Conditionally Active Peptide

Abstract

The present invention discloses a non-naturally occurring polypeptide or isolated polypeptide having an activity ratio of at least 1.3, wherein the activity ratio is the ratio of the activity in a test at a first pH in the presence of at least one substance having a molecular weight of less than 900 a.mu and a pKa that differs from the first pH by up to 4 pH units to the activity in a test at a second pH in the presence of the same at least one substance. The substance has a pKa between the first pH and the second pH and may be a small molecule. Pharmaceutical preparations comprising the polypeptide and uses thereof are also disclosed. Methods for producing conditionally active polypeptides are also disclosed.

Description

[0001] This application is a divisional application of a Chinese patent application filed on August 31, 2016, with Chinese patent application number 201680077540.7 and invention name “Conditionally Active Polypeptide”, and this application claims priority to US 62 / 249,907 and PCT / US2016 / 019242. Technical Field

[0002] The present invention relates to the field of providing improved polypeptides with desired activities. In particular, the present invention relates to methods for producing conditionally active polypeptides from a parent polypeptide, wherein the conditionally active polypeptide is more active under one condition than under another condition in the presence of a specific compound or ionic species. Background Art

[0003] There is a large literature describing methods of evolving various properties of proteins (particularly enzymes or antibodies) to make them active or stable under different conditions. For example, enzymes have been evolved to be stable at higher temperatures. In cases where enzyme activity is enhanced at higher temperatures, much of the increase in activity can be attributed to the higher kinetic activity generally described by the Q10 rule, which predicts that for enzymes, the turnover rate doubles for every 10°C increase in temperature.

[0004] In addition, there are natural mutations that render a protein unstable under its normal operating conditions, thereby reducing the activity of the protein under normal operating conditions. For example, there are known temperature mutants that are active at lower temperatures, but the activity is usually at a reduced level compared to the wild-type protein from which the mutant was derived.

[0005] It is desirable to generate polypeptides with conditional activity, for example, less active or almost inactive under one condition, but active under another condition. It is also desirable to generate polypeptides that are activated or inactivated in a particular environment, or that are activated or inactivated over time. In addition to temperature, other conditions that can evolve polypeptides or provide conditional activity include pH, osmotic pressure, weight molar osmotic pressure concentration, oxidative stress and electrolyte concentration. In addition to the activity of the polypeptide, it is generally desirable to improve other characteristics during the evolution process, including chemical resistance and proteolytic resistance.

[0006] Many strategies for evolving polypeptides have been described before. For example, US2005 / 0100985 discloses a fast and convenient method for generating a set of mutant polynucleotides from a parental template polynucleotide by replacing each original codon position in a template polynucleotide with a codon encoding 20 naturally occurring amino acids. This method is simply referred to as saturation mutagenesis, and can be used in combination with other mutagenesis methods, such as a method in which two or more related polynucleotides are introduced into a suitable host cell to generate a hybrid polynucleotide by recombination and reduction rearrangement.

[0007] Giver et al., "Directed evolution of athermostable esterase," Proc. Natl. Acad. Sci. USA, vol. 95, pp. 12809-12813 (1998), used in vitro evolution to study the relationship between stability and activity of mesophilic esterases. Six generations of random mutagenesis, recombination, and screening significantly stabilized the Bacillus subtilis para-nitrobenzyl esterase (Tm increase> 14°C) without compromising its catalytic activity at lower temperatures. The study found that mutations that increase thermal stability while maintaining low temperature activity are very rare. Improving one property by accumulating amino acid substitutions usually comes at the expense of another property, regardless of whether the two properties are anti-correlated or completely unrelated.

[0008] Evolving a parent polypeptide to be inactive or nearly inactive (less than 50%, 30% or 10% activity, particularly 1% activity) under its normal working conditions while maintaining equivalent or better activity under abnormal conditions may require the coexistence of destabilizing mutations and activity enhancing mutations that do not counteract the destabilizing effect. It is expected that destabilizing mutations will have a greater effect on the reduction of polypeptide activity than predicted by standard rules such as Q10, and therefore, the ability to evolve polypeptides that work effectively under abnormal conditions, for example, while being less active or inactive under their normal working conditions, will result in conditionally active polypeptides.

[0009] Therefore, the conditionally active polypeptide has enhanced activity compared to the parent protein under abnormal conditions, and decreased activity compared to the parent protein under normal physiological conditions. When used as a therapeutic protein, the conditionally active polypeptide therefore preferably acts on the location where the abnormal conditions exist, such as the tumor microenvironment. Due to this preferential action, the conditionally active polypeptide can potentially cause less damage to normal tissues / organs where normal physiological conditions exist, and therefore produces fewer side effects. This allows for longer treatments or higher doses of the conditionally active polypeptide to be used, resulting in higher efficacy of the treatment.

[0010] WO2010 / 104821 and WO2011 / 009058 disclose methods for evolving and screening conditionally active proteins.

[0011] There remains a need for conditionally active polypeptides that have increased activity and / or selectivity in specific environments and / or under specific conditions. Summary of the invention

[0012] In one embodiment, the invention relates to a non-naturally occurring polypeptide or isolated polypeptide having an activity ratio of at least 1.3, wherein the activity ratio is the ratio of the activity in a test at a first pH in the presence of at least one substance having a molecular weight of less than 900 a.mu and a pKa that differs from the first pH by up to 0.5, 1, 2, 3 or 4 pH units to the activity in a test at a second pH in the presence of the same at least one substance.

[0013] In one embodiment, the invention relates to a non-naturally occurring polypeptide or isolated polypeptide having an activity ratio of at least 1.3, wherein the activity ratio is the ratio of the activity in a test at a first pH in the presence of at least one substance having a molecular weight of less than 900 a.mu to the activity in a test at a second pH in the presence of the same at least one substance, and wherein the pKa of the substance is between the first pH and the second pH.

[0014] In one embodiment, the invention relates to a non-naturally occurring polypeptide or isolated polypeptide having an activity ratio of at least 1.3, wherein the activity ratio is the ratio of the activity in a test at a first pH in the presence of a substance selected from histidine, histamine, hydrogenated adenosine diphosphate, hydrogenated adenosine triphosphate, citrate, bicarbonate, acetate, lactate, hydrogen sulfide, hydrogen sulfide, ammonium, dihydrogen phosphate and any combination thereof to the activity in a test at a second pH in the presence of the same substances.

[0015] The ratio of the activity of the polypeptide described in any of the foregoing embodiments in a test at a first pH to the activity in a test at a second pH can be 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 described in the foregoing embodiments can be tested at a first pH that is an acidic pH and a second pH that is an alkaline pH or a neutral pH. The second pH can be a normal physiological pH, within the normal range of physiological conditions at the site where the polypeptide is administered to the individual or within the normal range of physiological conditions at the tissue or organ at the site of action of the polypeptide in the individual; the first pH can be an abnormal pH, which deviates from the normal range of physiological conditions at the site of polypeptide administration or the normal range of physiological conditions at the tissue or organ at the site of action of the polypeptide.

[0016] The first pH may be in the range of 5.5-7.2, or in the range of 6.2-6.8. The second pH may be in the range of 7.2-7.6. The first pH may be about 6.0, and the second pH may be about 7.4.

[0017] The polypeptide described in any of the foregoing embodiments can be a non-naturally occurring mutant polypeptide evolved from a parent polypeptide. The mutant polypeptide described in any of the foregoing embodiments can be derived from a wild-type parent polypeptide comprising a non-naturally occurring polypeptide. The mutant polypeptide described in any of the foregoing embodiments can contain at least one amino acid replacement compared to the parent polypeptide. The mutant polypeptide described in any of the foregoing embodiments can have a higher proportion of charged amino acid residues than the parent polypeptide.

[0018] The polypeptide or mutant polypeptide described in any of the foregoing embodiments may be a protein or a protein fragment. The polypeptide or mutant polypeptide described in any of the foregoing embodiments may be selected from antibodies, single-chain antibodies and antibody fragments, and the activity is the binding activity with antigens. The polypeptide or mutant polypeptide described in any of the foregoing embodiments may be the Fc region of an antibody. The polypeptide or mutant polypeptide may be an enzyme, and the activity may be an enzyme activity. The polypeptide or mutant polypeptide may be selected from receptors, regulatory proteins, soluble proteins, cytokines, and fragments of receptors, regulatory proteins, soluble proteins or cytokines.

[0019] The substance described in any of the preceding embodiments may be hydrogen sulfide, bicarbonate or hydrogen sulfide. The pKa of the substance described in any of the preceding embodiments may be greater than 6.2.

[0020] 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 functional domains may have pH-dependent activity. The polypeptide may be a bispecific antibody.

[0021] 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.

[0022] In another embodiment, the present invention relates to a method of treating a solid tumor, an inflamed joint, or a brain disease or disorder, the method comprising administering a polypeptide as described in any one of the preceding embodiments. The polypeptide can be administered as part of a chimeric antigen receptor comprising the polypeptide for a T cell or as an antibody-drug conjugate comprising the polypeptide.

[0023] In another embodiment, the invention relates to a chimeric antigen receptor for a T cell comprising said polypeptide.In each of the foregoing embodiments, said antibody may be linked to a nanoparticle.

[0024] In another embodiment, the present invention relates to an antibody-drug conjugate comprising the polypeptide.

[0025] In another embodiment, the present invention provides a pharmaceutical composition comprising a conditionally active biological protein and a pharmaceutically acceptable carrier.

[0026] In another embodiment, the present invention provides a method for producing a conditionally active polypeptide from a parent polypeptide, the method comprising the steps of:

[0027] (i) evolving the parent polypeptide by mutating at least one region outside the active site of the parent polypeptide to produce one or more mutant polypeptides;

[0028] (ii) subjecting the one or more polypeptides and a parent polypeptide to a first test under normal physiological conditions to measure the activity of the active site under normal physiological conditions, and to a second test under abnormal conditions to measure the activity of the active site under abnormal conditions, wherein normal physiological conditions and abnormal conditions are the same conditions with different values; and

[0029] (iii) selecting from the one or more mutant polypeptides a conditionally active polypeptide that exhibits both of the following properties: (a) a decrease in activity compared to the same activity of a parent polypeptide in a first assay, and (b) an increase in activity compared to the same activity of a parent polypeptide in a second assay. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] Figure 1 The conditionally active antibodies generated in Example 9 and their selectivity at pH 6.0 relative to pH 7.4 are shown.

[0031] Figure 2 The binding activity of conditionally active antibodies to antigens tested in different buffer solutions is shown.

[0032] Figure 3 The effect of changing the composition of Krebs buffer on the binding activity of conditionally active antibodies is shown.

[0033] Figure 4 It was shown that the binding activity of three different conditionally active antibodies as described in Example 12 was dependent on the presence and concentration of bicarbonate at pH 7.4.

[0034] Figure 5 It is a diagram showing the structure of a chimeric antigen receptor (CAR).

[0035] Figure 6 is a diagram showing the formation of salt bridges in deoxyhemoglobin, where three amino acid residues form two salt bridges that stabilize the T quaternary structure of deoxyhemoglobin, resulting in a lower affinity for oxygen.

[0036] Figure 7 The activity of conditionally active antibodies against Ror2 in different buffer solutions is shown.

[0037] Figure 8 The activity of conditionally active antibodies against Axl in different buffer solutions is shown.

[0038] Fig. 9 The activity of the conditionally active antibodies against Axl found using a test solution with 10 mM hydrogen sulfide ions is shown.

[0039] definition

[0040] In order to facilitate understanding of the examples provided herein, some frequently occurring methods and / or terms are defined herein. The definitions of the following terms are incorporated by reference to U.S. Patent No. 8,709,755B2: "reagent", "ambiguous base requirement", "amino acid", "amplification", "chimeric properties", "homologous", "comparison window", "conservative amino acid substitution", "corresponding to", "degradation effective", "determined sequence skeleton", "determined sequence kernel", "digestion", "directional connection", "DNA shuffling", "drug" or "drug molecule", "effective amount", "epitope", "enzyme", "evolution" or "evolution", "fragment" or "derivative" or "analog", "single amino acid full replacement", "gene", "genetic instability", "heterologous", "homologous" or "homeologous", "industrial application", "same" or "identity", "identity region", "isolated", "isolated nucleic acid", "ligand", "connection", "joint" or "spacer", "microenvironment", "molecular properties to be evolved", "Mutation", "N,N,G / T", "normal physiological conditions" or "wild-type working conditions", "nucleic acid molecule", "nucleic acid molecule", "nucleic acid sequence encoding..." or "DNA coding sequence" or "nucleotide sequence encoding...", "nucleic acid encoding an enzyme (protein)" or "DNA encoding an enzyme (protein)" or "polynucleotide encoding an enzyme (protein)", "specific nucleic acid molecule species", "assembling the working nucleic acid sample into a nucleic acid library", "nucleic acid library", "construct", "oligonucleotide" (or the synonym "oligo"), "homologous", "operably linked", "parental polynucleotide combination", "patient" or "individual", "physiological conditions", "population", "precursor form", "pseudo-random", "quasi-repeated units", "random peptide library", "random peptide sequence", "receptor", "recombinant" enzyme, "synthetic" enzyme, "related polynucleotides", "reduced rearrangement (Reductive" "reassortment), "reference sequence," "repeat index (RI)," "restriction site," "selectable polynucleotide," "sequence identity," "similarity," "specific binding," "specific hybridization," "specific polynucleotide," "stringent hybridization conditions," "substantially identical," "substantially pure enzyme," "substantially pure," "therapeutic," "variable segment," and "variant."

[0041] As used herein, the term "about" in relation to an assay quantity refers to the normal variation in the assay quantity that can be measured and implemented by one skilled in the art with a level of care commensurate with the purpose of the measurement and the precision of the measuring instrument to be expected. Unless otherwise indicated, "about" refers to a variation of + / - 10% of a given value.

[0042] As used herein, the term "activity" refers to any function that a protein can perform, including catalytic reactions and binding to a partner. For an enzyme, the activity may be enzymatic activity. For an antibody, the activity may be the binding activity between the antibody and its antigen (i.e., binding activity). For a receptor or ligand, the activity may be the binding activity between the receptor and its ligand.

[0043] As used herein, the term "antibody" refers to intact immunoglobulin molecules and fragments of immunoglobulin molecules, such as Fab, Fab', (Fab')2, Fv and SCA fragments, which are capable of binding to antigenic epitopes. These antibody fragments retain some ability to selectively bind to the antigen (e.g., polypeptide antigen) from which the antibody is derived, and these antibody fragments can be prepared using methods well known in the art (see, e.g., Harlow and Lane, supra), and are further described as follows. Antibodies can be used to separate prepared amounts of antigen by immunoaffinity chromatography. Various other uses of such antibodies are diagnosis of disease and / or staging of disease (e.g., neoplasia) and for therapeutic applications to treat disease, such as, for example: neoplasia, autoimmune disease, AIDS, cardiovascular disease, infection, etc. Chimeric, human-like, humanized or fully human antibodies are particularly useful for administration to human patients.

[0044] Fab fragments consist of a monovalent antigen-binding fragment of an antibody molecule and can be produced by digesting an intact antibody molecule with an enzyme such as papain to produce a fragment consisting of an entire light chain and a portion of a heavy chain.

[0045] Fab' fragments of antibody molecules can be obtained by treating intact antibody molecules with pepsin followed by reduction to produce molecules consisting of an intact light chain and a portion of a heavy chain. Two Fab' fragments are obtained per antibody molecule treated in this manner.

[0046] The (Fab')2 fragment of an antibody can be obtained by treating an intact antibody molecule with pepsin without subsequent reduction. The (Fab')2 fragment is a dimer of two Fab' fragments held together by two disulfide bonds.

[0047] The Fv fragment is defined as a genetically engineered fragment containing the light chain variable region and the heavy chain variable region, represented as two chains.

[0048] Single-chain antibodies ("SCA" or scFv) are genetically engineered single-chain molecules containing a light chain variable region and a heavy chain variable region, which are connected by a suitable flexible polypeptide linker and may contain additional amino acid sequences at the amino- and / or carboxyl-termini. For example, a single-chain antibody may include a tether segment for connecting an encoding polynucleotide. Functional single-chain antibodies generally contain a sufficient portion of a light chain variable region and a sufficient region of a heavy chain variable region to retain the properties of a full-length antibody binding to a specific target molecule or epitope.

[0049] 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), which enables these cytotoxic effector cells to specifically bind to antigen-bearing target cells and subsequently kill the target cells with cytotoxins. Ligand-specific high-affinity IgG antibodies directed to the surface of target cells stimulate cytotoxic cells and are required for this killing. Lysis of target cells is extracellular, requires direct cell-to-cell contact, and does not involve complement.

[0050] Any specific antibody can be tested for its ability to mediate target cell lysis by ADCC. In order to evaluate ADCC activity, the antibody of interest is added to the target cell, thereby displaying the target ligand combined with the immune effector cell, and the target ligand can be activated by the antigen-antibody complex, resulting in the cell lysis of the target cell. Cell lysis is usually detected by releasing a label (e.g., a radioactive substrate, a fluorescent dye, or a natural intracellular protein) from the lysed cells. Useful effector cells for this test include peripheral blood mononuclear cells (PBMC) and natural killer (NK) cells. Specific examples of in vitro ADCC tests are described in Bruggemann et al, 1987, J Exp Med, vol. 166, page 1351; Wilkinson et al, 2001, J Immunol. Methods, vol. 258, page 183; Patel et al, 1995 J. Immunol. Methods, vol. 184, page 29. Alternatively or additionally, ADCC activity of the antibody of interest can be assessed in vivo, e.g., in a animal model such as that disclosed in Clynes et al., 1998, PNAS USA, vol. 95, p. 652.

[0051] The term "antigen" or "Ag" as used herein is defined as a molecule capable of inducing an immune response. Such an immune response may involve antibody production, or activation of specifically immunocompetent cells, or both. It will be appreciated by those skilled in the art that any macromolecule (including nearly all proteins or peptides) may be used as an antigen. It is apparent that antigens may be produced, synthesized or derived from biological samples. Such biological samples may include, but are not limited to, tissue samples, tumor samples, cells or biological fluids.

[0052] The term "antisense RNA" as used herein refers to an RNA molecule that can form a duplex with a second RNA molecule by being complementary or partially complementary to a second RNA molecule. The antisense RNA molecule can be complementary to the translated region or the untranslated region of the second RNA molecule. The antisense RNA does not need to be completely complementary to the second RNA molecule. The antisense RNA may or may not have the same length as the second RNA molecule; the antisense RNA molecule may be longer or shorter than the second RNA molecule. If the second RNA molecule is an mRNA, the binding of the antisense RNA will completely or partially prevent the mRNA from being translated into a functional protein product.

[0053] The term "biosimilar" or "follow-on biologic" is used in a manner consistent with the working definition promulgated by the U.S. Food and Drug Administration (FDA), which defines a biosimilar as "closely similar" to a reference product (although there are minor clinically meaningful differences in inactive components). In practice, there are no clinically meaningful differences between the reference product and the biosimilar product in terms of safety, purity and potency (Public Health Service (PHS) Regulation 262). A biosimilar can also be a biosimilar that meets one or more of the guidelines adopted by the Committee for Medicinal Products for Human Use of the European Medicines Agency (CHMP) on 30 May 2012 and published by the European Union as "Guidance on similar biological medicinal products containing monoclonal antibodies - Nonclinical and clinical issues" (document reference EMA / CHMP / BMWP / 403543 / 2010). For example, an "antibody biosimilar" is a follow-on version of an innovator antibody (the reference antibody) that is usually manufactured by a different company. Differences between an antibody biosimilar and a reference antibody can include post-translational modifications, for example, by attaching other biochemical groups such as phosphates, various lipids, and carbohydrates to the antibody; by post-translational proteolytic cleavage; by changing the chemical properties of amino acids (e.g., formylation); or by many other mechanisms. Other post-translational modifications may be the result of manufacturing process manipulations—for example, glycosylation can occur as the product is exposed to reducing sugars. In some cases, storage conditions may allow certain degradation pathways to occur, such as oxidation, deamidation, or aggregation. Because all of these product-related variations may be included in the antibody biosimilar.

[0054] The terms "cancer" and "cancerous" refer to or describe a physiological condition in mammals that is typically characterized by unregulated cell growth / proliferation. A "tumor" comprises one or more cancer cells. Examples of cancer include, but are not limited to, carcinoma, lymphoma, blastoma, sarcoma, and leukemia or lymphoid malignancies. More specific examples of such cancers include squamous cell carcinoma (e.g., epithelial squamous cell carcinoma), lung cancer (including small cell lung cancer, non-small cell lung cancer ("NSCLC"), lung adenocarcinoma, and lung squamous cell carcinoma), peritoneal cancer, hepatocellular carcinoma, gastric cancer or stomach cancer (including gastrointestinal cancer), pancreatic cancer, glioblastoma, cervical cancer, ovarian cancer, liver cancer, bladder cancer, hepatoma, breast cancer, colon cancer, rectal cancer, colorectal cancer, endometrial cancer or uterine cancer, salivary gland cancer, kidney cancer or renal cancer, prostate cancer, vulvar cancer, thyroid cancer, liver cancer, anal cancer, penile cancer, and head and neck cancer.

[0055] The term "chimeric antigen receptor" or "CAR" used herein refers to an engineered receptor that transplants antigen specificity to cytotoxic cells, such as T cells, NK cells, and macrophages. The CAR of the present invention may include at least one antigen-specific targeting region (ASTR), an extracellular spacer domain (ESD), a transmembrane domain (TM), one or more co-stimulatory domains (CSD), and an intracellular signaling domain (ISD). In some embodiments, ESD and / or CSD are optional. In one embodiment, ASTR is bispecific and can recognize two different antigens or epitopes. After ASTR specifically binds to the target antigen, ISD activates intracellular signaling of cytotoxic cells. For example, depending on the antigen binding properties of CAR, ISD can redirect T cell specificity and cytotoxicity to a selected target in a non-MHC restricted manner. Non-MHC restricted antigen recognition enables cytotoxic cells expressing CAR to have the ability to recognize antigens without relying on antigen processing, thereby bypassing the main mechanism of tumor escape. Moreover, when expressed in T cells, CAR advantageously does not dimerize with endogenous T cell receptor (TCR) α chain and β chain.

[0056] 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 the term "conditionally active polypeptide" refers to a variant or mutant of a parent polypeptide, wherein the activity of the variant or mutant polypeptide under a first condition is at least 1.3 times the activity under a second condition. The conditionally active polypeptide can exhibit activity at one or more selected locations of the body and / or exhibit enhanced or reduced activity at another location of the body. For example, in one aspect, the evolved conditionally active biological protein is almost 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 including stress proteins, vault-associated proteins, neuronal proteins, digestive tract proteins, growth factors, mitochondrial proteins, cytoplasmic proteins, animal proteins, structural proteins, plant proteins, and fragments of any of these proteins. Each conditionally active polypeptide described herein is preferably a conditionally active biological polypeptide.

[0057] The term "cytokine" or "cytokine" used herein refers to a large class of biomolecules that affect immune system cells. This definition is intended to include, but is not limited to, those biomolecules that act to control or regulate individual immune responses locally or at other locations away from the secretion site through the blood circulation. Exemplary cytokines include, but are not limited to, interferon-α (IFN-α), interferon-β (IFN-β), and interferon-γ (IFN-γ), interleukins (e.g., IL-1 to IL-29, particularly IL-2, IL-5, IL-6, IL-7, IL-10, IL-12, IL-15, and IL-18), tumor necrosis factor (e.g., TNF-α and TNF-β), erythropoietin (M-CSF), MIP3a, monocyte chemotactic protein (MCP)-1, intracellular adhesion molecules (ICAM), macrophage colony stimulating factor (M-CSF), granulocyte colony stimulating factor (G-CSF), and granulocyte-macrophage colony stimulating factor (GM-CSF).

[0058] As used herein, the term "electrolyte" is used to define a mineral in blood or other body fluids that carries an electrical charge. For example, in one aspect, normal physiological conditions and abnormal conditions may have different values ​​of "electrolyte concentrations." Exemplary electrolytes include, but are not limited to, ionized calcium, sodium, potassium, magnesium, chloride, citrate, lactate, bicarbonate, and phosphate.

[0059] The term "full length antibody" refers to an antibody comprising an antigen-binding variable region (V H or V L) and a light chain constant domain (CL) and heavy chain constant domains CHI, 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 may be classified into different "classes" based on the amino acid sequence of the constant domain of their heavy chains. There are five main types of full-length antibodies: IgA, IgD, IgE, IgG and IgM, several of which may be further divided into "subclasses" (isotypes), such as IgG1, IgG2, IgG3, IgG4, IgA and IgA2. The heavy chain constant domains corresponding to different classes of antibodies are referred to as α, δ, ε, γ and μ, respectively.

[0060] The term "growth factor" used herein refers to a polypeptide molecule that can achieve cell differentiation. Examples of growth factors include, but are not limited to, epidermal growth factor (EGF), transforming growth factor-α (TGFα), transforming growth factor-β (TGF-β), human endothelial cell growth factor (ECGF), granulocyte macrophage colony stimulating factor (GM-CSF), bone morphogenetic protein (BMP), nerve growth factor (NGF), vascular endothelial growth factor (NEGF), fibroblast growth factor (FGF), insulin-like growth factor (IGF), cartilage-derived morphogenetic protein (CDMP) and platelet-derived growth factor (PDGF).

[0061] The term "hormone" used herein means a substance usually identified as a mediator, which is usually released by a cell or gland in a part of an organism to serve as a messenger for other parts of the organism. Exemplary hormones include endocrine hormones that are directly released into the bloodstream and exocrine hormones (or ectohormones) that are directly secreted into and secreted from the duct, which either flow into the bloodstream or diffuse from cell to cell by diffusion in a process called paracrine signaling. Vertebrate hormones can be divided into three chemical classes: hormones and monoamines derived from peptide hormones, lipids, and phospholipids. Peptide hormones are composed of polypeptide chains. Examples of peptide hormones include insulin and growth hormone. Hormones derived from lipids and phospholipids are derived from lipids (such as linoleic acid and arachidonic acid) and phospholipids. The main category is steroid hormones derived from cholesterol and eicosanoids. Examples of steroid hormones are testosterone and cortisol. Monoamines are derived from aromatics (such as phenylalanine, tyrosine, and tryptophan) by the action of aromatic amino acid decarboxylase. Examples of monoamines are thyroxine and adrenaline.

[0062] The term "immunomodulator" as used herein refers to an agent whose effect on the immune system causes the activity of at least one pathway involved in the immune response to be immediately enhanced or delayed or reduced. This reaction can occur naturally or be triggered artificially, as a part of the innate immune system or the adaptive immune system or both. 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, interferon-β and interferon-γ), stem cell growth factors called "S1 factors", erythropoietin and thrombopoietin. Examples of suitable immunomodulator moieties include IL-2, IL-6, IL-10, IL-12, IL-18, IL-21, interferons, TNF (e.g., TNF-α), and the like.

[0063] An "individual" or "subject" is a mammal. Mammals include, but are not limited to, domesticated animals (e.g., cows, sheep, cats, dogs, and horses), primates (e.g., humans and non-human primates such as monkeys), rabbits, and rodents (e.g., mice and rats).

[0064] The term "library" as used herein refers to a collection of proteins in a single pool. The library is preferably produced using recombinant DNA technology. For example, a collection of cDNA or any other protein encoding DNA can be inserted into an expression vector to produce a protein library. A collection of cDNA or protein encoding DNA can also be inserted into a phage genome to produce a phage display library of wild-type proteins. A collection of cDNAs can be generated from a selected cell population or tissue sample, for example, by the method disclosed by Sambrook et al. (Molecular Cloning, Cold Spring Harbor Laboratory Press, 1989). A collection of cDNAs produced by a selected cell type can also be obtained from a vector such as The wild-type protein library used in this article is not a collection of biological samples.

[0065] The term "ligand" as used herein refers to a molecule that is recognized by a specific receptor and specifically binds to the 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, sugars, polynucleotides, nucleic acids, oligosaccharides, proteins, and monoclonal antibodies. Typically, a ligand comprises two structural parts: a first part that participates in the binding of the ligand to its receptor and a second part that does not participate in such binding.

[0066] As used herein, "receptor" refers to a molecule that has an affinity for a given ligand. A receptor can be a naturally occurring or artificially synthesized molecule. A receptor can be used in its unaltered state or as an aggregate formed with other substances. A receptor can be covalently or non-covalently linked to a binding unit directly or through a specific binding substance. Examples of receptors include, but are not limited to, antibodies (which include monoclonal antibodies and can react with antisera to specific antigenic determinants (such as those located on viruses, cells, or other materials), cell membrane receptors, complexes of carbohydrates and glycoproteins, enzymes, and hormone receptors. The binding of a ligand to its receptor indicates that the ligand and its receptor molecule are combined to form a complex through specific molecular recognition, which can be detected by a variety of ligand receptor binding tests known to those skilled in the art.

[0067] The term "microRNA" or "miRNA" used herein refers to an unprocessed or processed RNA transcript from a miRNA gene. Unprocessed microRNA gene transcripts typically include RNA transcripts of about 70-100 nucleotides in length. The transcribed microRNA can be digested into an active RNA molecule of 19-25 nucleotides by using RNase (e.g., Dicer, Argonaut or RNase III). This active 19-25 nucleotide RNA molecule is also referred to as "processed" microRNA gene transcript or "mature" microRNA.

