complementarity determining regions may be carried out in one step or in multiple sequential steps, optionally with screening after each step.

[0190] In yet another embodiment, the parent polypeptide has several regions other than its active site. These several regions may be mutated sequentially in multiple evolutionary steps, optionally with screening after one or more of the evolutionary steps. For example, the evolutionary steps may involve evolving one of the regions of the polypeptide, followed by screening for a conditionally active polypeptide; then evolving another of the regions of the polypeptide, followed by screening for a conditionally active polypeptide; and then followed by another step of evolving yet another region of the polypeptide and screening for a conditionally active polypeptide.

[0191] In some circumstances, evolution of one or more regions of the parent polypeptide and / or mutant conditionally active polypeptide other than the active site (e.g., surrounding or distant regions) can alter the activity of the active site. By mutating surrounding or distant regions rather than the active site, in some circumstances, the active site of the mutant polypeptide can be more or less active than the active site of the parent polypeptide under certain conditions. In other embodiments, the desired conditional activity or selectivity is achieved when improved by evolution of one or more regions of the parent or mutant polypeptide other than the region containing the active site.

[0192] In some embodiments, the conditionally active polypeptide obtained by evolving a region of the parent polypeptide other than the region containing the active site can result in a preference of at least 2, or at least 3, or at least 5.

[0193] A suitable method for expressing the resulting mutant DNA to produce a mutant polypeptide is described in US Pat. No. 8,709,755 B2.

[0194] A method for screening mutant polypeptides to select for conditionally active polypeptides is described in US Pat. No. 8,709,755 B2.

[0195] Assay conditions for screening and selecting conditionally active polypeptides The first and second conditions, or normal and abnormal physiological conditions, of the assay used in the screening step can be selected from temperature, pH, osmolality, osmolality, oxidative stress, electrolyte concentration, and combinations of two or more such conditions. For example, the normal physiological condition of temperature can be 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 a tumor microenvironment, which can be 1-2°C higher than normal physiological temperature. In another example, the normal and abnormal physiological conditions can also be a normal physiological pH in the range of 7.2-7.8, or 7.2-7.6, and an abnormal pH present in a tumor microenvironment, such as a range of 5.5-7.2, 6-7, or 6.2-6.8.

[0196] Both assays under the first and second conditions, or under normal and abnormal physiological conditions, can be performed in an assay medium. The assay medium can be, for example, a solution that can contain buffers and other components. Common buffers that can be used in the assay medium include citrate buffers such as sodium citrate, phosphate buffers, bicarbonate buffers such as Krebs buffer, phosphate-buffered saline (PBS), Hank's buffer, Tris buffer, HEPES buffer, and the like. Other buffers known to those skilled in the art that are suitable for assays can also be used. These buffers can be used to mimic the compositional characteristics or components of human or animal body fluids, such as plasma or lymph.

[0197] The assay solution useful in the method of the present invention may contain at least one component selected from inorganic compounds, ions, and organic molecules, preferably those commonly found in the body fluids of mammals, such as humans or animals. Examples of such components include nutritional components and metabolites, as well as any other components that may be found in body fluids. The present invention contemplates that this component may or may not be part of a buffer system. For example, the assay solution may be a PBS buffer with added bicarbonate ions, where bicarbonate is not part of the PBS buffer. Alternatively, bicarbonate ions are a component of Krebs buffer.

[0198] While a component is present in substantially the same concentration in both assay solutions (for the first and second conditions), the two assay solutions may differ in other respects, such as pH, temperature, electrolyte concentration, or osmolality, and thus the component is used as a constant rather than a difference between the first and second conditions, or between two conditions, normal physiological conditions and abnormal conditions.

[0199] In some embodiments, a component is present in both assay solutions at a concentration that is close to or the same as the normal physiological concentration of that component in a mammal, particularly a human.

[0200] The inorganic compound or ion may be selected from one or more of boric acid, calcium chloride, calcium nitrate, diammonium phosphate, magnesium sulfate, monoammonium phosphate, monopotassium phosphate, potassium chloride, potassium sulfate, copper sulfate, iron sulfate, manganese sulfate, zinc sulfate, magnesium sulfate, calcium nitrate, calcium chelates, copper chelates, iron chelates, manganese chelates and zinc chelates, ammonium molybdate, ammonium sulfate, calcium carbonate, magnesium phosphate, potassium bicarbonate, potassium nitrate, hydrochloric acid, carbon dioxide, sulfuric acid, phosphoric acid, carbonic acid, uric acid, hydrogen chloride, urea, phosphorus ions, sulfate ions, chloride ions, magnesium ions, sodium ions, potassium ions, ammonium ions, iron ions, zinc ions and copper ions.

[0201] Examples of normal physiological concentrations of some inorganic compounds include uric acid in the concentration range of 2-7.0 mg / dL, calcium ion in the concentration range of 8.2-11.6 mg / dL, chloride ion in the concentration range of 355-381 mg / dL, iron ion in the concentration range of 0.028-0.210 mg / dL, potassium ion in the concentration range of 12.1-25.4 mg / dL, sodium ion in the concentration range of 300-330 mg / dL, carbonate in the concentration range of 15-30 mM, citrate ion at approximately 80 μM, histidine ion in the range of 0.05-2.6 mM, histamine in the range of 0.3-1 μM, HAPT ion (hydrogenated adenosine triphosphate) in the range of 1-20 μM, and HADP ion in the range of 1-20 μM.

[0202] In some embodiments, the ions present in the assay solutions under both the first and second conditions, or under both the normal physiological and abnormal conditions, are selected from hydroxide, halide (chloride, bromide, iodide), oxyhalide, sulfate, magnesium, calcium, bisulfate, carbonate, bicarbonate, sulfonate, oxyhalide, nitrate, nitrite, phosphate, hydrogen phosphate, dihydrogen phosphate, persulfate, monopersulfate, borate, ammonium, or organic ions, such as carboxylate, phenolate, sulfonate (organic sulfates such as methyl sulfate), vanadate, tungstate, borate, organoborate, citrate, oxalate, acetate, pentaborate, histidine, and phenolate.

[0203] The organic compounds present in the assay solutions under both the first and second conditions, or under both normal and abnormal physiological conditions, may be selected from amino acids such as histidine, alanine, isoleucine, arginine, leucine, asparagine, lysine, aspartic acid, methionine, cysteine, phenylalanine, glutamic acid, threonine, glutamine, tryptophan, glycine, valine, pyrrolysine, proline, selenocysteine, serine, tyrosine, and mixtures thereof.

[0204] Examples of normal physiological concentrations of some of the amino acids include 3.97 ± 0.70 mg / dL alanine, 2.34 ± 0.62 mg / dL arginine, 3.41 ± 1.39 mg / dL glutamic acid, 5.78 ± 1.55 mg / dL glutamine, 1.77 ± 0.26 mg / dL glycine, 1.42 ± 0.18 mg / dL histidine, 1.60 ± 0.31 mg / dL isoleucine, and 1.77 ± 0.26 mg / dL urea. These included 91±0.34 mg / dL leucine, 2.95±0.42 mg / dL lysine, 0.85±0.46 mg / dL methionine, 1.38±0.32 mg / dL phenylalanine, 2.02±6.45 mg / dL threonine, 1.08±0.21 mg / dL tryptophan, 1.48±0.37 mg / dL tyrosine, and 2.83±0.34 mg / dL valine.

[0205] The organic compounds present in the assay solutions under both the first and second conditions, or under both the normal and abnormal physiological conditions, may be selected from non-protein nitrogen-containing compounds such as creatine, creatinine, guanidinoacetic acid, uric acid, allantoin, adenosine, urea, ammonia, and choline. Examples of normal physiological concentrations of some of these compounds include 1.07±0.76 mg / dL creatine, 0.9-1.65 mg / dL creatinine, 0.26±0.24 mg / dL guanidinoacetic acid, 4.0±2.9 mg / dL uric acid, 0.3-0.6 mg / dL allantoin, 1.09±0.385 mg / dL adenosine, 27.1±4.5 mg / dL urea, and 0.3-1.5 mg / dL choline.

[0206] The organic compounds present in the assay solutions under both the first and second conditions, or under both normal and abnormal physiological conditions, may be selected from organic acids such as citric acid, α-ketoglutaric acid, succinic acid, malic acid, fumaric acid, acetoacetic acid, β-hydroxybutyric acid, lactic acid, pyruvic acid, α-ketonic acid, acetic acid, and volatile fatty acids. Examples of normal physiological concentrations of some of these organic acids include 2.5±1.9 mg / dL citric acid, 0.8 mg / dL α-ketoglutaric acid, 0.5 mg / dL succinic acid, 0.46±0.24 mg / dL malic acid, 0.8-2.8 mg / dL acetoacetic acid, 0.5±0.3 mg / dL β-hydroxybutyric acid, 8-17 mg / dL lactic acid, 1.0±0.77 mg / dL pyruvate, 0.6-2.1 mg / dL α-ketonic acid, and 1.8 mg / dL volatile fatty acids.

[0207] The organic compounds present in the assay solutions under both the first and second conditions, or under both normal and abnormal physiological conditions, can be selected from sugars (carbohydrates), such as glucose, pentoses, hexoses, xylose, ribose, mannose, and galactose, as well as disaccharides, including lactose, GlcNAcβ1-3Gal, Galα1-4Gal, Manα1-2Man, GalNAcβ1-3Gal, and O-, N-, C-, or S-glycosides. Examples of normal physiological concentrations of some of these sugars include 83±4 mg / dL glucose, 102±73 mg / dL polysaccharides (as hexoses), 77±63 mg / dL glucosamine, 0.4-1.4 mg / dL hexuronate (as glucuronic acid), and 2.55±0.37 mg / dL pentoses.

[0208] The organic compounds present in the assay solutions under both the first and second conditions, or under both the normal and abnormal physiological conditions, can be selected from lipids or their derivatives, such as cholesterol, lecithin, cephalin, sphingomyelin, and bile acids. Examples of normal physiological concentrations of some of these compounds include 40-70 mg / dL free cholesterol, 100-200 mg / dL lecithin, 0-30 mg / dL cephalin, 10-30 mg / dL sphingomyelin, and 0.2-0.3 mg / dL bile acids (as cholic acid).

[0209] The organic compounds present in the assay solutions under both the first and second conditions, or under both the normal and abnormal physiological conditions, can be selected from proteins such as fibrinogen, antihemophilic globulin, immune gamma globulin, immune euglobulin, isoagglutinin, beta-pseudoglobulin, glycoprotein, lipoprotein, and albumin. For example, the normal physiological concentration of mammalian serum albumin is 3.5 to 5.0 g / dL. In one embodiment, the albumin is bovine serum albumin.

[0210] The organic compounds present in the assay solutions under both the first and second conditions, or under both the normal and abnormal physiological conditions, may be selected from vitamins such as vitamin A, carotene, vitamin E, ascorbic acid, thiamine, inositol, folic acid, biotin, pantothenic acid, riboflavin, etc. Examples of normal physiological concentrations of some of these vitamins include vitamin A from 0.019 to 0.036 mg / dL, vitamin E from 0.90 to 1.59 mg / dL, inositol from 0.42 to 0.76 mg / dL, folic acid from 0.00162 to 0.00195 mg / dL, and biotin from 0.00095 to 0.00166 mg / dL.

[0211] The concentration of an inorganic compound, ion, or organic molecule in the assay solution (both assays under first and second conditions, or under both normal and abnormal physiological conditions) can be within the normal physiological range of that inorganic compound, ion, or organic molecule in human or animal serum. However, concentrations outside the normal physiological range can also be used. For example, the normal range for magnesium ions in human serum is 1.7 to 2.2 mg / dL, and for calcium ions is 8.5 to 10.2 mg / dL. The magnesium ion concentration in the assay solution can be from about 0.17 mg / dL to about 11 mg / dL. The calcium ion concentration in the assay solution can be from about 0.85 mg / dL to about 51 mg / dL. As a general rule, the concentration of an inorganic compound, ion, or organic molecule in the assay solution may be as low as 5%, or 10%, or 20%, or 30%, or 40%, or 50%, or 60%, or 70%, or 80% of the normal physiological concentration of that inorganic compound, ion, or organic molecule in human serum, or as high as 1.5, or 2, or 3, or 4, or 5, or 7, or 9, or 10, or even 20 times the normal physiological concentration of that inorganic compound, ion, or organic molecule in human serum. Various components of the assay solution may be used at concentration levels different from their respective normal physiological concentrations.

[0212] The activity of the mutant polypeptide is measured using an assay under first and second conditions, or under normal physiological and abnormal conditions. During the assay, both the mutant polypeptide and its binding partner are present in the assay solution. The relationship between the mutant polypeptide and its binding partner can be, for example, antibody-antigen, ligand-receptor, enzyme-substrate, or hormone-receptor. For the mutant polypeptide to exhibit its activity, it must be able to contact and bind to its binding partner. The activity of the mutant polypeptide toward its binding partner is then exhibited and measured following binding between the mutant polypeptide and its binding partner.

[0213] In some embodiments, ions used in the assay may function to form bridges between the mutant polypeptide being screened and its binding partner, particularly those containing charged amino acid residues. Thus, ions may be capable of binding to both the mutant polypeptide and its binding partner through hydrogen and / or ionic bonds. This may aid in binding between the mutant polypeptide and its binding partner by allowing ions to access sites that may be difficult for large molecules (mutant polypeptide or its binding partner) to access. In some cases, ions in the assay solution may increase the likelihood that the mutant polypeptide and its binding partner will bind to each other. Furthermore, ions may additionally or alternatively aid in binding between the mutant polypeptide and its binding partner by binding to the large molecule (mutant polypeptide or its binding partner). This binding may alter the conformation of the large molecule to and / or maintain it in a specific conformation that facilitates binding to the binding partner.

[0214] It has been observed that ions can assist in binding between a mutant polypeptide and its binding partner, possibly by forming ionic bonds between the mutant polypeptide and its binding partner. Thus, screening can be much more efficient and more hits (candidate conditionally active polypeptides) can be identified compared to the same assay without ions. Suitable ions can be selected from magnesium, sulfate, bisulfate, carbonate, citrate, HAPT, HADP, bicarbonate, nitrate, nitrite, phosphate, hydrogen phosphate, dihydrogen phosphate, persulfate, monopersulfate, borate, lactate, citrate, histidine, histamine, and ammonium.

[0215] Ions have been found to function to support binding between a mutant polypeptide and its binding partner at a pH close to the pKa of the ion, and such ions are preferably relatively small compared to the size of the mutant polypeptide.

[0216] In one embodiment, when the abnormal condition is a pH different from normal physiological pH under normal physiological conditions, ions suitable for increasing the number of hits for candidate conditionally active polypeptides can be selected from ions having a pKa close to the abnormal pH tested in the assay. For example, the pKa of the ion can be up to 2 pH units away from the abnormal pH, up to 1 pH unit away from the abnormal pH, up to 0.8 pH units away from the abnormal pH, up to 0.6 pH units away from the abnormal pH, up to 0.5 pH units away from the abnormal pH, up to 0.4 pH units away from the abnormal pH, up to 0.3 pH units away from the abnormal pH, up to 0.2 pH units away from the abnormal pH, or up to 0.1 pH units away from the abnormal pH.

[0217] Exemplary pKas of ions useful in the present invention (which may vary very slightly at different temperatures) are as follows: ammonium ion has a pKa of about 9.24, dihydrogen phosphate has a pKa of about 7.2, acetate has a pKa of about 4.76, histidine has a pKa of about 6.04, bicarbonate ion has a pKa of about 6.4, citrate has a pKa of 6.4, lactate ion has a pKa of about 3.86, histamine has a pKa of about 6.9, and HATP has a pKa of 6.95 (HATP 3- ⇔ATP 4- +H + ), and HADP has a pKa of 6.88 (HADP 3- ⇔ADP 4- +H + ).

[0218] In one embodiment, conditionally active polypeptides are assayed and selected in the presence of disulfide. Disulfide has a pKa of 7.05. In some embodiments, different disulfide concentrations can be used in assays corresponding to normal physiological conditions and assays corresponding to abnormal physiological conditions. Alternatively, the assay media for both normal and abnormal physiological conditions can have approximately the same disulfide concentration, but differ in some specific conditional values; for example, the assays can be performed at different pHs. The disulfide concentration used in the assay can be between 1 mM and 100 mM. Preferably, the assay media has a disulfide concentration of 2 to 500 nM, or 3 to 200 nM, or 5 to 100 nM. In some aspects, the disulfide concentration can be between 1 mM and 20 mM, or 2 mM and 10 mM. Assays performed in the presence of disulfide are known.

[0219] In certain embodiments, once the pH of the abnormal condition (i.e., the abnormal pH) is known, ions suitable for increasing candidate conditionally active polypeptide hits may be selected from ions having a pKa at or near the abnormal pH; for example, candidate ions may have a pKa that is up to 4 pH units away from the abnormal pH, up to 3 pH units away from the abnormal pH, up to 2 pH units away from the abnormal pH, up to 1 pH unit away from the abnormal pH, up to 0.8 pH units away from the abnormal pH, up to 0.6 pH units away from the abnormal pH, up to 0.5 pH units away from the abnormal pH, up to 0.4 pH units away from the abnormal pH, up to 0.3 pH units away from the abnormal pH, up to 0.2 pH units away from the abnormal pH, or up to 0.1 pH unit away from the abnormal pH.

[0220] As previously mentioned, ions are most effective at supporting binding between mutant polypeptides and their binding partners at a pH at or near their pKa. For example, in assay solutions with a pH of 7.2 to 7.6, bicarbonate ions (which have a pKa of approximately 6.4) have been found to be less effective at supporting binding between mutant polypeptides and their binding partners. As the pH of the assay solution is lowered to 6.7 or even to approximately 6.0, bicarbonate ions become increasingly effective at supporting binding between mutant polypeptides and their binding partners. As a result, assays at pH 6.0 may identify more hits than assays at pH 7.2 to 7.6. Similarly, histidine is less effective at supporting binding between mutant polypeptides and their binding partners at pH 7.4. As the pH of the assay solution is lowered to 6.7 or even to approximately 6.0, histidine becomes increasingly effective at supporting binding between mutant polypeptides and their binding partners, and more hits may be identified, for example, at a pH in the range of approximately 6.2 to 6.4.

[0221] In the present invention, it has been unexpectedly found that when the pH of an assay solution under normal physiological conditions (i.e., normal physiological pH) differs from the pH of an assay solution under abnormal conditions (i.e., abnormal pH), ions with pKas ranging from approximately the midpoint between normal physiological pH and abnormal pH to approximately the abnormal pH can significantly support the binding between the mutant polypeptide to be screened and its binding partner. As a result, this screening assay is much more efficient in finding more hit or candidate conditional polypeptides that are highly active under abnormal conditions.