[0068] The term "multispecific antibody" as used herein is an antibody that has binding specificity for at least two different epitopes. Exemplary multispecific antibodies can bind both BBB-R and brain antigens. Multispecific antibodies can be prepared as full-length antibodies or antibody fragments (e.g., F(ab')2 bispecific antibodies). Engineered antibodies with two, three or more (e.g., four) functional antigen binding sites are also contemplated (see, e.g., US2002 / 0004587A1).

[0069] The term "nanoparticle" used herein refers to a microscopic particle whose size is measured in nanometers (nm), with a maximum linear dimension of less than about 1000nm or less than about 500nm, or less than about 200nm, or less than about 100nm, or less than about 50nm. As used herein, linear dimension refers to the distance between any two points on the nanoparticle measured in a straight line. The nanoparticle of the present invention can be irregular, elliptical, spindle-shaped, rod-shaped, disc-shaped, flat, cylindrical, erythrocyte-shaped, spherical or substantially spherical in shape, as long as their shape and size allow for binding interactions. The nanoparticle of the present invention is preferably made of biocompatible materials (polymers or lipids).

[0070] As used herein, the term "naturally occurring" as applied to an object refers to the fact that an object can be found in nature. For example, a polypeptide or polynucleotide sequence present in an organism (including a virus) is naturally occurring, which can be isolated from a natural source and has not been intentionally modified by man in a laboratory. A polypeptide cut from a larger polypeptide is not a naturally occurring polypeptide because the terminal groups of the cut polypeptide will be different in the cut form from the terminal groups in the larger naturally occurring polypeptide because these terminal groups will no longer be associated with adjacent polypeptides. In general, the term "naturally occurring" refers to an object that is present in a non-pathological (non-diseased) individual, such as a species that is ubiquitous.

[0071] The terms "parent polypeptide" and "parent protein" as used herein refer to polypeptides or proteins that can be evolved using the methods of the present invention to produce conditionally active polypeptides or proteins. The parent polypeptide protein can be a wild-type protein including non-naturally occurring proteins. For example, a therapeutic polypeptide or protein or a mutant polypeptide 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.

[0072] As used herein, the term "pH-dependent" refers to a polypeptide having different properties or activities at different pH values.

[0073] The term "polypeptide" as used herein refers to a polymer in which the monomers are amino acids and the monomers 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, such as a selected region of an amino acid chain 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 and a synthetic amino acid chain. A fragment refers to an amino acid sequence that is a part of a full-length protein, and its length is generally about 8 amino acids to about 500 amino acids, preferably about 8 amino acids to about 300 amino acids, more preferably about 8 amino acids to about 200 amino acids, and more preferably about 10 amino acids to about 50 or 100 amino acids. In addition, amino acids other than naturally occurring amino acids (e.g., β-alanine, phenylglycine, and homoarginine) can be included in the polypeptide. Commonly encountered non-genetically encoded amino acids can also be included in the polypeptide. Amino acids can be D-optical isomers or L-optical isomers. The D-isomer is preferably used in specific situations further described below. In addition, other peptide mimetics are also useful, for example, in the linker sequence of a polypeptide (see Spatola, 1983, in Chemistry and Biochemistry of Amino Acids. Peptides and Proteins, Weinstein, ed., Marcel Dekker, New York, p. 267). Generally, the term "protein" is not intended to express any significant differences from the term "polypeptide", except that it includes structures of two or more polypeptide chains held together by covalent or non-covalent bonds.

[0074] As used herein, the term "recombinant antibody" refers to an antibody (e.g., a chimeric, humanized, or human antibody or an antigen-binding fragment thereof) expressed by a host cell comprising a nucleic acid encoding the antibody. Examples of "host cells" for preparing 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 yeast cells belonging to the genus Saccharomyces (e.g., Saccharomyces cerevisiae) or yeast cells belonging to the genus Aspergillus (e.g., Aspergillus niger); (5) bacterial cells, such as Escherichia coli cells or Bacillus subtilis cells, etc.

[0075] The term "regulatory protein" as used herein refers to any protein that has the following effects: enhancing or reducing the activity of another polypeptide or RNA molecule; increasing or reducing the abundance of another polypeptide or RNA molecule; changing the interaction between another polypeptide or RNA molecule and other polypeptides, DNA or RNA molecules or any other binding substrates; and / or changing the cellular location of another polypeptide or RNA molecule. When regulatory proteins increase or decrease the transcription rate of a gene, they are generally referred to as transcription factors that have an effect on the promoter or enhancer region of the gene. Examples of transcription factors include mammalian transcription factors such as NFkB, NF1, cyclic AMP response element binding protein (CREB), MyoD1, homeobox transcription factors, Sp1, oncogenes and jun, Mep-1, GATA-1, Isl-1, LFB1, NFAT, Pit-1, OCA-B, Oct-1 and Oct-2, yeast A / alpha, cErb-A, myc, mad and max, p53, mdml and other factors listed in Latchman, 1998, Eukaryotic Transcription Factors, 3rd. Ed., Academic Press: New York. Fusion protein derivatives of these or other transcription factors may also be used, in which the DNA binding motif of the fusion protein (at least providing the binding specificity) is fused to a small molecule modulator binding site.

[0076] The term "small interfering RNA" or "siRNA" as used herein refers to RNA or RNA-like molecules that can interact and cause destruction of mRNA molecules with sequence homology to siRNA (Elbashir et al., Genes Dev, vol.15, pp.188-200, 2001). It is believed that siRNA can be incorporated into a ribonucleoprotein complex called RNA-induced silencing complex (RISC). RISC uses siRNA sequences to identify mRNA molecules that are at least partially complementary to the incorporated siRNA chains, and then cuts these target mRNA molecules or inhibits their translation. Typical siRNA is a double-stranded nucleic acid molecule, each chain having about 19 nucleotides to about 28 nucleotides (i.e., about 19, 20, 21, 22, 23, 24, 25, 26, 27 or 28 nucleotides). siRNA can also be single-stranded RNA, although not as effective as double-stranded siRNA. Single-stranded siRNA has a length of about 19 nucleotides to about 49 nucleotides. Single-stranded siRNA has a 5' phosphate group or is phosphorylated in situ or in vivo at the 5' position. Single-stranded siRNA can be chemically synthesized or synthesized by in vitro transcription or endogenously expressed from an expression vector or expression cassette. The 5' phosphate group can be added by a kinase or can be the result of nuclease cleavage of the RNA.

[0077] The term "small molecule" refers to a molecule or ion having a molecular weight of less than 900 a.mu, or more preferably less than 500 a.mu, or more preferably less than 200 a.mu, or even more preferably less than 100 a.mu. In the assays and environments of the present invention, small molecules may generally exist as a mixture of molecules and deprotonated ions of molecules, depending primarily on the pH of the assay or environment.

[0078] The term "therapeutic protein" as used herein refers to any protein and / or polypeptide that is administered to a mammal by, for example, a researcher or clinician to elicit a biological or medical response in a tissue, system, animal or human being that is being sought. Therapeutic proteins can elicit more than one biological or medical response. Examples of therapeutic proteins include antibodies, enzymes, hormones, cytokines, regulatory proteins, and fragments thereof.

[0079] As used herein, the term "therapeutically effective amount" refers to any amount that results in, but is not limited to, a cure, prevention, or improvement of a disease, condition, or side effect, or a reduction in the rate of development of a disease or condition, compared to a corresponding individual not receiving the amount. The term also includes within its scope amounts effective to enhance normal physiological function and amounts effective to induce in a patient an amount of physiological function that enhances or contributes to the therapeutic effect of a second agent.

[0080] The term "tumor microenvironment" as used herein refers to the microenvironment in and around solid tumors that supports the growth and metastasis of tumor cells. The tumor microenvironment includes peripheral blood vessels, immune cells, fibroblasts, other cells, soluble factors, signaling molecules, extracellular matrix, and mechanical signals that can promote tumorigenic transformation, support tumor growth and infiltration, protect tumors from host immune attacks, promote treatment resistance, and provide microenvironments (niches) for dormant metastatic tumors. Tumors are closely related to the microenvironment around them and constantly interact with each other. Tumors can affect their microenvironment by releasing extracellular signals, promoting tumor angiogenesis, and inducing peripheral immune tolerance, and immune cells in the microenvironment can affect the growth and evolution of cancer cells. See Swarts et al., "Tumor Microenvironment Complexity: Emerging Roles in Cancer Therapy," Cancer Res, vol., 72, pp. 2473-2480, 2012; Weber et al., "Thetumor microenvironment," Surgical Oncology, vol. 21, pp. 172-177, 2012; Blagosklonny, "Antiangiogenic therapy and tumor progression," Cancer Cell, vol. 5, pp. 13-17, 2004; Siemann, "Tumor microenvironment," Wiley, 2010; and Bagley, "The tumor microenvironment," Springer, 2010.

[0081] The term "wild type" as used herein refers to a polynucleotide that does not contain any mutations. "Wild-type protein", "wild-type protein", "wild-type biological protein" or "wild-type biological protein" may refer to a protein that can be isolated from nature and has a certain level of activity under natural conditions, including amino acid sequences found under natural conditions. The terms "parent molecule" and "target protein" also include wild-type proteins. DETAILED DESCRIPTION

[0082] A. pH-dependent conditionally active polypeptides

[0083] In one aspect, the present invention relates to conditionally active polypeptides having pH-dependent activity in the presence of a substance whose pKa is within 0.5, 1, 2, or 4 units of the pH at which activity is desired. In another aspect, the present invention relates to conditionally active polypeptides having pH-dependent activity in the presence of a substance whose pKa is from about 4 to about 10, or from about 4.5 to about 9.5, or from about 5 to about 9, or from about 5.5 to about 8, or from about 6.0 to about 7.0. In another aspect, the present invention relates to conditionally active polypeptides having pH-dependent activity in the presence of a substance selected from histidine, histamine, hydrogenated adenosine diphosphate, hydrogenated adenosine triphosphate, citrate, bicarbonate, acetate, lactate, hydrogen sulfide, hydrogen sulfide, ammonium, dihydrogen phosphate, and any combination thereof.

[0084] Substances present in the test medium that have a significant effect on the activity of the conditionally active polypeptide tend to be substances that have at least two ionization states: an uncharged or less charged state and a charged or more charged state. As a result, the pKa of a substance that affects the activity of a conditionally active polypeptide can play a role in determining the extent of the effect of the substance on a specific activity of the polypeptide and / or at a specific pH.

[0085] A pH-dependent conditionally active polypeptide has a higher activity at a first pH than at a different second pH, both activities being tested in an assay in the presence of one or more of the above substances. To determine the pH dependency of a conditionally active polypeptide, the polypeptide is tested for the same activity in the same assay medium at two different pH values.

[0086] In the same test medium, the ratio of the activity at a first pH to the same activity at a second pH can be referred to as the selectivity of the pH-dependent conditionally active polypeptide. The selectivity of the pH-dependent conditionally active polypeptide is 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.

[0087] It has been observed that pH-dependent conditionally active polypeptides contain an increased number (or proportion) 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 pH-dependent conditionally active polypeptides compared to the parent polypeptide from which the pH-dependent conditionally active polypeptide is derived. As a result, due to the increased number of charged amino acid residues, the pH-dependent conditionally active polypeptide is more likely to interact with charged substances in the test medium. This in turn affects the activity of the conditionally active polypeptide.

[0088] It has also been observed that pH-dependent conditionally active polypeptides generally have different activities in the presence of different substances in the test medium. Substances with at least two ionization states (uncharged or less charged state and charged or more charged state) can dissociate to a greater extent at a specific pH (depending on the pKa value), thereby increasing the probability of interacting with charged amino acid residues present in the conditionally active polypeptide. This factor can be used to enhance the selectivity and / or pH-dependent activity of the conditionally active polypeptide.

[0089] The nature of the charge of the conditionally active polypeptide can be a factor in determining suitable substances that affect the activity of the conditionally active polypeptide. In some embodiments, the conditionally active polypeptide can have more positively charged amino acid residues: lysine, arginine and histidine compared to the parent polypeptide. Thus, the conditionally active polypeptide can be selected to have a desired level of interaction with a specific substance present in an environment where the activity is desired, and / or to have a desired level of interaction with a specific substance present in an environment where the desired activity is reduced. Similarly, the conditionally active polypeptide can have more negatively charged amino acid residues: aspartic acid and glutamic acid compared to the parent polypeptide.

[0090] The position of charged amino acid residues on the pH-dependent conditionally active polypeptide may also have an effect on activity. For example, the proximity of the charged amino acid residue to the binding site of the conditionally active polypeptide can be used to influence the activity of the polypeptide.

[0091] In some embodiments, the interaction of a charged environmental substance with a conditionally active polypeptide can block or hinder the activity of a pH-dependent conditionally active polypeptide. For example, a charged amino acid that interacts with a charged environmental substance can exhibit an allosteric effect on the binding site of the conditionally active polypeptide.

[0092] In other embodiments, it is possible that the interaction of charged environmental substances with conditionally active polypeptides can form salt bridges between different parts of the polypeptide, particularly charged or polarized parts. It is known that the formation of salt bridges stabilizes the structure of polypeptides (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 structure, which usually undergoes continuous small structural changes called "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 the binding of proteins to their partners, because structural fluctuations allow conditionally active proteins to effectively recognize and bind to their partners (Karplus, et al., "Molecular dynamics and protein functions," PNAS, vol. 102, pp. 6679-6685, 2015). By forming a salt bridge, the binding site on the conditionally active polypeptide, especially the binding pocket, is not easy to contact its partner, probably because the salt bridge can directly block the partner from entering the binding site. Even if the salt bridge is far away from the binding site, the allosteric effect can change the conformation of the binding site to inhibit binding. Therefore, after the salt bridge stabilizes (fixes) the structure of the conditionally active polypeptide, the polypeptide may become inactive in binding to its partner, resulting in reduced activity.

[0093] A known example of how a polypeptide and its structure can be stabilized by salt bridges is hemoglobin. Structural and chemical studies have shown that at least two groups of chemical groups are responsible for the formation of salt bridges: the amino termini and side chains of histidine β146 and α122, which have pKa values ​​close to pH 7. In deoxyhemoglobin, the terminal carboxyl group of β146 forms a salt bridge with a lysine residue in the α subunit of another αβ dimer. If the imidazole group of the histidine residue is protonated, this interaction locks the side chain of histidine β146 in a position where it can form a salt bridge with the negatively charged aspartic acid 94 in the same chain ( Figure 6 ). At high pH, ​​the side chain of histidine β146 is not protonated and no salt bridge is formed. However, as the pH decreases, the side chain of histidine β146 is protonated and a salt bridge is formed between histidine β146 and aspartic acid β94, which stabilizes the quaternary structure of deoxyhemoglobin, resulting in a greater tendency for oxygen to be released in active metabolic tissues (with lower pH). Hemoglobin shows pH-dependent binding activity for oxygen at low pH, and the binding activity for oxygen decreases due to the formation of salt bridges. On the other hand, at high pH, ​​the binding activity for oxygen is enhanced due to the absence of salt bridges.

[0094] Similarly, by forming salt bridges in conditionally active polypeptides, small molecules such as bicarbonate can reduce the binding activity of conditionally active polypeptides with their partners. For example, at a pH lower than that of bicarbonate with a pKa of 6.4, bicarbonate is protonated and therefore uncharged. Uncharged bicarbonate cannot form a salt bridge, and therefore has little effect on the binding of conditionally active polypeptides with their partners. Therefore, conditionally active polypeptides have high binding activity with their partners at low pH. On the other hand, at high pH higher than the pKa of bicarbonate, bicarbonate is ionized by losing protons, thereby becoming negatively charged. Negatively charged bicarbonate will form a salt bridge between the positively charged or polarized parts on the conditionally active polypeptide to stabilize the structure of the conditionally active polypeptide. This will block or reduce the binding of the conditionally active polypeptide with its partner. Therefore, conditionally active polypeptides have low activity at high pH. Therefore, conditionally active polypeptides are conditionally active in the presence of bicarbonate, and have higher binding activity at low pH than at high pH.

[0095] When a substance such as bicarbonate is not present in the test medium, the conditionally active polypeptide may lose its conditional activity. This may be due to the lack of a salt bridge on the conditionally active polypeptide to stabilize (fix) the structure of the polypeptide. Therefore, the partner will have similar accessibility to the binding site on the conditionally active polypeptide at any pH, resulting in similar activity at the first pH and the second pH.

[0096] In other embodiments, the interaction between the small molecule or ion and the conditionally active polypeptide can change the structure of the polypeptide in a way that enhances its activity. For example, the change in structure can increase the binding affinity of the conditionally active polypeptide by changing the location, steric hindrance, or binding energy of the binding site required for binding affinity. In this case, it may be necessary to select a small molecule that binds to the conditionally active polypeptide at the pH where activity is desired.

[0097] It should be understood that although salt bridges (ionic bonds) are the strongest and most common way for compounds and ions to affect the activity of conditionally active polypeptides, other interactions between these compounds and ions and conditionally active polypeptides may also help stabilize (fix) the structure of conditionally active polypeptides. Other interactions include hydrogen bonds, hydrophobic interactions, and van der Waals interactions.

[0098] In some embodiments, in order to select suitable compounds or ions, the conditionally active polypeptide is compared with the parent polypeptide from which it is evolved to determine whether the conditionally active polypeptide has a higher proportion of negatively charged amino acid residues or positively charged amino acid residues. Compounds with suitable charges at the second pH can then be selected to affect the activity of the conditionally active polypeptide. For example, when the conditionally active polypeptide has a higher proportion of positively charged amino acid residues than the parent polypeptide, suitable small molecules should generally be negatively charged at the second pH to interact with the conditionally active polypeptide. On the other hand, when the conditionally active polypeptide has a higher proportion of negatively charged amino acid residues than the parent polypeptide, suitable small molecules should generally be positively charged at the second pH to interact with the conditionally active polypeptide.

[0099] In other embodiments, the activity of a conditionally active polypeptide is controlled by the interaction of a small molecule or ion with a target polypeptide that serves as a binding partner for the conditionally active polypeptide. In this case, the same principles discussed above apply, except that the goal is to generate an interaction between the small molecule or ion and the target polypeptide. The target polypeptide can be, for example, an antigen of a conditionally active antibody or a ligand of a conditionally active receptor.

[0100] Suitable small molecules can be any inorganic or organic molecule that changes from an uncharged or less charged state at a first pH to a charged or more charged state at a second pH. Therefore, small molecules should generally have a pKa between the first pH and the second pH. For example, the pKa of bicarbonate is 6.4. Therefore, at a higher pH, such as pH 7.4, negatively charged bicarbonate will bind to the charged amino acid residues in the conditionally active polypeptide and reduce activity. On the other hand, at a lower pH, such as pH 6.0, less charged bicarbonate will not bind to the conditionally active polypeptide in the same amount, thereby allowing the conditionally active polypeptide to have a higher activity.

[0101] The pKa of hydrogen sulfide is 7.05. Therefore, at a higher pH, such as pH 7.4, more negatively charged hydrogen sulfide will bind to the positively charged amino acid residues in the conditionally active polypeptide and reduce its activity. On the other hand, at a lower pH, such as pH 6.2-6.8, less charged hydrogen sulfide / hydrogen sulfide will not bind to the conditionally active polypeptide at the same level, thereby allowing the conditionally active polypeptide to have a higher activity.

[0102] Small molecules with a pKa between the first pH and the second pH are preferably used in the present invention. Preferred substances are selected from hydrogen sulfide, hydrogen sulfide, histidine, histamine, citrate, bicarbonate, acetate and lactate. Each of these small molecules has a pKa between 6.2 and 7.0. In addition, other small molecules, such as N-tris(hydroxymethyl)methylglycine (tricine, pKa8.05) and N,N-dihydroxyethylglycine (bicine, pKa 8.26) can also be used. Other suitable small molecules can be found in textbooks using the principles of the present application, such as the CRC Handbook of Chemistry and Physics (CRC Handbook of Chemistry and Physics) published by CRC Press, the 96th edition; Chemical Properties Handbook (Chemical Properties Handbook), McGraw-Hill Education, 1998.

[0103] The concentration of the small molecule in the test medium or environment is preferably or close to the physiological concentration of the small molecule in the individual. For example, the physiological concentration of bicarbonate (in human serum) is in the range of 15mM to 30mM. Therefore, the concentration of bicarbonate in the test medium can be 10mM to 40mM, or 15mM to 30mM, or 20mM to 25mM, or about 20mM. The physiological concentration of hydrogen sulfide is also very low. The concentration of hydrogen sulfide in the test medium can be 3nM to 500nM, or 5nM to 200nM, or 10nM to 100nM, or 10nM to 50nM.

[0104] In the present invention, the concentration of the conditionally active polypeptide selected and adopted makes the normal physiological concentration of the specific substance in the environment have a significant effect on the activity of the conditionally active polypeptide in the pH range of interest. Therefore, in many therapies, it may be advantageous to make the conditionally active polypeptide have low activity in blood or human serum at pH 7.2-7.4 or so to allow treatment to be delivered by blood flow, while minimizing or preventing the activation of the conditionally active polypeptide. As a result, for such treatment, it will be advantageous to select a small molecule with a pKa lower than pH 7.2-7.4 to ensure that a sufficient amount of ionization of the small molecule occurs at the blood flow pH, thereby significantly affecting the activity of the conditionally active polypeptide. At the same time, the pKa of the small molecule should be equal to or higher than the pH at which the conditionally active polypeptide has the desired activity, to ensure that the binding site on the conditionally active polypeptide is released by the protonation of the small molecule to activate the conditionally active polypeptide.

[0105] Small molecules preferably have low molecular weight and / or relatively small conformation to ensure maximum accessibility to a small pocket on a target polypeptide or conditionally active polypeptide by minimizing steric hindrance. For this reason, the molecular weight of small molecules is generally 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, as shown in Examples 13 and 14 below, hydrogen sulfide, hydrogen sulfide, and bicarbonate all have low molecular weight and small structure, providing accessibility to a pocket on a target polypeptide or conditionally active polypeptide.

[0106] The small molecule can be present in the test or environment at substantially the same concentration, for example, bicarbonate is about 20 μM. In some embodiments, the small molecule can be present at different concentrations in different environments, so it may be desirable to simulate this in the test. For example, hydrogen sulfide has a higher concentration in the tumor microenvironment than in human serum. Therefore, one test can simulate a tumor microenvironment with an acidic pH and a higher concentration of hydrogen sulfide, while the second test can simulate human serum with a neutral or weakly alkaline pH and a lower concentration of hydrogen sulfide. The acidic pH can be in the range of 6.0 to 6.8, while the neutral or weakly alkaline pH can be about 7.4. The higher concentration of hydrogen sulfide for the first test can be 30 μM, while the lower concentration of hydrogen sulfide for the second buffer can be 10 μM or less, or 5 μM.

[0107] In some embodiments, the conditionally active polypeptide is pH dependent when two or more different small molecules are present (eg, a combination of bicarbonate and histidine).

[0108] When the small molecule is not present, the conditionally active polypeptide may lose its pH dependency. Thus, in the absence of the small molecule, the conditionally active polypeptide may have similar activity between the first pH and the second pH in the absence of the small molecule.

[0109] In some embodiments, the first pH is an acidic pH, and the second pH is an alkaline or neutral pH. In other embodiments, the first pH is an alkaline pH, and the second pH is an acidic or neutral pH. For example, the first pH can be a pH in the range of about 5.5-7.2, or about 6.0-7.0, or about 6.2-6.8. The second pH can be a pH in the range of about 7.0-7.8, or about 7.2-7.6.

[0110] Conditionally active polypeptides that are more active at acidic pH and less active at alkaline or neutral pH can be targeted to an acidic tumor microenvironment at a pH of about 5.5-7.2, or about 6.2-6.8.

[0111] In other embodiments, the first pH at which the pH-dependent polypeptide is more active may be, for example, an alkaline pH of 7.6-7.9, such as in synovial fluid (see Jebens et al., "On the viscosity and pH of synovial fluid and pH of blood," Journal of Bone and Joint Surgery, vol. 41B, pp. 388-400, 1959). The second pH may be the blood pH of about 7.2-7.6, at which the activity of the conditionally active polypeptide is low. These conditionally active polypeptides may be useful for targeting joint diseases, particularly inflammation of the joints.

[0112] In other embodiments, conditionally active polypeptides can be designed to target the brain. There is a pH difference between the two sides of the blood-brain barrier, with the pH on the brain side being 0.2 pH units lower than the blood pH. Thus, the first pH of the brain, where the conditionally active polypeptide is more active, can be about 7.0-7.2 (brain pH), while the second pH can be about 7.4 (blood pH).

[0113] The conditionally active polypeptide may be an enzyme, a cytokine, a receptor, in particular a cellular receptor, a regulatory polypeptide, a soluble polypeptide, an antibody or a hormone.

[0114] The conditionally active polypeptide can be a fragment of a parent polypeptide. For example, the conditionally active polypeptide can be an antibody fragment, a single-chain antibody, an enzyme fragment, a receptor fragment, a cytokine fragment or a hormone fragment. The antibody fragment can be an Fc fragment of an antibody.

[0115] Fc fragments can be used as parent polypeptides to produce conditionally active Fc fragments, and the binding activity of the conditionally active Fc fragment to complement at a first pH is higher than the binding activity to the same complement at a second pH. The combination of Fc fragments and complement can be used to provide antibody-dependent, cell-mediated cytotoxicity. The first pH can be acidic in the range of 5.5-7.2 or 6.2-6.8, such as the pH in the tumor microenvironment, and the second pH ranges from 7.2-7.6. The first pH is different from the pH in the lysosome, which is usually about 4.0. In addition, the lysosome is the location where the Fc fragment (like any other polypeptide) is targeted for degradation. There is no complement in the lysosome and there is no cell-mediated cytotoxicity caused by the lysosome.

[0116] Conditionally active polypeptides may have two functional domains, at least one of which, preferably both functional domains, has a pH-dependent activity. The two functional domains may be evolved simultaneously and selected to identify the two functional domains in the same mutant polypeptide. Alternatively, the two functional domains may be evolved independently and selected to identify the pH-dependent activity, respectively. If the two functional domains are not in the same mutant polypeptide, they may be fused into a chimeric polypeptide having two individually identified functional domains.

[0117] In one aspect, the conditionally active polypeptide exhibits enhanced activity at a first pH compared to the parent polypeptide in the presence of a factor such as a protein, and exhibits reduced activity at a second pH compared to the parent polypeptide. The protein may be a protein present in blood, human serum, or in a body microenvironment such as a tumor microenvironment, an inflamed area, etc. A suitable protein may be an albumin, particularly a mammalian albumin, such as bovine albumin or human albumin.

[0118] In one aspect, a protein such as albumin is present in a test solution used to screen and select conditionally active polypeptides from mutant polypeptides produced by the evolution step. In another aspect, a test solution with a protein such as albumin is also used to test the activity of the selected conditionally active polypeptides under the same or different conditions.

[0119] B. Engineering of conditionally active peptides

[0120] Conditionally active polypeptides can be engineered by one or more of the protein engineering techniques described herein. Non-limiting examples of protein engineering techniques include conjugating conditionally active polypeptides to nucleic acids, conjugating conditionally active polypeptides to nanoparticles, engineering conditionally active polypeptides in chimeric antigen receptors, and engineering masked conditionally active polypeptides.

[0121] The conditionally active polypeptides of the present invention can be conjugated to nucleic acid molecules, such as DNA or RNA molecules, via a linker. Conditionally active polypeptides can help deliver nucleic acid molecules to a target position in an individual that has a condition, where the conditionally active polypeptide is more active than at other positions where the condition is not present. For example, a conditionally active polypeptide can be a conditionally active antibody that has a higher binding activity to its antigen under conditions in a tumor microenvironment than under conditions in other positions such as human serum. This effect can be used to deliver nucleic acid molecules to a tumor microenvironment by conjugating the nucleic acid molecule to a conditionally active polypeptide and administering the conjugate to an individual.

[0122] In some embodiments, nucleic acid molecules can be reagents for regulating gene expression at target sites. Abnormal gene expression is relevant to many diseases. Therefore, correcting abnormal gene expression can help control or even cure these diseases. For example, abnormal gene expression is a feature of most cancer cells, some of which have elevated expression levels in cancer cells, such as many oncogenes (for example, epidermal growth factor receptor 2 (HER2) is overexpressed in breast cancer cells). Selective inhibition of the expression of the constitutive elevation of oncogenes provides an opportunity to inhibit cancer cell proliferation.

[0123] Nucleic acid molecules that can inhibit gene expression include antisense RNA, small interfering RNA (siRNA), microRNA, oligoDNA, and oligonucleotide mimetics having an uncharged achiral polyamide backbone linked to nucleobases (Pooga et al., Curr Cancer Drug Targets, 1(3):231-9, 2001; Pandey et al., Expert Opin Biol Ther., 9(8):975-89, 2009).

[0124] Antisense RNA is a short RNA molecule that can bind to a specific complementary region of mRNA by base pairing to inhibit the expression of mRNA in a sequence-specific manner. Antisense RNA can induce RNaseH (which cuts mRNA at the site bound to the antisense RNA), or can physically block translation or other steps in mRNA processing and protein synthesis.

[0125] Small interfering RNA (siRNA) is usually a short double-stranded RNA segment, at least a portion of which is complementary to the mRNA sequence whose translation is to be blocked. siRNA works by using post-transcriptional gene silencing mechanisms that use chromatin remodeling, inhibition of protein translation or direct mRNA degradation, which is common in eukaryotic cells (Caplen, "Gene therapy progress and prospects. Downregulating gene expression: the impact of RNA interference," Gene Ther., 11 (16): 1241-1248, 2004) and Bertrand et al., "Comparison of antisense oligonucleotides and siRNAs in cell culture and in vivo," Biochem Biophys Res Commun., 296 (4): 1000-1004, 2002).

[0126] In particular, through RISC, siRNA can initiate a potent cascade of sequence-specific degradation of mRNAs homologous to the siRNA (Fire et al., "Potent and specific genetic interference by double-stranded RNA in Caenorhabditis elegans," Nature 391: 806-811, 1998). When siRNA is introduced into cells, it is processed by an RNase III enzyme called Dicer, which cuts long siRNAs into short 21-23 nucleotide duplexes with symmetrical 2-3 nucleotide 3' overhangs and 5' phosphate and 3' hydroxyl groups (Tuschl et al., "Targeted mRNA degradation by double-stranded RNA in vitro," Genes Dev. 13: 3191-3197, 1999; Hamilton and Baulcombe, "A species of small antisense RNA in posttranscriptional gene silencing inplants," Science, 286: 950-952, 1999). Therefore, effective siRNA only requires a short continuous complementary sequence to pair with mRNA to trigger siRNA-mediated silencing (Jackson and Linsley, "Noise amidst the silence: off-target effects of siRNAs?" Trends Genet., 20: 521-524, 2004). siRNA does not integrate into the genome, thus providing higher safety than plasmid or viral vectors.

[0127] MicroRNA (miRNA) is a class of naturally occurring small non-coding RNA molecules with a length of 21-25 nucleotides. MicroRNAs are partially complementary to mRNA molecules on which microRNAs act. The main function of microRNAs is to reduce gene expression through translational inhibition, mRNA cleavage and deadenylation. The central online resource library of miRNA species, sequence data, annotations and target predictions hosted by the Sanger Institute in the UK is called miRBase. MicroRNA genes are transcribed by RNA polymerase II to produce pri-miRNAs with a 5' cap and a poly-A tail. In the nucleus, pri-miRNAs are processed by the microprocessing complex (composed of the RNAse III enzyme Drosha and double-stranded RNA Pasha / DGCR8) to produce pre-miRNAs. These pre-miRNAs are exported to the cytoplasm through the nuclear transport protein exportin (Exp5) and the Ran-GTPase complex, where Ran GTPase binds to Exp5 to form nuclear heterotrimers with pre-miRNAs. These pre-miRNAs are then processed by the RNAse III enzyme Dicer to produce mature microRNAs.

[0128] Another type of nucleic acid that can be delivered by conditionally active polypeptides is an oligonucleotide mimic, which comprises an uncharged non-chiral polyamide backbone connected to a core base. Oligonucleotide mimics are commonly referred to as peptide nucleic acids (PNAs). More specifically, PNA is a DNA analog in which N-(2-aminoethyl) glycine polyamide replaces a phosphoric acid-ribose ring backbone, and a methylene-carbonyl linker connects natural core bases and non-natural core bases to N-(2-aminoethyl) glycine. Although the backbone structure has undergone fundamental changes, PNA can carry out sequence-specific binding with DNA and mRNA according to the Watson-Crick base pairing principle.

[0129] PNA binds to complementary DNA / RNA with a higher affinity than natural nucleic acids, partly due to the lack of negative charge on the backbone and thus reduced charge-charge repulsion, as well as favorable geometric factors. The complex of PNA and DNA / mRNA is very stable in biological fluids, resulting in inhibition of transcription and translation of target genes by specific hybridization with DNA or mRNA. Typically, PNA is synthesized using a well-known solid phase peptide synthesis protocol. See Kim et al., J. Am. Chem. Soc., 115, 6477–6481, 1993; Hyrup et al., J. Am. Chem. Soc., 116, 7964–7970, 1994; Egholm et al., Nature, 365, 566–568, 1993; Dueholm et al. 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. al., J. Am. Chem. Soc., 118, 5544–5552, 1996). In contrast to DNA, which is depurinated when treated with strong acid and hydrolyzed in alkaline hydroxides, PNA is completely acid-stable and sufficiently stable to weak bases.

[0130] Another type of nucleic acid that can be delivered by conditionally active polypeptides is oligoDNA. OligoDNA is a short single-stranded segment of DNA that can selectively inhibit the expression of genes with sequences complementary to the oligoDNA after entering the plasma of cells. For antisense applications, the oligoDNA interacts with the target mRNA or pre-mRNA and forms a duplex, inhibiting its translation or processing, thereby inhibiting the biosynthesis of the protein. For antigenic applications, the oligoDNA must enter the nucleus, form a triplex with the double-stranded genomic DNA, inhibit the transcription of the gene, thereby producing less mRNA, resulting in less gene product of the protein produced.

[0131] Another type of nucleic acid that can be delivered by conditionally active polypeptides is spherical nucleic acids (SNA, see Zhang, J Am Chem Soc, 134 (40): 16488–16491, 2012). SNAs contain densely functionalized and highly oriented nucleic acids covalently attached to the surface of metallic, semiconducting or insulating inorganic or polymer core materials. They can also be coreless hollow structures composed almost entirely of nucleic acid molecules. Such spherical nucleic acids are able to bypass an individual's natural defenses against exogenous nucleic acids. Spherical nucleic acids utilize the unique properties of their densely packed, highly oriented nucleic acid shells to achieve protection and effective delivery of nucleic acids. This shell produces an area of ​​high local salt concentration, which, when combined with steric inhibition, is used to reduce nuclease activity and protect nucleic acids from enzymatic degradation. In addition, these spherical nucleic acids recruit scavenger proteins that promote endocytosis from the natural extracellular environment to their surfaces.

[0132] After entering the cytoplasm, spherical nucleic acids can inhibit the expression of target genes through antisense or siRNA approaches. Therefore, spherical nucleic acids provide several advantages over viral vectors and many other synthetic systems, including low toxicity, low immunogenicity, resistance to enzymatic degradation, and more durable gene knockdown. Conditionally active polypeptides, particularly conditionally active antibodies, can deliver spherical nucleic acids to target locations, such as diseased or inflamed tissues (e.g., tumors and inflamed joints).

[0133] Conditionally active polypeptides can also be conjugated to nanoparticles via linkers to help deliver the nanoparticles to target locations that have conditions that make the conditionally active polypeptide more active.Nanoparticles are known carriers for toxins, radioactive agents, or other therapeutic agents that are encapsulated in the nanoparticles.

[0134] The therapeutic agent encapsulated in the nanoparticle can be a protein that can dedifferentiate tumor cells and thus potentially revert tumor cells back to normal cells (Friedmann-Morvinski and Verma, "Dedifferentiation and reprogramming: origins of cancer stem cells," EMBO Reports, 15(3): 244-253, 2014). The nanoparticle can be linked to a conditionally active antibody to selectively deliver the linked nanoparticle and the encapsulated therapeutic agent to an environment where the conditionally active antibody is most active.

[0135] Several types of nanoparticles with different configurations can be used in the present invention.Nanoparticles can be made of a series of biocompatible materials, including biostable polymers, biodegradable polymers, fullerenes, lipids or combinations thereof.Biostable polymers refer to polymers that do not degrade in vivo.Biodegradable polymers refer to polymers that can be degraded after being delivered to a patient.For example, when polymers are exposed to body fluids such as blood, they can be gradually absorbed and / or eliminated by enzymes in the body.The method of producing nanoparticles with various degradation rates is known to those skilled in the art, see, for example, U.S. Patent No. 6,451,338, U.S. Patent No. 6,168,804 and U.S. Patent No. 6,258,378.

[0136] Exemplary nanoparticles of the present invention include liposomes, polymer vesicles and polymer particles. Liposomes refer to a chamber that is completely enclosed by a double layer that is usually composed of phospholipids. Liposomes can be prepared according to standard techniques known to those skilled in the art. One technique is to suspend a suitable lipid (e.g., phosphatidylcholine) in an aqueous medium and then ultrasonicate the mixture. Another technique is to rapidly mix a lipid solution in ethanol-water, such as by injecting lipids into a stirred ethanol-water solution using a needle. In some embodiments, additionally or alternatively, liposomes may also include other amphipathic substances, such as sphingomyelin or lipids containing polyethylene glycol (PEG).

[0137] Polymer vesicles include diblock or triblock copolymers modified to form a double-layer structure similar to liposomes. Depending on the length and composition of the block copolymer, polymer vesicles can be substantially more solid than liposomes. In addition, the ability to control the chemical properties of each block of the block copolymer allows the composition of the polymer vesicle to be adjusted to adapt to the desired application. For example, the film thickness of the polymer vesicle, i.e. the thickness of the double-layer structure, can be controlled by changing the chain length of a single block in the block copolymer. The glass transition temperature of each block in the copolymer is regulated to affect the fluidity of the polymer vesicle and therefore affect the permeability of its membrane. Even the release mechanism of the encapsulated reagent can be improved by changing the characteristics of the copolymer.

[0138] Polymer vesicles can be prepared by a method comprising the steps of: (i) dissolving the block copolymer in an organic solvent, (ii) applying the resulting solution to the surface of a container, and then (iii) removing the solvent, thereby leaving a film of the copolymer on the container wall. The film is then hydrated to form polymer vesicles. Alternatively, dissolving the block copolymer in a solvent and then adding a solvent that is a weak solvent for one block of the copolymer will also produce polymer vesicles.

[0139] A variety of techniques can be used to encapsulate therapeutic agents in polymer vesicles. For example, therapeutic agents can be mixed in water and then used to rehydrate copolymer films. Another example is to osmotically drive therapeutic agents into the core of preformed polymer vesicles, a process called force loading. Another example is to use double emulsion technology, which can generate polymer vesicles with relatively monodispersity and high loading efficiency. Double emulsion technology involves using microfluidics to produce double emulsions containing water droplets surrounded by an organic solvent layer. Then the structure of droplets (droplet-in-a-drop) in these droplets is dispersed in a continuous aqueous phase. Block copolymers are dissolved in organic solvents and self-assembled into original polymer vesicles on the concentric interfaces of double emulsions. Final polymer vesicles are formed after the organic solvent is completely evaporated from the shell of the original polymer vesicle. This technology allows the size of polymer vesicles to be well controlled. In addition, the ability to keep the internal fluid completely separated from the external fluid in the whole process allows very effective encapsulation of therapeutic agents.

[0140] In contrast to the shell structure of liposomes and polymersomes, polymer particles refer to solid or porous particles.Methods for attaching therapeutic agents to the surface of polymer particles or integrating biologically active agents into the structure of polymer particles are known to those skilled in the art.

[0141] Polymers that can be used to prepare 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, polyglycolic acid, polyglycolide, 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), and poly(D,L-lactide-co-caprolactone). , poly(glycolide-co-caprolactone), poly(trimethylene carbonate), polyesteramides, poly(glycolic acid-co-trimethylene carbonate), co-polyetheresters (e.g., PEO / PLA), polyphosphazenes, biomolecules (such as fibrin, fibrin glue, fibrinogen, cellulose, starch, collagen and hyaluronic acid, elastin and hyaluronic acid), polyurethanes, polysilicone, polyesters, polyolefins, polyisobutylene and ethylene-alpha olefin copolymers, acrylic polymers and copolymers other than polyacrylates, vinyl halide polymers and copolymers (such as polyvinyl chloride), polyvinyl ethers (such as polyvinyl methyl ether), polyvinylidene halides ... ether), polyvinylidene halides (such as polyvinyl chloride), polyvinyl ethers (such as polyvinyl methyl ether), polyvinylidene halides (such as polyvinyl ether), polyvinylidene halides (such as polyvinyl chloride), polyvinyl ethers (such as polyvinyl methyl ether), polyvinylidene halides (such as polyvinyl ether), polyvinylidene halides (such as polyvinyl ether), polyvinylidene halides (such as polyvinyl ether), polyvinylidene halides (such as poly halides (such as polyvinylidene chloride), polyacrylonitrile, polyvinyl ketone, polyvinyl aromatic compounds (such as polystyrene), polyvinyl esters (such as polyvinyl acetate), acrylonitrile-styrene copolymers, acrylonitrile butadiene styrene (ABS) resins, polyamides (such as nylon 66 and polycaprolactam), polycarbonates including tyrosine-based, polyoxymethylene, polyimides, polyethers, polyurethanes, rayon, rayon triacetates, cellulose, cellulose acetate, cellulose butyrate, cellulose acetate butyrate, cellophane, cellulose nitrate, cellulose propionate, cellulose ethers and carboxymethyl cellulose.

[0142] In some embodiments, in addition to the selectivity from conditionally active polypeptides, nanoparticles can also provide tissue selectivity through coatings. For example, nanoparticles can be coated with a poly (glutamic acid) peptide coating of electrostatic adsorption to change the external composition of the core particle. Negatively charged poly glutamic acid peptides containing arginine-glycine-aspartic acid (RGD) ligands can increase in vitro gene delivery to endothelial cells compared to particles coated with a scrambled sequence containing RDG instead of RGD. These peptides consist of three parts: a section of poly (glutamic acid) providing a negative charge, a linker of polyglycine, and a terminal sequence with charge changes and the potential to change the biophysical properties and tissue selectivity of the particles. The coating and the particles themselves are biodegradable through their amide bonds and ester bonds, respectively. See Harris et al. ("Tissue-Specific Gene Delivery via Nanoparticle Coating," Biomaterials, vol. 31, pp. 998-1006, 2010).

[0143] The mammalian immune system uses T cells to fight substances or cells with foreign antigens. When encountering solid tumors, T cells often fail to produce an effective response. Even when T cells arrive at the tumor site, they face a series of immunosuppressive factors that can enable cancer cells to escape the immune system. CAR-T technology uses genetic engineering methods to reprogram natural circulating T cells by inserting chimeric antigen receptors (CARs) into T cells to produce highly specific CAR-T cells, where CAR guides engineered CAR-T cells to target tissues by specifically binding to antigens on the surface of target tissues. Therefore, CAR-T cells can specifically target tumor cells, making CAR-T cells more effective than naturally circulating T cells. CAR-T cells can also be engineered to target other target tissues, such as inflamed joints and brain tissues.

[0144] The CAR of the present invention comprises at least one antigen-specific targeting region (ASTR), an extracellular spacer domain (ESD), a transmembrane domain (TM), one or more co-stimulatory domains (CSD) and an intracellular signaling domain (ISD), see Figure 5and Jensen et al., "Design and implementation of adoptive therapy with chimeric antigen receptor-modified T cells," Immunol Rev., vol. 257, pp. 127–144, 2014. After the ASTR specifically binds to the target antigen on the tumor or other target tissue, the ISD activates intracellular signaling in the CAR-T cell. For example, using the antigen binding properties of antibodies, the ISD can redirect the specificity and reactivity of CAR-T cells to the selected target (e.g., tumor cells or other targeted cells) in a non-MHC-restricted manner. Non-MHC-restricted antigen recognition enables CAR-T cells to recognize tumor cells and initiate antigen processing, thereby bypassing the main mechanism by which tumors escape immune system surveillance. In one embodiment, ESD and / or CSD are optional. In another embodiment, the ASTR has bispecificity, which allows it to specifically bind to two different antigens or epitopes.

[0145] The conditionally active polypeptide of the present invention can be engineered into an ASTR or a portion thereof so that the CAR has a higher activity of binding to the target antigen in a specific environment (e.g., a tumor microenvironment or synovial fluid) than in the blood or other parts of the body where there are different environments. Such a CAR can preferentially deliver T cells to the disease site, thereby significantly reducing the side effects caused by T cells attacking normal tissues. This enables the use of higher doses of T cells to increase the therapeutic effect and improve individual tolerance to treatment.

[0146] These CARs are particularly valuable for developing new therapies that are needed in a short or limited time in a subject. Examples of beneficial applications include high-dose systemic treatments and high-concentration local treatments. See Maher, "Immunotherapy of Malignant Disease Using Chimeric Antigen Receptor Engrafted T Cells," ISRN Oncology, vol. 2012, article ID 278093, 2012.

[0147] ASTRs may comprise 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 lead to recognition of cells bearing cytokine receptors), affibodies, ligand binding domains from naturally occurring receptors, and soluble protein / peptide ligands of receptors (e.g., receptors on tumor cells). In fact, almost any molecule that can bind to a given antigen with high affinity can be used in ASTRs.

[0148] In some embodiments, the CAR of the present invention includes at least two ASTRs targeting at least two different antigens or two epitopes on the same antigen. In one embodiment, the CAR comprises three or more ASTRs targeting at least three or more different antigens or epitopes. When there are multiple ASTRs in the CAR, the ASTRs can be arranged in series and can be separated by a linker peptide ( Figure 5 ).

[0149] In yet another embodiment, the ASTR comprises a diabody. In a diabody, a scFv is formed with a linker peptide that is too short for the two variable regions to fold together, thereby driving scFv dimerization. Shorter linkers (one or two amino acids) lead to the formation of trimers, so-called triabodies or tribodies. Tetrabodies can also be used in ASTRs.

[0150] The antigen targeted by CAR is present on the surface or inside of cells in the tissue targeted for removal, such as tumors, glandular (eg, prostate) hyperplasia, warts, and unwanted adipose tissue. When surface antigens are more effectively recognized and bound by the ASTR of CAR, intracellular antigens can also be targeted by CAR. In some embodiments, the target antigen is preferably unique to cancer, inflammatory diseases, neuronal disorders, diabetes, cardiovascular diseases, or infectious diseases. Examples of target antigens include antigens expressed by various immune cells, cancers, sarcomas, lymphomas, leukemias, germ cell tumors, germ cells, and cells associated with various blood diseases, autoimmune diseases, and / or inflammatory diseases.

[0151] Antigens specific to cancer that can be targeted by ASTR include one or more of the following: 4-IBB, 5T4, adenocarcinoma antigen, alpha-fetoprotein, BAFF, B-lymphoma cells, C242 antigen, CA-125, carbonic anhydrase 9 (CA-IX), C-MET, CCR4, CD152, CD19, CD20, CD200, CD22, CD221, CD23 (IgE receptor), CD28, CD30 (TNFRSF8), CD33, CD4, CD40, 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 αvβ3, MORAb-009, MS4A1, MUC1, mucin CanAg, N-glycolylneuraminic acid, NPC-1C, PDGF-R a, PDL192, phosphatidylserine, prostate cancer cells, RANKL, RON, ROR1, SCH900105, SDC1, SLAMF7, TAG-72, tenascin C, TGFβ2, TGF-β, TRAIL-R1, TRAIL-R2, tumor antigen CTAA16.88, VEGF-A, VEGFR-1, VEGFR2, or vimentin.

[0152] Antigens specific to inflammatory diseases that can be targeted by ASTR include one or more of the following: AOC3 (VAP-1), CAM-300, CCL11 (eotaxin-1), CD125, CD147 (basic immunoglobulin, 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-5, IL-6, IL-6 receptor, integrin α4, integrin α4β7, Lamaglama, LFA-1 (CD11a), MEDI-528, myostatin, OX-40, rhuMAb β7, scleroscin, SOST, TGFβ1, TNF-α or VEGF-A.

[0153] Antigens specific to neuronal disorders that can be targeted by the ASTRs of the present invention include one or more of amyloid beta or MABT5102A. Antigens specific to diabetes that can be targeted by the ASTRs of the present invention include one or more of L-Iβ or CD3. Antigens specific to cardiovascular diseases that can be targeted by the ASTRs of the present invention include one or more of C5, cardiac myosin, CD41 (integrin α-lib), fibrin II, β chain, ITGB2 (CD18), and sphingosine-1-phosphate.

[0154] Antigens specific to infectious diseases that can be targeted by the ASTRs of the present invention include one or more of the following: 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.

[0155] Other examples of target antigens include surface proteins present on cancer cells in a unique or amplified manner, such as the IL-14 receptor, CD19, CD20 and CD40 of B cell lymphoma, LewisY and CEA antigens of various cancers, Tag72 antigens of breast and colorectal cancer, EGF-R of lung cancer, folate binding protein and HER-2 protein commonly amplified in human breast cancer and ovarian cancer, or viral proteins, such as gp120 and gp41 envelope proteins of human immunodeficiency virus (HIV), envelope proteins of hepatitis B virus and hepatitis C virus, glycoprotein B and other envelope glycoproteins of human cytomegalovirus, and envelope proteins of tumor viruses such as Kaposi's sarcoma-associated herpes virus. Other possible target antigens include CD4, where the ligand is HIVgp120 envelope glycoprotein, and other viral receptors, such as ICAM, which is a receptor for human rhinovirus, and related receptor molecules for poliovirus.

[0156] In another embodiment, CAR can target antigens involved in cancer therapy cells such as NK cells to activate cancer therapy cells by acting as immune effector cells. One example is a CAR targeting CD16A antigen to enable NK cells to fight against malignant tumors expressing CD30. Bispecific tetravalent AFM13 antibodies are examples of antibodies that can deliver this effect. Further details of this type of embodiment can be found in, 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, Vl. 125, no. 26, pp. 4024-4031.

[0157] In some embodiments, the extracellular spacer domain and the transmembrane domain can be ubiquitylation-resistant, which can enhance CAR-T cell signaling and thereby enhance anti-tumor activity (Kunii et al., "Enhanced function of redirected human t cells expressing linkerfor activation of t cells that is resistant to ubiquitylation," Human Gene Therapy, vol. 24, pp. 27-37, 2013). In this region, the extracellular spacer domain is outside the CAR-T cell and is therefore exposed to different conditions and may conditionally produce ubiquitylation resistance.

[0158] C. Engineering Masked Conditionally Active Peptides

[0159] The conditionally active polypeptides of the present invention, in particular conditionally active antibodies, can mask their conditional activity and / or mask the activity of the agent conjugated thereto by means of a masking moiety. Once the masking moiety is removed or cleaved from the conditionally active polypeptide, the masked activity can be obtained. 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.

[0160] In some embodiments, the conditionally active antibody is connected to a masking portion, which masks the conditional activity and / or the activity of the agent conjugated thereto. For example, when the conditionally active antibody is connected to a masking portion, such connection or modification may cause a structural change that may reduce or inhibit the ability of the conditionally active antibody to specifically bind to its antigen. Once the conditionally active antibody reaches the target tissue or microenvironment, the masking portion 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 typically active in the tumor microenvironment, which may cleave the masking portion to release the conditionally active antibody that is active in the tumor tissue.

[0161] 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. For example, in certain embodiments, to ensure adequate delivery time, the masking effect is designed to last for at least 2, 4, 6, 8, 12, 28, 24, 36, 48, 60, 72, 84, 96 hours, or 5, 10, 15, 30, 45, 60, 90, 120, 150, 180 days, or 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 months or longer in target displacement for in vivo measurements or in vitro immunosorbent assays.

[0162] In certain embodiments, the masking portion is structurally similar to the natural binding partner (antigen) of the conditionally active antibody. The masking portion can be a modified natural binding partner of the conditionally active antibody, which contains amino acid changes that at least slightly reduce the affinity and / or antibody avidity of the conditionally active antibody. In some embodiments, the masking portion does not contain the natural binding partner of the conditionally active antibody or is substantially free of homology with the natural binding partner of the conditionally active antibody. In other embodiments, the sequence identity between the masking portion and the natural binding partner of the conditionally active antibody does not exceed 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75% or 80%.

[0163] The masking part can be provided in various forms. In certain embodiments, the masking part can be a known binding partner of a conditionally active antibody, provided that the binding affinity and / or avidity of the masking part to the conditionally active antibody is smaller than the binding affinity and / or avidity of the target protein targeted by the conditionally active antibody after the masking part is cut, so as to reduce the interference of the masking part to the desired combination with the target. Therefore, the masking part is preferably a masking part with the following characteristics: before the masking part is cut, the masking part masks the conditionally active antibody so that it does not bind to the target, but after the masking part has been cut from the antibody, it will not essentially or significantly interfere with the binding of the active molecule to the target or compete with the target. In a specific embodiment, the conditionally active antibody and the masking part do not include the amino acid sequence of a naturally occurring binding partner pair, which makes at least one of the conditionally active antibody and the masking part not have an amino acid sequence as a naturally occurring binding partner member.

[0164] Alternatively, the masking moiety may not specifically bind to the conditionally active antibody, but may interfere with the conditionally active antibody-target binding through non-specific interactions (e.g., steric hindrance). For example, the masking moiety may be positioned such that the structure or conformation of the antibody allows the masking moiety to mask the conditionally active antibody, such as through charge-based interactions, thereby interfering with the target's access to the conditionally active antibody.

[0165] In some embodiments, the masking moiety is attached to the conditionally active antibody by covalent bonding. In another embodiment, the conditionally active antibody is prevented from binding to its target by binding the masking moiety to the N-terminus of the conditionally active antibody. In another embodiment, the conditionally active antibody is attached to the masking moiety via a cysteine-cysteine ​​disulfide bond between the masking moiety and the conditionally active antibody.

[0166] In certain embodiments, the conditionally active antibody is also linked to a cleavable moiety (CM). The CM can be cleaved by an enzyme, or the CM can be reduced by a reducing agent, or the CM can be photolyzed. In one embodiment, the amino acid sequence of the CM can overlap with or be contained within the masking moiety. In another embodiment, the CM is between the conditionally active antibody and the masking moiety. It should be noted that all or part of the CM can help mask the conditionally active antibody before cleavage. When the CM is cleaved off, the activity of the conditionally active antibody binding to its antigen is enhanced.

[0167] The CM may be a substrate for an enzyme that is co-localized with the target antigen at the treatment site in the individual. Alternatively or additionally, the CM may have a cysteine-cysteine ​​disulfide bond that can be broken by reduction thereof. The CM may also be a photolabile substrate that is activatable by a light source.

[0168] The enzyme that cleaves the CM should be preferentially located in the desired target tissue of the conditionally active antibody, wherein the conditionally active antibody is more active under the conditions (abnormal conditions) provided by the target tissue (such as diseased tissue or tumor tissue). For example, there are known proteases that have increased levels in many cancers (such as solid tumors). See, for example, La Rocca et al, (2004) British J. of Cancer 90 (7): 1414-1421. Non-limiting examples of these 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 damage, local 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 an elevated level in tumor tissue compared to non-cancerous tissue.

[0169] In some embodiments, CM can be a substrate for an enzyme selected from myristin, plasmin, TMPRSS-3 / 4, MMP-9, MT1-MMP, cathepsin, caspase, human neutrophil elastase, β-secretase, uPA and PSA. The enzyme that cuts CM is present at a relatively high level in the target tissue of the treatment site (e.g., diseased tissue or tumor tissue; for example, for therapeutic treatment or diagnostic treatment) compared to the level present in the tissue of the non-treatment site (e.g., healthy tissue). Therefore, the conditional activity of the antibody is more active at the diseased tissue or tumor tissue, and the enzyme present in the diseased tissue or tumor tissue can cut the CM, which further enhances the activity of the conditionally active antibody or the activity of the agent conjugated thereto. Unmodified or uncut CM can effectively inhibit or mask the activity of the conditionally active antibody, so that the conditionally active antibody is less active in normal tissues (normal physiological conditions). The dual mechanism (conditional activity and masking part) of inhibiting the activity of the conditionally active antibody in normal tissues allows the use of high doses of conditionally active antibodies without causing significant adverse reactions.

[0170] In some embodiments, the CM may be a substrate for an enzyme selected from the enzymes listed in Table 1 below.

[0171] Table 1. Exemplary enzymes / proteases

[0172]

[0173] Alternatively or in addition, the CM may include disulfide bonds of cysteine ​​pairs that can be cleaved by reducing agents such as cellular reducing agents including glutathione (GSH), thioredoxin, NADPH, flavin, ascorbate, etc., which may be present in large quantities in or around the tissue of solid tumors.

[0174] In some embodiments, the conditionally active antibody comprises both CM and a masking moiety. When the enzyme cuts off the CM, the masking of the activity of the conditionally active antibody is removed. In some embodiments, one or more joints (e.g., flexible joints) may need to be inserted between the antibody, the masking moiety, and the CM to provide flexibility. For example, the masking moiety and / or the CM may not contain a sufficient number of residues (e.g., Gly, Ser, Asp, Asn, particularly Gly and Ser, particularly Gly) to provide the required flexibility. Therefore, it may be beneficial to introduce one or more amino acids to provide a flexible joint. For example, the masked conditionally active antibody can have the following structure (wherein the following formula represents the amino acid sequence in the N- to C- terminal direction or the C- to N- terminal direction):

[0175] (MM)-L1-(CM)-(AB)

[0176] (MM)-(CM)-L1-(AB)

[0177] (MM)-L1-(CM)-L2-(AB)

[0178] Ring [L1-(MM)-L2-(CM)-L3-(AB)]

[0179] Wherein MM is a masking moiety, AB is a conditionally active antibody; L1, L2 and L3 are each independently and optionally present or absent, and are the same or different flexible linkers comprising at least one flexible amino acid (e.g., Gly); in the case where a loop is present, the entire structure is in the form of a cyclic structure due to a disulfide bond between a pair of cysteines at or near the N-terminus and C-terminus of the structure.