[0222] In some embodiments, the pKa may even be at least one pH unit away from the abnormal pH. When the abnormal pH is an acidic pH, the pKa of a suitable ion may range from (abnormal pH -1) to the midpoint between the abnormal pH and normal physiological pH. When the abnormal pH is a basic pH, the pKa of a suitable ion may range from (abnormal pH +1) to the midpoint between the abnormal pH and normal physiological pH. Ions may be selected from those described herein. However, many more ions not explicitly described herein may also be used. It is understood that once the abnormal pH and normal physiological pH of a screening assay have been selected, one of skill in the art may use the guidance of the present invention to select any ion with a suitable pKa to increase the efficiency of the screening in identifying more hits with high activity under abnormal conditions.

[0223] For example, if the abnormal pH for one exemplary screen is 8.4 and the normal physiological pH is 7.4, any ion with a pKa in the range of about 7.9 (midpoint) to 9.4 (i.e., 8.4 + 1) can be used for screening. Some ions with pKas in this range include those derived from tricine (pKa 8.05), hydrazine (pKa 8.1), bicine (pKa 8.26), N-(2-hydroxyethyl)piperazine-N'-(4-butanesulfonic acid) (pKa 8.3), N-tris[hydroxymethyl]methyl-3-aminopropanesulfonic acid (pKa 8.4), and taurine (pKa 9.06). In another example, if the abnormal pH for one exemplary screen is 6 and the normal physiological pH is 7.4, any ion with a pKa in the range of about 5 (i.e., 6 - 1) to 6.7 (midpoint) can be used for screening. Some ions with pKas in this range include those derived from malate (pKa 5.13), pyridine (pKa 5.23), piperazine (pKa 5.33), cacodylate (pKa 6.27), succinate (pKa 5.64), 2-(N-morpholino)ethanesulfonic acid (pKa 6.10), citrate (pKa 6.4), histidine (pKa 6.04), and bis-tris (pKa 6.46). Those skilled in the art can consult numerous chemistry manuals and textbooks to identify known chemical compounds, including both inorganic and organic chemical compounds, that can be converted into ions with pKas within this range. Among chemical compounds with suitable pKas, those with smaller molecular weights may be preferred.

[0224] Thus, the present invention unexpectedly found that the generation of the ultimately identified conditionally active polypeptide not only depends on the generation of the correct polypeptide variant, but also on the use of ions with suitable pKa in the assay solution. Because ions can facilitate the efficient selection of highly active variants from a large library, the present invention believes that in addition to generating a large library of mutant polypeptides (e.g., by CPE and CPS), efforts should be directed toward finding suitable ions (with appropriate pKa) to use in the assay solution. Furthermore, the absence of suitable ions may result in low screening efficiency and a reduced likelihood of finding highly active variants. Consequently, multiple screening rounds may be required to obtain the same number of highly active variants without suitable ions.

[0225] Ions in the assay solution may be formed in situ from components of the assay solution or may be included directly in the assay solution. For example, CO2 from the air may be dissolved in the assay solution to provide carbonate and bicarbonate ions. In another example, sodium dihydrogen phosphate may be added to the assay solution to provide dihydrogen phosphate ions.

[0226] The concentration of this component in the assay solution (for both the first or normal physiological condition assay and the second or abnormal condition assay) may be the same or substantially the same as the concentration of the same component typically found in a naturally occurring body fluid of a mammal, such as a human. In other embodiments, the concentration of the component may be higher, particularly for components that are ions that may function to assist in binding between the mutant polypeptide and its binding partner, since it has been observed that higher concentrations of such ions may result in the formation of ionic bonds between the mutant polypeptide and its binding partner, effectively facilitating binding and increasing the likelihood of finding more hit or candidate conditionally active polypeptides.

[0227] In some embodiments, the concentration of ions in the assay solution can be positively correlated with the likelihood of finding more hits using the assay, especially when concentrations above normal physiological concentrations are used. For example, human serum has a bicarbonate ion concentration of approximately 15-30 mM. In one example, as the bicarbonate ion concentration in the assay solution was increased from 3 mM to 10 mM, 20 mM, 30 mM, 50 mM, and 100 mM, the number of hits in the assay also increased with each increase in bicarbonate concentration. Taking this into consideration, the assay solution can have a bicarbonate concentration in the range of about 3 mM to about 200 mM, or about 5 mM to about 150 mM, or about 5 mM to about 100 mM, or about 10 mM to about 100 mM, or about 20 mM to about 100 mM, or about 25 mM to about 100 mM, or about 30 mM to about 100 mM, or about 35 mM to about 100 mM, or about 40 mM to about 100 mM, or about 50 mM to about 100 mM.

[0228] In another embodiment, the citrate concentration in the assay solution can be about 30 μM to about 120 μM, or about 40 μM to about 110 μM, or about 50 μM to about 110 μM, or about 60 μM to about 100 μM, or about μM to about 90 μM, or about μM.

[0229] In one embodiment, normal physiological conditions are normal physiological pH in the range of 7.2 to 7.6, and abnormal conditions are abnormal pH in the range of 5.5 to 7.2, 6 to 7, or 6.2 to 6.8. Assay solutions for assays under normal physiological conditions have normal physiological pH and 50 mM bicarbonate ions. Assay solutions for assays under abnormal conditions have abnormal pH and 50 mM bicarbonate ions. Because the pKa of bicarbonate ions is approximately 6.4, bicarbonate ions can support binding between the mutant polypeptide and its binding partner at abnormal pHs of 6.0 to 6.4, e.g., pH 6.0 or 6.2.

[0230] In yet another embodiment, the normal physiological conditions are normal physiological pH in the range of 7.2 to 7.6, and the abnormal conditions are abnormal pH in the range of 5.5 to 7.2, 6 to 7, or 6.2 to 6.8. The assay solution for the assay under normal physiological conditions has normal physiological pH and 80 μM citrate ion. The assay solution for the assay under abnormal conditions has abnormal pH and 80 μM citrate ion. Because citrate ion has a pKa of 6.4, it can effectively support the binding between the mutant polypeptide and the binding partner in the assay solution under abnormal conditions of pH 6.0 to 6.4. Therefore, more candidate conditionally active polypeptides having higher binding activity under pH 6.0 to 6.4 and lower activity under pH 7.2 to 7.8 can be identified. Other ions, including acetate, histidine, bicarbonate, HATP, and HADP, function similarly, and assay solutions containing such ions allow for the effective screening of mutant polypeptides that have higher binding activity at pH values ​​around the ion's pKa and lower binding activity at pH values ​​different from the ion's pKa (e.g., normal physiological pH).

[0231] In yet another embodiment, the normal physiological conditions are a normal physiological temperature of 37°C and the abnormal conditions are an abnormal temperature of 38-39°C (temperatures present in some tumor microenvironments). The assay solution for assays under normal physiological conditions has a normal physiological temperature and 20 mM bicarbonate. The assay solution for assays under abnormal conditions has an abnormal temperature and 20 mM bicarbonate.

[0232] In yet another embodiment, the normal physiological condition is a particular concentration of an electrolyte in normal human serum, and the abnormal condition is a concentration of the same electrolyte at a different abnormal concentration that may exist in a different location in the animal or human or may be caused by a condition in the animal or human that alters the normal physiological electrolyte concentration in human serum.

[0233] Binding between a mutant polypeptide and / or its binding partner can also be affected in a number of other ways. Typically, this can be done by including one or more additional components in the assay solution. These additional components can be designed to interact with either the mutant polypeptide, the binding partner, or both. In addition, these additional components can affect binding using a combination of two or more interactions and a combination of two or more types of interactions.

[0234] In one embodiment, the binding interaction of interest is between an antibody and an antigen. In this embodiment, one or more additional components may be included in the assay solution to affect the antibody, the antigen, or both. In this manner, the desired binding interaction may be enhanced.

[0235] In addition to ions that can form ionic bonds with a mutant polypeptide and / or its binding partner to aid in binding between the mutant polypeptide and its binding partner, the present invention also encompasses other components that can be utilized to aid in binding between a mutant polypeptide and its binding partner. In one embodiment, molecules that can form hydrogen bonds with a mutant polypeptide and / or its binding partner can be utilized. In another embodiment, molecules capable of hydrophobic interactions with a mutant polypeptide and / or its binding partner can be used. In yet another embodiment, molecules capable of van der Waals interactions with a mutant polypeptide and / or its binding partner are contemplated.

[0236] As used herein, the term "hydrogen bond" refers to a relatively weak, non-covalent interaction between hydrogen covalently bonded to an electronegative atom such as carbon, nitrogen, oxygen, sulfur, chlorine, or fluorine (hydrogen bond donor) and the unshared pair of electrons of an electron-donating atom such as nitrogen, oxygen, sulfur, chlorine, or fluorine (hydrogen bond acceptor).

[0237] Components capable of forming hydrogen bonds with a mutant polypeptide and / or its binding partner include organic molecules with polar bonds as well as inorganic molecules. A mutant polypeptide and / or a binding partner of a mutant polypeptide typically contains amino acids capable of forming hydrogen bonds. Suitable amino acids have side chains with polar groups capable of forming hydrogen bonds. Non-limiting examples of suitable amino acids include glutamine (Gln), glutamic acid (Glu), arginine (Arg), asparagine (Asn), aspartic acid (Asp), lysine (Lys), histidine (His), serine (Ser), threonine (Thr), tyrosine (Tyr), cysteine ​​(Cys), methionine (Met), and tryptophan (Trp).

[0238] These amino acids can function as both hydrogen donors and hydrogen acceptors, for example, the oxygen atom of an -OH group, as found in Ser, Thr, and Tyr; the oxygen atom of a -C=O group, as found in Glu and Asp; the sulfur atom of an -SH group or -SC-, as found in Cys and Met; and the sulfur atom of an -NH3 group, as found in Lys and Arg. + Nitrogen atoms of groups, as well as nitrogen atoms of -NH- groups such as may be found in Trp, His, and Arg, can all function as hydrogen acceptors. Also, groups in this list that contain a hydrogen atom (e.g., -OH, -SH, NH3 + and -NH-) can also function as a hydrogen donor.

[0239] In some embodiments, the backbone of the variant polypeptide and / or its binding partner may also participate in the formation of one or more hydrogen bonds. For example, the backbone may have a repeating structure of -(C=O)-NH-, as in a peptide bond. The oxygen and nitrogen atoms of this structure may function as hydrogen acceptors, while the hydrogen atoms may participate in hydrogen bonds.

[0240] Inorganic compounds having at least one polar bond involving a hydrogen or oxygen atom that can be used for hydrogen bonding include, for example, HO, NH, HO, hydrazine, carbonates, sulfates, and phosphates. Organic compounds include alcohols; phenols; thiols; aliphatic amines, amides; epoxides, carboxylic acids; ketones, aldehydes, ethers, esters, organic chlorides, and organic fluorines. Compounds capable of forming hydrogen bonds are well known in the chemical literature, such as those discussed in "The Nature of the Chemical Bond," Linus Pauling, Cornell University Press, 1940, pages 284-334.

[0241] In some embodiments, alcohols include methanol, ethanol, propanol, isopropanol, butanol, pentanol, 1-hexanol, 2-octanol, l-decanol, cyclohexanol, and higher alcohols; diols such as ethylene glycol, propylene glycol, glycerol, diethylene glycol, and polyalkylene glycols. Suitable phenols include hydroquinone, resorcinol, catechol, phenol, o-, m-, and p-cresol, thymol, α- and β-naphthol, pyrogallol, guaiacol, and phloroglucinol. Suitable thiols include methanethiol, ethanethiol, 1-propanethiol, 2-propanethiol, butanethiol, tert-butyl mercaptan, pentanethiols, hexanethiol, thiophenol, dimercaptosuccinic acid, 2-mercaptoethanol, and 2-mercaptoindole. Suitable amines include methylamine, ethylamine, propylamine, isopropylamine, aniline, dimethylamine, methylethylamine, trimethylamine, aziridine, piperidine, N-methylpiperidine, benzidine, cyclohexylamine, ethylenediamine, hexamethylenediamine, o-, m-, and p-toluidine, and N-phenylpiperidine. Suitable amides include ethanamide, N,N-dimethylacetamide, N,N-dimethylformamide, N,N-dimethylmethoxyacetamide, and N-methyl-Np-cyanoethylformamide. Suitable epoxides include ethylene oxide, propylene oxide, tert-butyl hydroperoxide, styrene oxide, epoxide glycidol, cyclohexene oxide, di-tert-butyl peroxide, cumene hydroperoxide or ethylbenzene hydroperoxide, isobutylene oxide, and 1,2-epoxyoctane.Examples of carboxylic acids include terephthalic acid, isophthalic acid, phthalic acid, salicylic acid, benzoic acid, acetic acid, lauric acid, adipic acid, lactic acid, citric acid, acrylic acid, glycine, hexahydrobenzoic acid, o-, m-, and p-toluic acid, nicotinic acid, isonicotinic acid, and para-aminobenzoic acid. Examples of ketones include acetone, 3-propanone, butanone, pentanone, methyl ethyl ketone, diisobutyl ketone, ethyl butyl ketone, methyl isobutyl ketone, methyl tert-butyl ketone, cyclohexanone, acetone, methyl ethyl ketone, methyl propyl ketone, methyl butyl ketone, methyl amyl ketone, methyl hexyl ketone, diethyl ketone, ethyl butyl ketone, dipropyl ketone, diisobutyl ketone, diacetone alcohol, phorone, isophorone, cyclohexanone, methylcyclohexanone, and acetophenone. Aldehydes include formaldehyde, acetaldehyde, propionaldehyde, butyraldehyde, benzaldehyde, cinnamaldehyde, isobutyraldehyde, valeraldehyde, octaldehyde, benzaldehyde, cinnamaldehyde, cyclohexanone, salicylic aldehyde, and furfural.Esters include ethyl acetate, methyl acetate, ethyl formate, butyl acetate, ethyl lactate, ethyl butyrate, propyl acetate, ethyl formate, propyl formate, butyl formate, amyl formate, methyl acetate, ethyl acetate, propyl acetate, butyl acetate, amyl acetate, methyl isoamyl acetate, methoxybutyl acetate, hexyl acetate, cyclohexyl acetate, benzyl acetate, methyl propionate, ethyl propionate, butyl propionate, amyl propionate, methyl butyrate, ethyl butyrate, butyl butyrate, amyl butyrate, methyl acetoacetate, and ethyl acetoacetate. Ethers that can be used in the present invention include dimethyl ether, methyl ethyl ether, diethyl ether, methyl propyl ether, and dimethoxyethane. Ethers can also be cyclic, such as ethylene oxide, tetrahydrofuran, and dioxane.

[0242] The organic chlorides include chloroform, pentachloroethane, dichloromethane, trichloromethane, carbon tetrachloride, tetrachloromethane, tetrachloroethane, pentachloroethane, trichloroethylene, tetrachloroethylene, and ethylene dichloride.The organic fluorines include fluoromethane, difluoromethane, trifluoromethane, trifluoroethane, tetrafluoroethane, pentafluoroethane, difluoropropane, trifluoropropane, tetrafluoropropane, pentafluoropropane, hexafluoropropane, and heptafluoropropane.

[0243] Hydrogen bonds can be classified as strong, medium, or weak based on the strength of the bond (Jeffrey, George A.; An introduction to hydrogen bonding, Oxford University Press, 1997). Strong hydrogen bonds have a donor-acceptor distance of 2.2-2.5 Å and an energy range of 14-40 kcal / mol. Medium hydrogen bonds have a donor-acceptor distance of 2.5-3.2 Å and an energy range of 4-15 kcal / mol. Weak hydrogen bonds have a donor-acceptor distance of 3.2-4.0 Å and an energy range of <4 kcal / mol. Some examples of hydrogen bonds, along with their energy levels, are FH···:F (38.6 kcal / mol), OH···:N (6.9 kcal / mol), OH···:O (5.0 kcal / mol), NH···:N (3.1 kcal / mol), and NH···:O (1.9 kcal / mol). For more information, see Perrin et al., "Strong" hydrogen bonds in chemistry and biology, Annual Review of Physical Chemistry, vol. 48, pages 511-544, 1997; Guthrie, "Short strong hydrogen bonds: can they explain enzymic catalysis?" Chemistry & Biology, March 1996, 3:163-170.

[0244] In some embodiments, components used in the present invention can form strong hydrogen bonds with mutant polypeptides and / or their binding partners. These components tend to have atoms with high electronegativity. The atoms known to have the highest electronegativity are, in order, F>O>Cl>N. Therefore, the present invention preferably uses organic compounds containing fluorine, hydroxyl groups, or carbonyl groups in forming hydrogen bonds. In one embodiment, organic fluorines may be used in the present invention to form strong hydrogen bonds.

[0245] In another embodiment, moieties capable of hydrophobic interaction with the mutant polypeptide and / or its binding partner are utilized, including organic compounds with hydrophobic groups.

[0246] As used herein, the term "hydrophobic interaction" refers to a reversible attractive interaction between a hydrophobic compound or a hydrophobic region of a compound and another hydrophobic compound or a hydrophobic region of another compound. This type of interaction is described in "Hydrophobic Interactions," A. Ben-Nairn (1980), Plenum Press, New York.

[0247] Hydrophobic materials, due to their non-polar nature, experience the repulsive force of water molecules. When relatively non-polar molecules or groups in aqueous solutions associate with other non-polar molecules rather than with water, it is called a "hydrophobic interaction."

[0248] The variant polypeptides and their binding partners typically contain amino acids capable of hydrophobic interaction. These amino acids can typically be characterized by having at least one side chain with a non-polar group capable of hydrophobic interaction. Hydrophobic amino acids include, for example, alanine (Ala), isoleucine (Ile), leucine (Leu), phenylalanine (Phe), valine (Val), proline (Pro), glycine (Gly), and, to a lesser extent, methionine (Met), and tryptophan (Trp).

[0249] Components capable of hydrophobic interaction with a mutant polypeptide and / or its binding partner include organic compounds that are hydrophobic molecules or molecules containing at least one hydrophobic moiety. In some embodiments, these hydrophobic components can be hydrocarbons selected from aromatic hydrocarbons, substituted aromatic hydrocarbons, polycyclic aromatic hydrocarbons, aromatic or non-aromatic heterocycles, cycloalkanes, alkanes, alkenes, and alkynes. Hydrophobic groups can include aromatic groups, alkyl, cycloalkyl, alkenyl, and alkynyl groups. The terms "alkyl," "alkenyl," and "alkynyl," as used herein, refer to unsaturated aliphatic groups having 1 to 30 carbon atoms, including straight-chain alkenyl / alkynyl groups, branched-chain alkenyl / alkynyl groups, cycloalkenyl (alicyclic) groups, alkyl-substituted cycloalkyl groups, and cycloalkyl-substituted alkenyl / alkynyl groups. Such hydrocarbon moieties may also be substituted on one or more carbon atoms.

[0250] It can be understood that the strength of hydrophobic interactions is based on the amount of available "hydrophobes" that can interact with each other. Thus, hydrophobic interactions can be adjusted, for example, by increasing the amount and / or "hydrophobicity" of the hydrophobic moiety in the molecule involved in the hydrophobic interaction. For example, a hydrophobic moiety (which may comprise a hydrocarbon chain in its native form) can be modified to increase its hydrophobicity (its ability to increase the strength of the hydrophobic interaction involving that moiety) by adding a hydrophobic side chain to one of the carbons of its carbon skeleton. In preferred embodiments of the present invention, this can include the addition of various steroid compounds and / or their derivatives, such as sterol-based compounds, and more particularly various polycyclic compounds, including cholesterol. Generally, the side chain can be linear, aromatic, aliphatic, cyclic, polycyclic, or any other type of hydrophobic side chain as contemplated by those skilled in the art.

[0251] The types of moieties capable of van der Waals interactions with the variant polypeptide and / or its binding partner are usually, but not necessarily, compounds with polar moieties. As used herein, "van der Waals interactions" refers to the attractive forces between atoms, moieties, molecules, and surfaces that arise due to the interrelationship of the fluctuating polarities of adjacent atoms, moieties, or molecules as a result of dipole-dipole interactions and / or quantum dynamics.

[0252] Van der Waals interactions in the present invention are attractive forces between a mutant polypeptide or a binding partner and a component. Van der Waals interactions can arise from three sources. First, some molecules / moieties can be permanent electric dipoles, even though they are electrically neutral. Due to a fixed distortion of the electronic charge distribution in the structure of some molecules / moieties, one side of the molecule / moiety is always somewhat positive and the other side somewhat negative. The tendency of such permanent dipoles to align with each other results in a net attractive force. This is the interaction between two permanent dipoles (Keesom force).

[0253] Second, the presence of a molecule that is a permanent dipole can temporarily distort the electronic charges of other adjacent polar or nonpolar molecules, thereby inducing further polarization. An additional attractive force results when a permanent dipole interacts with an adjacent induced dipole. This is the interaction between a permanent dipole and a corresponding induced dipole and can be referred to as a Debye force. Third, even if the molecules involved are not permanent dipoles (e.g., the organic liquid benzene), there is still an attractive force between the molecules due to the two momentarily induced dipoles in the molecule. This is the interaction between two momentarily induced dipoles and can be referred to as a London dispersion force.

[0254] The variant polypeptide and / or binding partner may contain a number of amino acids capable of van der Waals interactions. These amino acids may have polar side chains, including glutamine (Gln), asparagine (Asn), histidine (His), serine (Ser), threonine (Thr), tyrosine (Tyr), cysteine ​​(Cys), methionine (Met), and tryptophan (Trp). These amino acids may also have side chains with nonpolar groups, including alanine (Ala), isoleucine (Ile), leucine (Leu), phenylalanine (Phe), valine (Val), proline (Pro), and glycine (Gly).

[0255] Components capable of van der Waals interactions with the mutant polypeptide and / or its binding partner include polar or non-polar inorganic compounds soluble in the assay solution. The assay solution is generally an aqueous solution, and therefore these polar or non-polar inorganic compounds are preferably soluble in water. Preferred materials for van der Waals interactions are polar, allowing for dipole-dipole interactions. For example, AlF3 has a polar Al-F bond and is soluble in water (approximately 0.67 g / 100 ml water at 20°C). HgCl2 has a polar Hg-Cl bond and is soluble in water at 7.4 g / 100 ml at 20°C. PrCl2 has a polar Pr-Cl bond and is soluble in water at approximately 1 g / 100 ml at 20°C.

[0256] Suitable polar compounds capable of van der Waals interactions include alcohols, thiols, ketones, amines, amides, esters, ethers, and aldehydes. Suitable examples of these compounds are described above in relation to hydrogen bonding. Suitable non-polar compounds capable of van der Waals interactions include aromatic hydrocarbons, substituted aromatic hydrocarbons, polycyclic aromatic hydrocarbons, aromatic or non-aromatic heterocycles, cycloalkanes, alkanes, alkenes, and alkynes.

[0257] Hydrogen-bonding components, hydrophobic components, and van der Waals components can be used to influence binding between a mutant polypeptide and its binding partner in several ways. In one embodiment, bridges can be formed between a mutant polypeptide and its binding partner by hydrogen bonding, hydrophobic interactions, and / or van der Waals interactions. Such bridges can bring the mutant polypeptide and the binding partner into close proximity to each other, facilitating binding, and / or position the mutant polypeptide and / or the binding partner relative to each other to facilitate binding.

[0258] In another embodiment, hydrogen bonding and / or hydrophobic interactions may increase the likelihood that a mutant polypeptide will bind to its binding partner, e.g., by causing the polypeptide and binding partner to assemble or associate with each other in a manner that increases the likelihood of binding. Thus, one or more of these interactions, alone or in combination, may be used to assemble the mutant polypeptide and binding partner closer together or position them in a manner that promotes binding, e.g., by drawing the binding sites closer to each other or by positioning the non-binding portions of the molecule further apart, thereby positioning the binding sites closer to each other.

[0259] In yet another embodiment, hydrogen bonding and / or hydrophobic interactions can affect the conformation of the mutant polypeptide and / or its binding partner, resulting in a conformation that is more conducive to binding between the mutant polypeptide and its binding partner. Specifically, binding or interaction with one or more amino acids of the mutant polypeptide and / or binding partner can result in one or more conformational changes in the mutant polypeptide or binding partner that are favorable to the mutant polypeptide / binding partner binding reaction.

[0260] The present invention performs two pairs of assays, one of which determines a decrease in the activity of a mutant polypeptide in an assay under normal physiological conditions compared to the original parent polypeptide from which the mutant polypeptide was derived under normal physiological conditions, and a second of which determines an increase in the activity of a mutant polypeptide in an assay under abnormal conditions compared to the original parent polypeptide from which the mutant polypeptide was derived under abnormal conditions.

[0261] The conditions used in the assay pairs of the present invention can be selected from temperature, pH, osmolality, osmolality, oxidative stress, electrolyte concentration, and the concentration of any other components of the assay solution or medium. Thus, a particular component of the assay medium can be used at substantially the same concentration in both assay pairs. In such cases, the component is typically present to mimic a particular environment in a human or animal, such as serum, a tumor microenvironment, a synovial fluid environment, a neural environment, or any other environment that may be encountered at the point of administration, through which an administered therapy may pass, or at the point of treatment. An important aspect of selecting one or more components that mimic these environments is that it can improve the results of the selection process performed using the assay pair. For example, mimicking a particular environment allows for the evaluation of different effects of particular components of that environment on mutant polypeptides during the selection process. A particular environmental component may, for example, alter or bind to the mutant polypeptide, inhibit the activity of the mutant polypeptide, inactivate the mutant polypeptide, etc.

[0262] In some embodiments, one or more components of the assay solution are preferably small compounds such as disulfide, hydrogen sulfide, histidine, histamine, citrate, bicarbonate, lactate, and acetate. In one embodiment, the small molecule components are preferably present in the assay solution at a concentration of about 100 μm to about 100 mM, or more preferably about 0.5 to about 50 mM, or about 1 to about 10 mM.

[0263] The concentration of a component in the assay solution may be the same as or substantially the same as the concentration of the same component typically found in a naturally occurring bodily fluid of a mammal, such as a human. This may be referred to as the normal physiological concentration of the component in the bodily fluid. In other embodiments, the concentration of a particular component in the assay solution may be lower or higher than the concentration of the same component typically found in a naturally occurring bodily fluid of a mammal, such as a human.

[0264] In another embodiment, the components may be present at substantially different concentrations in each of the assay pairs. In such cases, the presence, absence, or concentration of the components becomes the condition being assayed, since it is the concentration of the components that is the condition that distinguishes between assay solutions for assays under normal physiological conditions and those under abnormal conditions. Thus, the conditionally active polypeptides produced by this embodiment of the method of the invention can be selected for an activity that is at least partially dependent on the concentration of the components.

[0265] In some embodiments, a component may be present in one pair of assay solutions but completely absent from the other pair of assay solutions. For example, the lactate concentration in the abnormal condition assay solution may be set to a level that mimics the lactate concentration in the tumor microenvironment. Lactate may be absent from the normal physiological condition assay solution pair.

[0266] In one embodiment, the normal physiological condition is a first lactate concentration representative of normal physiological conditions, and the abnormal condition is a second lactate concentration representative of abnormal conditions present at a particular location in the body.

[0267] In another example, glucose may be absent in the abnormal conditions assay solution to mimic the absence of glucose as may be found in a tumor microenvironment, while glucose may be set to a level that mimics plasma glucose concentrations in the pair of normal physiological conditions assay solutions. This feature can be used to preferentially deliver a conditionally active polypeptide to a location or environment that is inactive or minimally active during transport, and activate the conditionally active polypeptide upon reaching an environment where concentrations of the components in the abnormal conditions assay solution are present.

[0268] For example, the tumor microenvironment typically has both low glucose concentrations and high lactate concentrations compared to human serum. Normal physiological glucose concentrations range from about 2.5 mM to about 10 mM in serum. On the other hand, glucose concentrations in the tumor microenvironment are typically very low, ranging from 0.05 mM to 0.5 mM. In one embodiment, assay solutions for assays under normal physiological conditions have a glucose concentration ranging from about 2.5 mM to about 10 mM, while assay solutions for assays under abnormal conditions have a glucose concentration ranging from about 0.05 mM to about 0.5 mM. The conditionally active polypeptides thus produced have higher activity in low-glucose environments (in the tumor microenvironment) than in high-glucose environments (in normal tissue or blood). While the conditionally active polypeptides are functional in the tumor microenvironment, they may have reduced activity while passing through the bloodstream.

[0269] Normal physiological concentrations of lactate in serum range from about 1 mM to about 2 mM. On the other hand, lactate concentrations in tumor microenvironments typically range from 10 mM to 20 mM. In one embodiment, assay solutions for assays under normal physiological conditions have lactate concentrations ranging from about 1 mM to about 2 mM, while assay solutions for assays under abnormal conditions have lactate concentrations ranging from about 10 mM to about 20 mM. The conditionally active polypeptides thus prepared have higher activity in high lactate environments (tumor microenvironments) than in low lactate environments (normal tissues or blood). Therefore, these conditionally active polypeptides are functional in tumor microenvironments but may have reduced activity while passing through the bloodstream.

[0270] Similarly, it is known that muscle pain results in higher-than-normal (abnormal) lactate concentrations. Therefore, when searching for mutant polypeptides that may be active in a muscle pain environment, an abnormal condition assay pair can be performed in the presence of higher lactate concentrations to mimic the muscle pain environment, while a normal physiological condition assay pair can be performed with lower lactate concentrations or in the absence of lactate. In this way, mutant polypeptides whose activity improves with increasing lactate concentrations in a muscle pain environment can be selected. Such conditionally active polypeptides may be useful, for example, as anti-inflammatory agents.

[0271] In another embodiment, two or more components may be used in both pairs of assay solutions. In this type of assay, conditionally active polypeptides may be selected using features of both of the two types of assays described above. Alternatively, two or more components may be used to increase the selectivity of the conditionally active polypeptide. For example, returning to the tumor microenvironment, the abnormal condition assay pair may be performed in an assay medium comprising both a high lactate concentration and a low glucose concentration, while the corresponding normal physiological condition assay pair may be performed in an assay medium comprising both a relatively low lactate concentration and a relatively high glucose concentration.

[0272] The present invention contemplates that components selected from inorganic compounds, ions, and organic molecules may be used alone or in combination to select conditionally active polypeptides that are more active at a given concentration of the component compared to a different concentration of the same component.

[0273] Assays that rely on differential concentrations of one or more metabolites as conditions that distinguish between a normal environment (normal physiological conditions) and an abnormal environment (abnormal conditions) can be particularly suitable for selecting conditionally active polypeptides that are more active in the tumor microenvironment than in plasma, because the tumor microenvironment typically has many metabolites that have different concentrations compared to the concentrations of the same metabolites in plasma.

[0274] Kinoshita et al., "Absolute Concentrations of Metabolites in Human Brain Tumors Using In Vitro Proton Magnetic Resonance Spectroscopy," NMR IN BIOMEDICINE, vol. 10, pp. 2-12, 1997, compared metabolites in normal brain and brain tumors. This group found that N-acetylaspartic acid has a concentration of 5000-6000 μM in normal brain, but this concentration is only 300-400 μM in glioblastoma, 1500-2000 μM in astrocytoma, and 600-1500 μM in anaplastic astrocytoma. Furthermore, inositol has a concentration of 1500-2000 μM in normal brain, but this concentration is 2500-4000 μM in glioblastoma, 2700-4500 μM in astrocytoma, and 3800-5800 μM in anaplastic astrocytoma. Phosphorylethanolamine has a concentration of 900-1200 μM in normal brain, but this concentration is 2000-2800 μM in glioblastoma, 1170-1370 μM in astrocytoma, and 1500-2500 μM in anaplastic astrocytoma. Glycine has a concentration of 600-1100 μM in normal brain, but this concentration is 4500-5500 μM in glioblastoma, 750-1100 μM in astrocytoma, and 1900-3500 μM in anaplastic astrocytoma. Alanine has a concentration of 700-1150 μM in normal brain, but this concentration is 2900-3600 μM in glioblastoma, 800-1200 μM in astrocytoma, and 300-700 μM in anaplastic astrocytoma. These metabolites may also have different concentrations in the blood; for example, N-acetylaspartic acid has a blood concentration of approximately 85000 μM; inositol has a blood concentration of approximately 21700 μM; glycine has a blood concentration of approximately 220-400 μM; and alanine has a blood concentration of approximately 220-300 μM.

[0275] Therefore, these metabolites, including at least N-acetylaspartic acid, inositol, glycine, and alanine, can be used at different concentrations in assay solutions to select conditionally active polypeptides that are active in brain tumors but not in blood or normal brain tissue. For example, to select conditionally active polypeptides that are active in the tumor microenvironment of glioblastoma but not active, or at least less active, in blood or normal brain tissue, an assay solution containing 85,000 μM N-acetylaspartic acid can be used in an assay pair under normal physiological conditions, and an assay solution containing 350 μM N-acetylaspartic acid can be used in an assay pair under abnormal conditions.

[0276] Mayers et al., "Elevated circulating branched-chain amino acids are an early event in pancreatic adenocarcinoma development," Nature Medicine, Vol. 20, pp. 1193-1198, 2014, examined the concentrations of various different metabolites, including branched-chain amino acids, in the prediagnostic plasma of pancreatic patients. Patients with pancreatic tumors were found to have several metabolites present at different concentrations in their bloodstream compared to the concentrations of the same metabolites in the blood of people without pancreatic cancer. Mayers et al. also found that pancreatic cancer patients had significantly elevated branched-chain amino acids in their plasma compared to normal subjects. Branched-chain amino acids present at elevated concentrations include isoleucine, leucine, and valine (Table 1 in Mayers et al.). Other metabolites, shown in Figure 1 in Mayers' study, were present at significantly different concentrations in the plasma of pancreatic cancer patients compared to normal healthy individuals. These metabolites include at least acetylglycine, glycine, phenylalanine, tyrosine, 2-aminoadipic acid, taurodeoxycholate / taurochenodeoxycholate, aconitate, isocitrate, lactate, α-glycerophosphate, and urate. Therefore, based on the finding that certain metabolites are present at different concentrations in the plasma of pancreatic cancer patients and normal healthy patients, it can be predicted that the tumor microenvironment of pancreatic cancer will also have different concentrations of these metabolites than may be present in the pancreatic microenvironment of healthy patients.

[0277] Thus, in one embodiment, one or more of these metabolites may be used in an assay solution under normal physiological conditions in an amount that approximates the concentration of these metabolites in the plasma of a healthy individual (i.e., the normal physiological concentration of the metabolites). For example, known normal physiological concentrations in the plasma of a healthy individual are about 1.60±0.31 mg / dL for isoleucine, about 1.91±0.34 mg / dL for leucine, and about 2.83±0.34 mg / dL for valine. The assay solution under normal physiological conditions may have normal physiological concentrations of one or more of these branched-chain amino acids within these ranges. The assay solution under abnormal conditions may have a concentration of the same branched-chain amino acid that is about 5-fold, or about 10-fold, or about 20-fold, or about 50-fold, or about 70-fold, or about 100-fold, or about 150-fold, or about 200-fold, or about 500-fold higher than the normal physiological concentration of the corresponding branched-chain amino acid in a healthy individual. This may reflect that the higher concentrations of these branched-chain amino acids found in plasma by Mayers et al. arise from the tumor microenvironment and are diluted in the bloodstream, and therefore, based on the findings of Mayers et al., one would expect the concentrations of these branched-chain amino acids to be significantly elevated in the pancreatic tumor microenvironment. Similarly, assays under abnormal conditions may reflect the concentrations of other metabolites in the blood of pancreatic cancer patients, even if the concentrations of certain metabolites are significantly lower in cancer patients compared to normal individuals. In this way, one can ensure that the screen mimics the actual environment, thereby selecting the most active variants for the particular environment of interest.

[0278] In some other embodiments, the assay solution under normal physiological conditions may contain one or more branched amino acids at concentrations that mimic the concentrations in the plasma of pancreatic cancer patients to mimic the actual plasma environment of these patients. In such embodiments, the assay solution under abnormal conditions may have a concentration of the same branched amino acid that is about 2-fold, or about 3-fold, or about 4-fold, or about 5-fold, or about 7-fold, or about 8-fold, or about 10-fold, or about 15-fold, or about 20-fold, or about 50-fold higher than the concentration of the corresponding branched amino acid in the plasma of pancreatic cancer patients, to reflect the fact that these higher concentrations occur in the tumor microenvironment and that concentrations in the bloodstream represent a dilution of the actual concentrations in the tumor microenvironment. Similarly, other metabolites may also have different concentrations in the assay solutions under normal physiological conditions and abnormal conditions, which may reflect actual differences expected from data collected on the bloodstream. In some cases, a particular metabolite may be deficient in the bloodstream of a pancreatic patient, in which case a concentration reflective of the measured bloodstream concentration can be used in the assay for normal physiological conditions, and a lower concentration can be used in the assay for abnormal conditions to take into account the likelihood that the metabolite will be consumed in the tumor microenvironment. Conditionally active polypeptides selected using this assay solution may be more active in the pancreatic cancer microenvironment than in the plasma of pancreatic cancer patients.

[0279] In some embodiments, the present invention may utilize whole plasma from a pancreatic cancer patient. For example, in one embodiment, assay solutions for one or both of the assays under normal physiological conditions and abnormal conditions may include a simulation of one or more components of plasma from a pancreatic cancer patient. In an exemplary embodiment, the assay solution for normal physiological conditions has a pH in the range of 7.2-7.6 and is spiked with 30 wt.% plasma from a pancreatic cancer patient, while the assay solution for abnormal conditions has a pH in the range of 6.2-6.8 and is spiked with 30 wt.% plasma from a pancreatic cancer patient. In this embodiment, the plasma from the pancreatic cancer patient is present to both (1) ensure that the conditionally active polypeptide is not activated in blood at a pH of 7.2-7.6, and (2) also ensure that the conditionally active polypeptide can be activated in the tumor microenvironment at a pH of 5.5-7.2, 6-7, or 6.2-6.8, even in the presence of this metabolic composition found in the blood of pancreatic cancer patients. This allows treatment to be tailored to the pancreatic cancer patient.