[0180] The joint that is suitable for the present invention is usually a joint that provides flexibility to suppress the activity of conditionally active antibodies for the masking part. Such a joint is generally referred to as a flexible joint. Suitable joints can be easily selected and can have any suitable different lengths, such as 1 amino acid (such as Gly) to 20 amino acids, 2 amino acids to 15 amino acids, 3 amino acids to 12 amino acids, including 4 amino acids to 10 amino acids, 5 amino acids to 9 amino acids, 6 amino acids to 8 amino acids or 7 amino acids to 8 amino acids, and can be 1,2,3,4,5,6 or 7 amino acids.

[0181] Exemplary flexible linkers include glycine polymers (G) n , glycine-serine polymers (including, for example, (GS) n 、(GSGGS) n and (GGGS) n, wherein n is an integer of at least 1), glycine-alanine polymers, alanine-serine polymers, and other flexible linkers known in the art. Exemplary flexible linkers include, but are not limited to, Gly-Gly-Ser-Gly, Gly-Gly-Ser-Gly-Gly, Gly-Ser-Gly-Ser-Gly, Gly-Ser-Gly-Gly-Gly, Gly-Gly-Gly-Ser-Gly, Gly-Ser-Ser-Gly, and the like.

[0182] Some techniques for masking the activity of conditionally active antibodies are described in WO2010081173A2.

[0183] The present invention 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 comprises the following steps: evolving DNA encoding a parent polypeptide using one or more evolution techniques to produce a mutant DNA; expressing the mutant DNA to obtain a mutant polypeptide; subjecting the mutant polypeptide and the parent polypeptide to a test under a first condition and a test under a second condition; and selecting a conditionally active polypeptide having the following two characteristics from the mutant polypeptide: (a) decreased activity compared to the parent polypeptide in a test under the first condition, and (b) enhanced activity compared to the parent polypeptide in a test under the second condition. The test under the first condition and the test under the second condition are performed in a test solution containing at least one component selected from an inorganic compound, an ion, and an organic molecule. In some embodiments, the first condition is a normal physiological condition and the second condition is an abnormal condition.

[0184] Conditionally active polypeptides are reversibly or irreversibly inactivated under a first condition or normal physiological condition, but are active under a second condition or abnormal condition at the same or comparable level as the first condition or normal physiological condition. These conditionally active polypeptides and methods for producing these polypeptides have been described in U.S. Patent No. 8,709,755B2. Conditionally active polypeptides are particularly valuable for developing new therapies that are active in a host for a short time or a limited period of time. This is particularly valuable in cases where the prolonged action of a given dose of a therapeutic agent is harmful to the host but its limited activity is required for the desired treatment. Examples of beneficial applications include high-dose local or systemic treatments, as well as high-concentration local treatments. Inactivation under a first condition or normal physiological conditions can be determined by a combination of dosing and polypeptide inactivation rate. This condition-based inactivation is particularly important for enzyme therapeutics, where catalytic activity causes large negative effects in a relatively short period of time.

[0185] The present invention also relates to methods of engineering or evolving parent polypeptides to produce conditionally active polypeptides that are reversibly or irreversibly activated or inactivated over time, or that are activated or inactivated only when they are in certain microenvironments in the body, including specific organs in the body, such as tumor microenvironments, synovial fluid, bladder, or kidneys. In some embodiments, the conditionally active polypeptide is an antibody or antibody fragment directed against one or more target proteins (antigens) described herein.

[0186] Conditionally active polypeptides can be isolated polypeptides with pH-dependent activity, wherein in the presence of a substance selected from histidine, histamine, hydrogenated adenosine diphosphate, hydrogenated adenosine triphosphate, citrate, bicarbonate, acetate, lactate, hydrogen sulfide, hydrogen sulfide, ammonium, dihydrogen phosphate, and any combination thereof, the activity at a first pH is at least about 1.3 times the activity at a second pH. In the absence of small molecules, the same activity is not pH-dependent. 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 times the same activity at a second pH. The first pH may be an abnormal pH in the range of about 5.5-7.2 or about 6.2-6.8, and the second pH may be a normal physiological pH in the range of about 7.2-7.6.

[0187] D. Parent polypeptide

[0188] The parent polypeptide can be a wild-type polypeptide including a non-naturally occurring polypeptide, a mutant polypeptide derived from a wild-type polypeptide such as a therapeutic polypeptide, a chimeric polypeptide derived from a different wild-type polypeptide, or even a synthetic polypeptide. The parent polypeptide can be selected from antibodies, enzymes, cytokines, regulatory proteins, hormones, receptors, ligands, biosimilars, immunomodulators, growth factors, and fragments of these polypeptides.

[0189] A description of suitable wild-type polypeptides and the manner in which they can be evolved and selected for the production of conditionally active polypeptides has been described in US Pat. No. 8,709,755 B2.

[0190] In some embodiments, the parent polypeptide can be selected from a library of wild-type polypeptide or mutant polypeptide, such as a phage display library. In such an embodiment, a large amount of candidate polypeptides are expressed in a phage library, particularly expressed by surface display technology. Candidate polypeptides from the library are screened to obtain suitable parent polypeptides. Typical phage libraries can contain phages expressing thousands or even millions of candidate polypeptides in a bacterial host. In one embodiment, a phage library can comprise a plurality of phages.

[0191] In order to construct a phage library, a filamentous phage, such as filamentous E. coli phage M13, is usually genetically modified by inserting an oligonucleotide encoding a candidate polypeptide into the coding sequence of one of the phage coat proteins. The coat protein of the phage is then expressed together with the candidate polypeptide so that the candidate polypeptide is displayed on the surface of the phage particle. The displayed candidate polypeptide can then be screened to obtain a suitable parent polypeptide.

[0192] A common technique for screening suitable parent polypeptides is by fixing phage particles with desired candidate polypeptides on a support. The support can be a plastic plate coated with a "bait" that can bind to the desired candidate polypeptide. Unbound phage particles can be washed off the plate. The phage particles (with the desired candidate) bound to the plate are washed and eluted, and the eluted phage particles are amplified in bacteria. The sequence encoding the candidate polypeptide in the selected phage 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.

[0193] Another common technique for screening suitable parent polypeptides is to use an enzyme test method to test the desired enzyme activity of each phage clone, which is the enzyme activity displayed by the candidate polypeptide. According to the specific enzyme activity, those skilled in the art can design a suitable test method to screen for candidate polypeptides with a desired level of enzyme activity.

[0194] In some embodiments, the phage library is provided as an array so that each phage clone occupies a specific position on the array. This array can be provided on a solid support, such as a film, an agar plate or a microtiter plate, and each phage clone in the library is placed on these solid supports or adheres to a specific predetermined position of the solid support. In the case of an agar plate, such a plate preferably includes a bacterial growth medium to support bacterial growth. When the array is arranged on a film (such as nitrocellulose or a nylon membrane), the bacterial culture is applied to the film, and the film is soaked with a nutrient growth medium. In addition, phage clones can also be provided on beads, in which case a single phage clone can be adhered to a single bead. Alternatively, each phage clone can be arranged on the end of an optical fiber, in which case the optical fiber is used to optically transmit ultraviolet radiation from a light source.

[0195] A typical phage library may contain 10 6 Up to 10 10Species of phage, each of which is characterized by a coat protein carrying a different candidate polypeptide (e.g., gp3 or gp8 of phage M113). The bacterial host of the phage library can be selected from a bacterial genus, including, for example, Salmonella, Staphylococcus, Streptococcus, Shigella, Listeria, Campylobacter, Klebsiella, Yersinia, Pseudomonas, and Escherichia.

[0196] The oligonucleotide encoding the candidate polypeptide can be a collection of cDNA encoding a wild-type polypeptide. Known methods for synthesizing cDNA from biological samples can express suitable parental polypeptides. Any genetic information that shows physiological activity through transcripts can be harvested as cDNA. When producing cDNA, full-length cDNA must be synthesized. There are several methods that can be used to synthesize full-length cDNA. For example, suitable methods include a method of marking a 5' cap site using a cap-binding protein of yeast or Hela cells (I. Edery et al., "An Efficient Strategy To Isolate Full-length cDNAs Based on a mRNA Cap Retention Procedure (CAPture)", Mol. Cell. Biol., vol. 15, pp. 3363-3371, 1995); and a method of removing phosphates from an incomplete cDNA that does not have a 5' cap by using alkaline phosphatase and then treating the complete cDNA with a decapping enzyme of tobacco mosaic virus so that only the full-length cDNA has 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. 243-250, 1995).

[0197] In the embodiment where the parent polypeptide is an antibody, a recombinant antibody derived from the complete antibody spectrum of an organism can be used to produce a candidate polypeptide library. The genetic information representing the antibody spectrum is compiled into a large collection of complete antibodies, which can be screened to obtain suitable parent antibodies with desired antigen binding activity and / or one or more other functional characteristics. In some embodiments, the B cells from animals (such as immunized people, mice or rabbits) immunized with antigens are separated. The mRNA from the separated B cells is collected and converted into cDNA, and then sequenced. The most common cDNA fragments encoding light chains and the most common cDNA fragments encoding heavy chains are assembled into antibodies. In one embodiment, the 100 most common cDNA fragments encoding light chains and the 100 most common cDNA fragments encoding heavy chains are assembled to produce candidate antibodies. In another embodiment, the most common cDNA fragments encoding only the variable regions of heavy chains and the variable regions of light chains are assembled to produce antibody fragments that only comprise variable regions and do not comprise constant regions.

[0198] In some embodiments, the cDNA fragment encoding the IgG heavy chain variable region is assembled with the most common variable region of IgK or IgK light chain. The assembled antibody contains the heavy chain variable region from IgG and the light chain variable region from IgK or IgK.

[0199] The cDNA encoding the assembled antibody is then cloned and expressed, preferably in a plate-based format. The binding activity of the expressed antibody can be tested using an ELISA test based on magnetic beads (beads), and suitable parent antibodies can be selected based on the ELISA test. The cDNA encoding the assembled antibody can also be expressed in a phage display library, and one or more desired parent antibodies can then be screened by any of the techniques disclosed herein.

[0200] In the embodiment where the parental polypeptide is an antibody, the parental antibody preferably has at least one feature that makes it easier to evolve the parental antibody into a conditionally active antibody. In certain embodiments, the parental antibody may have similar binding activity and / or features under both normal physiological conditions and abnormal conditions. In such embodiments, the parental antibody is selected based on a combination of the most similar binding activity and / or the most similar one or more features under both normal physiological conditions and abnormal conditions. For example, if the normal physiological condition and the abnormal condition are pH 7.4 and pH 6.0, respectively, then relative to antibodies with less similar binding activity at pH 7.4 and pH 6.0, the parental antibody with the most similar binding activity at pH 7.4 and pH 6.0 can be selected.

[0201] E. Identification of conditionally active peptides

[0202] After selecting the parental polypeptide, the DNA encoding the parental polypeptide is evolved using suitable mutagenesis techniques to produce mutant DNA, which can then be expressed to produce mutant polypeptides, and the mutant polypeptides are screened to determine conditionally active polypeptides. In some embodiments, evolution may be minimal, for example, in order to produce mutant polypeptides with required conditional activity, only a small amount of mutations are introduced into the parental polypeptide. For example, by comprehensive positional evolution (CPE, comprehensive positionalevolution) at each site, introducing less than about 20 changes (possibly less than about 18 changes) may be enough to produce suitable conditionally active polypeptides. For comprehensive positional synthesis (CPS, comprehensive positional synthesis), in the parental polypeptide, less than about 6 improved mutations, or less than about 5 improved mutations, or less than about 4 improved mutations, or less than about 3 improved mutations, or less than about 2 improved mutations may be enough to produce ideal conditionally active polypeptides.

[0203] In some embodiments, when the candidate polypeptide library (e.g., phage library and / or recombinant antibody library) is large enough, the evolution and expression steps may be unnecessary. Such a large library may contain parent polypeptides with conditional activity characteristics (low activity in tests under normal physiological conditions and high activity in tests under abnormal conditions compared to reference polypeptides, or lower activity in tests under normal physiological conditions than in tests under abnormal conditions). In these embodiments, the parent polypeptides in the library are subjected to a selection step to find conditional active polypeptides with the following characteristics: their activity in tests under normal physiological conditions is lower than their activity in tests under abnormal conditions. In one embodiment, the parent polypeptides in the library and the reference polypeptides are tested under normal physiological conditions and tested under abnormal conditions, respectively. The conditional active polypeptides selected from the library are conditional active polypeptides that show lower activity under normal physiological conditions compared to the reference polypeptides and show higher activity under abnormal conditions compared to the reference polypeptides. In this embodiment, since the library is already large enough, there are candidate polypeptides with conditional activity characteristics in the library. So for the purpose of finding conditional active polypeptides, it is not necessary to evolve the parent polypeptides.

[0204] In some embodiments, the reference polypeptide may not have conditional activity because it has similar or identical activity under normal physiological conditions and abnormal conditions. The reference polypeptide is a polypeptide of the same type as the candidate polypeptide in the library, such as the same type of enzyme, cytokine, regulatory protein, antibody, hormone or functional peptide. The reference polypeptide may also be the same type of 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, when the library contains a large number of candidate antibodies against an antigen, the reference polypeptide is an antibody against the same antigen, which has the same or similar binding activity to the antigen under normal physiological conditions and abnormal conditions.

[0205] Therefore, in one embodiment, the candidate polypeptides in the library are tested under normal physiological conditions and under abnormal conditions respectively with respect to the reference polypeptides. The conditionally active polypeptides selected from the library show the following two properties: (a) reduced activity compared to the reference polypeptide under normal physiological conditions, and (b) enhanced activity compared to the reference polypeptide under abnormal conditions.

[0206] F. Methods for Producing Conditionally Active Peptides

[0207] One or more mutagenesis techniques are used to evolve DNA encoding a parent polypeptide to produce mutant DNA; the mutant DNA is expressed to produce a mutant polypeptide; and the mutant polypeptide is subjected to a screening test under a first condition, which may be a normal physiological condition, and a screening test under a second condition, which may be an abnormal condition. Conditionally active polypeptides are selected from those mutant polypeptides that exhibit the following two properties: (a) reduced activity compared to the parent polypeptide when tested under the first condition, and (b) increased activity compared to the parent polypeptide when tested under the second condition. The reduced activity of the conditionally active polypeptide in the first condition or the normal physiological condition may be reversible or irreversible.

[0208] In some embodiments, the polypeptide to be evolved can 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 can be a polypeptide selected from a mutant polypeptide generated by a mutagenesis method that selects a polypeptide with a desired property, such as high binding activity, high expression level, or humanization. The selected polypeptide can be used as the parent polypeptide to be evolved in the methods disclosed herein.

[0209] A method for generating mutant DNA from a DNA encoding a parent polypeptide has been described in US Pat. No. 8,709,755 B2.

[0210] The DNA encoding the parental polypeptide is evolved to produce a mutant DNA and can be carried out using point mutations (displacements, insertions and / or deletions) or mutations of large segments in the DNA. In some aspects, the evolution step does not change the active site of the parental polypeptide, but only changes one or more regions around the active site and / or one or more regions away from the active site.

[0211] In one aspect, the evolution step involves converting the parental full-length antibody into a single-chain antibody. In this case, although the active site, i.e., the variable region, particularly the CDR, may not have any mutation relative to the parental antibody, the environment in which the active site exists has changed due to the elimination of the constant region. In one embodiment, the parental full-length antibody is an IgG antibody, and the mutant antibody is a single-chain antibody derived therefrom.

[0212] In some aspects, single-chain antibodies are bispecific antibodies with two arms, each of which binds to a different epitope. A mutation on one arm may affect the activity of the other arm. Therefore, the evolution step may involve mutating only one arm of the parent polypeptide of the bispecific antibody. In one embodiment, the arm may be shortened by deletion or extended by insertion to evolve the length of an arm. Alternatively, the evolution step may evolve the two arms of the bispecific antibody in the same evolution step or in a continuous evolution step, optionally screening after each step.

[0213] In another aspect, the parent polypeptide is an antibody or antibody fragment. The evolution step can mutate the Fc region. The mutation in the Fc region can be a replacement, insertion and / or deletion. The Fc region can be shortened by deleting a fragment of the Fc region, or extended by inserting a fragment into the Fc region.

[0214] In yet another aspect, the parent polypeptide comprises a plurality of complementary determining regions interrupted by a framework region. For example, such a parent polypeptide may be a variable region of an antibody, a light chain, or a heavy chain. In certain embodiments, the evolution step may mutate only the framework region or a combination of the complementary determining regions and the framework region. The evolution of the framework region and the complementary determining regions may be performed in a single step or in a plurality of consecutive steps, optionally with screening after each step.

[0215] In another aspect, the parent polypeptide has several regions outside of its active site. These regions can be mutated sequentially in multiple evolution steps, optionally followed by screening after one or more evolution steps. For example, an evolution step can evolve one region of a polypeptide, followed by screening for conditionally active polypeptides; then evolve another region of the polypeptide, followed by screening for conditionally active polypeptides; then further evolve yet another region of the polypeptide, followed by yet another step of screening for conditionally active polypeptides.

[0216] In some cases, one or more regions (for example, surrounding regions or remote regions) outside the active site of parental polypeptide and / or mutant conditional active polypeptide are evolved to change the activity of the active site. Mutation surrounding regions or remote regions rather than the active site can make the active site of the mutant polypeptide have more or less activity than the active site of the parental polypeptide under certain conditions in some cases. In other embodiments, by evolving one or more regions outside the region where the parental polypeptide or mutant polypeptide comprises the active site, the desired conditional activity is achieved, or selectivity is improved.

[0217] In some aspects, a conditionally active polypeptide derived from evolution of a region of a parent polypeptide other than the region containing the active site can result in at least 2, or at least 3, or at least 5 selectivities.

[0218] Suitable methods for expressing the generated mutant DNA to produce mutant polypeptides have been described in US Pat. No. 8,709,755 B2.

[0219] Methods for screening mutant polypeptides to select conditionally active polypeptides have been described in US Pat. No. 8,709,755 B2.

[0220] Test conditions for screening and selecting conditionally active peptides

[0221] The first and second conditions or normal physiological conditions and abnormal conditions used in the screening step for testing can be performed using conditions selected from temperature, pH, osmotic pressure, weight molar osmotic pressure concentration, oxidative stress, electrolyte concentration, and a combination of two or more such conditions. For example, the normal physiological condition of temperature can be a normal human body temperature of 37.0°C, and the abnormal condition of temperature can be a temperature different from the temperature of 37.0°C, such as the temperature in the tumor microenvironment can be 1-2°C higher than the normal physiological temperature. In another example, the normal physiological condition and the abnormal condition can also be a normal physiological pH in the range of 7.2-7.8 or 7.2-7.6 and an abnormal pH in the range of 5.5-7.2, 6-7, or 6.2-6.8, such as presented in the tumor microenvironment.

[0222] The test under the first condition and the second condition or the normal physiological condition and the abnormal condition can be carried out in the test medium. The test medium can be a solution, and the solution can include, for example, a buffer and other components. The common buffer that can be used in the test medium includes a citrate buffer (e.g., sodium citrate), a phosphate buffer, a bicarbonate buffer (e.g., Krebs buffer), a phosphate buffered saline (PBS), a Hank's buffer, a Tris buffer, a HEPES buffer, etc. Other buffers suitable for the test known to those skilled in the art can be used. These buffers can be used to simulate the characteristics or components of the body fluids (e.g., blood plasma or lymph) of humans or animals.

[0223] The test solution used in the method of the present invention may contain at least one component selected from inorganic compounds, ions and organic molecules, preferably components that are commonly present in the body fluids of mammals (e.g., humans or animals). Examples of these components include nutrients and metabolites, as well as any other components that may be present in body fluids. The present invention contemplates that the component may or may not be part of a buffer system. For example, the test solution may be a PBS buffer to which bicarbonate ions are added, wherein the bicarbonate ions are not part of the PBS buffer. Alternatively, the bicarbonate ions are part of a bicarbonate buffer.

[0224] The component may be present in two test solutions (for the first condition and the second condition) at substantially the same concentration, while the two test solutions differ in other aspects, such as pH, temperature, electrolyte concentration or osmotic pressure. Thus, the component is used as a constant rather than the difference between the two conditions of the first condition and the second condition or the normal physiological condition and the abnormal condition.

[0225] In some embodiments, the component is present in both test solutions at a concentration close to or identical to the normal physiological concentration of the component in a mammal, particularly a human.

[0226] The inorganic compound or ion can 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, chelates of calcium, copper, iron, manganese and zinc, ammonium molybdate, ammonium sulfate, calcium carbonate, magnesium phosphate, potassium bicarbonate, potassium nitrate, hydrochloric acid, carbon dioxide, sulfuric acid, phosphoric acid, carbonic acid, uric acid, hydrogen chloride, urea, phosphorus ion, sulfate ion, chloride ion, magnesium ion, sodium ion, potassium ion, ammonium ion, iron ion, zinc ion and copper ion.

[0227] Examples of normal physiological concentrations of some inorganic compounds include: uric acid in the range of 2-7.0 mg / dL, calcium ions in the range of 8.2-11.6 mg / dL, chloride ions in the range of 355-381 mg / dL, iron ions in the range of 0.028-0.210 mg / dL, potassium ions in the range of 12.1-25.4 mg / dL, sodium ions in the range of 300-330 mg / dL, carbonic acid in the range of 15-30 mM, citrate ions at about 80 μM, histidine ions at 0.05-2.6 mM, histamine at 0.3-1 μM, HAPT ions (hydrogenated adenosine triphosphate) at 1-20 μM, and HADP ions at 1-20 μM.

[0228] In some embodiments, the ions present in the test solution for testing under the first condition and the second condition or under normal physiological conditions and abnormal conditions are selected from hydroxide ions, halide ions (chloride ions, bromide ions, iodide ions), oxyhalide ions, sulfate ions, magnesium ions, calcium ions, hydrogen sulfate ions, carbonate ions, bicarbonate ions, sulfonate ions, oxyhalide ions, nitrate ions, nitrite ions, phosphate ions, hydrogen phosphate ions, dihydrogen phosphate ions, persulfate ions, monopersulfate ions, borate ions, ammonium ions, or organic ions, such as carboxylate ions, phenolate ions, sulfonate ions (organic sulfates such as methyl sulfate), vanadate ions, tungstate ions, borate ions, organic borate ions, citrate ions, oxalate ions, acetate ions, pentaborate ions, histidine ions, and phenolate ions.

[0229] The organic compound present in the test solution under the first condition and the second condition or under normal physiological conditions and abnormal conditions can be selected from, for example, amino acids such as histidine, alanine, isoleucine, arginine, leucine, asparagine, lysine, aspartic acid, methionine, cysteine, phenylalanine, glutamic acid, threonine, glutamine, tryptophan, glycine, valine, pyrrolysine, proline, selenocysteine, serine, tyrosine, and mixtures thereof.

[0230] Examples of normal physiological concentrations of some amino acids include: 3.97±0.70 mg / dL alanine, 2.34±0.62 mg / dL arginine, 3.41±1.39 mg / dL glutamate, 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, 1.91±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.

[0231] The organic compound present in the test solution for testing under the first condition and the second condition or the normal physiological condition and the abnormal condition can be selected from non-protein nitrogen-containing compounds such as creatine, creatinine, guanidinoacetic acid, uric acid, allantoin, adenosine, urea, ammonia and choline. Examples of normal physiological concentrations of some of these compounds include: 1.07±0.76 mg / dL of creatine, 0.9-1.65 mg / dL of creatinine, 0.26±0.24 mg / dL of guanidinoacetic acid, 4.0±2.9 mg / dL of uric acid, 0.3-0.6 mg / dL of allantoin, 1.09±0.385 mg / dL of adenosine, 27.1±4.5 mg / dL of urea and 0.3-1.5 mg / dL of choline.

[0232] The organic compound present in the test solution for testing under the first condition and the second condition or the normal physiological condition and the abnormal condition can be an organic acid, such as citric acid, α-ketoglutaric acid, succinic acid, malic acid, fumaric acid, acetoacetic acid, β-hydroxybutyric acid, lactic acid, pyruvic acid, α-keto 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 pyruvic acid, 0.6-2.1 mg / dL α-keto acid, 1.8 mg / dL volatile fatty acids.

[0233] The organic compound present in the test solution for testing under the first condition and the second condition or under normal physiological conditions and abnormal conditions can be selected from sugars (carbohydrates), such as glucose, pentose, hexose, xylose, ribose, mannose and galactose, and disaccharides including lactose, GlcNAcβ1-3Gal, Galα1-4Gal, Manα1-2Man, GalNAcβ1-3Gal and O-glycoside, N-glycoside, C-glycoside or S-glycoside. Examples of normal physiological concentrations of some of these sugars include: 83±4 mg / dL of glucose, 102±73 mg / dL of polysaccharides (such as hexose), 77±63 mg / dL of glucosamine, 0.4-1.4 mg / dL of hexuronate (as glucuronic acid) and 2.55±0.37 mg / dL of pentose.

[0234] The organic compound present in the test solution for testing under the first condition and the second condition or the normal physiological condition and the abnormal condition can be selected from fats or derivatives thereof, such as cholesterol, lecithin, cephalin, sphingomyelin and bile acid. Examples of normal physiological concentrations of some of these compounds include: 40-70 mg / dL of free cholesterol, 100-200 mg / dL of lecithin, 0-30 mg / dL of cephalin, 10-30 mg / dL of sphingomyelin and 0.2-0.3 mg / dL of bile acid (as cholic acid).

[0235] The organic compound present in the test solution for testing under the first condition and the second condition or under normal physiological conditions and abnormal conditions can be selected from proteins, such as fibrinogen, antihemophilic globulin, immunogamma globulin, immunoeuglobulin, isoagglutinin, beta-pseudoglobulin, glycoprotein, lipoprotein and albumin. For example, the normal physiological concentration of mammalian serum albumin is 3.35-5.0 g / dL. In one embodiment, the albumin is bovine serum albumin.

[0236] The organic compound present in the test solution for testing under the first condition and the second condition or the normal physiological condition and the abnormal condition can be selected from vitamins, such as vitamin A, carotene, vitamin E, ascorbic acid, thiamine, inositol, folic acid, biotin, pantothenic acid, riboflavin. Examples of normal physiological concentrations of some of these vitamins include: 0.019-0.036 mg / dL of vitamin A, 0.90-1.59 mg / dL of vitamin E, 0.42-0.76 mg / dL of inositol, 0.00162-0.00195 mg / dL of folic acid, and 0.00095-0.00166 mg / dL of biotin.

[0237] The concentration of the inorganic compound, ion or organic molecule in the test solution (for testing under the first condition and the second condition or under normal physiological conditions and under abnormal conditions) can be within the normal physiological concentration range of the 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 of magnesium ions in human serum is 1.7-2.2 mg / dL, and calcium is 8.5-10.2 mg / dL. The concentration of magnesium ions in the test solution can be about 0.17 mg / dL to about 11 mg / dL. The concentration of calcium ions in the test solution can be about 0.85 mg / dL to about 51 mg / dL. Typically, the concentration of the inorganic compound, ion or organic molecule in the test solution can 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 the inorganic compound, ion or organic molecule in human serum, or 1.5 times, or 2 times, or 3 times, or 4 times, or 5 times, or 7 times, or 9 times, or 10 times, or even 20 times the normal physiological concentration of the inorganic compound, ion or organic molecule in human serum. Different components of the test solution can be used at different concentration levels relative to their respective normal physiological concentrations.

[0238] The test under the first condition and the second condition or the normal physiological condition and the abnormal condition is used to measure the activity of the mutant polypeptide.During the test, the mutant polypeptide and its binding partner are present in the test solution.The relationship between the mutant polypeptide and its binding partner can be, for example, antibody-antigen, ligand-receptor, enzyme-substrate or hormone-receptor.In order to make the mutant polypeptide express its activity, the mutant polypeptide should be able to contact and combine with its binding partner.Then prove and measure the activity of the mutant polypeptide to its binding partner after the mutant polypeptide is combined with its binding partner.

[0239] In some embodiments, the ions used in the test can play a role in forming the bridge between the mutant polypeptide screened and its binding partner, particularly those ions comprising charged amino acid residues. Therefore, ions can be combined with both mutant polypeptide and its binding partner by hydrogen bonds and / or ionic bonds. This can help the combination between mutant polypeptide and its binding partner by enabling ions to reach sites that may be difficult to reach for macromolecules (mutant polypeptide or its binding partner). In some cases, the ions in the test solution can increase the possibility that mutant polypeptide and its binding partner are combined with each other. In addition, ions can additionally or alternatively assist the combination between mutant polypeptide and its binding partner by combining with larger molecules (mutant polypeptide or its binding partner). This combination can change the conformation of macromolecules and / or keep larger molecules in a specific conformation that is conducive to combining with their binding partners.

[0240] It has been observed that ions can assist the binding between a mutant polypeptide and its binding partner, and this effect may be achieved by forming ionic bonds with the mutant polypeptide and its binding partner. Therefore, compared with the same test without ions, screening can be much more effective and more hits (candidate conditionally active polypeptides) can be identified. Suitable ions can be selected from magnesium ions, sulfate ions, bisulfate ions, carbonate ions, citrate ions, HAPT ions, HADP ions, carbonate ions, nitrate ions, nitrite ions, phosphate ions, hydrogen phosphate ions, dihydrogen phosphate ions, persulfate ions, monosulfate ions, borate ions, lactate ions, citrate ions, histidine ions, histamine ions and ammonium ions.

[0241] Ions have been found to facilitate binding between a mutant polypeptide and its binding partner at a pH close to its pKa. Preferably, such ions are relatively small relative to the size of the mutant polypeptide.

[0242] In one embodiment, when the abnormal condition is a pH different from the normal physiological pH under normal physiological conditions, the ions suitable for increasing the number of hits for the candidate conditionally active polypeptide can be selected from ions with a pKa close to the abnormal pH to be tested in the test. For example, the pKa of the ion can be up to 2 pH units different from the abnormal pH, up to 1 pH unit different from the abnormal pH, up to 0.8 pH units different from the abnormal pH, up to 0.6 pH units different from the abnormal pH, up to 0.5 pH units different from the abnormal pH, up to 0.4 pH units different from the abnormal pH, up to 0.3 pH units different from the abnormal pH, up to 0.2 pH units different from the abnormal pH, or up to 0.1 pH units different from the abnormal pH.

[0243] The pKa of the ions useful in the present invention may vary slightly at different temperatures, exemplary pKas of the ions are as follows: ammonium ion with a pKa of about 9.24, dihydrogen phosphate with a pKa of about 7.2, acetate with a pKa of about 4.76, histidine with a pKa of about 6.04, bicarbonate ion with a pKa of about 6.4, citrate with a pKa of 6.4, lactate ion with a pKa of about 3.86, histamine with a pKa of about 6.9, HATP with a pKa of 6.95 HADP with a pKa of 6.88

[0244] In one embodiment, conditionally active polypeptides are tested and selected in the presence of hydrosulfide. The pKa of hydrosulfide is 7.05. In some embodiments, different concentrations of hydrosulfide can be used in tests representing normal and abnormal physiological conditions. Alternatively, the test medium for normal physiological conditions and abnormal conditions has approximately the same hydrosulfide concentration, with some differences in the values ​​for specific conditions, for example, the test can be performed at different pH. The concentration of hydrosulfide used in the test can be 1 mM to about 100 mM. Preferably, the concentration of hydrosulfide used in the test medium is 2-500 nM, 3-200 nM or 5-100 nM. In some aspects, the hydrosulfide concentration can be 1 mM to 20 mM or 2 mM to 10 mM. Tests performed in the presence of hydrosulfide are known.

[0245] In certain embodiments, once the pH of the abnormal condition (i.e., the abnormal pH) is known, ions suitable for increasing hits of the candidate conditionally active polypeptide can be selected from ions having a pKa that is the same as or close to the abnormal pH, for example, the pKa of the candidate ion can differ by up to 4 pH units from the abnormal pH, by up to 3 pH units from the abnormal pH, by up to 2 pH units from the abnormal pH, by up to 1 pH unit from the abnormal pH, by up to 0.8 pH units from the abnormal pH, by up to 0.6 pH units from the abnormal pH, by up to 0.5 pH units from the abnormal pH, by up to 0.4 pH units from the abnormal pH, by up to 0.3 pH units from the abnormal pH, by up to 0.2 pH units from the abnormal pH, or by up to 0.1 pH units from the abnormal pH.

[0246] As described above, under conditions where the pH is the same or similar to the pKa of the ion, the ion most effectively assists the binding between the mutant polypeptide and its binding partner. For example, it has been found that in a test solution with a pH of 7.2-7.6, bicarbonate ions (pKa of about 6.4) are not very effective in assisting the binding between the mutant polypeptide and its binding partner. When the pH in the test solution is reduced to 6.7 and further reduced to about 6.0, bicarbonate ions become more and more effective in assisting the binding between the mutant polypeptide and its binding partner. As a result, more hits can be identified in a test with a pH of 6.0 compared to a test with a pH of 7.2-7.6. Similarly, at pH 7.4, histidine is not very effective in assisting the binding between the mutant polypeptide and its binding partner. When the pH of the test solution is reduced to 6.7 and further reduced to about 6.0, histidine becomes more and more effective in assisting the binding between the mutant polypeptide and its binding partner, making it possible to identify more hits in a pH range of, for example, about 6.2-6.4.

[0247] The present invention surprisingly found that when the pH of the test solution under normal physiological conditions (i.e., normal physiological pH) and abnormal conditions (i.e., abnormal pH) is different, ions with the following pKa can greatly assist the binding between the mutant polypeptide being screened and its binding partner: the pKa is from about the middle point between normal physiological pH and abnormal pH to about abnormal pH. As a result, the screening test is more efficient in discovering more hits or candidate conditionally active polypeptides with high activity under abnormal conditions.

[0248] In some embodiments, the pKa may even differ from the abnormal pH by at least one pH unit. When the abnormal pH is an acidic pH, the pKa of a suitable ion may be in the range from (abnormal pH-1) to the midpoint between the abnormal pH and normal physiological pH. When the abnormal pH is an alkaline pH, the pKa of a suitable ion may be in the range from the midpoint between the abnormal pH and normal physiological pH to (abnormal pH+1). The ions may be selected from those described in the present application. However, more ions that have not been explicitly described in the present application may also be used. It should be understood that once the abnormal pH and normal physiological pH for the screening test are selected, those skilled in the art can use the guiding principles of the present invention to select any ions with a suitable pKa to improve the screening efficiency in identifying more hits with high activity under abnormal conditions.

[0249] For example, when the abnormal pH of the exemplary screening 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 pKa in this range include ions derived from N-tris(hydroxymethyl)methylglycine (pKa 8.05), hydrazine (pKa 8.1), N,N-dihydroxyethylglycine (pKa 8.26), N-(2-hydroxyethyl)piperazine-N'-(4-butanesulfonic acid) (pKa 8.3), N-tris[hydroxymethyl]methyl-3-aminopropanesulfonic acid (pKa 8.4), taurine (pKa 9.06). As another example, when the abnormal pH of the exemplary screening 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 pKa in this range include those derived from malate (pKa 5.13), pyridine (pKa 5.23), piperazine (pKa 5.33), cacodylate (pKa 6.27), succinate (pKa 5.64), 2-(N-morpholino)ethanesulfonic acid (pKa 6.10), citrate (pKa 6.4), histidine (pKa 6.04), and bis-tris (6.46). Those skilled in the art will be able to refer to a large number of chemical handbooks and textbooks to determine known compounds, both inorganic and organic, that can be converted to ions with pKa in the range. Among compounds with suitable pKa, compounds with smaller molecular weights may be preferred.

[0250] Thus, the present invention unexpectedly discovered that the production of conditionally active polypeptides that are ultimately identified depends not only on the generation of the correct polypeptide mutants, but also on the use of ions with a suitable pKa in the test solution. The present invention contemplates that in addition to generating large libraries of mutant polypeptides (e.g., by CPE and CPS), efforts should be made to find suitable ions (with appropriate pKa) for use in the test solution, because ions can facilitate the efficient selection of highly active mutants from large libraries. It is further contemplated that in the absence of suitable ions, the screening efficiency is low and the likelihood of finding mutants with high activity is reduced. Therefore, in the absence of suitable ions, multiple rounds of screening may be required to obtain the same number of mutants with high activity.

[0251] The ions in the test solution can be formed in situ by the components of the test solution, or directly contained in the test solution. For example, CO2 from air can be dissolved in the test solution to provide carbonate ions and bicarbonate ions. As another example, sodium dihydrogen phosphate can be added to the test solution to provide dihydrogen phosphate ions.

[0252] The concentration of the component in the test solution (for testing under the first condition or normal physiological conditions and testing under the second condition or abnormal conditions) can be the same or substantially the same as the concentration of the same component typically found in the naturally occurring body fluids of mammals (e.g., humans). In other embodiments, the concentration of the component may be higher, especially when the component is an ion that can play a role in assisting the binding between the mutant polypeptide and its binding partner, because it has been observed that higher concentrations of such ions can form ionic bonds with the mutant polypeptide and its binding partner, so they can actually promote binding and increase the possibility of discovering more hits or candidate conditionally active polypeptides.

[0253] In some embodiments, the concentration of the ion in the test solution is positively correlated with the probability of finding more hits using the test, particularly when concentrations exceeding normal physiological concentrations are used. For example, the concentration of bicarbonate ions in human serum is about 15-30 mM. In one example, as the concentration of bicarbonate ions in the test solution increases from 3 mM to 10 mM, to 20 mM, to 30 mM, to 50 mM, and to 100 mM, the number of hits in the test also increases with each increase in bicarbonate concentration. In view of this, the test solution can use a bicarbonate concentration ranging from about 3mM to about 200mM, or about 5mM to about 150mM, or about 5mM to about 100mM, or about 10mM to about 100mM, or about 20mM to about 100mM, or about 25mM to about 100mM, or about 30mM to about 100mM, or about 35mM to about 100mM, or about 40mM to about 100mM, or about 50mM to about 100mM.

[0254] In another embodiment, the concentration of citrate in the test 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.

[0255] In one embodiment, the normal physiological condition is a normal physiological pH of 7.2-7.6, and the abnormal condition is an abnormal pH in the range of 5.5-7.2, 6-7 or 6.2-6.8. The test solution for testing under normal physiological conditions has a normal physiological pH and 50 mM bicarbonate ions. The test solution for testing under abnormal conditions has an abnormal pH and 50 mM bicarbonate ions. Since the pKa of bicarbonate ions is about 6.4, bicarbonate ions can assist the binding between the mutant polypeptide and its binding partner at an abnormal pH of 6.0-6.4 (such as pH 6.0 or 6.2).

[0256] In another embodiment, the normal physiological condition is a normal physiological pH of 7.2-7.6, and the abnormal condition is an abnormal pH of 5.5-7.2, 6-7 or 6.2-6.8. The test solution for testing under normal physiological conditions has a normal physiological pH and 80 μM of citrate ions. The test solution for testing under abnormal conditions has an abnormal pH and 80 μM of citrate ions. Since the pKa of citrate ions is 6.4, citrate ions can effectively assist in the binding between mutant polypeptides and binding partners in the test solution for abnormal conditions of pH 6.0-6.4. Therefore, more candidate conditionally active polypeptides with higher binding activity under conditions of pH 6.0-6.4 and lower activity under conditions of pH 7.2-7.8 can be identified. Other ions including acetate, histidine, bicarbonate, HATP and HADP function in a similar manner, such that test solutions containing the ions can effectively screen for mutant polypeptides that have higher binding activity at a pH near the pKa of the ion and lower binding activity at a pH different from the pKa of the ion (e.g., normal physiological pH).

[0257] In another embodiment, the normal physiological condition is a normal physiological temperature at 37°C and the abnormal condition is an abnormal temperature at 38-39°C (temperature in certain tumor microenvironments). The test solution for testing under normal physiological conditions has a normal physiological temperature and 20 mM bicarbonate ions. The test solution for testing under abnormal conditions has an abnormal temperature and 20 mM bicarbonate ions.

[0258] In another embodiment, the normal physiological condition is a specific concentration of an electrolyte in normal human serum and the abnormal condition is a different, abnormal concentration of the same electrolyte, which may be located 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 concentration of the electrolyte in human serum.

[0259] The combination between mutant polypeptide and / or its binding partner also may be affected in many other ways.Usually, this impact will be realized by comprising one or more other components in the test solution.These other components can be designed to interact with mutant polypeptide, binding partner or both.In addition, these other components can use two or more interactive combinations and two or more types of interactive combinations to affect the combination.

[0260] In one embodiment, the binding interaction of interest is between an antibody and an antigen. In this embodiment, one or more other components may be included in the test solution to exert an influence on the antibody, the antigen, or both. In this way, the desired binding interaction may be enhanced.

[0261] In addition to the ions that can form ionic bonds with mutant polypeptide and / or its binding partner to assist the binding between mutant polypeptide and binding partner, the present invention also includes other components that can be used to assist mutant polypeptide and its binding partner to bind. In one embodiment, molecules that can form hydrogen bonds with mutant polypeptide and / or its binding partner are used. In another embodiment, molecules that can carry out hydrophobic interactions with mutant polypeptide and / or its binding partner can be used. In another embodiment, molecules that can carry out van der Waals interactions with mutant polypeptide and / or its binding partner are considered.

[0262] The term "hydrogen bond" as used herein refers to a relatively weak non-covalent interaction between a hydrogen covalently bonded to an electronegative atom (such as carbon, nitrogen, oxygen, sulfur, chlorine, or fluorine) (a hydrogen bond donor) and an electron donor atom (e.g., nitrogen, oxygen, sulfur, chlorine, or fluorine) with an unshared electron pair (a hydrogen bond acceptor).

[0263] The components that can form hydrogen bonds with mutant polypeptide and / or its binding partner include organic molecules and inorganic molecules with polar bonds. The binding partner of mutant polypeptide and / or mutant polypeptide generally contains amino acids that can form hydrogen bonds. Suitable amino acids have side chains containing polar groups that can form 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).

[0264] These amino acids can act as hydrogen donors and hydrogen acceptors. For example, the oxygen atom in an -OH group (such as can be found in Ser, Thr and Tyr), the oxygen atom in a -C=O group (such as can be found in Glu and Asp), the sulfur atom in a -SH group or -SC- (such as can be found in Cys and Met), the -NH3 + Nitrogen atoms in groups (such as those found in Lys and Arg) and nitrogen atoms in -NH- groups (such as those found in Trp, His, and Arg) can act as hydrogen acceptors. In addition, groups in this list that contain hydrogen atoms (such as -OH, -SH, NH3 + and -NH-) can serve as hydrogen donors.

[0265] In some embodiments, the backbone of the mutant polypeptide and / or its binding partner may also participate in the formation of one or more hydrogen bonds. For example, the backbone may have a repeating structure of -(C=O)-NH-, such as in a peptide bond. The oxygen atoms and nitrogen atoms in this structure may serve as hydrogen acceptors, and the hydrogen atoms may participate in hydrogen bonds.

[0266] Inorganic compounds having at least one polar bond containing a hydrogen atom or an oxygen atom available for hydrogen bonding may include, for example, H2O, NH3, H2O2, hydrazines, carbonates, sulfates, and phosphates. Organic compounds such as alcohols; phenols; thiols; aliphatics, amines, amides; epoxides, carboxylic acids; ketones; aldehydes; ethers, esters; organic chlorides and organic fluorides. Compounds that can form hydrogen bonds are well known in the chemical literature, such as those discussed in, for example, "The Nature of the Chemical Bond," by Linus Pauling, Cornell University Press, 1940, pp. 284-334.

[0267] In some embodiments, alcohols may include methanol, ethanol, propanol, isopropanol, butanol, amyl alcohol, 1-hexanol, 2-octanol, 1-decanol, cyclohexanol and higher alcohols; glycols such as ethylene glycol, propylene glycol, glycerol, diethylene glycol and polyalkylene glycols. Suitable phenols include hydroquinone, resorcinol, catechol, phenol, o-cresol, m-cresol and p-cresol, thymol, α-naphthol and β-naphthol, pyrogallol, guaiacol and phloroglucinol. Suitable thiols include methyl mercaptan, ethanethiol, 1-propanethiol, 2-propanethiol, butyl mercaptan, tert-butyl mercaptan, pentanethiol, hexanethiol, thiophenol, dimercaptosuccinic acid, 2-mercaptoethanol and 2-mercaptoindole. Suitable amines include methylamine, ethylamine, propylamine, isopropylamine, aniline, dimethylamine and methylethylamine, trimethylamine, aziridine, piperidine, N-methylpiperidine, benzidine, cyclohexylamine, ethylenediamine, hexamethylenediamine, o-toluidine, m-toluidine and p-toluidine and N-phenylpiperidine. Suitable amides include acetamide, N,N-dimethylacetamide, N,N-dimethylformamide, N,N-dimethylmethoxyacetamide and N-methyl-N-to-cyanoethylformamide. Epoxides may 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-octane oxide. Carboxylic acids may include terephthalic acid, isophthalic acid, phthalic acid, salicylic acid, benzoic acid, acetic acid, lauric acid, adipic acid, lactic acid, citric acid, acrylic acid, glycine, hexahydrobenzoic acid, o-toluic acid, m-toluic acid and p-toluic acid, nicotinic acid, isonicotinic acid and p-aminobenzoic acid. Ketones may include acetone, 3-acetone, butanone, amyl ketone, 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 can include formaldehyde, acetaldehyde, propionaldehyde, butyraldehyde, benzaldehyde, cinnamaldehyde, sobutyraldehyde, valeraldehyde, octanal, benzaldehyde, cinnamaldehyde, cyclohexanone, salicylaldehyde and furfural. Esters include ethyl acetate, methyl acetate, ethyl formate, butyl acetate, ethyl lactate, ethyl butyrate, propyl acetate, ethyl formate, propyl formate, butyl formate, amyl formate, methyl acetate, ethyl acetate, propyl acetate, butyl acetate, amyl acetate, methyl isopentyl acetate, methoxybutyl acetate, hexyl acetate, cyclohexyl acetate, benzyl acetate, methyl propionate, ethyl propionate, butyl propionate, methyl butyrate, ethyl butyrate, butyl butyrate, amyl butyrate, methyl acetoacetate and ethyl acetoacetate, etc. Ethers that can be used in the present invention include dimethyl ether, methyl ethyl ether, ethyl ether, methyl propyl ether and dimethoxyethane. The ether may be cyclic, such as ethylene oxide, tetrahydrofuran and dioxane.

[0268] Organic chlorides include chloroform, pentachloroethane, dichloromethane, trichloromethane, carbon tetrachloride, tetrachloromethane, tetrachloroethane, pentachloroethane, trichloroethylene, tetrachloroethylene and dichloroethylene. Organic fluorines may include fluoromethane, difluoromethane, trifluoromethane, trifluoroethane, tetrafluoroethane, pentafluoroethane, difluoropropane, trifluoropropane, tetrafluoropropane, pentafluoropropane, hexafluoropropane and heptafluoropropane.

[0269] Hydrogen bonds can be classified into strong, medium or weak hydrogen bonds according to the strength of the bond (Jeffrey, George A.; An introduction to hydrogen bonding, Oxford University Press, 1997). The donor-acceptor distance of a strong hydrogen bond is The energy is in the range of 14-40 kcal / mol. The donor-acceptor distance of a medium hydrogen bond is The energy is in the range of 4-15 kcal / mol. The donor-acceptor distance of a weak hydrogen bond is The energy is in the range of <4 kcal / mol. Some examples of hydrogen bonds with 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 examples, see Perrin et al. "Strong" hydrogen bonds in chemistry and biology, Annual Review of Physical Chemistry, vol. 48, pp. 511-544, 1997; Guthrie, "Short strong hydrogen bonds: can they explain enzymic catalysis?" Chemistry & Biology March 1996, 3: 163-170.

[0270] In some embodiments, the components used in the present invention can form strong hydrogen bonds with mutant polypeptides and / or their binding partners. These components often contain atoms with strong electronegativity. The atoms known to have the strongest electronegativity are F>O>Cl>N in turn. Therefore, the present invention preferably uses organic compounds containing fluorine, hydroxyl or carbonyl to form hydrogen bonds. In one embodiment, organic fluorine can be used in the present invention to form strong hydrogen bonds.

[0271] In another embodiment, components capable of hydrophobic interactions with the mutant polypeptide and / or its binding partner are used. These components include organic compounds with hydrophobic groups.

[0272] The term "hydrophobic interaction" as used herein 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 interaction has been described in the literature "Hydrophobic Interactions," A. Ben-Nairn (1980), Plenum Press, New York.

[0273] Hydrophobic substances are repelled by water molecules due to their non-polar nature. When relatively non-polar molecules or groups in aqueous solution associate with other non-polar molecules instead of with water, it is called "hydrophobic interaction".

[0274] Mutant polypeptide and its binding partner usually comprise amino acid capable of hydrophobic interaction.Usually these amino acid are characterized by having at least one side chain containing 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), to a lesser extent, methionine (Met) and tryptophan (Trp).

[0275] The component that can carry out hydrophobic interaction with mutant protein and / or its binding partner includes organic compound, and this organic compound is hydrophobic molecule or the molecule containing at least one hydrophobic part.In some embodiments, these hydrophobic components can be the hydrocarbon selected from aromatic hydrocarbon, substituted aromatic hydrocarbon, polyaromatic hydrocarbon, aromatic or non-aromatic heterocycle, cycloalkane, alkane, alkene and alkyne.Hydrophobic group can include aryl, alkyl, cycloalkyl, alkenyl and alkynyl.Term " alkyl ", " alkenyl " and " alkynyl " used in this article refer to the unsaturated aliphatic group with one to thirty carbon atoms, including straight chain alkenyl / alkynyl, branched chain alkenyl / alkynyl, cycloalkenyl (alicyclic) group, alkyl substituted cycloalkyl and cycloalkyl substituted alkenyl / alkynyl.Such hydrocarbon part can also be substituted on one or more carbon atoms.

[0276] It is understood that the intensity of hydrophobic interaction is based on the available amount of " hydrophobes " that can interact with each other.Therefore, hydrophobic interaction can be regulated by, for example, increasing the amount and / or " hydrophobic " property of the hydrophobic part in the molecule participating in hydrophobic interaction.For example, hydrophobic part (its original form can include hydrocarbon chain) can be modified, by making it have the hydrophobic side chain of one of the carbon atom that is connected to its carbon skeleton to increase its hydrophobicity (increasing the ability of the intensity of the hydrophobic interaction that this part participates in).In a preferred embodiment of the present invention, this can include the connection of various polycyclic compounds, including, for example, various steroid compounds and / or derivatives thereof, for example sterol type compounds, more specifically cholesterol.Usually, side chain can be the hydrophobic side chain of straight chain, aromatic, aliphatic, cyclic, polycyclic or any various other types that those skilled in the art think of.

[0277] The types of components capable of van der Waals interactions with mutant polypeptides and / or their binding partners are typically, but not always, compounds with polar moieties. "Van der Waals interactions" as used herein refers to attractive forces between atoms, moieties, molecules and surfaces that result from dipole-dipole interactions and / or correlations that occur as a result of quantum dynamics in the fluctuating polarization of neighboring atoms, moieties or molecules.

[0278] The van der Waals interaction of the present invention is an attractive force between a mutant polypeptide or binding partner and a component. Van der Waals interactions may come from three sources. First, some molecules / parts, although electrically neutral, may be permanent electric dipoles. Due to fixed deformations in the electronic charge distribution in the structure of some molecules / parts, one side of the molecule / part is always slightly positively charged, while the opposite side is slightly negatively charged. The tendency of these permanent dipoles to align with each other results in a net attractive force. This is an interaction between two permanent dipoles (Keesom force).

[0279] Second, the presence of a molecule that is a permanent dipole can temporarily deform the electronic charge in other nearby polar or non-polar molecules, causing further polarization. The interaction between the permanent dipole and the adjacent induced dipole creates another attractive force. This is the interaction between the permanent dipole and the corresponding induced dipole, which can be called the Debye force. Third, although the participating molecules are not permanent dipoles (such as organic liquid benzene), there is an attractive force between molecules with two transient induced dipoles in the molecule. This is the interaction between the two transient induced dipoles, which can be called the London dispersion force.

[0280] There are many amino acids that can perform van der Waals interactions in the mutant polypeptide and / or binding partner. These amino acids can have polar side chains, including glutamine (Gln), asparagine (Asn), histidine (His), serine (Ser), threonine (Thr), tyrosine (Tyr), cysteine ​​(Cys), methionine (Met), tryptophan (Trp). These amino acids can also have side chains containing non-polar groups, including alanine (Ala), isoleucine (Ile), leucine (Leu), phenylalanine (Phe), valine (Val), proline (Pro), glycine (Gly).

[0281] The components that can carry out van der Waals interactions with mutant polypeptide and / or its binding partner include polar or non-polar inorganic compounds that are soluble in the test solution. The test solution is typically an aqueous solution, so these polar or non-polar inorganic compounds are preferably soluble in water. The preferred material for carrying out van der Waals interactions is a polar substance, so that they can carry out dipole-dipole interactions. For example, AIF3 has a polar Al-F bond and is soluble in water (about 0.67g / 100ml water at 20°C). HgCl2 has a polar Hg-Cl bond and can be dissolved in water with an amount of 7.4g / 100ml water at 20°C. PrCl2 has a polar Pr-Cl bond and can be dissolved in water with an amount of about 1g / 100ml water at 20°C.

[0282] Suitable polar compounds capable of van der Waals interactions include alcohols, thiols, ketones, amines, amides, esters, ethers and aldehydes. Suitable examples of these compounds have been described above in connection with hydrogen bonding. Suitable non-polar compounds capable of van der Waals interactions include aromatic hydrocarbons, substituted aromatic hydrocarbons, polyaromatic hydrocarbons, aromatic or non-aromatic heterocycles, cycloalkanes, alkanes, olefins, alkynes.

[0283] The components, hydrophobic components and van der Waals components that form hydrogen bonds can be used to influence the combination of mutant polypeptide and its binding partner in various ways. In one embodiment, hydrogen bonds, hydrophobic interactions and / or van der Waals interactions can form bridges between mutant polypeptide and its binding partner. Such bridges can make mutant polypeptide and binding partner closer to each other, to promote combination and / or mutant polypeptide and / or binding partner are positioned relative to each other in a manner that promotes combination.

[0284] In another embodiment, hydrogen bond formation and / or hydrophobic interactions can increase the likelihood of binding of a mutant polypeptide to its binding partner, for example by causing the polypeptide and binding partner to aggregate or associate with each other in a manner that increases the likelihood of binding. Thus, one or more of these interactions can be used alone or in combination to bring the mutant polypeptide and binding partner closer together, or to arrange the mutant polypeptide and binding partner in a manner that promotes binding, for example by bringing the binding sites closer together or arranging the non-binding portions of the molecule further away from each other, thereby bringing the binding sites closer together.

[0285] In another embodiment, hydrogen bond formation and / or hydrophobic interactions can affect the conformation of the mutant polypeptide and / or its binding partner to provide a conformation that is more conducive to the binding of the mutant polypeptide to its binding partner. Specifically, binding or interaction with one or more amino acids of the mutant polypeptide and / or binding partner may cause one or more conformational changes in the mutant polypeptide or binding partner that are conducive to the mutant polypeptide / binding partner binding reaction.

[0286] The present invention conducts two pairs of tests. One pair of tests is to find mutant polypeptides with the following characteristics: in tests under normal physiological conditions, the activity of the mutant polypeptide is reduced compared with the activity of the parent polypeptide from which the mutant polypeptide is derived under the normal physiological conditions; the second pair of tests is to find mutant polypeptides with the following characteristics: in tests under abnormal conditions, the activity of the mutant polypeptide is enhanced compared with the activity of the parent polypeptide from which the mutant polypeptide is derived under the abnormal conditions.

[0287] The conditions used in two pairs of tests of the present invention can be selected from the concentration of any other component of temperature, pH, osmotic pressure, weight molar osmotic pressure concentration, oxidative stress, electrolyte concentration and test solution or culture medium. Therefore, the specific component of test culture medium can be used with substantially the same concentration in two pairs of tests. In this case, the presence of this component is generally used to simulate the specific environment in humans or animals, such as serum, tumor microenvironment, muscle environment, neural environment or any other environment that may be encountered at the point of application, any other environment that the applied treatment may experience or any other environment that may be encountered at the treatment point. An important aspect of selecting one or more components of these environments to simulate is that it can improve the result of the selection process carried out using two pairs of tests. For example, simulating a specific environment makes it possible to obtain the specific component of this environment in the selection process to treat the various effects of mutant polypeptides to be evaluated. The component of a specific environment can for example change mutant polypeptides or be combined with mutant polypeptides, suppress the activity of mutant polypeptides, inactivate mutant polypeptides, etc.

[0288] In some embodiments, one or more components of the test solution are preferably small compounds, such as hydrogen sulfide, hydrogen sulfide, histidine, histamine, citrate, bicarbonate, lactate and acetate. In one embodiment, the small molecule component is preferably present in the test solution at a concentration of about 100 μM to about 100 mM, or more preferably about 0.5 mM to about 50 mM, or about 1 mM to about 10 mM.

[0289] The concentration of a component in a test solution 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 (e.g., a human). This concentration may be referred to as the normal physiological concentration of the component in the body fluid. In other embodiments, the concentration of a particular component in a test solution may be less than or greater than the concentration of the same component typically present in a naturally occurring body fluid of a mammal (e.g., a human).

[0290] In another embodiment, the components may be present at significantly different concentrations in each pair of tests. In this case, the presence, absence, or concentration of the component becomes the condition being tested, since it is the concentration of the component that is the condition for which there is a difference between the test solution used for testing under normal physiological conditions and the test solution used for testing under abnormal conditions. Thus, the conditionally active polypeptides produced according to this embodiment of the method of the invention will be selected to obtain conditionally active polypeptides whose activity depends at least in part on the concentration of the component.

[0291] In some embodiments, a component may be present in one pair of test solutions but completely absent in another pair of test solutions. For example, the lactate concentration in the test solution for abnormal conditions may be set to a level that mimics the lactate concentration in the tumor microenvironment. Lactate may not be present in a pair of test solutions for normal physiological conditions.