[0280] In another exemplary embodiment, the assay solution under normal physiological conditions has a pH in the range of 7.2 to 7.6 and is added with 30 wt.% plasma from a pancreatic cancer patient, while the assay solution under abnormal conditions has a pH in the range of 5.5 to 7.2 or 6.2 to 6.8 and is not added with any plasma from a pancreatic cancer patient.

[0281] Each of the several types of assays discussed above may use the same component selected from inorganic compounds, ions, and organic molecules. For example, in the case of lactate, lactate may be used at substantially the same concentration in a pair of assay solutions for both normal and abnormal physiological conditions. The normal and abnormal physiological conditions will in turn differ in one or more other aspects, such as temperature, pH, concentration of other components, etc. In different embodiments, lactate may be used as one of the factors distinguishing between normal and abnormal physiological conditions, thereby reflecting that lactate is at a higher concentration in an abnormal tumor microenvironment compared to normal physiological conditions (non-tumor microenvironment).

[0282] In some embodiments, two or more components are added to both the assay solutions under normal physiological conditions and those under abnormal conditions at substantially the same concentrations. For example, both citrate and bovine serum albumin (BSA) are added to these assay solutions. The citrate concentration in both assay solutions may be about 80 μM, and the BSA concentration may be about 10-20%. More specifically, the assay solution for the assay pair under normal physiological conditions may have a pH in the range of 7.2-7.6, with citrate at a concentration of about 80 μM and BSA at a concentration of about 10-20%. The assay solution for the assay pair under abnormal conditions may have a pH in the range of 6.2-6.8, with citrate at a concentration of about 80 μM and BSA at a concentration of about 10-20%.

[0283] In one embodiment, serum may be added to both the assay solutions under normal physiological conditions and those under abnormal conditions at substantially the same concentration. Because serum contains numerous inorganic compounds, ions, and organic molecules (including polypeptides), the assay solution may contain multiple components selected from inorganic compounds, ions, and organic molecules present at substantially the same concentrations between the two assay solutions. The assay solution may contain 5-30 vol.%, or 7-25 vol.%, or 10-20 vol.%, or 10-15 vol.% serum. In some other embodiments, the assay solutions under both normal physiological conditions and those under abnormal conditions are serum-free. The serum may be human serum, bovine serum, or serum from any other mammal. In some other embodiments, the assay solution is serum-free.

[0284] Assay solutions under normal and abnormal physiological conditions may have different pHs, and the pH of such assay solutions may be adjusted using CO and O levels in the buffer using bicarbonate.

[0285] In some other embodiments, at least one of two or more components is added at different concentrations to the assay solutions under normal and abnormal physiological conditions. For example, both lactate and bovine serum albumin (BSA) are added to the assay solutions. The lactate concentration may differ between the assay solutions under normal and abnormal physiological conditions, while BSA may have the same concentration in both assay solutions. Lactate may have a concentration in the range of 30-50 mg / dL in the assay solution under abnormal physiological conditions and a concentration in the range of 8-15 mg / dL in the assay solution under normal physiological conditions. On the other hand, BSA has the same concentration in both assay solutions, such as about 10-20%. The conditionally active polypeptide thus selected using these assay solutions is more active at high lactate concentrations of 30-50 mg / dL compared to low lactate concentrations of 8-15 mg / dL in the presence of BSA.

[0286] In some embodiments, assay solutions may be designed to select for conditionally active polypeptides whose activity depends on two or more conditions. In one exemplary embodiment, a conditionally active polypeptide may have activity that is dependent on both pH and lactate. An assay solution for selecting such conditionally active polypeptides may have an assay solution with a pH of 7.2-7.6 and a lactate concentration of 8-15 mg / dL for normal physiological conditions. An assay solution for abnormal conditions may have a pH of 6.2-6.8 and a lactate concentration of 30-50 mg / dL. Optionally, both assay solutions for normal and abnormal physiological conditions may also contain ions that support binding between the mutant polypeptide and its binding partner, thereby increasing the number of hits for candidate biologically active polypeptides.

[0287] In yet another exemplary embodiment, the conditionally active polypeptide may have activity dependent on pH, glucose, and lactate. An assay solution for selecting such conditionally active polypeptides may have a pH of 7.2-7.6, a glucose concentration ranging from 2.5-10 mM, and a lactate concentration ranging from 8-15 mg / dL for normal physiological conditions. An assay solution for abnormal conditions may have a pH of 6.2-6.8, a glucose concentration ranging from 0.05-0.5 mM, and a lactate concentration ranging from 30-50 mg / dL. Optionally, both the assay solutions for normal and abnormal physiological conditions may also contain ions that support binding between the mutant polypeptide and its binding partner, thereby increasing the number of candidate biologically active polypeptides that bind to the binding partner at pH 6.2-6.8. Conditionally active polypeptides selected using such assay solutions are more active in an environment of pH 6.2-6.8, glucose concentration 0.05-0.5 mM, and lactate concentration 30-50 mg / dL than in an environment of pH 7.2-7.6, glucose concentration 2.5-10 mM, and lactate concentration 8-15 mg / dL.

[0288] The two or more components selected from inorganic compounds, ions, and organic molecules are intended to create an abnormal condition assay solution that mimics the environment of the location / site (i.e., target site) to which the selected conditionally active polypeptide is to be delivered. In some embodiments, at least three components in the environment of the target site may be added to the assay solution, or at least four components in the environment of the target site may be added to the assay solution, or at least five components in the environment of the target site may be added to the assay solution, or at least six components in the environment of the target site may be added to the assay solution.

[0289] In one embodiment, bodily fluid collected from a target site (where a conditionally active polypeptide may be more active) may be used directly as an assay solution for an assay under abnormal conditions. For example, synovial fluid may be collected from a subject, preferably a subject with a joint disease in need of treatment. The collected synovial fluid may be optionally diluted and used as an assay solution in an assay pair under abnormal conditions to select a conditionally active polypeptide. By using the collected synovial fluid, optionally diluted, as an assay solution for an assay under abnormal conditions and as an assay solution simulating human plasma for an assay under normal physiological conditions, the selected conditionally active polypeptide (e.g., TNF-α) may be more active in the joint than in other locations or organs. For example, a subject with an inflamed joint (such as arthritis) may be treated with TNF-α. However, TNF-α typically has severe side effects that damage other tissues and organs. Conditionally active TNF-α, which is highly active in synovial fluid but not or less active in the blood, may deliver the activity of TNF-α to the joints while reducing or potentially eliminating the side effects of TNF-α on other parts of the body.

[0290] The development of conditionally active polypeptides with activity dependent on multiple conditions will result in improved selectivity of the conditionally active polypeptide for target sites within a subject's body. Ideally, the conditionally active polypeptide is inactive, or at least significantly less active, at other locations where only some of the conditions are present. In one embodiment, a conditionally active polypeptide active at a pH of 6.2-6.8, a glucose concentration of 0.05-0.5 mM, and a lactate concentration of 30-50 mg / dL can be delivered specifically to the tumor microenvironment, since all of these conditions are present in the tumor microenvironment. Other tissues or organs may only have one or two of these conditions, but not all three, which may be insufficient to fully activate the conditionally active polypeptide in other tissues or organs. For example, post-exercise muscle may have a low pH in the range of 6.2-6.8, but may not have other assay conditions. Thus, the conditionally active polypeptide is inactive, or at least less active, in post-exercise muscle.

[0291] In some embodiments, a step can be performed to confirm that the activity of the conditionally active polypeptide is indeed dependent on the conditions used to select the conditionally active polypeptide. For example, a conditionally active polypeptide can be selected that is dependent on three conditions: pH 6.2-6.8, glucose concentration 0.05-0.5 mM, and lactate concentration 30-50 mg / dL. The selected conditionally active polypeptide can then be tested under each of these three conditions individually and in an environment containing a pair of the three conditions to confirm that the conditionally active polypeptide is inactive or has low activity under these test conditions or environments.

[0292] In some embodiments, certain serum components are intentionally minimized or excluded from the assay medium. For example, when screening for antibodies, serum components that bind to or adsorb the antibody can be minimized or excluded from the assay medium. Such bound antibodies can generate false positives, thereby including binding mutant antibodies that are not conditionally active but rather simply bind to components present in serum under a variety of different conditions. Therefore, careful selection of assay components that minimizes or eliminates components that could potentially bind to the variants in the assay can reduce the number of non-functional variants that may falsely identify as positive for conditional activity due to binding to components in the assay other than the desired binding partner. For example, in some embodiments screening for mutant polypeptides that tend to bind to components in human serum, BSA can be used in the assay solution to reduce or eliminate the possibility of false positives caused by mutant polypeptides binding to components in human serum. Other similar substitutions can also be made in the detailed examples to achieve the same goal.

[0293] In some embodiments, the assay conditions mimic the environment near the cell membrane, such as inside, on, or outside the cell membrane, or the environment of the joint. Some factors that may affect binding activity during screening in the cell membrane environment include receptor expression, internalization, antibody-drug conjugate (ADC) potency, etc.

[0294] The assay format can be any suitable assay known to those skilled in the art. Examples include ELISA, enzyme activity assays, in vitro (organ, etc.) real tissue screening, tissue slides, whole animals, cell lines, and the use of 3D systems. For example, suitable cell-based assays are described in WO 2013 / 040445, tissue-based assays are described in U.S. Pat. No. 7,993,271, whole animal-based screening methods are described in U.S. Pat. App. Pub. No. 2010 / 0263599, and 3D system-based screening methods are described in U.S. Pat. App. Pub. No. 2011 / 0143960.

[0295] In some embodiments, the evolution process may generate variant polypeptides that may simultaneously possess other desirable properties in addition to the conditionally active properties discussed above. Suitable other desirable properties that may be evolved include binding activity, expression, humanization, etc. Thus, the present invention may be used to generate conditionally active polypeptides that also have improved at least one or more of these other desirable properties.

[0296] In some embodiments, the present invention generates conditionally active polypeptides. The selected conditionally active polypeptides can be further mutated, for example, in a second evolutionary step using one of the mutagenesis techniques disclosed herein, thereby improving another property of the selected conditionally active polypeptide, such as binding activity, expression, humanization, etc. After this second evolutionary step, the mutant polypeptides can be screened for both conditional activity and the improved property.

[0297] In some embodiments, after evolving a parent polypeptide to generate mutant polypeptides, a first conditionally active polypeptide is selected that exhibits both (a) a decrease in the first activity compared to the parent polypeptide in an assay under normal physiological conditions, and (b) an increase in the first activity compared to the parent polypeptide in an assay under abnormal conditions. The first conditionally active polypeptide can then be subjected to one or more additional evolution, expression, and selection steps to select at least a second conditionally active polypeptide that exhibits both (1) a decrease in the second activity compared to the parent polypeptide in an assay under normal physiological conditions, and (b) an increase in the second activity compared to the parent polypeptide in an assay under abnormal conditions, or (2) a greater ratio of the first activity under abnormal conditions to the first activity under normal physiological conditions, compared to the first conditionally active polypeptide and / or the parent polypeptide. Note that the second conditionally active polypeptide can have both higher first and second activities under abnormal conditions compared to the parent polypeptide, and lower first and second activities under normal physiological conditions compared to the parent polypeptide.

[0298] In certain embodiments, the present invention aims to produce conditionally active polypeptides that have a large ratio of activity between their activity under abnormal conditions (or second conditions) and their activity under normal physiological conditions (or first conditions) (e.g., greater selectivity between abnormal and normal physiological conditions). The ratio of activity under abnormal conditions (or second conditions) to activity under normal physiological conditions (or first conditions), i.e., selectivity, can be at least about 2:1, or at least about 3:1, or at least about 4:1, or at least about 5:1, or at least about 6:1, or at least about 7:1, or at least about 8:1, or at least about 9:1, or at least about 10:1, or at least about 11:1, or at least about 12:1, or at least about 13:1, or at least about 14:1, or at least about 15:1, or at least about 16:1, or at least about 17:1, or at least about 18:1, or at least about 19:1, or at least about 20:1, or at least about 30:1, or at least about 40:1, or at least about 50:1, or at least about 60:1, or at least about 70:1, or at least about 80:1, or at least about 90:1, or at least about 100:1.

[0299] In one embodiment, the conditionally active polypeptide is an antibody, which may have a ratio of activity under abnormal conditions to activity under normal physiological conditions of at least about 5:1, or at least about 6:1, or at least about 7:1, or at least about 8:1, or at least about 9:1, or at least about 10:1, or at least about 15:1, or at least about 20:1, or at least about 40:1, or at least about 80:1. In one embodiment, the conditionally active polypeptide is used to target tumor sites, where the conditionally active polypeptide is active at tumor sites (in the tumor microenvironment) and is significantly less active or inactive at non-tumor sites (normal physiological conditions).

[0300] In one embodiment, the conditionally active polypeptide is an antibody intended to be conjugated to another agent, such as those disclosed elsewhere herein. This conditionally active antibody may have a higher ratio of activity under abnormal conditions to activity under normal physiological conditions. For example, a conditionally active antibody conjugated to another agent may have a ratio of activity under abnormal conditions to activity under normal physiological conditions of at least about 10:1, or at least about 11:1, or at least about 12:1, or at least about 13:1, or at least about 14:1, or at least about 15:1, or at least about 16:1, or at least about 17:1, or at least about 18:1, or at least about 19:1, or at least about 20:1. This may be particularly important when the conjugated agent is, for example, toxic or radioactive, because it is desirable to localize the conjugated agent at the disease or treatment site (where the abnormal condition exists).

[0301] G. Creation of Conditionally Active Polypeptides Selected conditionally active polypeptides with reversible or irreversible activity may be generated for therapeutic, diagnostic, research and related purposes and / or may be subjected to one or more additional evolution and selection cycles.

[0302] Conditionally active polypeptides can be produced using polypeptide-expressing cellular production hosts or organisms. To make the production process more efficient, DNA encoding the conditionally active polypeptide can be codon-optimized for the cellular production host or organism. Codon optimization has been described previously; for example, Narum et al., "Codon optimization of gene fragments encoding Plasmodium falciparum merzoite proteins enhances DNA vaccine protein expression and immunogenicity in mice," Infect. Immun. 2001 December, 69(12):7250-3, describes codon optimization in a mouse system. Outchkourov et al., "Optimization of the expression of Equistatin in Pichia pastoris, protein expression and purification," Protein Expr. Purif. 2002 February;24(1):18-24, describes codon optimization in a yeast system. Feng et al., "High level expression and mutagenesis of recombinant human phosphatidylcholine transfer protein using a synthetic gene: evidence for a C-terminal membrane binding domain," Biochemistry 2000 Dec. 19, 39(50):15399-409, describes codon optimization in Escherichia coli (E. coli).Humphreys et al., "High-level periplasmic expression in Escherichia coli using a eukaryotic signal peptide: importance of codon usage at the 5' end of the coding sequence," Protein Expr. Purif. 2000 Nov. 20(2):252-64, describes how codon usage affects protein secretion in Escherichia coli (E. coli).

[0303] The cellular production host may be a mammalian system selected from the group consisting of CHO, HEK293, IM9, DS-I, THP-I, Hep G2, COS, NIH 3T3, C33a, A549, A375, SK-MEL-28, DU 145, PC-3, HCT 116, Mia PACA-2, ACHN, Jurkat, MM1, Ovcar 3, HT 1080, Panc-1, U266, 769P, BT-474, Caco-2, HCC 1954, MDA-MB-468, LnCAP, NRK-49F, and SP2 / 0 cell lines; and mouse splenocytes and rabbit PBMCs. In one embodiment, the mammalian system is selected from a CHO or HEK293 cell line. In a specific aspect, the mammalian system is a CHO-S cell line. In another embodiment, the mammalian system is a HEK293 cell line.

[0304] In some embodiments, the cellular production host is a yeast cell line, such as S. cerevisiae or Pichia yeast cells. In some embodiments, the cellular production host is a prokaryotic cell, such as E. coli (Owens RJ and Young RJ, J. Immunol. Meth., vol. 168, p. 149, 1994; Johnson S and Bird RE, Methods Enzymol., vol. 203, p. 88, 1991). Conditionally active polypeptides can also be produced in plants (Firek et al., Plant Mol. Biol., vol. 23, p. 861, 1993).

[0305] Conditionally active polypeptides can also be produced synthetically using chemical methods well known in the art (e.g., Caruthers, "New chemical methods for synthesizing polynucleotides," Nucleic Acids Res. Symp. Ser. 215-223, 1980; Horn, "Synthesis of oligonucleotides on cellulose. Part II: design and synthetic strategy to the synthesis of 22 oligodeoxynucleotides coding for Gastric Inhibitory Polypeptide (GIP)"). 1", Nucleic Acids Res. Symp. Ser. 225-232, 1980; Banga, A.K., Therapeutic Peptides and Proteins, Formulation, Processing and Delivery Systems Technomic Publishing Co., Lancaster, Pa., 1995. For example, peptide synthesis can be carried out using various solid-phase techniques (see, e.g., Roberge "A strategy for a convergent synthesis of N-linked glycopeptides on a solid support", Science 269:202, 1995; Merrifield "Concept and early development of solid-phase peptide synthesis", Methods Enzymol. 289:3-13, 1997), or automated synthesis may be achieved using, for example, an ABI 43 IA peptide synthesizer (Perkin Elmer) following instructions provided by the manufacturer.

[0306] Solid-phase chemical peptide synthesis methods have been known in the art since the early 1960s (Merrifield, R.B., "Solid-phase synthesis I. The synthesis of a tetrapeptide", J. Am. Chem. Soc., 85:2149-2154, 1963) (see also Stewart, J.M. and Young, J.D., Solid Phase Peptide Synthesis, 2nd Ed., Pierce Chemical Co., Rockford, 111., pp. 11-12)) and have recently been used in commercially available laboratory peptide design and synthesis kits (Cambridge Research Biochemicals). Such commercially available laboratory kits generally utilize the teachings of H.M. Geysen et al., "Use of peptide synthesis to probe viral antigens for epitopes to a resolution of a single amino acid," Proc. Natl. Acad. Sci., USA, 81:3998, 1984, and provide for the synthesis of peptides at the tips of numerous "rods" or "pins," all attached to a single plate. In using such a system, the plate of rods or pins is inverted and inserted into corresponding wells or reservoirs in a second plate, which contains a solution for attaching or immobilizing the appropriate amino acids to the tips of the pins or rods. By repeating this process step, i.e., inverting and inserting the tips of the rods and pins into the appropriate solutions, amino acids are built up into the desired peptide. Additionally, many FMOC peptide synthesis systems are available. For example, assembly of polypeptides or fragments can be performed on a solid support using an Applied Biosystems, Inc., Model 431 A™ automated peptide synthesizer. Such equipment provides ready access to the peptides of the present disclosure, either by direct synthesis or by synthesis of a series of fragments that can be coupled using other known techniques.

[0307] The conditionally active polypeptides can also be glycosylated. Glycosylation can be added chemically or post-translationally by the cellular biosynthetic machinery, which incorporates the use of known glycosylation motifs, which may be native to the sequence or added as a peptide or to the nucleic acid coding sequence. Glycosylation can be O-linked or N-linked.