[0292] In one embodiment, the normal physiological condition is a first lactate concentration representative of a normal physiological condition and the abnormal condition is a second lactate concentration representative of an abnormal condition existing at a particular location in the body.

[0293] In another example, glucose may not be present in the test solution for abnormal conditions to simulate the glucose deficiency that may be found in the tumor microenvironment, while in a pair of test solutions for normal physiological conditions, glucose may be set to a level that simulates plasma glucose concentration. This feature can be used to preferentially deliver the conditionally active polypeptide to the location or environment, while the conditionally active polypeptide has no or minimal activity during transport, and activates the conditionally active polypeptide when it reaches the environment where the concentration of the component in the test solution for abnormal conditions is present.

[0294] For example, compared with human serum, the tumor microenvironment generally has a lower glucose concentration and a higher lactate concentration. The normal physiological concentration of glucose in serum is in the range of about 2.5mM to about 10mM. On the other hand, the concentration of glucose in the tumor microenvironment is generally very low, in the range of 0.05mM to 0.5mM. In one embodiment, the test solution for testing under normal physiological conditions has a glucose concentration in the range of about 2.5mM to about 10mM, and the test solution for testing under abnormal conditions has a glucose concentration in the range of about 0.05mM to about 0.5mM. The conditionally active polypeptide thus produced has a higher activity in a low glucose environment (in the tumor microenvironment) than in a higher glucose environment (in normal tissue or blood). The conditionally active polypeptide will work in the tumor microenvironment, but the activity is low during transportation in the bloodstream.

[0295] The normal physiological concentration of lactate in serum is in the range of about 1mM to about 2mM. On the other hand, in the tumor microenvironment, the lactate concentration is generally in the range of 10mM to 20mM. In one embodiment, the test solution for testing under normal physiological conditions has a lactate concentration of about 1mM to about 2mM, and the test solution for testing under abnormal conditions has a lactate concentration of about 10mM to about 20mM. The conditionally active polypeptide thus produced has a higher activity in a high lactate environment (in the tumor microenvironment) than in a lower lactate environment (in normal tissue or blood). Therefore, the conditionally active polypeptide works in the tumor microenvironment, but is less active during transport in the bloodstream.

[0296] Similarly, it is known that painful muscles have higher (abnormal) lactate concentrations than normal muscles. Therefore, when seeking mutant polypeptides that are active in the painful muscle environment, a pair of tests under abnormal conditions can be performed in the presence of a higher concentration of lactate to simulate the painful muscle environment, while a pair of tests under normal physiological conditions can be performed under conditions of lower concentrations of lactate or in the absence of lactate. In this way, mutant polypeptides with enhanced activity in the painful muscle environment with increased lactate concentrations can be selected. Such conditionally active polypeptides can be used as, for example, anti-inflammatory agents.

[0297] In another embodiment, two or more components can be used in two pairs of test solutions. In this type of test, the characteristics of the above two tests can be used to select conditionally active polypeptides. Alternatively, two or more components can be used to increase the selectivity of conditionally active polypeptides. For example, with respect to the tumor microenvironment, a pair of tests under abnormal conditions can be performed in a test medium with a high lactate concentration and a low glucose concentration, while a corresponding pair of tests under normal physiological conditions can be performed in a test medium with a relatively low lactate concentration and a relatively high glucose concentration.

[0298] The present invention contemplates that each component selected from the group consisting of inorganic compounds, ions, and organic molecules can be used alone or in combination to select a conditionally active polypeptide that is more active at one concentration of the component than at a different concentration of the same component.

[0299] Tests that rely on different concentrations of one or more metabolites as a discriminating condition between a normal environment (normal physiological conditions) and an abnormal environment (abnormal conditions) may be particularly useful for selecting conditionally active polypeptides that are more active in the tumor microenvironment than in plasma, because the tumor microenvironment typically has a significant number of metabolites that have concentrations that differ from the concentration of the same metabolites in plasma.

[0300] The literature Kinoshita et al., "Absolute Concentrations of Metabolites in Human Brain Tumors Using In Vitro Proton Magnetic Resonance Spectroscopy," NMR INBIOMEDICINE, vol. 10, pp. 2-12, 1997 compared metabolites in normal brain and brain tumors. The team found that the concentration of N-acetylaspartate in normal brain was 5000-6000 μM, but the concentration in glioblastoma was only 300-400 μM, the concentration in astrocytoma was 1500-2000 μM, and the concentration in anaplastic astrocytoma was 600-1500 μM. In addition, the concentration of inositol in normal brain is 1500-2000 μM, the concentration in glioblastoma is 2500-4000 μM, the concentration in astrocytoma is 2700-4500 μM, and the concentration in anaplastic astrocytoma is 3800-5800 μM. The concentration of phosphatidylethanolamine in normal brain is 900-1200 μM, but the concentration in glioblastoma is 2000-2800 μM, the concentration in astrocytoma is 1170-1370 μM, and the concentration in anaplastic astrocytoma is 1500-2500 μM. Glycine has a concentration of 600-1100 μM in normal brain, but 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 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-acetylaspartate has a concentration of about 85000 μM in the blood, inositol has a concentration of about 21700 μM in the blood, glycine has a concentration of about 220-400 μM in the blood, and alanine has a concentration of about 220-300 μM in the blood.

[0301] Therefore, these metabolites, including at least N-acetylaspartate, inositol, glycine and alanine, can be used in different concentrations in the test solution to select conditionally active polypeptides that are active in brain tumors but inactive in blood or normal brain tissue. For example, a test solution with an N-acetylaspartate concentration of 85000 μM can be used for a pair of tests under normal physiological conditions, and a test solution with an N-acetylaspartate concentration of 350 μM can be used for a pair of tests under abnormal conditions to select conditionally active polypeptides that are active in the tumor microenvironment of glioblastoma but inactive or at least less active in blood or normal brain tissue.

[0302] The literature Mayers et al., "Elevated circulating branched chain amino acids are an early event in pancreatic adenocarcinoma development," Nature Medicine, vol. 20, pp. 1193-1198, 2014 studied the concentrations of various metabolites, including branched-chain amino acids, in the pre-diagnosis plasma of pancreatic patients. It was found that the concentrations of several metabolites in the bloodstream of pancreatic tumor patients were different from the concentrations of the same metabolites in the blood of people who did not have pancreatic cancer. Mayers et al. also found that compared with normal individuals, the branched-chain amino acids in the plasma of pancreatic cancer patients were significantly increased. Branched-chain amino acids present in elevated concentrations include isoleucine, valine, and valine (Table 1 of Mayers et al.). Mayers Figure 1 The concentrations of other metabolites shown in the plasma of pancreatic cancer patients are significantly different from those in normal healthy people. These metabolites include at least acetylglycine, glycine, phenylalanine, tyrosine, 2-aminoadipic acid, taurodeoxycholic acid / taurochenodeoxycholate, aconitate, isocitrate, lactic acid, α-glycerophosphate and uric acid. Therefore, based on the finding that certain metabolites exist at different concentrations in the plasma of pancreatic cancer patients and normal healthy individuals, it can be predicted that the concentrations of these metabolites in the tumor microenvironment of pancreatic cancer will also be different from the concentrations in the pancreatic microenvironment of healthy individuals.

[0303] Therefore, in one embodiment, one or more of these metabolites can be used in a test solution under normal physiological conditions, and its dosage is close to the concentration of these metabolites in the plasma of healthy individuals (i.e., metabolites at normal physiological concentrations). For example, the known normal physiological concentration of isoleucine in the plasma of healthy individuals is about 1.60 ± 0.31 mg / dL, leucine is about 1.91 ± 0.34 mg / dL, and valine is about 2.83 ± 0.34 mg / dL. The test solution under normal physiological conditions can have a normal physiological concentration within the above range of one or more of these branched-chain amino acids. The concentration of the same branched-chain amino acid in the test solution under abnormal conditions is about 5 times, or about 10 times, or about 20 times, or about 50 times, or about 70 times, or about 100 times, or about 150, or about 200 times, or about 500 times the normal physiological concentration of the corresponding branched-chain amino acid in a healthy individual. Since the high concentrations of these branched-chain amino acids found in the plasma tested by Mayers et al. are derived from the tumor microenvironment and diluted in the bloodstream, based on the findings of Mayers et al., it can be reflected that the concentrations of these branched-chain amino acids in the pancreatic tumor microenvironment are expected to be significantly elevated. Similarly, even if the concentration of a particular metabolite in cancer patients is significantly lower than that in normal individuals, testing under abnormal conditions can also reflect the concentration of other metabolites in the blood of pancreatic cancer patients. In this way, screening can simulate the actual environment, thereby ensuring the selection of mutants with the highest activity in this specific environment.

[0304] In some other embodiments, the test solution for normal physiological conditions may include one or more branched-chain amino acids at a concentration that simulates the concentration in the plasma of pancreatic cancer patients, thereby simulating the actual plasma environment of these patients. In such embodiments, the test solution for abnormal conditions may include the same branched-chain amino acids at a concentration that is about 2 times, or about 3 times, or about 4 times, or about 5 times, or about 7 times, or about 8 times, or about 10 times, or about 15 times, or about 20 times, or about 50 times greater than the concentration of the corresponding branched-chain amino acid in the plasma of pancreatic cancer patients, to reflect the fact that these higher concentrations are derived from the tumor microenvironment and that the concentration in the bloodstream represents a dilution of the actual concentration in the tumor microenvironment. Similarly, other metabolites may also have different concentrations in the test solution for normal physiological conditions and the test solution for abnormal conditions to reflect the actual differences expected based on the data collected from the bloodstream. In some cases, a deficiency of a particular metabolite in the bloodstream of pancreatic patients may be noted, in which case a concentration reflecting the concentration measured in the bloodstream may be used in tests under normal physiological conditions, and a lower concentration may be used in tests under abnormal conditions to account for the expectation that the metabolite may be depleted in the tumor microenvironment. Conditionally active polypeptides thus selected using these test solutions will be more active in the pancreatic cancer microenvironment than in the plasma of pancreatic cancer patients.

[0305] In some embodiments, the whole plasma of pancreatic cancer patients can be used in the present invention. For example, in one embodiment, in the test solution for one or both tests in the test under normal physiological conditions and the test under abnormal conditions, one or more components in the plasma of pancreatic cancer patients can be simulated. In an exemplary embodiment, the pH of the test solution of normal physiological conditions is in the range of 7.2-7.6, to which 30wt% of the plasma of pancreatic cancer patients is added, and the pH of the test solution of abnormal conditions is in the range of 6.2-6.8, to which 30wt% of the plasma of pancreatic cancer patients is added. In this embodiment, there is the plasma of pancreatic cancer patients, so that even in the presence of a combination of metabolites found in the blood of pancreatic cancer patients (1) it is ensured that the conditionally active polypeptide is not activated in the blood at a pH of 7.2-7.6, and (2) it is also ensured 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. This will be customized for pancreatic cancer patients.

[0306] In another exemplary embodiment, the pH of the test solution under normal physiological conditions is in the range of 7.2-7.6, to which 30 wt % of plasma from pancreatic cancer patients is added, and the pH of the test solution under abnormal conditions is in the range of 5.5-7.2 or 6.2-6.8, to which no plasma from pancreatic cancer patients is added.

[0307] The same component selected from inorganic compounds, ions and organic molecules can be used for each of the above-mentioned types of tests. For example, in the case of lactate, lactate can be used at substantially the same concentration in the test solution pairs under normal physiological conditions and abnormal conditions. However, normal physiological conditions and abnormal conditions will differ in one or more other aspects (e.g., temperature, pH, concentration of another component, etc.). In various embodiments, lactate can be used as one of the distinguishing factors between normal physiological conditions and abnormal conditions to reflect the fact that the concentration of lactate in abnormal tumor microenvironments is higher than that in normal physiological conditions (non-tumor microenvironments).

[0308] In some embodiments, two or more components are added to two test solutions of normal physiological conditions and abnormal conditions at substantially the same concentration. For example, citrate and bovine serum albumin (BSA) are added to the test solution. In the two test solutions, the concentration of citrate can be about 80 μM, and the concentration of BSA can be about 10-20%. More specifically, the test solution for a pair of tests under normal physiological conditions can have a pH in the range of 7.2-7.6, the concentration of citrate is about 80 μM, and the concentration of BSA is about 10-20%. The test solution for a pair of tests under abnormal conditions can have a pH in the range of 6.2-6.8, the concentration of citrate is about 80 μM, and the concentration of BSA is about 10-20%.

[0309] In one embodiment, human serum can be added to two test solutions of normal physiological conditions and abnormal conditions at substantially the same concentration. Since human serum contains a large amount of inorganic compounds, ions, organic molecules (including polypeptides), the test solution will have a variety of and a large number of components selected from inorganic compounds, ions, organic molecules, and the concentrations of these components in the two test solutions are substantially the same. The test solution can have 5-30% by volume, or 7-25% by volume, or 10-20% by volume, or 10-15% by volume of serum. In some other embodiments, the test solution for normal physiological conditions and the test solution for abnormal conditions do not contain serum. The serum can be human serum, bovine serum albumin, or serum from any other mammal. In some other embodiments, the test solution does not contain serum.

[0310] The test solution for normal physiological conditions and the test solution for abnormal conditions may have different pH. By using bicarbonate, the pH of such an assay solution can be adjusted using the CO2 and O2 levels in the buffer.

[0311] In some other embodiments, at least one of two or more components is added to the test solution of normal physiological conditions and the test solution of abnormal conditions at different concentrations. For example, lactate and bovine serum albumin (BSA) are added to the test solution. The lactate concentration of the test solution of normal physiological conditions and the test solution of abnormal conditions can be different, and BSA can have the same concentration in two test solutions. In the test solution of abnormal conditions, the concentration of lactate can be in the range of 30-50mg / dL, and in the test solution of normal physiological conditions, the concentration of lactate can be in the range of 8-15mg / dL. On the other hand, BSA has the same concentration in two test solutions, such as about 10-20%. In the presence of BSA, the conditionally active polypeptide selected using these test solutions is more active at a high lactate concentration of 30-50mg / dL than at a low lactate concentration of 8-15mg / dL.

[0312] In some embodiments, a test solution can be designed to select conditionally active polypeptides with an activity that depends on two or more conditions. In an exemplary embodiment, a conditionally active polypeptide may have an activity that depends on both pH and lactate. The test solution for selecting such conditionally active polypeptides can be a test solution of normal physiological conditions with a pH of 7.2-7.6, and its lactate concentration is within the range of 8-15 mg / dL. The pH of the test solution of abnormal conditions can be 6.2-6.8, and the concentration of lactate is within the range of 30-50 mg / dL. Optionally, both the test solution of normal physiological conditions and the test solution of abnormal conditions can also contain ions to assist the binding between the mutant polypeptide and its binding partner, thereby increasing the number of hits of the candidate bioactive polypeptide.

[0313] In another exemplary embodiment, conditionally active polypeptides can have an activity that depends on pH, glucose and lactate. The test solution for selecting such conditionally active polypeptides can be a test solution of normal physiological conditions with a pH of 7.2-7.6, with a glucose concentration in the range of 2.5-10mM and a lactate concentration in the range of 8-15mg / dL. The pH of the test solution of abnormal conditions can be 6.2-6.8, with a glucose concentration in the range of 0.05-0.5mM and a lactate concentration in the range of 30-50mg / dL. Optionally, the test solution of normal physiological conditions and the test solution of abnormal conditions can also contain ions to assist the binding between the mutant polypeptide and its binding partner, thereby increasing the number of candidate bioactive polypeptides that bind to the binding partner under the condition of pH 6.2-6.8. Conditionally active polypeptides selected using this test solution 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.

[0314] Two or more components selected from inorganic compounds, ions and organic molecules are used to prepare a test solution for abnormal conditions, which simulates the environment at the position / site (i.e., target site) to which the selected conditionally active polypeptide will be delivered. In some embodiments, at least three components present in the environment at the target site can be added to the test solution, or at least four components present in the environment at the target site can be added to the test solution, or at least five components present in the environment at the target site can be added to the test solution, or at least six components present in the environment at the target site can be added to the test solution.

[0315] In one embodiment, the fluid removed from the target site (where the conditionally active polypeptide will be more active) can be used directly as a test solution for testing under abnormal conditions. For example, synovial fluid can be removed from an individual, preferably an individual with a joint disease that needs treatment. The removed synovial fluid, optionally diluted, can be used as a test solution in a pair of tests under abnormal conditions to select conditionally active polypeptides. By using the removed synovial fluid, optionally diluted, as a test solution for testing under abnormal conditions and a test solution for testing under normal physiological conditions that simulates human plasma, the selected conditionally active polypeptide (e.g., TNF-α) is more active at the joints than at other locations or organs. For example, individuals with inflamed joints (e.g., arthritis) can be treated with TNF-α. However, TNF-α generally has serious side effects that damage other tissues and organs. Conditionally active TNF-α that is more active in synovial fluid but inactive or less active in blood can deliver the activity of TNF-α to the joints while reducing or possibly eliminating the side effects of TNF-α on the rest of the body.

[0316] The development of conditionally active polypeptides with activity that depends on multiple conditions will increase the selectivity of conditionally active polypeptides to target sites in the individual body. Ideally, in other locations where only some of these conditions exist, conditionally active polypeptides are inactive or at least significantly reduced in activity. In one embodiment, conditionally active polypeptides that are active under conditions of pH 6.2-6.8, glucose concentration of 0.05-0.5mM and lactate concentration of 30-50mg / dL can be specifically delivered to the tumor microenvironment because all these conditions exist in the tumor microenvironment. One or two of these conditions may exist in other tissues or organs, but not all three conditions exist, so it is not enough to fully activate the conditionally active polypeptides in other tissues or organs. For example, muscles after exercise can have a low pH within the range of 6.2-6.8. However, there is no other test condition. Therefore, conditionally active polypeptides are inactive or at least less active in muscles after exercise.

[0317] In some embodiments, steps can be taken to confirm that the activity of the conditionally active polypeptide does depend on the conditions for selecting the conditionally active polypeptide. For example, the conditionally active polypeptide is selected to depend on the following three conditions: pH 6.2-6.8, 0.05-0.5mM glucose concentration and 30-50mg / dL lactate concentration. The selected conditionally active polypeptide can then be tested individually under each of the three conditions and in multiple environments with two of the three conditions to confirm that the conditionally active polypeptide is inactive or less active in these tests.

[0318] In some embodiments, some components of serum can be minimized or removed from the test medium on purpose. For example, when screening antibodies, the serum components of the binding or adsorption antibodies can be minimized or removed from the test medium. Such combined antibodies may produce false positives, thereby comprising mutant antibodies that do not have conditional activity and are only bound to the combination of components present in serum under various conditions. Therefore, carefully select test components to minimize or remove the components that may be combined with mutants in the test, and the method can be used to reduce the quantity of non-functional mutants, because the non-functional mutants are combined with components rather than with desired binding partners in the test, and therefore may be unintentionally identified as conditional activity positive. For example, in some embodiments of the mutant polypeptides that screening tends to be combined with the components in human serum, BSA can be used for test solutions to reduce or eliminate the possibility of false positives caused by the combination of mutant polypeptides with the components of human serum. Other similar replacements can also be carried out in specific cases to achieve the same goal.

[0319] In some embodiments, the screening environment simulates the environment near the cell membrane, such as the environment inside the cell membrane, at the cell membrane, or outside the cell membrane, or the environment in the joint. When screening in a cell membrane environment, some factors that may affect binding activity include receptor expression, internalization, antibody drug complex (ADC) titer, etc.

[0320] The test format can be any suitable test known to those skilled in the art. Examples include ELISA, enzyme activity test, in vitro real tissue screening (organs, etc.), tissue sections, whole animals, cell lines, and the use of 3D systems. For example, WO2013 / 040445 describes a test based on suitable cells, US 7,993,271 describes a test based on tissues, US2010 / 0263599 describes a screening method based on whole animals, and US2011 / 0143960 describes a screening method based on 3D systems.

[0321] In some embodiments, the evolution step can produce mutant polypeptides that can have other desired properties in addition to the above-mentioned conditional active characteristics. Suitable other desired properties that can be evolved can include binding activity, expression, humanization, etc. Therefore, the present invention can be used to produce conditionally active polypeptides that also have at least one or more of these other desired properties that are improved.

[0322] In some embodiments, the present invention produces conditionally active polypeptides. In, for example, a second evolution step, the selected conditionally active polypeptides can be further mutated using a mutagenesis technique disclosed herein to improve another property of the selected conditionally active polypeptide, such as binding activity, expression, humanization, etc. After the second evolution step, the mutant polypeptides can be screened for conditional activity and improved properties.

[0323] In some embodiments, after evolving the parent polypeptide to produce a mutant polypeptide, a first conditionally active polypeptide is selected that exhibits both of the following properties: (a) a decrease in the first activity compared to the parent polypeptide in a test under normal physiological conditions, and (b) an increase in the first activity compared to the parent polypeptide in a test under abnormal conditions. The first conditionally active polypeptide can then be further subjected to one or more additional evolution, expression, and selection steps to select at least a second conditionally active polypeptide that (1) exhibits both of the following properties: (a) a decrease in the second activity compared to the parent polypeptide in a test under normal physiological conditions, and (b) an increase in the second activity compared to the parent polypeptide in a test 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 a higher first activity and a lower second activity under abnormal conditions than the parent polypeptide, and a lower first activity and a lower second activity under normal physiological conditions than the parent polypeptide.

[0324] In certain embodiments, the present invention is directed to producing conditionally active polypeptides having a greater ratio of activity under abnormal conditions (or second conditions) to activity under normal physiological conditions (or first conditions) (e.g., greater selectivity between abnormal conditions and normal physiological conditions). The ratio or selectivity of activity under abnormal conditions (or second conditions) to activity under normal physiological conditions (or first conditions) 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.

[0325] In one embodiment, the conditionally active polypeptide is an antibody that has 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 a tumor site, wherein the conditionally active polypeptide is active at the tumor site (tumor microenvironment) and has significantly less activity or no activity at non-tumor sites (normal physiological conditions).

[0326] In one embodiment, the conditionally active polypeptide is an antibody intended to be conjugated to another agent (e.g., those disclosed elsewhere herein). The activity of the conditionally active antibody under abnormal conditions is higher than the activity under normal physiological conditions. For example, the ratio of the activity of the conditionally active antibody to be conjugated to another agent under abnormal conditions to the activity under normal physiological conditions is 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 is particularly important when the conjugated agent is, for example, toxic or radioactive, because it is desired that the conjugated agent be concentrated at the diseased site or treatment site (where the abnormal condition exists).

[0327] G. Production of conditionally active peptides

[0328] Selected conditionally active polypeptides having reversible or irreversible activity can be generated for therapeutic, diagnostic, research, and related purposes, and / or these conditionally active polypeptides can be subjected to one or more additional cycles of evolution and selection.

[0329] Conditionally active polypeptides can be produced using polypeptide-expressing cell production hosts or organisms. In order to make the production process more efficient, the DNA encoding the conditionally active polypeptide can be codon-optimized for the cell production host or organism. Codon optimization has been described previously, for example, Narum et al., "Codon optimization of gene fragment sensing Plasmodium falciparum merzoite proteins enhances DNA vaccine protein expression and immunogenicity in mice," Infect. Immun. 2001 December, 69(12):7250-3, which describes codon optimization in a mouse expression 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, which 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 (using artificially synthesized genes for efficient expression of human phospholipid transfer protein recombinant and mutagenesis: evidence for the C-terminal membrane binding domain) "Biochemistry 2000 Dec. 19, 39 (50): 15399-409, which describes codon optimization in Escherichia coli; Humphreys et al.,"High-level periplasmic expression in Escherichia coli using aeukaryotic signal peptide:importance of codon usage at the 5'end of the coding sequence",Protein Expr. Purif. 2000 Nov. 20(2):252-64, which describes how codon usage affects protein secretion in Escherichia coli. .

[0330] Cell production host can be selected from CHO, HEK293, IM9, DS-1, THP-1, Hep G2, COS, NIH 3T3, C33a, A549, A375, SK-MEL-28, DU145, PC-3, HCT116, MiaPACA-2, ACHN, Jurkat, MM1, ovarian cancer cells (Ovcar) 3, HT 1080, pancreatic cancer cells -1 (Panc-1), U266, 769P, BT-474, Caco-2, HCC1954, MDA-MB-468, LnCAP, NRK-49F and SP2 / 0 mammalian cell lines; and mouse spleen cells and rabbit PBMC. In one embodiment, the mammalian system is selected from CHO or HEK293 cell lines. In a specific aspect, the mammalian system is a CHO-S cell line. In another embodiment, the mammalian system is a HEK293 cell line.

[0331] In some embodiments, the cell production host is a yeast cell system, such as S. cerevisiae yeast cells or Pichia yeast cells. In some embodiments, the cell production host is a prokaryotic cell such as Escherichia coli (Owens RJ and Young RJ, J. Immunol. Meth., vol. 168, p. 149, 1994; Johnson S and Bird RE, Methods Enzymol., vol. 203, p. 88, 1991). Conditionally active polypeptides can also be produced in plants (Firek et al. Plant MoI. Biol., vol. 23, p. 861, 1993).

[0332] Conditionally active polypeptides can also be produced by synthetic methods using chemical methods known in the art. See, for example, Caruthers, "New chemical methods for synthesizing polynucleotides," Nucleic Acids Res. Symp. Ser. 215-223, 1980; Horn, "Synthesis of oligonucleotides on cellulose. Part II: design and synthetic strategy to the synthesis of 22 oligodeoxynucleotides coding for Gastric Inhibitory Polypeptide (GIP), " Nucleic Acids Res. Symp. Ser. 225-232, 1980; Banga, AK, Therapeutic Peptides and Proteins, Formulation, Processing and Delivery Systems, Technomic Publishing Co., Lancaster, Pa, 1995. For example, peptide synthesis can be performed 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); and automated synthesis can be achieved, for example, using an ABI 43IA peptide synthesizer (Perkin Elmer) according to the instructions provided by the manufacturer.

[0333] Solid phase chemical peptide synthesis methods have been known in the art since the early 1960s (Merrifield, RB, "Solid-phase synthesis I. The synthesis of atetrapeptide", J. Am. Chem. Soc, 85:2149-2154, 1963) (see also Stewart, JM and Young, JD, Solid Phase Peptide Synthesis, 2nd Ed., Pierce Chemical Co., Rockford, 111., pp. 11-12)) and have recently been used in commercially available laboratory peptide design and synthesis kits (Cambridge Research Institute of Biochemistry). Such commercially available laboratory kits generally utilize the teachings of HM Gysen et al., "Use of peptide synthesis to probe viral antigens for epitopes to a resolution of a single amino acid," Proc. Natl. Acad. Sci., USA, 81: 3998, 1984, and provide for the synthesis of peptides at the tips of a plurality of "rods" or "needles," all of which are connected to a single plate. When such a system is used, the plate to which the rods or needles are connected is inverted and inserted into a second plate of a corresponding well or reservoir, which contains a solution for attaching or anchoring the appropriate amino acids to the tips of the needles or rods. By repeating this process step, i.e., inverting the tips of the rods and needles and inserting them into a suitable solution, the amino acids are combined into the desired peptides. In addition, there are a large number of FMOC peptide synthesis systems available. For example, Applied Biosystems, Inc. 431A can be used. TM Automated peptide synthesizers perform the assembly of polypeptides or fragments on a solid support. Such equipment can make it easy to obtain the peptides of the invention by direct synthesis or by synthesizing a series of fragments that can be linked using other known techniques.

[0334] Conditionally active polypeptides can also be glycosylated. Glycosylation can be added post-translationally chemically or by cellular biosynthetic mechanisms, wherein the latter includes the use of known glycosylation motifs that can be native to the sequence or can be added as a peptide or in a nucleic acid encoding sequence. Glycosylation can be O-linked or N-linked.

[0335] Conditionally active polypeptides include all "simulations" and "peptidomimetics" forms. The terms "simulations" and "peptidomimetics" refer to synthetic compounds having substantially the same structure and / or functional properties as the polypeptide of the present invention. Simulations can be composed entirely of synthetic, non-natural amino acid analogs, or chimeric molecules of partially natural peptide amino acids and partially non-natural amino acid analogs. Simulations can also include any amount of such substitutions as long as the conservative substitutions of natural amino acids do not substantially change the structure and / or activity of the mimics. With regard to polypeptides of the present invention as conservative variants, routine experiments will determine whether the mimics are within the scope of the present invention, i.e., determine whether their structure and / or function have not been substantially changed.

[0336] The polypeptide mimetic compositions of the present invention may comprise any combination of non-natural structural components. On the other hand, the mimetic compositions of the present invention include one or all of the following three structural groups: a) a residue linking group other than the natural amide bond ("peptide bond") connection; b) a non-natural residue that replaces naturally occurring amino acid residues; or c) a residue that induces secondary structure simulation, i.e., a residue that induces or stabilizes secondary structure (e.g., β turn, γ turn, β fold, α helical conformation, etc.). For example, when all or some residues of the polypeptide of the present invention are connected by chemical means other than the natural peptide bond, it may be referred to as a mimetic. Individual peptide mimetic residues may be connected by peptide bonds, other chemical bonds, or coupling methods, such as glutaraldehyde, N-hydroxysuccinimide esters, bifunctional maleimide, N, N'-dicyclohexylcarbodiimide (DCC) or N, N'-diisopropylcarbodiimide (DIC). Linking groups that can be used as alternatives to the traditional amide bond ("peptide bond") include, for example, ketomethylene (e.g., -C(=O)-CH2- instead of -C(=O)-NH-), aminomethylene (CH2-NH), ethylene, olefin (CH=CH), ether (CH2-O), thioether (CH2-S), tetrazole (CN4-), thiazole, retroamide, thioamide, or ester (see, e.g., Spatola (1983) in Chemistry and Biochemistry of Amino Acids, Peptides and proteins, Vol. 7, pp 267-357, "Peptide Backbone Modifications," Marcell Dekker, NY).

[0337] When a polypeptide of the invention comprises all or some non-natural residues in place of naturally occurring amino acid residues, the polypeptide of the invention may also be referred to as a mimetic. Non-natural residues are well described in the scientific and patent literature; several exemplary non-natural compositions and guidelines for use as mimetics of natural amino acid residues are described below. Mimetics of aromatic amino acids can be generated by substitution with, for example, the following: D- or L-naphthylalanine; D- or L-phenylglycine; D- or L-2-thienylalanine; D- or L-1, -2, 3- or 4-pyrenoylalanine; D- or L-3-thienylalanine; D- or L-(2-pyridyl)-alanine; D- or L-(3-pyridyl)-alanine; D- or L-(2-pyrazinyl)-alanine; D- or L-(4-isopropyl)-phenylglycine. The aromatic rings of non-natural amino acids include, for example, thiazolyl, thienyl, pyrazolyl, benzimidazolyl, naphthyl, furanyl, pyrrolyl, and pyridinyl aromatic rings.

[0338] The analogue of acidic amino acid can be by replacing with non-carboxylated amino acid and keeping negative charge at the same time, by replacing with (phosphono) alanine, or by sulphated threonine replacement generation.Carboxyl side group (for example, aspartyl or glutamyl) can also be by reacting with carbodiimide (R'~N—C--N--R') such as 1-cyclohexyl-3-(2-morpholinyl-(4-ethyl)) carbodiimide or 1-ethyl-3 (4-nitrogen cation-4,4-dimethylpentyl) carbodiimide to selectively modify.By reacting with ammonium ion, aspartyl or glutamyl can also be converted into asparaginyl and glutaminyl residue.Alkalogenic analogue can be produced by replacing with following amino acid for example (except lysine and arginine): ornithine, citrulline or (guanidino)-acetic acid or (guanidino) alkyl-acetic acid replacement, wherein alkyl is defined as above. Nitrile derivatives (e.g., containing a CN- moiety replacing COOH) can be substituted to obtain asparagine or glutamine. Asparaginyl and glutaminyl residues can be deamidated to obtain the corresponding asparagine or glutamyl residues. Arginine residue mimetics can be produced by reacting arginyl with, for example, one or more conventional reagents (including, for example, phenylglyoxal, 2,3-dione, 1,2-cyclohexanedione or ninhydrin), the reaction preferably being carried out under alkaline conditions. Tyrosine residue mimetics can be produced by reacting tyrosyl with, for example, aromatic diazo compounds or tetranitromethane. N-acetylimidazole and tetranitromethane can be used to form O-acetyl tyrosyl substances and 3-nitro derivatives, respectively. Cysteinyl residue mimetics are produced by reacting cysteinyl residues with, for example, α-haloacetates (such as 2-chloroacetic acid or chloroacetamide) and corresponding amines; to obtain carboxymethyl or carboxyaminomethyl derivatives. Cysteine ​​residue mimetics can also be produced by reacting cysteinyl residues with, for example, bromotrifluoroacetone, α-bromo-β-(5-imidazolyl) propionic acid, chloroacetyl phosphate, N-alkyl maleimide, 3-nitro-2-pyridyl disulfide, methyl 2-pyridyl disulfide, p-chloromercuryl benzoate, 2-chloromercuryl-4-nitrophenol; or chloro-7-nitrobenzo-oxa-1,3-diazole. Lysine mimetics can be produced (and amino terminal residues altered) by reacting lysyl with, for example, succinic anhydride or other carboxylic anhydrides. Lysine and other α-amino-containing residue mimetics can also be produced by reacting with imidoesters such as pyridine imidomethyl ester, pyridoxal phosphate, pyridoxal, chloroborohydride, trinitrobenzenesulfonic acid, O-methylisourea, 2,4-pentanedione, and transamidase-catalyzed reactions with glyoxylate. Methionine mimetics can be produced by reaction with, for example, methionine sulfoxide. Proline mimetics include, for example, pipecolic acid, thiazolidine carboxylic acid, 3- or 4-hydroxyproline, dehydroproline, 3- or 4-methylproline or 3,3-dimethylproline. Histidine residue mimetics can be produced by reaction of histidyl with, for example, diethyl orthocarbonate or p-bromophenacyl bromide.Other mimetics include, for example, those produced by hydroxylation of proline and lysine; those produced by phosphorylation of the hydroxyl groups of seryl or threonyl residues; those produced by methylation of the α-amino groups of lysine, arginine and histidine; those produced by acetylation of the N-terminal amine; those produced by methylation of backbone amide residues or substitution with N-methyl amino acids; or those produced by amidation of the C-terminal carboxyl group.

[0339] The residues (e.g., amino acids) of the polypeptides of the present invention may also be replaced by amino acids (or peptidomimetic residues) of opposite chirality. Thus, any amino acid naturally present in the L-configuration (which may also be referred to as R or S, depending on the structure of the chemical entity) may be replaced by an amino acid of the same chemical structure type but of opposite chirality or a peptidomimetic of opposite chirality, referred to as a D-amino acid, which may also be referred to as an R- or S-form.

[0340] The invention 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 may occur anywhere in the polypeptide, including the peptide backbone, amino acid side chains, and the amino or carboxyl termini. It should be understood that the same type of modification may be present at several sites in a given polypeptide to the same or varying degrees. A given polypeptide may also have many 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 a phosphatidylinositol, cross-linking cyclization, disulfide bond formation, demethylation, formation of covalent cross-links, formation of cysteine, formation of pyroglutamate, formylation, gamma-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 protein, such as arginylation. See, e.g., Creighton, TE, as—Structure and Molecular Properties 2nd Ed., WH Freeman and Company, New York (1993); Posttranslational Covalent Modification of proteins, BC Johnson, Ed., Academic Press, New York, pp. 1-12 (1983).

[0341] H. Engineering of conditionally active antibodies

[0342] The conditionally active antibodies of the present invention can be engineered by one or more antibody engineering techniques described herein. Non-limiting examples of antibody engineering techniques include antibody conjugation, engineered multispecific antibodies, engineered bispecific conditionally active antibodies against immune effector cell surface antigens and target antigens, and engineered antibody Fc regions.

[0343] Suitable methods for conjugating conditionally active antibodies are described in WO2015 / 175375. In one embodiment, the ratio of the activity of the conditionally active antibodies for conjugation under abnormal conditions to the activity under normal physiological conditions disclosed herein is preferably 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.

[0344] In some embodiments, conditionally active antibodies can be conjugated to the Fc region of these antibodies. The above-mentioned conjugated molecules, compounds or drugs can be conjugated to the Fc region, as described in U.S. Patent No. 8,362,210. For example, the Fc region can be conjugated to a cytokine or toxin to be delivered to the site where the conditionally active antibody shows preferential activity. Methods for conjugating polypeptides to the Fc region of antibodies are known in the art. See, e.g., U.S. Pat. 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. al., Proc.Natl.Acad.Sci.USA, vol.88, pp.10535-10539, 1991; Traunecker et al., Nature, vol.331, pp.84-86, 1988; Zheng et al., J. Immunol., vol.154, pp.5590-5600, 1995; and ViIet al., Proc. Natl. Acad. Sci. USA, vol.89, pp.11337-11341, 1992.

[0345] In some embodiments, the conditionally active antibody can be covalently linked to the conjugated reagent through an intermediate joint having at least two reactive groups, one of which reacts with the conditionally active antibody and the other reacts with the conjugated reagent. A joint that can include any compatible organic compound can be selected so that the reaction with the conditionally active antibody or with the conjugated reagent does not adversely affect the reactivity and / or selectivity of the conditionally active antibody. In addition, the connection of the joint to the conjugated reagent cannot destroy the activity of the conjugated reagent. The ratio of the anticancer agent molecule conjugated to the conditionally active antibody to the conditionally active polypeptide molecule reaches 3:1, or 4:1, or 5:1, or 6:1. In one example, the ratio of the anticancer agent to the conditionally active polypeptide is about 4:1.

[0346] Suitable linkers for oxidized conditionally active antibodies include linkers containing groups selected from primary amines, secondary amines, hydrazines, hydrazides, hydroxylamines, phenylhydrazides, semicarbazides, and thiosemicarbazide groups. Suitable linkers for reduced conditionally active antibodies include linkers having certain reactive groups that can react with the sulfhydryl groups of the reduced conditionally active antibodies. Such reactive groups include, but are not limited to, reactive haloalkyl groups (including, for example, haloacetyl groups), p-mercurybenzoate groups, and groups capable of Michael-type addition reactions (including, for example, maleimides and the group types described in Mitra and Lawton, J. Amer. Chem. Soc. Vol. 101, pp. 3097-3110, 1979).

[0347] Suitable methods for engineering multispecific conditionally active antibodies have been described in WO2015 / 175375.

[0348] Conditionally active antibodies can be engineered to produce bispecific conditionally active antibodies against immune effector cell surface antigens and target antigens. The bispecific conditionally active antibodies of the present invention can attract immune effector cells to disease sites where target antigens are present. Bispecific conditionally active antibodies are antibodies that can specifically bind to two different antigens: immune effector cell surface antigens and target antigens. Bispecific antibodies can be full-length antibodies that include two arms, one arm binds to immune effector cell surface antigens, and the other arm binds to target antigens. Bispecific antibodies can be antibodies that only include heavy chain variable domains (V H ) and the light chain variable domain (V L ). In one embodiment, the antibody fragment comprises at least two V H V L Unit: one for binding to the surface antigen of immune effector cells and the other arm for binding to the target antigen. In another embodiment, the antibody fragment comprises at least two single variable domains (V H or V L): one for binding to an immune effector cell surface antigen, and the other for binding to a target antigen. In some embodiments, the bispecific conditionally active antibody comprises two scFvs: one that binds to an immune effector cell surface antigen, and the other that binds to a target antigen.

[0349] The attracted immune effector cells have binding activity to the target antigens on the immune effector cells and the diseased cells or diseased tissues, which can attract the immune effector cells to the virus-stricken cells or diseased tissues containing the target antigens. The attracted immune effector cells will then attack the diseased cells or diseased tissues, thereby helping to cure the disease, because the immune effector cells can inhibit or even destroy the diseased cells or diseased tissues. 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.

[0350] Bispecific conditionally active antibodies have two binding activities, one for immune effector cell surface antigens and the other for target antigens. In one embodiment, both binding activities are conditional, which means that the binding activity of the bispecific conditionally active antibody to immune effector cell surface antigens and target antigens is lower than the binding activity of the parent antibody under normal physiological conditions, and higher than the binding activity of the parent antibody under abnormal conditions. In one embodiment, only one of the two binding activities is conditional, which means that the binding activity of the bispecific conditionally active antibody to immune effector cell surface antigens or the binding activity of the bispecific conditionally active antibody to the target antigen is conditional. In this case, the binding activity of the bispecific conditionally active antibody to immune effector cell surface antigens or the binding activity of the bispecific conditionally active antibody to the target antigen is lower than the corresponding activity of the parent antibody under normal physiological conditions, and higher than the corresponding activity of the parent antibody under abnormal conditions.

[0351] The two arms (e.g., two V H V L As is well known in the art, the smallest antibody fragment containing a complete antigen binding site has a heavy chain variable domain and a light chain variable domain (V H and V L This structure corresponds to that found in natural antibodies, in which the three complementarity determining regions (CDRs) of each variable domain interact to define V H -V LThe antigen binding site on the surface of the dimer. A total of six CDRs give the antibody antigen binding specificity. The framework (FR) flanking the CDRs has a tertiary structure that is essentially conserved in natural immunoglobulins of species such as humans and mice. These FRs serve to keep the CDRs in their proper orientation. The constant domains are not required for binding function, but help stabilize the V H -V L Interaction. Even 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 the binding affinity is usually lower than that of 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 pp. 1327-1337, 1972). Therefore, the domains of the binding site of a bispecific conditionally active antibody can be constructed as a pair of V domains of different immunoglobulins. H -V L 、V H -V H or V L -V L Structural domain.

[0352] In some embodiments, the bispecific conditionally active antibody can be constructed as a continuous polypeptide chain by recombinant DNA technology, for example, in the following manner: a nucleic acid molecule encoding the bispecific conditionally active antibody is expressed to construct a continuous polypeptide chain (see, for example, Mack et al., "A small bispecific antibody construct expressed as a functional single-chain molecule with high tumor cell cytotoxicity," Proc. Natl. Acad. Sci. USA, vol. 92, pp. 7021-7025, 2005). H and V L The order of the domains is not critical to the present invention, as long as the arrangement V H and V L The domain structure enables the antigen binding site to fold appropriately to form one binding site for the surface antigen of the immune effector cell and one binding site for the target antigen.

[0353] Some of the techniques described herein for engineering multispecific conditionally active antibodies can be used to generate bispecific conditionally active antibodies directed against an immune effector cell surface antigen and a target antigen.

[0354] Bispecific antibodies can be constructed as a single polypeptide chain, such as described in WO99 / 54440; Mack, J. Immunol. (1997), 158, 3965-3970; Mack, PNAS, (1995), 92, 7021-7025; Kufer, Cancer Immunol. Immunother., (1997), 45, 193-197; Loftier, Blood, (2000), 95, 6, 2098-2103; Bruhl, J. Immunol., (2001), 166, 2420-2426. A particularly preferred configuration of a bispecific antibody is a polypeptide construct in which V H Area and V L The regions are connected to each other through the linker domain. H Area and V L The order of the regions is not important. In one embodiment, a single polypeptide chain is constructed as V H1 -Linker domain-V L1 -Linker domain-V H2 -Linker domain-V L2 In another embodiment, a single polypeptide chain is constructed as V L1 -Linker domain-V H1 -Linker domain-V L2 -Linker domain-V H2 In another embodiment, a single polypeptide chain is constructed as V H1 -Linker domain-V H2 -Linker domain-V L1 -Linker domain-V L2 In another embodiment, a single polypeptide chain is constructed as V H1 -Linker domain-V L2 -Linker domain-V L1 -Linker domain-V H2 A single polypeptide chain can fold into two arms, each of which is capable of binding to an antigen on the surface of an immune effector cell or a target antigen.

[0355] The linker domain in a bispecific conditionally active antibody is long enough to allow these V H Domain and V LThe peptide fragment of intermolecular connection by the structural domain. The design of the linker suitable for this purpose is described in the prior art, for example EP623679B1, U.S. Patent No. 5,258,498, EP 573551B1 and U.S. Patent No. 5,525,491. The linker domain is preferably a hydrophilic flexible linker of 1-25 amino acids, selected from glycine, serine and / or glycine / serine. In one embodiment, the linker domain is a 15 amino acid linker sequence (Gly4Ser)3.

[0356] Other linker domains include oligomerization domains. The oligomerization domain can promote the binding of two or more V H Domain and V L The combination of domains folds into two arms, each of which can bind to an immune effector cell surface antigen or a target antigen. Non-limiting examples of oligomerization domains include leucine zippers (such as jun-fos, GCN4, E / EBP; Kostelny, J. Immunol. 148 (1992), 1547-1553; Zeng, Proc. Natl. Acad. Set 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 a tetramerization domain such as GCN4-LI (Zerangue, Proc. Natl. Acad. Sci. 97 (2000), 3591-3595).

[0357] In some embodiments, the knob-in-hole technique can be used to stabilize the folding of a single polypeptide chain bispecific conditional antibody. Ridgway et al. ("'Knobs-into-holes' engineering of antibody CH3domains for heavy chain heterodimerization," Protein Eng. 1996 Jul; 9(7): 617-21) described the knob-in-hole technique. This method has been used for the stacking of amino acid side chains between adjacent α-helices, where the side chains of the residues in the α-helix are represented as spaced knobs that are alternately arranged with a hole on the surface of a cylinder, and the hole may accommodate the knob of an adjacent α-helix (O'Shea et al., (1991) Science, 254, 539-544).

[0358] The immune effector cell surface antigen should be specific to one or a class of immune effector cells.Many immune effector cell surface antigens are known. Natural killer cells have surface antigens, including CD56, CD8, CD16, KIR family receptors, NKp46, NKp30, CD244 (2B4), CD161, CD2, CD7, CD3, and killer cell immunoglobulin-like receptors (Angelis et al., “Expansion of CD56-negative, CD16-positive, KIR-expressing natural killer cells after T cell-depleted haploidentical hematopoietic stem cell transplantation,” Acta Haematol. 2011; 126(1):13-20; Dalle et al., “Characterization of Cord Blood Natural Killer Cells: Implications for Transplantation and Neonatal Infections,” Pediatric Research (2005) 57, 649–655; Agarwal et al., “Roles and Mechanism of Natural Killer Cells in Clinical and Experimental Transplantation,” Expert Rev Clin Immunol. 2008;4(1):79-91).

[0359] Macrophages have surface antigens including CD11b, F4 / 80, CD68, CSF1R, MAC2, CD11c, LY6G, LY6C, IL-4Rα, CD163, CD14, CD11b, F4 / 80 (mouse) / EMR1 (human), CD68 and MAC-1 / MAC-3, PECAM-1 (CD31), CD62, CD64, CD45, Yml, 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, ...Macrophage receptors and immune recognition," Annu Rev Immunol 2005; 23: 901-44; Pilling, et al., "Macrophage receptors and immune recognition," Annu Rev Immunol 2005; 23: 901-44; Pilling, et al., "Macrophage receptors and immune recognition," Annu Rev Immunol 20 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).

[0360] Lymphokine-activated killer (LAK) cells have surface antigens, including T3, T4, T11, T8, TII, Leu7, and Leul1 (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. 3977-982).

[0361] T cells, especially 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 TAND B LYMPHOCYTES," JOURNAL OF EXPERIMENTAL MEDICINE, VOLUME 136, 1972, 207-215; Mingari ... al., "Surface markers of human Tlymphocytes," Ric ClinLab. 1982Jul-Sep; 12(3):439-448).

[0362] Bispecific conditionally active antibodies can bring immune effector cells to cells or tissues where target antigens are present after binding to immune effector cells, preferably target antigens are present on the surface. Once bispecific conditionally active antibodies (and immune effector cells) are combined with target antigens, immune effector cells can attack diseased cells or diseased tissues. Immune effector cells such as natural killer cells, macrophages, LAK cells, T cells (cytotoxicity) can kill and / or destroy diseased cells or tissues, for example, destroy tumor tissue.

[0363] Diseased cells or diseased tissues can be selected from cancer, inflammatory diseases, neuronal diseases, diabetes, cardiovascular diseases or infectious diseases. Examples of target antigens include antigens expressed by various immune cells, cancers, sarcomas, lymphomas, leukemias, germ cell tumors, blastomas and cells associated with various blood diseases, autoimmune diseases and / or inflammatory diseases.

[0364] Cancer-specific target antigens that can be targeted by bispecific conditionally active antibodies include 4-IBB, 5T4, adenocarcinoma antigen, α-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, CD44 v6, CD51, CD52, CD56, CD74, CD80, CEA, CNT0888, CTLA-4, DR5, EGFR, EpCAM, CD3, FAP, fibronectin superdomain B, folate receptor 1, GD2, GD3 ganglioside, glycoprotein 75, GPNMB, HER2 / neu, HGF, human scatter factor receptor kinase, IGF-1 receptor, IGF-1, 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-IC, 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.

[0365] The types of cancers treated with the genetically engineered cytotoxic cells or pharmaceutical compositions of the present invention include carcinomas, germ cell tumors and sarcomas, as well as certain leukemias or lymphoid malignancies, benign and malignant tumors, and malignant tumors, such as sarcomas, carcinomas, and melanomas. Cancers may be non-solid tumors (such as hematological tumors) or solid tumors. Also included are adult tumors / cancers and pediatric tumors / cancers.

[0366] Hematological cancer is a cancer of the blood or bone marrow. Examples of hematological (or hematological) cancers include leukemias, including acute leukemias (e.g., acute lymphocytic leukemia, acute myeloid leukemia, acute myeloid leukemia, and myeloblastic leukemia, promyelocytic leukemia, myelomonocytic leukemia, monocytic leukemia, and erythroleukemia), chronic leukemias (e.g., chronic myelogenous (granulocytic) leukemia, chronic myeloid leukemia, and chronic lymphocytic leukemia), polycythemia vera, lymphoma, Hodgkin's disease, non-Hodgkin's lymphoma (indolent and advanced forms), multiple myeloma, Waldenstrom's macroglobulinemia, heavy chain disease, myelodysplastic syndrome, hairy cell leukemia, and myelodysplasia.

[0367] Solid tumors are abnormal masses of tissue that usually do not contain cysts or areas of fluid. Solid tumors can be benign or malignant. Different types of solid tumors (such as sarcomas, carcinomas, and lymphomas) are named after the type of cells that form the solid tumor. Examples of solid tumors that may be treated include sarcomas and carcinomas, including 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, bile duct carcinoma, choriocarcinoma, Wilms' tumor, cervical cancer, testicular tumors, seminoma, bladder cancer, melanoma, and CNS tumors (e.g., gliomas (such as brain stem gliomas and mixed gliomas), glioblastomas (also known as glioblastoma multiforme) multiforme)), astrocytoma, CNS lymphoma, germ cell tumor, medulloblastoma, schwannoma, ependymoma, pinealoma, hemangioblastoma, acoustic neuroma, oligodendroglioma, meningioma, neuroblastoma, retinoblastoma, and brain metastasis).

[0368] Target antigens that can be targeted by bispecific conditionally active antibodies and are specific to inflammatory diseases include AOC3 (VAP-1), CAM-3001, CCL11 (eotaxin-1), CD125, CD147 (basic immunoglobulin), CD154 (CD40L), CD2, CD20, CD23 (IgE receptor), CD25 (IL-2 receptor chain), CD3, CD4, CD5, IFN-α, IFN-γ, IgE, IgE Fc region, IL-1, IL-12, IL-23, IL-13, IL-17, IL-17A, IL-22, IL-4, IL-5, IL-5, IL-6, IL-6 receptor, integrin α4, integrin α4β7, Lama glama, LFA-1 (CD1 la), MEDI-528, myostatin, OX-40, rhuMAb β7, sclerostin, SOST, TGFβ1, TNF-α, or VEGF-A.

[0369] Target antigens specific to neuronal disorders that can be targeted by the bispecific conditionally active antibodies of the present invention include one or more of amyloid beta 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), fibrin II, β chain, ITGB2 (CD18) and sphingosine-1-phosphate.

[0370] 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, aggregation 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.

[0371] Other examples of target antigens include surface proteins that are present on cancer cells in a characteristic or amplified manner, such as the IL-14 receptor, CD19, CD20, and CD40 of B-cell lymphomas, LewisY and CEA antigens of various cancers, Tag72 antigens of breast and colorectal cancers, EGF-R of lung cancer, folate binding protein and HER-2 protein that are often amplified in human breast cancer and ovarian cancer, or viral proteins, such as the gp120 and gp41 envelope proteins of human immunodeficiency virus (HIV), envelope proteins of hepatitis B and C viruses, glycoprotein B and other envelope glycoproteins of human cytomegalovirus, and envelope proteins from methyl viruses such as Kaposi's sarcoma-associated herpes virus. Other possible target antigens include CD4, where the ligand is the HIV gp120 envelope glycoprotein, and other viral receptors, such as ICAM, which is a receptor for human rhinovirus, and related receptor molecules for poliovirus.

[0372] Human immunodeficiency virus (HIV) cannot enter human cells unless it first binds to two key molecules on the cell surface, CD4 and a co-receptor. The first co-receptor recognized was CCR5, and later in the life cycle of the virus, another chemokine receptor CXCR4 became a co-receptor for HIV-1 (D'Souza, Nature Med.2, 1293 (1996); Premack, Nature Med.2, 1174; Fauci, Nature 384, 529 (1996)). HIV-1 strains that carry out most of the virus transmission through sexual contact are called M-tropic viruses. These HIV-1 strains (also called non-syncytial induction (NSI) primary viruses) can be found in primary CD4 + Replicate in T cells and macrophages and use the chemokine receptor CCR5 (and less commonly CCR3) as its co-receptor. T-tropical viruses (sometimes referred to as syncytium-inducing (SI) primary viruses) can also replicate in primary CD4+T cells, but can also infect established CD4+T cell lines in vitro, and they achieve the above functions through the chemokine receptor CXCR4 (fusion viral protein). At least under certain in vitro conditions, many of these T-tropical virus strains can use CCR5 in addition to CXCR4, and some can enter macrophages through CCR5 (D'Souza, Nature Med.2, 1293 (1996); Premack, Nature Med.2, 1174; Fauci, Nature 384, 529 (1996)). Since M-tropical HIV-1 strains are involved in about 90% of HIV sexual transmission, CCR5 is the main co-receptor of the patient's virus.

[0373] The number and identity of co-receptor molecules on target cells and the ability of HIV-1 strains to enter cells via different co-receptors appear to be key determinants of disease progression. High expression of CCR3 and CCR5 was also observed in lymph node T cells and B cells derived from Hodgkin's disease patients. Type I diabetes is considered to be a T cell-mediated autoimmune disease. In related animal models, the expression of CCR5 receptors in the pancreas is associated with the progression of type I diabetes (Cameron (2000) J.Immunol.165,1102-1110). In one embodiment, bispecific conditionally active antibodies bind to CCR5 as a target antigen, which can be used to inhibit HIV infection of host cells and slow down the progression of other diseases.

[0374] 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). Therefore, a bispecific conditionally active antibody preferably comprises, for example, a V or V-binding region of an antibody that is specific for CCR5 (in particular, human CCR5). L Domain and V H domain (i.e., the second domain from Ig) and the V domain of an antibody specific for the CD3 antigen on T cells H Domain and V L Structural domain.

[0375] In another embodiment, the present invention provides a bispecific conditionally active antibody with CD3 on T cells and CD19 as target antigens. It has been shown that CD19 is a very useful medical target. CD19 is expressed in the entire B cell lineage from precursor B cells to mature B cells, and is uniformly expressed on all lymphoma cells, but is not present in stem cells (Haagen, Clin Exp Immunol 90 (1992), 368-75; Uckun, Proc. Natl. Acad. Sci. USA 85 (1988), 8603-7). Combination therapy using antibodies against CD19 and other immunomodulatory antibodies has been disclosed for the treatment of B cell malignancies (WO02 / 04021, US2002006404, US2002028178) and autoimmune diseases (WO02 / 22212, US2002058029). WO00 / 67795 discloses the use of antibodies to CD19 for the treatment of indolent and aggressive B-cell lymphomas, and the formation of acute and chronic lymphocytic leukemias. WO02 / 80987 discloses the therapeutic use of immunotoxins based on antibodies to the antigen CD19 for the treatment of diseases such as B-cell non-Hodgkin's lymphoma, Hodgkin's lymphoma or B-cell leukemias (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)).

[0376] In another embodiment, the present invention provides a bispecific conditionally active antibody against CD3 on T cells, and CD20 as a target antigen. CD20 is one of the cell surface proteins present on B lymphocytes. The CD20 antigen is present in normal and malignant precursor B lymphocytes and mature B lymphocytes, including those in more than 90% of B cell non-Hodgkin's lymphomas (NHL). The antigen is not present in hematopoietic stem cells, activated B lymphocytes (plasma cells) and normal tissues. Several major murine antibodies 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).

[0377] CD20 has been described in immunotherapy strategies using vaccination with DNA encoding scFv linked to a carrier protein to treat plasma cell malignancies (Treon et al., 2000, Semin Oncol 27(5), 598), and immunotherapy using a CD20 antibody (IDEC-C2B8) has been shown to be effective in treating non-Hodgkin's B-cell lymphoma.

[0378] In some embodiments, bispecific conditionally active antibodies are single polypeptide chains encoded by polynucleotide molecules. The polynucleotides can be, for example, DNA, cDNA, RNA, or synthetically produced DNA or RNA, or recombinantly produced chimeric nucleic acid molecules comprising any polynucleotide in those polynucleotides in a single or combined form. The polynucleotides can be a part of a vector, such as an expression vector, including any expression system conventionally used in plasmids, cosmids, viruses and phages, or genetic engineering. The vector can include other genes, such as marker genes, which allow the vector to be selected under suitable conditions in a suitable host cell.

[0379] In one aspect, the polynucleotide is operably linked to an expression control sequence that allows expression in a prokaryotic or eukaryotic cell. An expression vector derived from a virus (such as a retrovirus, vaccinia virus, adeno-associated virus, herpes virus, or bovine papilloma virus) can be used to deliver the polynucleotide or vector to a mammalian cell. The vector comprising the polynucleotide of the present invention can be transformed into a host cell by well-known methods, which vary according to the type of cell host. For example, calcium chloride transfection is commonly used for prokaryotic cells, and calcium phosphate treatment or electroporation can be used for other cell hosts.