[0308] Conditionally active polypeptides include all forms of "mimetic" and "peptidomimetic." The terms "mimetic" and "peptidomimetic" refer to synthetic chemical compounds having substantially the same structural and / or functional properties as the polypeptides of the present disclosure. Mimetics can be composed entirely of synthetic, non-natural amino acid analogs, or they are chimeric molecules of partly natural peptide amino acids and partly non-natural amino acid analogs. Mimetics can also incorporate any amount of natural amino acid conservative substitutions, as long as the substitutions do not also substantially alter the mimetic's structure and / or activity. As with the polypeptides of the present disclosure that are conservative variants, routine experimentation will determine whether a mimetic is within the scope of the present disclosure, i.e., its structure and / or function are not substantially altered.

[0309] The polypeptide mimetic compositions of the present disclosure may contain any combination of non-natural structural components. In alternative embodiments, the mimetic compositions of the present disclosure include one or all of the following three structural groups: a) residue linkages other than natural amide bond ("peptide bond") linkages; b) non-natural residues in place of naturally occurring amino acid residues; or c) residues that induce secondary structure mimicry, i.e., thereby inducing or stabilizing secondary structure, such as β-turn, γ-turn, β-sheet, α-helical conformation, etc. For example, a polypeptide of the present disclosure can be characterized as a mimetic when all or some of its residues are joined by chemical means other than natural peptide bonds. Individual peptidomimetic residues can be joined by peptide bonds or other chemical bonds or coupling means, such as glutaraldehyde, N-hydroxysuccinimide esters, bifunctional maleimides, N,N'-dicyclohexylcarbodiimide (DCC), or N,N'-diisopropylcarbodiimide (DIC). Linking groups that can be used in place of the traditional amide bond ("peptide bond") linkage include, for example, ketomethylene (e.g., -C(C=O)CH2- as opposed to -(C=O)-NH-), aminomethylene (CH2-NH), ethylene, olefin (CH=CH), ether (CHO), thioether (CH2S), tetrazole (CN4--), thiazole, retroamide, thioamide, or ester (see, e.g., Spatola (1983), Chemistry and Biochemistry of Amino Acids, Peptides and Proteins, Vol. 7, pp 267-357, "Peptide Backbone Modifications," Marcell Dekker, NY).

[0310] Polypeptides of the present disclosure can also be characterized as mimetics by containing all or some non-natural residues in place of naturally occurring amino acid residues. Non-natural residues are well described in the scientific and patent literature; some exemplary non-natural compositions and guidelines useful as mimetics of natural amino acid residues are set forth below. Mimetics of aromatic amino acids include, for example, D- or L-naphylalanine; D- or L-phenylglycine; D- or L-2 thieneylalanine; D- or L-1,-2, 3-, or 4-pyreneylalanine; D- or L-3 thieneylalanine; D- or L-(2-pyridinyl)-alanine; D- or L-(3-pyridinyl)-alanine; D- or L-(2-pyrazinyl)-alanine; D- or L-(4-isopropyl)-phenylglycine; D-(trifluoromethyl ... D-(methyl)-phenylglycine; D-(trifluoromethyl)-phenylalanine; Dp-fluoro-phenylalanine; D- or Lp-biphenylphenylalanine; D- or Lp-methoxy-biphenylphenylalanine; D- or L-2-indole(alkyl)alanine; and D- or L-alkylanines, where alkyl may be substituted or unsubstituted methyl, ethyl, propyl, hexyl, butyl, pentyl, isopropyl, isobutyl, sec-isotyl, iso-pentyl, or a non-acidic amino acid. Aromatic rings of unnatural amino acids include, for example, thiazolyl, thiophenyl, pyrazolyl, benzimidazolyl, naphthyl, furanyl, pyrrolyl, and pyridyl aromatic rings.

[0311] Mimetics of acidic amino acids can be generated, for example, by substitution with noncarboxylated amino acids; (phosphono)alanine; sulfated threonine while maintaining the negative charge. Carboxyl side groups (e.g., aspartyl or glutamyl) can also be selectively modified by reaction with carbodiimides (R'-NCN-R'), such as 1-cyclohexyl-3(2-morpholinyl-(4-ethyl)carbodiimide or 1-ethyl-3(4-azonia-4,4-dimethylpentyl)carbodiimide. Aspartyl or glutamyl can also be converted to asparaginyl and glutaminyl residues by reaction with ammonium ions. Mimetics of basic amino acids can be generated, for example, by substitution with the amino acids ornithine, citrulline, or (guanidino)acetic acid (in addition to lysine and arginine), or (guanidino)alkylacetic acids, where alkyl is defined above. Nitrile derivatives (e.g., containing a CN moiety instead of COOH) can be substituted for asparagine or glutamine. Asparaginyl and glutaminyl residues can be converted to the corresponding Deamination can be performed to aspartyl or glutamyl residues. Arginine residue mimetics can be generated by reacting arginyl with, for example, one or more conventional reagents, such as phenylglyoxal, 2,3-butanedione, 1,2-cyclohexanedione, or ninhydrin, preferably under alkaline conditions. Tyrosine residue mimetics can be generated by reacting tyrosyl with, for example, aromatic diazonium compounds or tetranitromethane. N-acetylimidizol and tetranitromethane can be used to form O-acetyltyrosyl species and 3-nitro derivatives, respectively. Cysteine ​​residue mimetics can be generated by reacting cysteinyl residues with, for example, α-haloacetic acids, such as 2-chloroacetic acid or chloroacetamide, and the corresponding amines to give carboxymethyl or carboxyamidomethyl derivatives.Cysteine ​​residue mimetics can also be generated by reacting cysteinyl residues with, for example, bromotrifluoroacetone, α-bromo-β-(5-imidozoyl)propionic acid; chloroacetyl phosphate, N-alkylmaleimides, 3-nitro-2-pyridyl disulfide; methyl 2-pyridyl disulfide; p-chloromercuribenzoate; 2-chloromercuri-4 nitrophenol; or chloro-7-nitrobenzo-oxa-1,3-diazole. Lysine mimetics can be generated (and the amino-terminal residue can be modified) by reacting lysinyl with, for example, succinic or other carboxylic acid anhydrides. Lysine and other α-amino-containing residue mimetics can also be generated by reaction with imidoesters such as methylpicolinimidate, pyridoxal phosphate, pyridoxal, chloroborohydride, trinitrobenzenesulfonic acid, O-methylisourea, 2,4-pentanedione, and transamidase-catalyzed reactions with glyoxylate. Methionine mimetics can be generated, for example, by reaction with methionine sulfoxide. Proline mimetics include, for example, pipecolic acid, thiazolidinecarboxylic acid, 3- or 4-hydroxyproline, dehydroproline, 3- or 4-methylproline, or 3,3-dimethylproline. Histidine residue mimetics can be generated by reaction of histidyl with, for example, diethylprocarbonate or parabromophenacyl bromide. Other mimetics include those created by, for example, hydroxylation of proline and lysine; phosphorylation of the hydroxyl group of seryl or threonyl residues; methylation of the α-amino group of lysine, arginine, and histidine; acetylation of the N-terminal amine; methylation or substitution of backbone amide residues with N-methyl amino acids; or amidation of the C-terminal carboxyl group.

[0312] Residues, e.g., amino acids, of the polypeptides of the present disclosure may also be replaced by amino acids (or peptidomimetic residues) of the opposite chirality. Thus, any amino acid that naturally occurs in the L-configuration (which may also be referred to as R or S, depending on the structure of the chemical entity), referred to as a D-amino acid, can be replaced with a peptidomimetic amino acid of the same chemical structure type but of the opposite chirality, which may also be referred to as the R or S form.

[0313] The present disclosure also provides methods for modifying conditionally active polypeptides by natural processes, such as post-translational processing (e.g., phosphorylation, acylation, etc.), or by chemical modification techniques. Modifications can occur anywhere in a polypeptide, including the peptide backbone, the amino acid side-chains, and the amine or carboxyl termini. It will be understood that the same type of modification may be present in the same or varying degrees at several sites in a given polypeptide, and that a given polypeptide may contain many different types of modifications. Modifications include acetylation, acylation, PEGylation, ADP-ribosylation, amidation, covalent attachment of flavin, covalent attachment of a heme moiety, covalent attachment of a nucleotide or nucleotide derivative, covalent attachment of a lipid or lipid derivative, covalent attachment of phosphatidylinositol, cyclization cross-linking, disulfide bond formation, demethylation, formation of covalent cross-links, formation of cysteine, formation of pyroglutamate, formylation, γ-carboxylation, glycosylation, GPI anchor formation, hydroxylation, iodination, methylation, myristoylation, oxidation, PEGylation, proteolytic processing, phosphorylation, prenylation, racemization, selenoylation, sulfation, and transfer-RNA-mediated addition of amino acids to proteins such as arginylation. See Creighton, TE, a s-Structure and Molecular Properties 2nd Ed., W.H. Freeman and Company, New York (1993); Posttranslational Covalent Modification of Proteins, B.C. Johnson, Ed., Academic Press, New York, pp. 1-12 (1983).

[0314] H. Engineering Conditionally Active Antibodies The conditionally active antibodies of the present invention may be engineered by one or more of the antibody engineering techniques described herein, including, but not limited to, antibody conjugation, engineering multispecific antibodies, engineering bispecific conditionally active antibodies against an immune effector cell surface antigen and a target antigen, and engineering the Fc region of an antibody.

[0315] Suitable methods for conjugating conditionally active antibodies are described in WO 2015 / 175375. In one embodiment, the conditionally active antibodies used in the conjugations disclosed herein preferably have a ratio of activity under abnormal conditions to activity under normal physiological conditions of at least about 10:1, or at least about 12:1, or at least about 14:1, or at least about 16:1, or at least about 18:1, or at least about 20:1, or at least about 22:1, or at least about 24:1, or at least about 26:1.

[0316] In some embodiments, the conditionally active antibody can be conjugated to the Fc region of the antibody. The above-described conjugate molecules, compounds, or drugs can be conjugated to the Fc region as described in U.S. Pat. No. 8,362,210. For example, the Fc region can be conjugated to a cytokine or toxin that is delivered to the site where the conditionally active antibody exhibits preferential activity. Methods for conjugating polypeptides to the Fc region of an antibody are known in the art. See, for example, U.S. Patent Nos. 5,336,603, 5,622,929, 5,359,046, 5,349,053, 5,447,851, 5,723,125, 5,783,181, 5,908,626, 5,844,095, and 5,112,946. ;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, vol. 88, pages 10535-10539, 1991; Traunecker et al., Nature, vol. 331, pages 84-86, 1988; Zheng et al., J. Immunol., vol. 154, pages 5590-5600, 1995; and ViI et al., Proc. Natl. Acad. Sci. USA, vol. 89, pages 11337-11341, 1992.

[0317] In some embodiments, the conditionally active antibody can be covalently attached to the conjugated drug via an intermediate linker having at least two reactive groups, one reactive with the conditionally active antibody and one reactive with the conjugated drug. The linker, which can include any compatible organic compound, can be selected so that reaction with the conditionally active antibody or the conjugated drug does not adversely affect the reactivity and / or selectivity of the conditionally active antibody. Furthermore, the addition of the linker to the conjugated drug must not destroy the activity of the conjugated drug. The ratio of conjugated anti-cancer drug molecules to conditionally active polypeptide molecules is up to 3:1, or 4:1, or 5:1, or 6:1. In one example, the ratio of anti-cancer drug to conditionally active polypeptide is about 4:1.

[0318] Suitable linkers for oxidized conditionally active antibodies include those containing a group selected from primary amine, secondary amine, hydrazine, hydrazide, hydroxylamine, phenylhydrazine, semicarbazide, and thiosemicarbazide groups. Suitable linkers for reduced conditionally active antibodies include those containing specific reactive groups capable of reacting with sulfhydryl groups on reduced conditionally active antibodies. Such reactive groups include, but are not limited to, reactive haloalkyl groups (including, for example, haloacetyl groups), p-mercuribenzoate groups, and groups capable of Michael-type addition reactions (including, for example, maleimides and groups of the type described by Mitra and Lawton, J. Amer. Chem. Soc. Vol. 101, pages 3097-3110, 1979).

[0319] Suitable methods for engineering multispecific conditionally active antibodies are described in WO 2015 / 175375.

[0320] Conditionally active antibodies can be engineered to generate bispecific conditionally active antibodies against an immune effector cell surface antigen and a target antigen. The bispecific conditionally active antibodies of the present invention are capable of attracting immune effector cells to disease sites where the target antigen is present. A bispecific conditionally active antibody is an antibody that can specifically bind to two different antigens: an immune effector cell surface antigen and a target antigen. A bispecific antibody may be a full-length antibody comprising two arms, one arm binding to an immune effector cell surface antigen and the other arm binding to a target antigen. Bispecific antibodies are antibodies that bind to a heavy chain variable domain (V H ) and the light chain variable domain (V L In one embodiment, the antibody fragment may comprise at least two V(II) arms, one for binding to an immune effector cell surface antigen and the other arm binding to a target antigen. H V L In another embodiment, the antibody fragment comprises at least two single variable domains (Vs), one for binding to an immune effector cell surface antigen and the other arm binding to a target antigen. H or V L In some embodiments, the bispecific conditionally active antibody comprises two scFvs, one of which binds to an immune effector cell surface antigen and the other of which binds to a target antigen.

[0321] The attracted immune effector cells can attract immune effector cells to diseased cells or tissues containing the target antigen by their binding activity to both the immune effector cells and the target antigen on the diseased cells or tissues. Because the immune effector cells have the ability to suppress or even destroy the diseased cells or tissues, the attracted immune effector cells can then attack the diseased cells or tissues, thus helping to cure the disease. For example, immune effector cells can destroy tumor cells or infected cells. Immune effector cells include natural killer cells, macrophages, lymphokine-activated killer (LAK) cells, and T cells.

[0322] A bispecific conditionally active antibody has two binding activities, one for an immune effector cell surface antigen and one for a target antigen. In one embodiment, both binding activities are conditional, meaning that the binding activity of the bispecific conditionally active antibody for both the immune effector cell surface antigen and the target antigen is lower than that of the wild-type antibody under normal physiological conditions and higher than that of the wild-type antibody under abnormal conditions. In one embodiment, only one of the two binding activities is conditional, meaning that either the binding activity of the bispecific conditionally active antibody for the immune effector cell surface antigen or the binding activity of the bispecific conditionally active antibody for the target antigen is conditional. In this case, either the binding activity of the bispecific conditionally active antibody for the immune effector cell surface antigen or the binding activity of the bispecific conditionally active antibody for the target antigen is lower than the corresponding activity of the wild-type antibody under normal physiological conditions and higher than the corresponding activity of the wild-type antibody under abnormal conditions.

[0323] The two arms of a bispecific conditionally active antibody (e.g., two V H V L The fragments (units or two scFvs) can be joined together using conventional methods. As is well known in the art, the minimum antibody fragment containing a complete antigen-binding site comprises one non-covalently associated heavy chain variable domain and one light chain variable domain (V H and V L This configuration is achieved by the interaction of the three complementarity-determining regions (CDRs) of each variable domain to form a V H -V L The CDRs correspond to those found in natural antibodies, which define an antigen-binding site on the surface of the dimer. Collectively, the six CDRs confer antigen-binding specificity to the antibody. The framework regions (FRs) flanking the CDRs have a tertiary structure that is essentially conserved in natural immunoglobulins from species as diverse as human and mouse. These FRs serve to hold the CDRs in their proper orientation. The constant domains are not required for binding function, but are important for the V H -V LEven a single variable domain (or half of an Fv containing only three CDRs specific for an antigen) has the ability to recognize and bind to an antigen, but usually with lower affinity than the entire binding site (Painter et al., "Contributions of heavy and light chains of rabbit immunoglobulin G to antibody activity. I. Binding studies on isolated heavy and light chains," Biochemistry, vol. 11, pages 1327-1337, 1972). Thus, the domains in the binding site of a bispecific conditionally active antibody can be composed of V chains from different immunoglobulins. H -V L , V H -V H or V L -V L It can be structured as a pair of domains.

[0324] In some embodiments, the bispecific conditionally active antibody can be constructed as a continuous polypeptide chain using recombinant DNA technology, e.g., by expressing a nucleic acid molecule encoding the bispecific conditionally active antibody to construct a continuous polypeptide chain (see, e.g., Mack et al., "A small bispecific antibody construct expressed as a functional single-chain molecule with high tumor cell cytotoxicity," Proc. Natl. Acad. Sci. USA, vol. 92, pages 7021-7025, 2005). H and V L The order of the domains is such that the antigen-binding site can be properly folded to form one binding site for the immune effector cell surface antigen and one binding site for the target antigen. H and V L As long as the domains are arranged, it is not critical to the present invention.

[0325] In generating bispecific conditionally active antibodies against an immune effector cell surface antigen and a target antigen, some of the techniques described herein for engineering multispecific conditionally active antibodies can be used.

[0326] Bispecific antibodies configured as a single polypeptide chain are known and are described in WO 99 / 54440, Mack, J. Immunol. (1997), 158, 3965-3970, Mack, PNAS, (1995), 92, 7021-7025, Kufer, Cancer Immunol. Immunother., (1997), 45, 193-197, Loffler, Blood, (2000), 95, 6, 2098-2103, and Bruhl, J. Immunol., (2001), 166, 2420-2426. A particularly preferred configuration for bispecific antibodies is V H and V L A polypeptide construct in which V domains are linked together by linker domains. H and V L The order of the regions is not important. In one embodiment, the single polypeptide chain comprises H1 -Linker domain-V L1 -Linker domain-V H2 -Linker domain-V L2 In another embodiment, the single polypeptide chain is configured as V L1 -Linker domain-V H1 -Linker domain-V L2 -Linker domain-V H2 In another embodiment, the single polypeptide chain is configured as V H1 -Linker domain-V H2 -Linker domain-V L1 -Linker domain-V L2 In another embodiment, the single polypeptide chain is configured as V H1 -Linker domain-V L2 -Linker domain-V L1 -Linker domain-V H2The single polypeptide chain can be folded into two arms, each capable of binding to an immune effector cell surface antigen or a target antigen.

[0327] The linker domain of the bispecific conditionally active antibody is H and V L The linker domain is a peptide fragment of sufficient length to allow intermolecular association between the domains. The design of linkers suitable for this purpose is described in the prior art, e.g., EP 623 679 B1, U.S. Pat. No. 5,258,498, EP 573 551 B1, and U.S. Pat. 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 15-amino acid linker of the sequence (Gly4Ser)3.

[0328] The additional linker domain comprises an oligomerization domain. The oligomerization domain is a domain consisting of two or more V H and V LThe combination of domains can be promoted to fold into two arms, each capable of binding to an immune effector cell surface antigen or a target antigen. Non-limiting examples of oligomerization domains include leucine zippers (jun-fos, GCN4, E / EBP, etc.; Kostelny, J. Immunol. 148 (1992), 1547-1553; Zeng, Proc. Natl. Acad. Sci. 94 (1997), 3673-3678; Williams, Genes Dev. 5 (1991), 1553-1563; Suter, "Phage Display of Peptides and Proteins", Chapter 11, (1996), Academic Press), antibody-derived oligomerization domains such as constant domains CH1 and CL (Mueller, FEBS Letters 422 (1998), 259-264) and / or tetramerization domains such as GCN4-LI (Zerangue, Proc. Natl. Acad. Sci. 97 (2000), 3591-3595).

[0329] In some embodiments, the folding of single polypeptide chain bispecific conditional antibodies may be stabilized using knobs-into-holes technology, as described by Ridgway et al. ("'Knobs-into-holes' engineering of antibody CH3 domains for heavy chain heterodimerization," Protein Eng. 1996 Jul;9(7):617-21). This technique involves packing amino acid side chains between adjacent a-helices, where the side chains of a-helices are represented on the surface of a cylinder as knobs spaced alternately with holes into which the knobs of adjacent a-helices can fit (O'Shea et al., (1991) Science, 254, 539-544).

[0330] Immune effector cell surface antigens should be specific to one or a class of immune effector cells. Surface antigens for many 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)

[0331] 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).

[0332] Lymphokine-activated killer (LAK) cells possess surface antigens including T3, T4, T11, T8, T11, 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).

[0333] T cells, particularly cytotoxic T cells, have surface antigens including CD2, CD3, CD4, CD5, CD6, CD8, CD28, T58, CD27, CD45, CD84, CD25, CD127, and CD196 (CCR6), CD197 (CCR7), CD62L, CD69, TCR, T10, T11, and CD45RO (Ledbetter et al., "Enhanced transmembrane signaling activity of monoclonal antibody heteroconjugates suggests molecular interactions between receptors on the T cell surface," Mol Immunol. 1989 Feb;26(2):137-45; Jondal et al., "SURFACE MARKERS ON HUMAN T AND B LYMPHOCYTES," JOURNAL OF EXPERIMENTAL MEDICINE, VOLUME 136, 1972, 207-215; Mingari et al. al., “Surface markers of human T lymphocytes,” Ric Clin Lab.1982 Jul-Sep;12(3):439-448).

[0334] After binding to an immune effector cell, the bispecific conditionally active antibody can direct the immune effector cell to a cell or tissue where the target antigen is preferably present on its surface. When the bispecific conditionally active antibody (having immune effector cells) binds to the target antigen, the immune effector cell can attack the diseased cell or tissue. Immune effector cells such as natural killer cells, macrophages, LAK cells, and T cells (cytotoxic) all have the ability to kill and / or destroy diseased cells or tissues, for example, the ability to destroy tumor tissue.

[0335] The diseased cells or tissues may be selected from cancer, inflammatory diseases, neurological disorders, diabetes, cardiovascular diseases, or infectious diseases. Examples of target antigens include antigens expressed by various immune cells, carcinomas, sarcomas, lymphomas, leukemias, germ cell tumors, blastomas, and cells associated with various hematological, autoimmune, and / or inflammatory diseases.

[0336] Cancer-specific target antigens that can be targeted by bispecific conditionally active antibodies include 4-IBB, 5T4, adenocarcinoma antigen, alpha-fetoprotein, BAFF, B-lymphoma cells, C242 antigen, CA-125, carbonic anhydrase 9 (CA-IX), C-MET, CCR4, CD152, CD19, CD20, CD200, CD22, CD221, CD23 (IgE receptor), CD28, CD30 (TNFRSF8), CD33, CD4, CD40, and CD44. v6, CD51, CD52, CD56, CD74, CD80, CEA, CNT0888, CTLA-4, DR5, EGFR, EpCAM, CD3, FAP, fibronectin extra domain-B, folate receptor 1, GD2, GD3 ganglioside, glycoprotein 75, GPNMB, HER2 / neu, HGF, human scatter factor receptor kinase, IGF-1 receptor, IGF-I, IgG1, LI-CAM, IL-13, IL-6, insulin-like growth factor I receptor, integrin α5β1, integrin ανβ3, MORAb-009, MS4A1, MUC1, mucin CanAg, N-glycolylneuraminic acid, NPC-1C, PDGF-Rα, PDL192, phosphatidylserine, prostate cancer cells, RANKL, RON, ROR1, SCH 900105, SDC1, SLAMF7, TAG-72, tenascin C, TGFβ2, TGF-β, TRAIL-R1, TRAIL-R2, tumor antigen CTAA16.88, VEGF-A, VEGFR-1, VEGFR2 or vimentin.

[0337] The types of cancer that can be treated with the genetically engineered cytotoxic cells or pharmaceutical compositions of the present invention include carcinomas, blastomas, and sarcomas, as well as certain leukemias or lymphoid malignancies, benign and malignant tumors, and malignant lesions, such as sarcomas, carcinomas, and melanomas. The cancer may be a non-solid tumor (such as a blood tumor) or a solid tumor. Adult tumors / cancers and pediatric tumors / cancers are also included.

[0338] Hematological cancers are cancers of the blood or bone marrow. Examples of hematological cancers (or hematopoietic cancers) include leukemias, such as acute leukemias (such as acute lymphocytic leukemia, acute myelocytic leukemia, acute myelogenous leukemia, and myeloblastic, promyelocytic, myelomonocytic, monocytic, and erythroleukemia), chronic leukemias (such as chronic myelocytic (granulocytic) leukemia, chronic myelogenous leukemia, and chronic lymphocytic leukemia), polycythemia vera, lymphoma, Hodgkin's disease, non-Hodgkin's lymphoma (indolent and aggressive types), multiple myeloma, Waldenstrom's macroglobulinemia, heavy chain disease, myelodysplastic syndromes, hairy cell leukemia, and myelodysplasia.

[0339] A solid tumor is an abnormal mass of tissue that usually does not contain cysts or liquid areas. Solid tumors can be benign or malignant. Different types of solid tumors are named after the type of cells that form them (such as sarcoma, carcinoma, and lymphoma). Examples of solid tumors that can be treated include fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma, osteosarcoma, and other sarcomas, synovioma, mesothelioma, Ewing's tumor, leiomyosarcoma, rhabdomyosarcoma, colon cancer, lymphoid malignancies, pancreatic cancer, breast cancer, lung cancer, ovarian cancer, prostate cancer, hepatocellular carcinoma, squamous cell carcinoma, basal cell carcinoma, adenocarcinoma, sweat gland carcinoma, medullary thyroid carcinoma, papillary thyroid carcinoma, pheochromocytoma, sebaceous gland carcinoma, papillary carcinoma, papillary adenocarcinoma, medullary carcinoma, bronchogenic carcinoma, renal cell carcinoma, hepatocellular carcinoma, cholangiocarcinoma, choriocarcinoma, Wilms' tumor, cervical carcinoma, These include testicular tumors, seminomas, bladder cancer, melanoma, and sarcomas and carcinomas, including CNS tumors (such as gliomas (such as brain stem gliomas and mixed gliomas), glioblastomas (also known as glioblastoma multiforme), astrocytomas, CNS lymphomas, germinomas, medulloblastomas, schwannomas, craniopharyogiomas, ependymomas, pinealomas, hemangioblastomas, acoustic neuromas, oligodendrogliomas, meningiomas, neuroblastomas, retinoblastomas, and brain metastases).

[0340] Target antigens specific to inflammatory diseases that can be targeted by bispecific conditionally active antibodies include AOC3 (VAP-1), CAM-3001, CCL11 (eotaxin-1), CD125, CD147 (basigin), CD154 (CD40L), CD2, CD20, CD23 (IgE receptor), CD25 (IL-2 receptor chain), CD3, CD4, CD5, IFN-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 α4, integrin α4β7, and Lama gramin B. glama), LFA-1 (CD11a), MEDI-528, myostatin, OX-40, rhuMAb β7, scleroscin, SOST, TGFβ1, TNF-α, or VEGF-A.

[0341] Target antigens specific to neurological disorders that can be targeted by the bispecific conditionally active antibodies of the present invention include one or more of β-amyloid or MABT5102A. Antigens specific to diabetes that can be targeted by the bispecific conditionally active antibodies of the present invention include one or more of L-Iβ or CD3. Antigens specific to cardiovascular diseases that can be targeted by the bispecific conditionally active antibodies of the present invention include one or more of C5, cardiac myosin, CD41 (integrin α-lib), fibulin II, β chain, ITGB2 (CD18), and sphingosine-1-phosphate.

[0342] Target antigens specific to infectious diseases that can be targeted by the bispecific conditionally active antibodies of the present invention include one or more of anthrax toxin, CCR5, CD4, clumping factor A, cytomegalovirus, cytomegalovirus glycoprotein B, endotoxin, Escherichia coli, hepatitis B surface antigen, hepatitis B virus, HIV-1, Hsp90, influenza A hemagglutinin, lipoteichoic acid, Pseudomonas aeruginosa, rabies virus glycoprotein, respiratory syncytial virus, and TNF-α.

[0343] Further examples of target antigens include surface proteins found in specific or amplified form on cancer cells, such as IL-14 receptor, CD19, CD20, and CD40 for B-cell lymphoma, Lewis Y and CEA antigens for various carcinomas, Tag72 antigen for breast and colorectal cancer, EGF-R for lung cancer, folate-binding protein and HER-2 protein, which are often amplified in human breast and ovarian cancers, or viral proteins, such as the gp120 and gp41 envelope proteins of HIV, envelope proteins from hepatitis B and C viruses, glycoprotein B and other envelope glycoproteins of human cytomegalovirus, and envelope proteins from oncoviruses such as Kaposi's sarcoma-associated herpesvirus. Other potential target antigens include CD4 (the ligand is the HIV gp120 envelope glycoprotein), and other viral receptors, such as ICAM, the receptor for human rhinovirus, and the related receptor molecule for poliovirus.

[0344] Human immunodeficiency virus (HIV) cannot enter human cells unless it first binds to two key molecules on the cell surface: CD4 and a coreceptor. The first coreceptor recognized is CCR5, and later in the viral life cycle, another chemokine receptor, CXCR4, becomes a coreceptor for HIV-1 (D'Souza, Nature Med. 2, 1293 (1996); Premack, Nature Med. 2, 1174; Fauci, Nature 384, 529 (1996)). HIV-1 strains that cause most sexually transmitted viral infections are called M-tropic viruses. These HIV-1 strains (also known as non-syncytial-inducing (NSI) primary viruses) replicate in primary CD4+ T cells and macrophages and can use the chemokine receptor CCR5 (and, less frequently, CCR3) as their coreceptor. T-tropic viruses (sometimes called syncytium-inducing (SI) primary viruses) can also replicate in primary CD4+ T cells and infect established CD4+ T cell lines in vitro via the chemokine receptor CXCR4 (fusin). Many of these T-tropic strains can use CCR5 in addition to CXCR4, and some can enter macrophages via CCR5, at least under certain in vitro conditions (D'Souza, Nature Med. 2, 1293 (1996); Premack, Nature Med. 2, 1174; Fauci, Nature 384, 529 (1996)). Because M-tropic HIV-1 strains are thought to be responsible for approximately 90% of sexual transmissions of HIV, CCR5 is the predominant coreceptor for this virus in patients.

[0345] The number and identity of coreceptor molecules on target cells, as well as the ability of HIV-1 strains to enter cells via different coreceptors, appear to be determinants of disease progression. High expression of CCR3 and CCR5 has also been observed in T and B cells in lymph nodes from Hodgkin's disease patients. Type I diabetes is thought to be a T cell-mediated autoimmune disease. In a relevant animal model, expression of the CCR5 receptor in the pancreas has been associated with the progression of type I diabetes (Cameron (2000) J. Immunol. 165, 1102-1110). In one embodiment, a bispecific conditionally active antibody binds to CCR5 as a target antigen and can be used to suppress HIV infection of host cells and slow the progression of other diseases.

[0346] Several antibodies that specifically bind to (human) CCR5 are known in the art, including MC-1 (Mack (1998) J. Exp. Med. 187, 1215-1224) or MC-5 (Blanpain, (2002) Mol. Biol. Cell. 13:723-37; Segerer (1999) Kidney Int. 56:52-64; Kraft (2001) Biol. Chem. 14;276:34408-18). Thus, bispecific conditionally active antibodies can be prepared, for example, by combining the V of an antibody specific for CCR5, preferably human CCR5. L and V H domain (i.e., the second domain from Ig), and the V domain of antibodies specific for the CD3 antigen on T cells. H and V L Preferably, it includes a domain.

[0347] In another embodiment, the present invention provides a bispecific, conditionally active antibody against CD19 as a target antigen and CD3 on T cells. CD19 has been shown to be a highly useful medical target. CD19 is expressed in the entire B cell lineage, from pro-B cells to mature B cells, and is uniformly expressed on all lymphoma cells, but is absent from stem cells (Haagen, Clin Exp Immunol 90 (1992), 368-75; Uckun, Proc. Natl. Acad. Sci. USA 85 (1988), 8603-7). Combination therapy using both antibodies against CD19 and additional immunomodulatory antibodies has been disclosed for the treatment of B-cell malignancies (WO 02 / 04021, U.S. Patent Application Publication Nos. 2002006404, 2002028178) and autoimmune diseases (WO 02 / 22212, 2002058029). 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 lymphocytic leukemia. WO 02 / 80987 discloses the therapeutic use of antibody-based immunotoxins directed against the antigen CD19 for the treatment of diseases such as B-cell non-Hodgkin's lymphoma, Hodgkin's lymphoma or B-cell leukemia (e.g., B-cell acute lymphoblastic leukemia (B-ALL), (e.g., hairy cell lymphoma), B-cell precursor acute lymphoblastic leukemia (pre-B-ALL), B-cell chronic lymphocytic leukemia (B-CLL)).

[0348] In a further embodiment, the present invention provides a bispecific, conditionally activating antibody directed against CD20 as the target antigen and CD3 on T cells. CD20 is a cell surface protein present on B lymphocytes. The CD20 antigen is found on normal and malignant pre-B lymphocytes and mature B lymphocytes, including those found in over 90% of B-cell non-Hodgkin's lymphomas (NHLs). This antigen is absent from hematopoietic stem cells, activated B lymphocytes (plasma cells), and normal tissues. Several antibodies, mostly of murine origin, have been described: 1F5 (Press et al., 1987, Blood 69 / 2, 584-591), 2B8 / C2B8, 2H7, 1H4 (Liu et al., 1987, J. Immunol. 139, 3521-3526; Anderson et al., 1998, U.S. Pat. No. 5,736,137; Haisma et al., 1998, Blood 92, 184-190; Shan et al., 1999, J. Immunol. 162, 6589-6595).

[0349] CD20 has been described in immunotherapeutic strategies for the treatment of plasma cell malignancies using vaccination with DNA encoding scFv linked to a carrier protein (Treon et al., 2000, Semin Oncol 27(5), 598), and immunotherapeutic treatment with a CD20 antibody (IDEC-C2B8) has been shown to be effective in the treatment of non-Hodgkin's B-cell lymphoma.

[0350] In some embodiments, the bispecific conditionally active antibody is a single polypeptide chain encoded by a polynucleotide molecule. The polynucleotide may be, for example, DNA, cDNA, RNA, or a recombinantly produced chimeric nucleic acid molecule comprising synthetically produced DNA or RNA, or any of these polynucleotides, alone or in combination. The polynucleotide may be part of a vector, such as an expression vector, including a plasmid, cosmid, virus, and bacteriophage, or any expression system conventionally used in genetic engineering. The vector may contain additional genes, such as marker genes, that allow for the selection of the vector under suitable conditions in a suitable host cell.

[0351] In one embodiment, the polynucleotide is operably linked to an expression control sequence that allows expression in prokaryotic or eukaryotic cells. Expression vectors derived from viruses such as retroviruses, vaccinia viruses, adeno-associated viruses, herpes viruses, or bovine papilloma viruses can be used to deliver the polynucleotide or vector to mammalian cells. The vectors containing the polynucleotides of the present invention can be transferred into host cells by well-known methods, which vary depending on the type of cellular host. For example, calcium chloride transfection is commonly used for prokaryotic cells, while calcium phosphate treatment or electroporation can be used for other cellular hosts.

[0352] In another embodiment, a conditionally active polypeptide can be engineered to produce a bispecific conditionally active polypeptide. The method for engineering a bispecific conditionally active polypeptide is similar to the method for engineering a bispecific conditionally active antibody as described in International Publication No. 2015 / 175375. For example, a bispecific conditionally active polypeptide can have two active sites, each of which has a conditional activity, i.e., less activity than the parent site under normal physiological conditions and more activity than the parent site under abnormal conditions. These two conditionally active sites can be independently evolved and screened, and then the two active sites can be linked to the same bispecific conditionally active polypeptide via a linker. In one embodiment, linkers that can be used in bispecific conditionally active antibodies are known linkers suitable for generating bispecific conditionally active polypeptides by linking two conditionally active sites of a conditionally active polypeptide.

[0353] Suitable methods for engineering the Fc region of conditionally active antibodies are described in WO 2015 / 175375.

[0354] Suitable methods for engineering conditionally active viral particles are described in WO 2015 / 175375.

[0355] In some embodiments, the conditionally active polypeptide may be inserted into a viral particle that is an oncolytic virus using the methods described in WO 2015 / 175375. Oncolytic viruses are viruses that have the ability to kill tumor cells upon contact with those cells. The conditionally active polypeptide inserted into the oncolytic virus may be more active in the tumor microenvironment and less active at other sites in the subject. For example, the conditionally active polypeptide may be more active at pH or other conditions present in the tumor microenvironment (e.g., pH 6.2-6.8) and less active at pH or other conditions present at other sites in the subject, such as normal physiological conditions (e.g., pH 7.2-7.6). The conditionally active polypeptide inserted into the oncolytic virus can be used to facilitate delivery of the oncolytic virus to the tumor, where it can target and kill tumor cells.

[0356] Oncolytic viruses of interest include adenovirus, herpes simplex virus type 1, vaccinia virus, parvovirus, reovirus, Newcastle disease virus, etc. Vaccinia virus is of particular interest.

[0357] In one embodiment, the oncolytic virus is selected from the group consisting of paramyxovirus, reovirus, herpesvirus, adenovirus, and Semliki Forest virus. In a further embodiment, the paramyxovirus is selected from the group consisting of Newcastle disease virus (NDV), measles virus, and mumps virus. In another embodiment, the NDV is derived from a strain selected from the group consisting of MTH68 / H, PV-701, and 73-T.

[0358] In another embodiment, the oncolytic virus is selected from herpesvirus, reovirus, E1B-deleted adenovirus, vesicular stomatitis virus, and poxvirus, which not only destroy tumor cells but also have the potential to release antigens from the destroyed tumor cells, thereby eliciting an immune response.

[0359] Specific examples of oncolytic viruses include, without limitation, adenoviruses (e.g., Delta-24, Delta-24-RGD, ICOVIR-5, ICOVIR-7, Onyx-015, ColoAd1, H101, AD5 / 3-D24-GMCSF), reoviruses, herpes simplex viruses (HSV; OncoVEX GMCSF), Newcastle disease virus, measles virus, retroviruses (e.g., influenza viruses), poxviruses (e.g., vaccinia viruses, including Copenhagen, Western Reserve, and Wyeth strains), myxoma viruses, rhabdoviruses (e.g., vesicular stomatitis virus (VSV)), picornaviruses (e.g., Seneca Valley virus; SW-001), coxsackieviruses, and parvoviruses.

[0360] In one aspect, the oncolytic virus is an adenovirus, including any member of its 57 human serotypes (HAdV-1 to 57). In one embodiment, the adenovirus is the Ad5 serotype. Alternatively, the adenovirus may be a hybrid serotype that may or may not contain Ad5 components. Non-limiting examples of suitable adenoviruses include Δ-24, Δ-24-RGD, ICOVIR-5, ICOVIR-7, ONYX-015, ColoAd1, H101, and AD5 / 3-D24-GMCSF. ONYX-015 is a hybrid of the Ad2 and Ad5 serotypes with deletions in the E1B-55K and E3B regions, enhancing cancer selectivity. H101 is a modified version of Onyx-015. ICOVIR-5 and ICOVIR-7 contain deletions of the Rb binding site in E1A and replacement of the E1A promoter with the E2F promoter. Colo Ad 1 is a chimeric Addl lp / Ad3 serotype. AD5 / 3-D24-GMCSF (CGTG-102) is a serotype 5 / 3 capsid-modified adenovirus encoding GM-CSF (the Ad5 capsid protein knob has been replaced with the knob domain from serotype 3).

[0361] In a particularly preferred embodiment, the oncolytic virus is a Delta-24 or Delta-24-RGD adenovirus. Delta-24 is described in U.S. Patent Application Publication Nos. 2003 / 0138405 A1 and 2006 / 0147420 A1. Delta-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. Delta-24-RGD further contains an insertion of the RGD-4C sequence (which binds strongly to ανβ3 and ανβ5 integrins) into the HI loop of the fibrous knob protein (Pasqualini R. et al., Nat Biotechnol., 15:542-546, 1997).

[0362] Oncolytic adenoviruses may also be further modified to improve their ability to treat cancer. Such modifications of oncolytic adenoviruses are described by Jiang et al. (Curr. Gene Ther. 2009 Oct 9(5):422-427), see also U.S. Patent Application Publication No. 2006 / 0147420 A1.

[0363] The oncolytic virus comprising the conditionally active polypeptide may be administered locally or systemically. For example, without limitation, the oncolytic virus may be administered intravascularly (intra-arterially or intravenously), intratumorally, intramuscularly, intradermally, intraperitoneally, subcutaneously, orally, parenterally, intranasally, intratracheally, transdermally, intraspinally, intraocularly, or intracranially.

[0364] The oncolytic virus may be administered in a single dose or multiple doses. The virus is administered in a concentration of at least 1 x 10 5 Plaque-forming units (PFU), at least 5 × 10 5 PFU, at least 1 × 10 6 PFU, at least 5 × 10 6 Or at least 5 x 10 6 PFU, 1 × 10 7 , at least 1 x 10 7 PFU, at least 1 × 10 8 or at least 1 × 10 8PFU, at least 1 × 10 8 PFU, at least 5 × 10 8 PFU, at least 1 × 10 9 or at least 1 × 10 9 PFU, at least 5 × 10 9 Or at least 5 x 10 9 PFU, at least 1 × 10 10 PFU or at least 1 × 10 10 PFU, at least 5 × 10 10 Or at least 5 x 10 10 PFU, at least 1 × 10 11 PFU or at least 1 × 10 11 PFU, at least 1 × 10 12 PFU, or at least 1 × 10 13 For example, the oncolytic virus may be administered in a dosage of about 10 PFU. 7 ~10 13 PFU, approx. 10 8 ~10 13 PFU, approx. 10 9 ~10 12 PFU, or approximately 10 8 ~10 12 It may be administered in a dosage of PFU.

[0365] In certain embodiments, cancers treated with oncolytic viruses include any solid tumor, such as lung cancer, ovarian cancer, breast cancer, cervical cancer, pancreatic cancer, gastric cancer, colon cancer, skin cancer, laryngeal cancer, bladder cancer, and prostate cancer. In one embodiment, the cancer is a cancer of the central nervous system. The cancer can be a neuroepithelial tumor, e.g., an astrocytic tumor (e.g., astrocytoma, anaplastic astrocytoma, glioblastoma, gliosarcoma, pilocytic astrocytoma, giant cell astrocytoma, pleomorphic xanthoastrocytoma), oligodendroglioma, ependymoma, oligoastrocytoma, spongioblastoma, astroblastoma, choroid plexus papiloma, choroid plexus carcinoma, ganglioneuroma, ganglioglioma, neurocytoma, neuroepithelial tumor, neuroblastoma, pineal tumor (such as pineocytoma, pineoblastoma, or mixed pineocytoma / pineoblastoma), medulloepithelioma, medulloblastoma, neuroblastoma or ganglioneuroblastoma, retinoblastoma, or ependymoblastoma. The cancer may be a central nervous system neoplasm, such as a sellar tumor (such as a pituitary adenoma, pituitary carcinoma, or craniopharyngioma), a hematopoietic tumor (such as a primary malignant lymphoma, plasmacytoma, or granulocytic sarcoma), a germ cell tumor (such as a germinoma, embryonal carcinoma, yolk sac tumor, choriocarcinoma, teratoma, or mixed germ cell tumor), a meningioma, a mesenchymal tumor, a melanoma, or a cranial or spinal nerve tumor (such as a schwannoma or neurofibroma). The cancer may be a low-grade glioma (e.g., an ependymoma, astrocytoma, oligodendroglioma, or mixed glioma) or a high-grade (malignant) glioma (e.g., glioblastoma multiforme). The cancer may be a primary or metastatic brain tumor. The conditionally active polypeptide, or a product engineered from the conditionally active polypeptide, may be used in a pharmaceutical composition. Some suitable pharmaceutical compositions are described in US Pat. No. 8,709,755 B2.

[0366] The pharmaceutical compositions can be used to treat various cancers, including carcinomas, blastomas, and sarcomas, and certain leukemias or lymphoid malignancies, benign and malignant tumors, and malignant lesions, such as sarcomas, carcinomas, and melanomas. The cancers can be non-solid tumors (such as blood tumors) or solid tumors. Adult tumors / cancers and pediatric tumors / cancers are also included.

[0367] Hematological cancers are cancers of the blood or bone marrow. Examples of hematological cancers (or hematopoietic cancers) include leukemias, such as acute leukemias (such as acute lymphocytic leukemia, acute myelocytic leukemia, acute myelogenous leukemia, and myeloblastic, promyelocytic, myelomonocytic, monocytic, and erythroleukemia), chronic leukemias (such as chronic myelocytic (granulocytic) leukemia, chronic myelogenous leukemia, and chronic lymphocytic leukemia), polycythemia vera, lymphoma, Hodgkin's disease, non-Hodgkin's lymphoma (indolent and aggressive types), multiple myeloma, Waldenstrom's macroglobulinemia, heavy chain disease, myelodysplastic syndromes, hairy cell leukemia, and myelodysplasia.

[0368] A solid tumor is an abnormal mass of tissue that usually does not contain cysts or liquid areas. Solid tumors can be benign or malignant. Different types of solid tumors are named after the type of cells that form them (such as sarcoma, carcinoma, and lymphoma). Examples of solid tumors that can be treated include fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma, osteosarcoma, and other sarcomas, synovioma, mesothelioma, Ewing's tumor, leiomyosarcoma, rhabdomyosarcoma, colon cancer, lymphoid malignancies, pancreatic cancer, breast cancer, lung cancer, ovarian cancer, prostate cancer, hepatocellular carcinoma, squamous cell carcinoma, basal cell carcinoma, adenocarcinoma, sweat gland carcinoma, medullary thyroid carcinoma, papillary thyroid carcinoma, pheochromocytoma, sebaceous gland carcinoma, papillary carcinoma, papillary adenocarcinoma, medullary carcinoma, bronchogenic carcinoma, renal cell carcinoma, hepatocellular carcinoma, cholangiocarcinoma, choriocarcinoma, Wilms' tumor, cervical carcinoma, These include testicular tumors, seminomas, bladder cancer, melanoma, and sarcomas and carcinomas, including CNS tumors (such as gliomas (such as brain stem gliomas and mixed gliomas), glioblastomas (also known as glioblastoma multiforme), astrocytomas, CNS lymphomas, germinomas, medulloblastomas, schwannomas, craniopharyogiomas, ependymomas, pinealomas, hemangioblastomas, acoustic neuromas, oligodendrogliomas, meningiomas, neuroblastomas, retinoblastomas, and brain metastases).

[0369] Pharmaceutical compositions containing the conditionally active polypeptide or products engineered from the conditionally active polypeptide can be formulated by known methods for preparing pharmaceutical compositions, in which the conditionally active polypeptide is typically combined with a mixture, solution, or composition containing a pharmaceutically acceptable carrier.

[0370] Pharmaceutically acceptable carriers are materials that can be tolerated by recipient patients. Sterile phosphate-buffered saline is an example of a pharmaceutically acceptable carrier. Other suitable pharmaceutically acceptable carriers are well known to those skilled in the art (see, for example, Gennaro (ed.), Remington's Pharmaceutical Sciences (Mack Publishing Company, 19th ed. 1995)). The formulation may further comprise one or more excipients, preservatives, solubilizers, buffers, albumin to prevent protein loss on the surface of the vial, etc.

[0371] The form, route of administration, dosage and regimen of the pharmaceutical composition will necessarily depend on the condition to be treated, the severity of the disease, the age, weight and sex of the patient, etc. These matters can be taken into consideration by those skilled in the art to formulate a suitable pharmaceutical composition. The pharmaceutical composition of the present invention can be formulated for topical, oral, parenteral, intranasal, intravenous, intramuscular, subcutaneous or intraocular administration, etc.

[0372] Preferably, the pharmaceutical composition contains a pharmaceutically acceptable medium for an injectable formulation, which may in particular be isotonic sterile saline (monosodium or disodium phosphate, sodium, potassium, calcium or magnesium chloride, etc. or a mixture of such salts), or a dried, in particular freeze-dried, composition that can be reconstituted into a solution for injection, for example, by adding sterile water or saline.

[0373] In some embodiments, a tonicity agent, sometimes known as a "stabilizer," is present to adjust or maintain the osmotic pressure of the liquid in the composition. When used with large, charged biomolecules such as proteins and antibodies, tonicity agents are often referred to as "stabilizers" because they can interact with the charged groups on amino acid side chains, thereby reducing the likelihood of inter- and intra-molecular interactions. The tonicity agent may be present in any amount between 0.1% and 25%, preferably between 1 and 5%, by weight of the pharmaceutical composition. Preferred tonicity agents include polyhydric sugar alcohols, preferably trihydric or higher sugar alcohols, such as glycerin, erythritol, arabitol, xylitol, sorbitol, and mannitol.

[0374] Additional excipients include agents that can act as one or more of the following: (1) bulking agents, (2) solubility enhancers, (3) stabilizers, and (4) agents that prevent denaturation or adhesion to container walls. Such excipients include polyhydric sugar alcohols (as listed above); amino acids such as alanine, glycine, glutamine, asparagine, histidine, arginine, lysine, ornithine, leucine, 2-phenylalanine, glutamic acid, threonine, and the like; organic sugars or sugar alcohols such as sucrose, lactose, lactitol, trehalose, stachyose, mannose, sorbose, xylose, ribose, ribitol, myoinisitose, myoinisitol, galactose, galactitol, glycerol, cyclitols (e.g., inositol, threonine ... Examples of suitable reducing agents include cellulose, cellulose acetate, cellulose acetate esters ...

[0375] Non-ionic surfactants or detergents (also known as "wetting agents") can be used to help solubilize the therapeutic agent and protect the therapeutic protein from agitation-induced aggregation, which also allows the formulation to be exposed to shear surface stress without denaturing the active therapeutic protein or antibody. The non-ionic surfactant may be present in a concentration range of about 0.05 mg / ml to about 1.0 mg / ml, preferably about 0.07 mg / ml to about 0.2 mg / ml.

[0376] Suitable nonionic surfactants include polysorbates (20, 40, 60, 65, 80, etc.), poloxamers (184, 188, etc.), PLURONIC® polyols, TRITON®, polyoxyethylene sorbitan monoethers (TWEEN®-20, TWEEN®-80, etc.), lauromacrogol 400, polyoxyl 40 stearate, polyoxyethylene hydrogenated castor oil 10, 50, and 60, glycerol monostearate, sucrose fatty acid esters, methyl cellulose, and carboxymethyl cellulose. Anionic detergents that can be used include sodium lauryl sulfate, dioctyle sodium sulfosuccinate, and dioctyle sodium sulfonate. Cationic detergents include benzalkonium chloride or benzethonium chloride.

[0377] The dosage used for administration can be adapted depending on various parameters, and in particular depending on the method of administration used, the pathology involved, or the desired duration of treatment. To prepare a pharmaceutical composition, an effective amount of the conditionally active polypeptide, or a product further engineered from the conditionally active polypeptide, can be dissolved or dispersed in a pharmaceutically acceptable carrier or aqueous medium.

[0378] Pharmaceutical preparations suitable for injection use include sterile aqueous solutions or dispersions; carriers such as sesame oil, peanut oil, or aqueous propylene glycol; and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions. In all cases, the preparation must be sterile and fluid to the extent that easy syringability exists. The preparation must be stable under the conditions of manufacture and storage and must be protected from the contaminating action of microorganisms such as bacteria and fungi.

[0379] Solutions of the conditionally active polypeptide as a free base or pharmacologically acceptable salt can be prepared in water suitably mixed with a surfactant. Dispersions can also be prepared in glycerol, liquid polyethylene glycols, and mixtures thereof, and in oils. Under ordinary conditions of storage and use, these preparations contain a preservative to prevent the growth of microorganisms.

[0380] Conditionally active polypeptides and products engineered from conditionally active polypeptides can be formulated into compositions in the form of salts. Pharmaceutically acceptable salts include acid addition salts (formed with the protein's free amino groups) and those formed with inorganic acids such as hydrochloric acid or phosphoric acid, or organic acids such as acetic acid, oxalic acid, tartaric acid, mandelic acid, and the like. Salts formed with free carboxyl groups can also be derived from inorganic bases such as sodium, potassium, ammonium, calcium, or ferric hydroxide, and organic bases such as isopropylamine, trimethylamine, histidine, procaine, and the like.

[0381] The carrier can also be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol, and the like), suitable mixtures thereof, and vegetable oils. Proper fluidity can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersions, and by the use of surfactants. Prevention of microbial action can be brought about by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, thimerosal, and the like. In many cases, it is preferable to include isotonic agents, for example, sugars or sodium chloride. Prolonged absorption of injectable compositions can be brought about by the use in the compositions of agents delaying absorption, for example, aluminum monostearate and gelatin.

[0382] Sterile injectable solution is prepared by mixing the required amount of conditionally active polypeptide in a suitable solvent with one or more of the other components listed above as necessary, and then sterilize by filtration.Generally, dispersion is prepared by mixing various sterilized active ingredients in a sterile medium that contains a basic dispersion medium and other necessary components listed above.For the preparation of sterile injectable solution, the preferred method of preparing sterile powder is vacuum drying and freeze-drying technology, which obtains powder of active ingredient plus any additional desired components from the solution that has been previously sterilized and filtered.

[0383] The preparation of more concentrated or highly concentrated solutions for direct injection is also contemplated, where the use of dimethyl sulfoxide (DMSO) as a solvent is expected to result in extremely rapid penetration, delivering high concentrations of active agent to small tumor areas.

[0384] Upon formulation, solutions may be administered in a manner compatible with the dosage formulation and in such amount as is therapeutically effective. The formulations are easily administered in a variety of dosage forms, such as the type of injectable solutions described above, although drug release capsules and the like can also be employed.

[0385] For parenteral administration in an aqueous solution, for example, the solution should be suitably buffered if necessary, and the diluent first rendered isotonic with sufficient saline or glucose. These particular aqueous solutions are particularly suitable for intravenous, intramuscular, subcutaneous, and intraperitoneal administration. In this regard, available sterile aqueous media will be known to those of skill in the art in light of the present disclosure. For example, a dosage can be dissolved in 1 ml of isotonic NaCl solution and either added to 1000 ml of subcutaneous infusion fluid or injected at the proposed infusion site (see, e.g., "Remington's Pharmaceutical Sciences," 15th Edition, pages 1035-1038 and 1570-1580). Some variation in dosage will necessarily occur depending on the condition of the subject being treated. The person responsible for administration will, in any event, determine the appropriate dose for the individual subject.

[0386] The conditionally active polypeptides and products engineered from the conditionally active polypeptides may be formulated in therapeutic mixtures to deliver about 0.0001 to 10.0 milligrams, or about 0.001 to 5 milligrams, or about 0.001 to 1 milligrams, or about 0.001 to 0.1 milligrams, or about 0.1 to 1.0 or even about 10 milligrams per dose. Multiple doses may also be administered at selected time intervals.

[0387] In addition to compounds formulated for parenteral administration, such as intravenous or intramuscular injection, other pharmaceutically acceptable forms include, for example, tablets or other solids for oral administration; time-release capsules; and any other form currently in use.

[0388] In certain embodiments, the use of liposomes and / or nanoparticles to introduce the conditionally active polypeptide, or further engineered products of the conditionally active polypeptide, into host cells is contemplated. The formation and use of liposomes and / or nanoparticles is known to those of skill in the art.

[0389] Nanocapsules can generally entrap compounds in a stable and reproducible manner. To avoid side effects due to intracellular polymer overload, such ultrafine particles (approximately 0.1 μm in size) are generally designed using polymers that are degradable in vivo. Biodegradable polyalkylcyanoacrylate nanoparticles that meet these requirements are contemplated for use in the present invention.

[0390] Liposomes are formed from phospholipids that spontaneously form multilamellar concentric bilayer vesicles (also called multilamellar vesicles (MLVs)) when dispersed in aqueous media. MLVs generally have diameters between 25 nm and 4 μm. Sonication of MLVs results in the formation of small unilamellar vesicles (SUVs) with diameters in the range of 200-500 Å that contain aqueous solution in their cores. The physical properties of liposomes depend on pH, ionic strength, and the presence of divalent cations.

[0391] Pharmaceutical formulations containing the conditionally active polypeptides described herein or products engineered from the conditionally active polypeptides are prepared in the form of lyophilized formulations or aqueous solutions by mixing with one or more optional pharmaceutically acceptable carriers (Remington's Pharmaceutical Sciences 16th edition, Osol, A. Ed. (1980)). Pharmaceutically acceptable carriers include, but are not limited to, buffers such as phosphate, citrate, and other organic acids; antioxidants including ascorbic acid and methionine; preservatives (such as octadecyldimethylbenzylammonium chloride; hexamethonium chloride; benzalkonium chloride; benzethonium chloride; phenol, butyl, or benzyl alcohol; alkyl parabens such as methyl or propyl paraben; catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol); low molecular weight (less than about 10 residues) polypeptides; serum albumin; These include proteins such as gelatin or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, histidine, arginine, or lysine; monosaccharides, disaccharides, and other carbohydrates, including glucose, mannose, or dextrins; chelating agents such as EDTA; sugars such as sucrose, mannitol, trehalose, or sorbitol; salt-forming counterions such as sodium; metal complexes (e.g., Zn-protein complexes); and / or non-ionic surfactants such as polyethylene glycol (PEG).

[0392] Exemplary pharmaceutically acceptable carriers herein further include interstitial drug dispersants, such as soluble neutral-active hyaluronidase glycoproteins (sHASEGPs), e.g., human soluble PH-20 hyaluronidase glycoproteins, e.g., rHuPH20 (HYLENEX®, Baxter International, Inc.). Certain exemplary sHASEGPs and methods of use, including rHuPH20, are described in U.S. Patent Application Publication Nos. 2005 / 0260186 and 2006 / 0104968. In one embodiment, a sHASEGP is combined with one or more additional glycosaminoglycanases, such as chondroitinase.

[0393] Exemplary lyophilized antibody formulations are described in U.S. Pat. No. 6,267,958. Aqueous antibody formulations include those described in U.S. Pat. No. 6,171,586 and WO 2006 / 044908, the latter formulations comprising a histidine acetate buffer.

[0394] The formulations herein may also contain two or more active ingredients as needed for the particular indication being treated. Preferably, ingredients with complementary activities that do not adversely affect each other are combined in a single formulation. For example, in addition to the conditionally active antibody, antibody fragment, or immunoconjugate of the present invention, it may be desirable to provide an EGFR antagonist (e.g., erlotinib), an anti-angiogenic agent (e.g., a VEGF antagonist, which may be an anti-VEGF antibody), or a chemotherapeutic agent (e.g., a taxoid or platinum agent). Such active ingredients are suitably present in combination in amounts effective for the intended use.

[0395] The active ingredient may be encapsulated in microcapsules prepared, for example, by coacervation techniques or by interfacial polymerization. For example, hydroxymethylcellulose or gelatin microcapsules and poly(methylmethacylate) microcapsules in colloidal drug delivery systems (e.g., liposomes, albumin microspheres, microemulsions, nanoparticles, and nanocapsules) or macroemulsions, respectively, may be utilized. Such techniques are disclosed in Remington's Pharmaceutical Sciences, 16th edition, Osol, A. Ed. (1980).

[0396] Sustained-release preparations may also be prepared. Suitable examples of sustained-release preparations include semipermeable matrices of solid hydrophobic polymers containing the antibody or antibody fragment, which matrices may be in the form of shaped articles, e.g., films, or microcapsules.

[0397] In some embodiments, the conditionally active polypeptides, or products engineered from the conditionally active polypeptides, can be used to make articles of manufacture containing materials useful for the treatment, prevention, and / or diagnosis of the described disorders. The articles of manufacture include a container and a label or package insert on or associated with the container. Suitable containers include, for example, bottles, vials, syringes, IV solution bags, etc. The container can be formed from a variety of materials, such as glass or plastic. The container holds a composition effective for the treatment, prevention, and / or diagnosis of a condition, alone or in combination with another composition, and can have a sterile access port (e.g., the container can be an intravenous solution bag or vial with a stopper pierceable by a hypodermic needle). At least one active agent in the composition is a conditionally active polypeptide of the invention, or a product further engineered from a conditionally active polypeptide. The label or package insert indicates that the composition is for use in treating the condition of choice. Additionally, the article of manufacture may include a first container having a composition in (a) (wherein the composition comprises a conditionally active polypeptide or a product engineered from the conditionally active polypeptide); and a second container having a composition in (b) (wherein the composition comprises an additional cytotoxic or other therapeutic agent). The article of manufacture in this embodiment of the invention may further include a package insert indicating that the composition may be used to treat a particular condition. Alternatively, or in addition, the article of manufacture may further include a second (or third) container containing a pharmaceutically acceptable buffer, such as bacteriostatic water for injection (BWFI), phosphate-buffered saline, Ringer's solution, and dextrose solution. Further, the article of manufacture may include other materials desirable from a commercial and user standpoint, including other buffers, diluents, filters, needles, and syringes.

[0398] The product may optionally include a container as a component of a parenteral, subcutaneous, intramuscular, intravenous, intraarticular, intrabronchial, intraperitoneal, intracapsular, intrachondral, intrasinus, intracavity, intracerebellar, intraventricular, intracolonic, intracervical, intragastric, intrahepatic, intramyocardial, intraosseous, intrapelvic, intrapericardial, intraperitoneal, intrapleural, intraprostatic, intrapulmonary, intrarectal, intrarenal, intraretinal, intraspinal, intrasynovial, intrathoracic, intrauterine, intravesical, bolus, vaginal, rectal, buccal, sublingual, nasal, or transdermal delivery device or system.

[0399] The conditionally active polypeptide, or a product further engineered from the conditionally active polypeptide, may be comprised in a medical device, wherein the device is suitable for contacting or administering the conditionally active polypeptide, or a product further engineered from the conditionally active polypeptide, by at least one method selected from parenteral, subcutaneous, intramuscular, intravenous, intraarticular, intrabronchial, intraperitoneal, intracapsular, intrachondral, intrasinus, intracavity, intracerebellar, intraventricular, intracolonic, intracervical, intragastric, intrahepatic, intramyocardial, intraosseous, intrapelvic, intrapericardial, intraperitoneal, intrapleural, intraprostatic, intrapulmonary, intrarectal, intrarenal, intraretinal, intraspinal, intrasynovial, intrathoracic, intrauterine, intravesical, bolus, vaginal, rectal, buccal, sublingual, intranasal, or transdermal.

[0400] In some further embodiments, the conditionally active polypeptide or a product engineered from the conditionally active polypeptide may be contained in a first container in lyophilized form in the kit, and the optional second container contains at least one preservative selected from the group consisting of phenol, m-cresol, p-cresol, o-cresol, chlorocresol, benzyl alcohol, phenylmercuric nitrite, phenoxyethanol, formaldehyde, chlorobutanol, magnesium chloride, alkyl parabens, benzalkonium chloride, benzethonium chloride, sodium dehydroacetate, and thimerosal, or mixtures thereof, in sterile water, sterile buffered water, or an aqueous diluent. In one aspect, in this kit, the concentrate of the conditionally active polypeptide or a product engineered from the conditionally active polypeptide in the first container is reconstituted with the contents of the second container to a concentration of about 0.1 mg / ml to about 500 mg / ml. In another aspect, the second container further contains a tonicity agent. In another embodiment, the second container further comprises a physiologically acceptable buffer. In one embodiment, the present disclosure provides a method of treating at least one condition mediated by a parent protein, comprising administering to a patient in need thereof a formulation provided in the kit and reconstituted prior to administration.

[0401] The following examples are illustrative of the present disclosure, but not limiting. Other suitable modifications and adaptations of a variety of conditions and parameters normally encountered in the field, which are obvious to those skilled in the art, are within the scope of the present disclosure. [Example]

[0402] Examples 1-5 relating to the production of conditionally active polypeptides are described in US Pat. No. 8,709,755 B2, hereby incorporated by reference.

[0403] Example 6: Evolving antibody light or heavy chains The heavy and light chains of antibody F1-10F10 were evolved separately using CPE. Screening of the light chain mutants identified 26 light chain mutants with conditional activity, where the mutants were more active than the wild type at pH 6.0 and less active than the wild type at pH 7.4. The 26 light chain mutants had the mutations at eight different positions in the light chain. Three of these eight positions were found in six or more of the 26 light chain mutants. These three positions were considered hot spots in the light chain. Screening of the heavy chain mutants identified 28 heavy chain mutants with conditional activity. These 28 heavy chain mutants had the mutations at eight different positions in the heavy chain. Three of these eight positions were found in six or more of the 28 heavy chain mutants. These three positions were considered hot spots in the heavy chain. The conditional activities of the light and heavy chain mutants were confirmed by ELISA analysis.

[0404] The best conditionally active antibodies generated in this example showed a 17-fold difference in their activity at pH 6.0 relative to their activity at pH 7.4. In addition, many of the conditionally active antibodies had reversible activity at pHs between the normal physiological pH of 7.4 and the abnormal pH of 6.0. Interestingly, when the activity of the conditionally active antibodies was tested in the pH range of 5.0 to 7.4 by ELISA analysis, most of the conditionally active antibodies generated in this example exhibited optimal binding activity at a pH of approximately 5.5 to 6.5.

[0405] The activity of the conditionally active antibodies produced in this example was also confirmed by FACS (fluorescence-activated cell sorting) analysis using whole cells, in which CHO cells were used to express the antibody antigen at pH 6.0 and pH 7.4. Binding activity was measured by adding the conditionally active antibodies to the CHO cells. FACS analysis confirmed the general trend in the ELISA analysis results regarding the selectivity of the conditionally active antibodies at pH 6.0 compared to pH 7.4.

[0406] Example 7: Selection of conditionally active antibodies in special buffers The mutant antibodies generated by the evolution process of the present invention were subjected to analysis at a normal physiological pH of 7.4 and an abnormal pH of 6.0. Both analyses were performed using a phosphate-buffered saline (PBS) solution containing bicarbonate, which is found in human serum. The bicarbonate concentration in the solution was the typical bicarbonate concentration in human serum, i.e., the physiological concentration. A comparison test was performed using the same PBS solution without bicarbonate.

[0407] The assay for measuring the binding activity of the mutant antibodies or conditionally active antibodies in this example was an ELISA assay, which was performed as follows: 1. The day before ELISA: Coat wells with 100ul of antibody Ab-A ECD his tag (2.08mg / ml) antigen at 1ug / ml in PBS. 3. The buffer solution was shaken off the 96-well plate coated with antibody Ab-A-His antigen and blotted dry on a paper towel. 4. The plate was washed three times with buffer N or PBS. 5. The plate was blocked with 200 ul of the specified buffer for 1 hour at room temperature. 6. The selected CPE / CPS mutants and wild-type proteins were diluted to 75 ng / ml in the designated buffer solution according to the layout. The pH of the buffer solution was set to either 6.0 or 7.4 (hereinafter referred to as the "designated buffer solution"). 6. The buffer was shaken off and 100ul of 75ng / ml sample was added to each well according to the plate layout. 7. The plate was incubated at room temperature for 1 hour. 8. The buffer was shaken off from the 96-well plate and blotted on a paper towel. 9. The plate was washed a total of three times with 200 ul of the designated buffer solution according to the layout. 10. Prepare anti-Flag HRP at 1:5000 dilution in the specified buffer solution and add 100ul of anti-Flag horseradish peroxide (HRP) to each well according to the layout. 11. The plate was incubated at room temperature for 1 hour. 13. The plate was washed a total of three times with 200 ul of the designated buffer solution. 14. The plate was developed with 50 ul of 3,3',5,5;-tetramethylbenzidine (TMB) for 1.5 minutes.

[0408] It was found that analysis in bicarbonate-containing PBS buffer solution resulted in a significantly higher success rate for selecting conditiona...

Claims

1. A non-naturally occurring polypeptide or isolated polypeptide having a ratio of activity assayed at a first pH in the presence of at least one species having a molecular weight of less than 900 a.m.u. and a pKa at most 0.5, 1, 2, 3, or 4 pH units away from the first pH to activity assayed at a second pH in the presence of the same at least one species of at least 1.

3.

2. 1. A non-naturally occurring or isolated polypeptide having a ratio of activity assayed at a first pH in the presence of at least one species having a molecular weight of less than 900 a.m.u. to activity assayed at a second pH in the presence of the same at least one species of at least 1.3, wherein said species has a pKa between said first pH and said second pH.

3. A non-naturally occurring polypeptide or isolated polypeptide, wherein the ratio of activity assayed at a first pH in the presence of a species selected from histidine, histamine, hydrogenated adenosine diphosphate, hydrogenated adenosine triphosphate, citrate, bicarbonate, acetate, lactate, disulfide, hydrogen sulfide, ammonium, dihydrogen phosphate, and any combination thereof to the activity assayed at a second pH in the presence of the same species is at least 1.

3.

4. 4. The polypeptide of any one of claims 1 to 3, wherein the ratio of the activity of the assay at the first pH to the activity of the assay at the second pH is at least 1.5, or at least 1.7, or at least 2.0, or at least 3.0, or at least 4.0, or at least 6.0, or at least 8.0, or at least 10.0, or at least 20.0, or at least 40.0, or at least 60.0, or at least 100.

0.

5. The polypeptide according to any one of claims 1 to 4, wherein the first pH is an acidic pH and the second pH is an alkaline pH or a neutral pH.

6. The polypeptide of any one of claims 1 to 5, wherein the second pH is a normal physiological pH within the normal range of physiological conditions at the site of administration of the polypeptide to a subject or in a tissue or organ at the site of action of the polypeptide in a subject, and the first pH is an abnormal pH that deviates from the normal range of physiological conditions at the site of administration of the polypeptide or in the tissue or organ at the site of action of the polypeptide.

7. The polypeptide of claim 6, wherein the first pH is in the range of 5.5 to 7.2, or in the range of 6.2 to 6.

8.

8. The polypeptide of claim 6 or 7, wherein the second pH is in the range of 7.2 to 7.

6.

9. 7. The polypeptide of claim 6, wherein the first pH is about 6.0 and the second pH is about 7.

4.

10. 10. The polypeptide of any one of claims 1 to 9, which is a non-naturally occurring mutant polypeptide evolved from a parent polypeptide.

11. The mutant polypeptide of claim 10 , wherein the parent polypeptide is a wild-type polypeptide.

12. The mutant polypeptide of claim 10 , wherein the parent polypeptide is a non-naturally occurring polypeptide.

13. A mutant polypeptide according to any one of claims 10 to 12, comprising at least one amino acid substitution compared to the parent polypeptide.

14. A variant polypeptide according to any one of claims 10 to 13, having a higher proportion of charged amino acid residues than the parent polypeptide.

15. A polypeptide or variant polypeptide according to any one of claims 1 to 14 which is a protein or protein fragment.

16. A polypeptide or mutant polypeptide described in any one of claims 1 to 14, wherein the polypeptide or mutant polypeptide is selected from an antibody, a single-chain antibody, and an antibody fragment, and the activity is binding activity to an antigen.

17. 17. The polypeptide or variant polypeptide of claim 16, which is an Fc region of an antibody.

18. The polypeptide or variant polypeptide according to any one of claims 1 to 14, wherein the polypeptide or variant polypeptide is an enzyme and the activity is an enzymatic activity.

19. 15. A polypeptide or variant polypeptide according to any one of claims 1 to 14, selected from a receptor, a regulatory protein, a soluble protein, a cytokine, and a fragment of a receptor, a regulatory protein, a soluble protein or a cytokine.

20. 19. The polypeptide or variant polypeptide of any one of claims 1 to 18, wherein the species is bicarbonate.

21. 20. The polypeptide or variant polypeptide of any one of claims 1 to 19, wherein the species has a pKa higher than 6.

2.

22. 22. The polypeptide of any one of claims 1 to 21, wherein the polypeptide has two functional domains, and the activity is an activity of one of the two functional domains.

23. 23. The polypeptide of claim 22, wherein both of the two functional domains have pH-dependent activity.

24. 24. The polypeptide of claim 22 or 23, which is a bispecific antibody.

25. A pharmaceutical composition comprising an effective amount of the polypeptide of any one of claims 1 to 24 and a pharmaceutically acceptable carrier.

26. Use of a polypeptide according to any one of claims 1 to 24 for the treatment of solid tumors, inflamed joints, or brain diseases or disorders.

27. A method for treating a solid tumor, an inflamed joint, or a brain disease or disorder, comprising the step of administering a polypeptide according to any one of claims 1 to 24.

28. 28. The method of claim 27, wherein the polypeptide is administered as part of a chimeric antigen receptor of a T cell comprising the polypeptide.

29. 28. The method of claim 27, wherein the polypeptide is administered linked to a nanoparticle.

30. 28. The method of claim 27, wherein the polypeptide is administered as an antibody-drug conjugate comprising the polypeptide.

31. A chimeric antigen receptor for T cells comprising the polypeptide of any one of claims 1 to 24.

32. A polypeptide according to any one of claims 1 to 24 linked to a nanoparticle.

33. An antibody-drug conjugate comprising the polypeptide of any one of claims 1 to 24.

33. A bispecific antibody comprising at least one binding domain containing a polypeptide according to any one of claims 1 to 24.

34. A chimeric protein containing two different binding or catalytic domains, one or both of said domains comprising a polypeptide according to any one of claims 1 to 24.

35. An oncolytic virus comprising a polypeptide according to any one of claims 1 to 24.

36. A polypeptide according to any one of claims 1 to 24, conjugated to a nucleic acid.

37. 37. The polypeptide of claim 36, wherein the nucleic acid is non-covalently or covalently conjugated and the nucleic acid is a nucleic acid comprising at least one non-natural nucleotide.

38. The polypeptide of claim 1 or 2, wherein the at least one species interacts with the polypeptide through hydrophilic interactions, hydrophobic interactions, or covalent interactions.

39. A polypeptide according to any one of claims 1 to 24, comprising two or more species.

40. 1. A method for producing a conditionally active polypeptide from a parent polypeptide, comprising: (i) evolving the parent polypeptide by mutating at least one region outside its active site, thereby generating one or more mutant polypeptides; (ii) subjecting the one or more polypeptides and the parent polypeptide to a first assay under normal physiological conditions to measure the activity of the active site under the normal physiological conditions, and to a second assay under abnormal conditions to measure the activity of the active site under the abnormal conditions, wherein the normal physiological conditions and the abnormal conditions are the same conditions but have different values; (iii) selecting from the one or more mutant polypeptides the conditionally active polypeptide that exhibits both: (a) a decreased activity compared to the same activity of the parent polypeptide in the first assay; and (b) an increased activity compared to the same activity of the parent polypeptide in the second assay. A method comprising:

41. 41. The method of claim 40, wherein the physiological and abnormal conditions are selected from pH, temperature, pH, osmolality, osmolality, oxidative stress, and electrolyte concentration.

42. 42. The method of claim 40 or 41, wherein the at least one region outside the active site is a region adjacent to the active site.

43. 42. The method of claim 40 or 41, wherein the at least one region outside the active site is a region away from the active site.

44. 44. The method of any one of claims 40 to 43, wherein the parent polypeptide is a heavy or light chain of an antibody, and the active site is a complementarity determining region of the parent polypeptide.

45. 45. The method of claim 44, wherein the at least one region outside the active site is an Fc region of the parent polypeptide.

46. 46. ​​The method of claim 45, wherein the evolution step comprises substituting the Fc region for the variable region of a separate antibody to form a bispecific antibody.

47. 46. ​​The method of claim 45, wherein the evolving step comprises substituting the Fc region for the variable region of a separate antibody to form a single chain antibody.

48. 45. The method of claim 44, wherein the at least one region outside the active site is a framework region of the parent polypeptide.

49. 48. The method of any one of claims 40 to 47, wherein the at least one region outside the active site is a plurality of regions, and the evolving step comprises sequentially evolving the plurality of regions.

50. 49. The method of any one of claims 40 to 48, wherein the evolution step comprises substitution, insertion, deletion, or a combination thereof.

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