[0380] In another aspect, conditional active polypeptides can be engineered to produce bispecific conditional active polypeptides. The method for engineering conditional active polypeptides is similar to the method for engineering bispecific conditional active antibodies as described in WO2015 / 175375. For example, a bispecific conditional active polypeptide may have two active sites, each of which has conditional activity, i.e., lower activity than the parent site under normal physiological conditions and higher activity than the parent site under abnormal conditions. The two conditional active sites can evolve and screen independently, and then the two active sites are connected to the same bispecific conditional active polypeptide by a joint. In one aspect, the joint that can be used in a bispecific conditional active antibody is a known joint that is suitable for producing a bispecific conditional active polypeptide by connecting two conditional active sites in a conditional active polypeptide.

[0381] Suitable methods for engineering Fc regions of conditionally active antibodies have been described in WO2015 / 175375.

[0382] Suitable methods for engineering conditionally active viral particles have been described in WO2015 / 175375.

[0383] In some aspects, using the method described in WO2015 / 175375, conditional active polypeptides can be inserted into viral particles as oncolytic viruses. Oncolytic viruses are viruses that can kill those tumor cells when in contact with tumor cells. The conditional active polypeptides inserted into oncolytic viruses can be more active in tumor microenvironments, but are less active in other parts of individuals. For example, conditional active polypeptides may be more active under pH (e.g., pH 6.2-6.8) or other conditions present in the tumor microenvironment, but are less active (e.g., pH 7.2-7.6) under pH or other conditions present in another position of an individual, such as normal physiological conditions. The conditional active polypeptides inserted into oncolytic viruses can be used to promote delivery of oncolytic viruses to tumors, where oncolytic viruses can target and kill tumor cells.

[0384] Oncolytic viruses of interest include adenovirus, herpes simplex virus-1, vaccinia virus, parvovirus, reovirus, Newcastle disease virus, etc. Vaccinia virus is of particular interest.

[0385] In one aspect, the oncolytic virus is selected from paramyxovirus, reovirus, herpes virus, adenovirus and Semliki Forest virus. In another aspect, the paramyxovirus is selected from Newcastle disease virus (NDV), measles virus and mumps virus. In another aspect, the NDV is from a strain selected from MTH68 / H, PV-701 and 73-T.

[0386] In another aspect, the oncolytic virus is selected from the group consisting of herpes virus, reovirus, E1B-deleted adenovirus, vesicular stomatitis virus, and poxvirus. These oncolytic viruses have the potential to not only destroy tumor cells but also release antigens from the destroyed tumor cells, thereby inducing an immune response.

[0387] Specific examples of oncolytic viruses include, but are not limited to, adenoviruses (e.g., Delta-24, Delta-24-RGD, ICOVIR-5, ICOVIR-7, Onyx-015, ColoAdl, H101, AD5 / 3-D24-GMCSF), reoviruses, herpes simplex virus (HSV, OncoVEX GM-CSF), Newcastle disease virus, measles virus, retroviruses (e.g., influenza virus), poxviruses (e.g., vaccinia virus, including Copenhagen, Western Reserve, Wyeth strains), myxoma virus, rhabdoviruses (vesicular stomatitis virus (VSV)), picornaviruses (e.g., Seneca Valley virus, SW-001), coxsackieviruses, and parvoviruses.

[0388] In one aspect, the oncolytic virus is an adenovirus comprising a member of any of its 57 human serotypes (HAdV-1 to 57). In one embodiment, the adenovirus is an 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 viral serotypes Ad2 and Ad5, with deletions in the E1B-55K and E3B regions to enhance cancer selectivity. HI 01 is a modified version of Onyx-015. ICOVIR-5 and ICOVIR-7 include a deletion of the Rb binding site of E1A and a replacement of the E2F promoter for the E1A promoter. ColoAd 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 is replaced by the knob domain of serotype 3).

[0389] In a particularly preferred embodiment, the oncolytic virus is δ-24 or δ-24-RGD adenovirus. δ-24 is described in US2003 / 0138405 A1 and US2006 / 0147420A1. δ-24 adenovirus is derived from adenovirus type 5 (Ad-5) and contains a 24 base pair deletion in the CR2 portion of the E1A gene. δ-24-RGD further comprises an RGD-4C sequence (which strongly binds αvβ3 and αvβ5 integrins) inserted into the H1 loop of the fiber knob protein (Pasqualini R.et al., Nat Biotechnol., 15:542-546, 1997).

[0390] The oncolytic adenovirus can also be further modified to improve the ability of the oncolytic adenovirus to treat cancer. Jiang et al. (Curr. Gene Ther. 2009 Oct 9 (5): 422-427) have described such oncolytic adenovirus modifications, see also US2006 / 0147420A1.

[0391] Oncolytic viruses including conditionally active polypeptides can be administered locally or systemically. For example, but not limited to, oncolytic viruses can be administered intravascularly (intra-arterially or intravenously), intratumorally, intramuscularly, intradermally, intraperitoneally, subcutaneously, orally, parenterally, intranasally, intratracheally, transdermally, intraspinally, intraocularly, or intracranially.

[0392] The oncolytic virus can be administered in a single administration or multiple administrations. The virus can be administered in an amount of at least 1×10 5Plaque forming units (PFU), at least 5×10 5 PFU, at least 1×10 6 PFU, at least 5×10 6 or at least 5×10 6 PFU, 1×10 7 , at least 1×10 7 PFU, at least 1×10 8 or at least 1×10 8 PFU, at least 1×10 8 PFU, at least 5×10 8 PFU, at least 1×10 9 or at least 1×10 9 PFU, at least 5×10 9 or at least 5×10 9 PFU, at least 1×10 10 PFU or at least 1 × 10 10 PFU, at least 5×10 10 or at least 5×10 10 PFU, at least 1×10 11 PFU or at least 1×l0 11 PFU, at least 1×10 12 PFU or at least 1 × 10 13 For example, an oncolytic virus can be administered at a dose of about 10 7 -10 13 PFU, about 10 8 -10 13 PFU, about 10 9 -10 12 PFU or about 10 8 -10 12 PFU dose administration.

[0393] In certain aspects, cancers to be treated with oncolytic cells include any solid tumor, such as lung cancer, ovarian cancer, breast cancer, cervical cancer, pancreatic cancer, stomach cancer, colon cancer, skin cancer, laryngeal cancer, bladder cancer, and prostate cancer. In one aspect, the cancer is a cancer of the central nervous system. The cancer can be a neuroepithelial tumor, such as an astrocytic tumor (e.g., astrocytoma, anaplastic astrocytoma, glioblastoma, gliosarcoma, pilocytic astrocytoma, giant cell astrocytoma, pleomorphic xanthoastrocytoma), oligodendroglioma, ependymoma, oligoastrocytoma, glioblastoma, astrocytoma, choroid plexus papilloma, choroid plexus carcinoma, gangliocytoma, ganglioglioma, neurocytoma, neuroepithelioma, neuroblastoma, pineal region tumor (e.g., pinealoblastoma, pineoblastoma or mixed pinealoblastoma / pineoblastoma), medulloepithelioma, medulloblastoma, neuroblastoma or ganglioblastoma, retinoblastoma or ependymoma. The cancer may be a tumor of the central nervous system, such as a sellar tumor (e.g., a pituitary adenoma, a pituitary carcinoma, or a craniopharyngioma), a hematopoietic tumor (e.g., a primary malignant lymphoma, a plasma cell tumor, or a granulocytic tumor sarcoma), a germ cell tumor (e.g., a germ cell tumor, an embryonal carcinoma, a yolk sac tumor, a choriocarcinoma, a teratoma, or a mixed germ cell tumor), a meningioma, a mesenchymal tumor, a melanocytoma, or a skull or spinal nerve (e.g., a schwannoma or a neurofibroma). The cancer may be a low-grade glioma (e.g., an ependymoma, an astrocytoma, an oligodendroglioma, or a mixed glioma) or a high-grade (malignant) glioma (e.g., a glioblastoma multiforme). The cancer may be a primary or metastatic brain tumor. 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 U.S. Pat. No. 8,709,755B2.

[0394] The pharmaceutical composition can be used to treat various types of cancer, including carcinomas, germ cell tumors and sarcomas, and certain leukemias or lymphoid malignancies, benign and malignant tumors (malignanttumors) and malignancies (malignancies), such as sarcomas, carcinomas and melanomas. Cancers may be non-solid tumors (such as blood tumors) or solid tumors. Adult tumors / cancers and pediatric tumors / cancers are also included.

[0395] Hematological cancer is a cancer of the blood or bone marrow. Examples of hematological (or hematological) cancers include leukemias, including acute leukemias (e.g., acute lymphocytic leukemia, acute myeloid leukemia, acute myeloid leukemia, and myeloblastic leukemia, promyelocytic leukemia, myelomonocytic leukemia, monocytic leukemia, and erythroleukemia), chronic leukemias (e.g., chronic myelogenous (granulocytic) leukemia, chronic myeloid leukemia, and chronic lymphocytic leukemia), polycythemia vera, lymphoma, Hodgkin's disease, non-Hodgkin's lymphoma (indolent and advanced forms), multiple myeloma, Waldenstrom's macroglobulinemia, heavy chain disease, myelodysplastic syndrome, hairy cell leukemia, and myelodysplasia.

[0396] Solid tumors are abnormal tissue masses that do not usually contain cysts or fluid areas. Solid tumors can be benign or malignant. Different types of solid tumors (such as sarcomas, carcinomas and lymphomas) have been named according to the cell type that forms the solid tumor. Examples of solid tumors that can be treated include sarcomas and carcinomas, including fibrosarcomas, 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, bronchial carcinoma, renal cell carcinoma , hepatocellular carcinoma, bile duct cancer, choriocarcinoma, Wilms' tumor, cervical cancer, testicular tumors, seminoma, bladder cancer, melanoma, and CNS tumors (e.g., gliomas (such as brain stem gliomas and mixed gliomas), glioblastomas (also known as glioblastoma multiforme), astrocytomas, CNS lymphomas, germ cell tumors, medulloblastomas, schwannomas, ependymomas, pinealomas, hemangioblastomas, acoustic neuromas, oligodendrogliomas, meningiomas, neuroblastomas, retinoblastomas, and brain metastases).

[0397] Pharmaceutical compositions comprising conditionally active polypeptides or products engineered from conditionally active polypeptides can be formulated according to known methods for preparing pharmaceutical compositions. In these methods, conditionally active polypeptides are typically combined with a mixture, solution or composition containing a pharmaceutically acceptable carrier.

[0398] A pharmaceutically acceptable carrier is a substance that can be tolerated by the patient receiving it. 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, e.g., Gennaro (ed.), Remington's Pharmaceutical Sciences (Mack Publishing Company, 19th ed. 1995)). The formulation may also include one or more excipients, preservatives, solubilizers, buffers, albumin to prevent protein loss on the surface of the vial, etc.

[0399] The form of the pharmaceutical composition, route of administration, dosage and regimen will naturally depend on the condition to be treated, the severity of the disease, the age, weight and sex of the patient, etc. The skilled person can take these considerations into account 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.

[0400] Preferably, the pharmaceutical composition contains a pharmaceutically acceptable carrier for an injectable formulation. These carriers may be in particular isotonic sterile saline solutions (mono- or disodium phosphate, sodium chloride, potassium chloride, calcium chloride or magnesium chloride, etc. or mixtures of these salts), or dry, in particular lyophilized compositions which, after addition of, for example, sterile water or physiological saline, allow the constitution of injectable solutions.

[0401] In some embodiments, there is a tonicity agent (sometimes referred to as a "stabilizer") to adjust or maintain the tonicity of the liquid in the composition. When used with large charged biomolecules such as proteins and antibodies, they are often referred to as "stabilizers" because they can interact with the charged groups of the amino acid side chains, thereby reducing the possibility of intermolecular and intramolecular interactions. The tonicity agent can be present in any amount of 0.1% to 25% by weight, preferably 1% to 5% by weight of the pharmaceutical composition. Preferred tonicity agents include polyols, preferably trivalent or higher sugar alcohols, such as glycerol, erythritol, arabitol, xylitol, sorbitol, and mannitol.

[0402] Other excipients include one or more agents that can be used as: (1) fillers, (2) solubilizers, (3) stabilizers, and (4) agents that prevent denaturation or adhesion to the container wall. Such excipients may include: polysaccharide alcohols (listed above); amino acids such as alanine, glycine, glutamine, asparagine, histidine, arginine, lysine, ornithine, leucine, 2-phenylalanine, glutamic acid, threonine, etc.; organic sugars or sugar alcohols, such as sucrose, lactose, lactitol, trehalose, stachyose, mannose, sorbitol, xylose, ribose, ribitol, inositol, galactose, galactitol, glycerol, cyclitols (e.g., inositol), polyethylene glycol; sulfur-containing reducing agents such as urea, glutathione, lipoic acid, sodium thioglycolate, thioglycerol, α-thioglycerol and sodium thiosulfate; low molecular weight proteins such as human serum albumin, bovine serum albumin, gelatin or other immunoglobulins; hydrophilic polymers such as polyvinyl pyrrolidone; monosaccharides (e.g., xylose, mannose, fructose, glucose); disaccharides (e.g., lactose, maltose, sucrose); trisaccharides such as raffinose; and polysaccharides such as dextrin or dextran.

[0403] Nonionic surfactants or detergents (also referred to as "wetting agents") can be used to help solubilize the therapeutic agent and protect the therapeutic protein from aggregation induced by agitation, which also allows the formulation to be exposed to shear surface stress without causing denaturation of the active therapeutic protein or antibody. The nonionic surfactant can 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.

[0404] Suitable nonionic surfactants include polysorbates (20, 40, 60, 65, 80, etc.), poloxamers (184, 188, etc.), Polyols, Polyoxyethylene sorbitan monoether ( etc.), lauromacrogol 400, polyoxyethylene 40 stearate, polyoxyethylene hydrogenated castor oil 10, 50 and 60, glyceryl monostearate, sucrose fatty acid ester, methylcellulose and carboxymethylcellulose. Anionic detergents that can be used include sodium lauryl sulfate, dioctyl sodium sulfosuccinate and dioctyl sodium sulfonate. Cationic detergents include benzalkonium chloride or benzethonium chloride.

[0405] The dosage for administration can be adjusted as a function of various parameters of the relevant pathology or the desired duration of treatment, in particular as a function of the mode of administration used. To prepare a pharmaceutical composition, an effective amount of a conditionally active polypeptide or a product further engineered from a conditionally active polypeptide can be dissolved or dispersed in a pharmaceutically acceptable carrier or aqueous medium.

[0406] Pharmaceutical preparations suitable for injectable use include sterile aqueous solutions or dispersions; carriers such as sesame oil, peanut oil, or aqueous propylene glycol solutions; and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions. In all cases, the preparation must be sterile and must be fluid to a degree that allows easy injection. It must be stable under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms such as bacteria and fungi.

[0407] Solutions of conditionally active polypeptides as free bases or pharmaceutically acceptable salts can be prepared in water suitably mixed with a surfactant. Dispersions can also be prepared in glycerol, liquid polyethylene glycols, mixtures thereof, and oils. Under normal conditions of storage and use, these preparations contain preservatives to prevent the growth of microorganisms.

[0408] Conditionally active polypeptides and products obtained from the engineering of conditionally active polypeptides can be formulated into compositions in the form of salts. Pharmaceutically acceptable salts include acid addition salts (formed with free amino groups of proteins) formed with inorganic acids (such as hydrochloric acid or phosphoric acid) or organic acids (such as acetic acid, oxalic acid, tartaric acid, mandelic acid, etc.). Salts formed with free carboxyl groups can also be derived from inorganic bases (such as sodium hydroxide, potassium hydroxide, ammonium hydroxide, calcium hydroxide or ferric hydroxide), and organic bases (such as isopropylamine, trimethylamine, histidine, procaine, etc.).

[0409] Carrier can also be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol and liquid polyethylene glycol, etc.), its suitable mixture and vegetable oil. For example, by using a coating such as lecithin, by maintaining the required particle size and by using a surfactant to maintain suitable fluidity in the case of dispersion. The effect of microorganisms can be prevented by various antibacterial and antifungal agents, such as parabens, chlorobutanol, phenol, sorbic acid, thimerosal, etc. In many cases, it is preferred to include isotonic agents, such as sugar or sodium chloride. The absorption of the composition can be extended by using an agent such as aluminum monostearate and gelatin that delays absorption in an injectable composition.

[0410] Sterile injectable solutions are prepared by incorporating the required amount of the conditionally active polypeptide into a suitable solvent along with one or more of the other ingredients listed above (if necessary), followed by filtration sterilization. Typically, dispersions are prepared by incorporating the various sterile active ingredients into a sterile vehicle containing a basic dispersion medium and the required other ingredients from those listed above. In the case of sterile powders for the preparation of sterile injectable solutions, preferred methods of preparation are vacuum drying and freeze drying techniques, which produce a powder of the active ingredient plus any other desired ingredients from a previously sterile filtered solution.

[0411] The preparation of more concentrated or highly concentrated solutions for direct injection is also contemplated, with the use of dimethyl sulfoxide (DMSO) as a solvent expected to result in extremely rapid penetration, delivering high concentrations of active agent to small tumor regions.

[0412] After formulation, the solution will be administered in a manner compatible with the dosage formulation and in a therapeutically effective amount. The preparation is easily administered in a variety of dosage forms, such as the type of injectable solution described above, but drug release capsules and the like may also be used.

[0413] For parenteral administration in the form of, for example, aqueous solutions, the solution should be appropriately buffered (if necessary) and the liquid diluent should first be isotonic with enough saline or glucose. These specific aqueous solutions are particularly suitable for intravenous, intramuscular, subcutaneous and intraperitoneal administration. In this regard, according to the present disclosure, the sterile aqueous medium that can be used is known to those skilled in the art. For example, a dose can be dissolved in 1ml isotonic NaCl solution, or added to 1000ml subcutaneous perfusion solution or injected at the infusion site of suggestion (see, for example, " Remington's Pharmaceutical Sciences " 15th Edition, pp.1035-1038 and 1570-1580). Depending on the individual illness treated, some dosage changes will inevitably occur. In any case, the personnel responsible for administration will determine the appropriate dose for a single individual.

[0414] Conditionally active polypeptides and products derived from the engineering of conditionally active polypeptides can be formulated in therapeutic mixtures to deliver about 0.0001-10.0 mg, or about 0.001-5 mg, or about 0.001-1 mg, or about 0.001-0.1 mg, or about 0.1-1.0 mg, or even about 10 mg per dose. Multiple doses can also be administered at selected time intervals.

[0415] 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 used.

[0416] In certain embodiments, it is contemplated that liposomes and / or nanoparticles are used to introduce conditionally active polypeptides or products obtained from further engineering of conditionally active polypeptides into host cells. The formation and use of liposomes and / or nanoparticles are known to those skilled in the art.

[0417] Nanocapsules can generally encapsulate compounds in a stable and reproducible manner. In order to avoid side effects caused by intracellular polymer overload, such ultrafine particles (about 0.1 μm in size) are generally designed using polymers that can degrade in vivo. It is envisioned that biodegradable polyalkyl cyanoacrylate nanoparticles that meet these requirements can be used in the present invention.

[0418] Liposomes are formed from phospholipids that are dispersed in an aqueous medium and spontaneously form multilamellar concentric bilayer vesicles, also known as multilamellar vesicles (MLVs). MLVs typically have diameters ranging from 25 nm to 4 μm. Sonication of MLVs results in the formation of liposomes with diameters ranging from 200 to 4 μm. Small unilamellar vesicles (SUVs) of the size 1.4 to 2.5 mm2 contain an aqueous solution in their core. The physical properties of liposomes depend on pH, ionic strength, and the presence of divalent cations.

[0419] The pharmaceutical preparations containing the conditionally active polypeptides or products obtained from further engineering of the conditionally active polypeptides as described in the present invention are prepared in the form of lyophilized preparations 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 phosphates, citrates and other organic acids; antioxidants including ascorbic acid and methionine; preservatives (e.g., octadecyldimethylbenzyl ammonium chloride; hexamethonium chloride; benzalkonium chloride; benzethonium chloride; phenolic alcohols, butyl alcohols or benzyl alcohols; alkyl parabens such as methyl paraben or propyl paraben; catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol); low molecular weight (less than about 10 residues) polyols. peptides; proteins such as serum albumin, 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 nonionic surfactants such as polyethylene glycol (PEG).

[0420] Exemplary pharmaceutically acceptable carriers herein also include interstitial drug dispersants, such as soluble neutral active hyaluronidase glycoprotein (sHASEGP), such as human soluble PH-20 hyaluronidase glycoprotein, such as rHuPH20 ( Baxter International, Inc.). Certain exemplary sHASEGPs and methods of use, including rHuPH20, are described in U.S. Patent Publication Nos. 2005 / 0260186 and 2006 / 0104968. In one aspect, sHASEGP is combined with one or more additional glycosaminoglycanases, such as chondroitinase.

[0421] Exemplary lyophilized antibody formulations are described in US Pat. No. 6,267,958. Aqueous antibody formulations include those described in US Pat. No. 6,171,586 and WO 2006 / 044908, the latter formulations comprising a histidine acetate buffer.

[0422] The formulations herein may also contain more than one active ingredient as needed for the specific indication being treated. Preferably, ingredients with complementary activities that do not adversely affect each other can be combined into a single formulation. For example, in addition to the conditionally active antibodies, antibody fragments or immunoconjugates of the invention, it may be necessary to provide an EGFR antagonist (e.g., erlotinib), an anti-angiogenic agent (e.g., a VEGF antagonist that can be an anti-VEGF antibody) or a chemotherapeutic agent (e.g., a taxane or a platinum agent). These active ingredients are suitable for being present in combination in an amount effective for the intended purpose.

[0423] The active ingredient can be encapsulated in microcapsules prepared, for example, by coacervation techniques or by interfacial polymerization. For example, hydroxymethylcellulose or gelatin microcapsules and poly(methyl methacrylate) microcapsules in colloidal drug delivery systems (e.g., liposomes, albumin microspheres, microemulsions, nanoparticles, and nanocapsules) or in macroemulsions can be used. These techniques are disclosed in Remington's Pharmaceutical Sciences 16th edition, Osol, A. Ed. (1980).

[0424] 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, eg, films, or microcapsules.

[0425] In some embodiments, conditionally active polypeptides or products obtained from the engineering of conditionally active polypeptides can be used to produce an article containing a substance for treating, preventing and / or diagnosing the disease. The article includes 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 of a variety of materials, such as glass or plastic. The container holds the composition itself or a combination of the composition and another composition that is effective in treating, preventing and / or diagnosing the condition, and can have a sterile access port (for example, the container can be an intravenous solution bag or a vial with a stopper pierceable by a hypodermic needle). At least one active agent in the composition is a conditionally active polypeptide of the present invention or a product obtained by further engineering of the conditionally active polypeptide. The label or package insert indicates that the composition is used to treat the selected condition. In addition, the article may include (a) a first container containing a composition, wherein the composition contains a conditionally active polypeptide or a product engineered from the conditionally active polypeptide; and (b) a second container containing a composition, wherein the composition contains an additional cytotoxin or other therapeutic agent. The article in this embodiment of the invention may also include a package insert indicating that the composition can be used to treat a specific condition. Alternatively or additionally, the article of manufacture may further comprise a second (or third) container comprising a pharmaceutically acceptable buffer, such as bacteriostatic water for injection (BWFI), phosphate-buffered saline, Ringer's solution, and dextrose solution. It may also include other materials desirable from a commercial and user standpoint, including other buffers, diluents, filters, needles, and syringes.

[0426] The article of manufacture can optionally comprise a container as part of a parenteral, subcutaneous, intramuscular, intravenous, intraarticular, intrabronchial, intraperitoneal, intracapsular, intracartilaginous, intracavitary, intracerebellar, intraventricular, intracolonic, intracervical, intragastric, intrahepatic, intramyocardial, intraosseous, intrapelvic, intrapericardial, intraperitoneal, intrapleural, intraprostatic, intrapulmonary, intrarectal, intrarenal, intraretinal, intraspinal, intrasynovial, intrathoracic, intrauterine, intravesical, pill, vaginal, rectal, oral, sublingual, intranasal, or transdermal delivery device or system.

[0427] The conditionally active polypeptide or a product derived from the engineering of a conditionally active polypeptide can be contained in a medical device, wherein the device is suitable for contacting or administering the conditionally active polypeptide or a product derived from the further engineering of a conditionally active polypeptide by at least one mode selected from the following: parenteral, subcutaneous, intramuscular, intravenous, intraarticular, intrabronchial, intraperitoneal, intracapsular, intracartilaginous, intracavitary, intracavitary, 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, oral, sublingual, intranasal, or transdermal absorption.

[0428] In other embodiments, the conditionally active polypeptide or the product obtained from the engineering of the conditionally active polypeptide can be included in the kit in a lyophilized form in a first container, and an optional second container containing sterile water, sterile buffered water, or at least one preservative selected from the following compounds: 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 a mixture thereof in a diluent aqueous solution. In one aspect, in the kit, the concentration of the conditionally active polypeptide or the product obtained from the engineering of 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 also includes an isotonic agent. In another aspect, the second container also includes a physiologically acceptable buffer. In one aspect, the present invention provides a method for treating a disorder mediated by at least one parent protein, comprising administering a preparation provided in the kit to a patient in need thereof, and reconstitution prior to administration.

[0429] The following examples are illustrative but not limiting of the present invention. Other suitable modifications and adaptations of the various conditions and parameters normally encountered in the art and apparent to those skilled in the art are within the scope of the present disclosure.

[0430] Example

[0431] Examples 1-5: Examples 1-5 for preparing conditionally active polypeptides have been described in US Pat. No. 8,709,755 B2, which is incorporated herein by reference.

[0432] Example 6: Evolution of light or heavy chains of antibodies

[0433] The heavy chain and light chain of antibody F1-10F10 were evolved separately using CPE. Light chain mutants were screened to find 26 li...

Claims

1. A method for producing a conditionally active antibody, single-chain antibody or antibody fragment from a parent antibody, single-chain antibody or antibody fragment, the method for producing a conditionally active antibody, single-chain antibody or antibody fragment comprising the following steps: (i) evolving the parent antibody, single-chain antibody or antibody fragment by mutating at least one amino acid to produce one or more mutated antibodies, single-chain antibodies or antibody fragments, wherein the antibody fragment is selected from Fab, Fab', (Fab')2, Fv and an SCA fragment capable of binding to an epitope of an antigen; (ii) subjecting the one or more mutant antibodies, single chain antibodies or antibody fragments to a first ELISA test and a second ELISA test, wherein the first ELISA test is performed at a first pH of 6.0-6.8 in the presence of a small molecule or an ion to measure the binding activity of the one or more mutant antibodies, single chain antibodies or antibody fragments, and the second ELISA test is also performed at a second pH of 7.2-7.8 in the presence of the small molecule or ion to measure the binding activity of the one or more mutant antibodies, single chain antibodies or antibody fragments, wherein the small molecule or ion has a molecular weight of less than 100 a.mu and a pKa that differs from the first pH by at most 4 pH units; (iii) selecting a mutant antibody, single chain antibody or antibody fragment having a ratio of binding activity in the first assay to binding activity in the second assay of at least 3.0; (iv) obtaining the conditionally active antibody, single chain antibody or antibody fragment by selecting a mutant antibody, single chain antibody or antibody fragment whose binding activity in the absence of the small molecule or ion is not dependent on pH from among the mutant antibodies, single chain antibodies or antibody fragments selected in step (iii), The small molecules or ions and their concentrations are selected from 3mM-200mM bicarbonate ions, 10mM-100mM hydrogen sulfide ions and 100μm-100mM hydrogen sulfide.

2. The method of claim 1, wherein the parent antibody, single-chain antibody or antibody fragment is a heavy chain or light chain of an antibody, and the at least one amino acid is located within a complementarity determining region of the parent antibody, single-chain antibody or antibody fragment.

3. The method of claim 2, wherein the at least one amino acid is located within the Fc region of the parent antibody, single chain antibody or antibody fragment.

4. The method of claim 3, wherein the evolving step comprises replacing the Fc region with a variable region of a different antibody to form a bispecific antibody.

5. The method of claim 3, wherein the evolving step comprises replacing the Fc region with a variable region of a different antibody to form a single chain antibody.

6. The method of claim 2, wherein the at least one amino acid is located within the framework region of the parent antibody, single chain antibody or antibody fragment.

7. The method according to any one of claims 1-6, wherein the evolving step comprises substitution, insertion, deletion or a combination thereof.

8. The method according to claim 1, wherein step (ii) further comprises subjecting the one or more mutated antibodies, single-chain antibodies or antibody fragments to the following tests: a test for measuring the binding activity of the one or more mutated antibodies, single-chain antibodies or antibody fragments at the first pH in the absence of the small molecule or ion, and a test for measuring the binding activity of the one or more mutated antibodies, single-chain antibodies or antibody fragments at the second pH in the absence of the small molecule or ion; and step (iii) further comprises selecting a mutated antibody, single-chain antibody or antibody fragment having pH-independent binding activity in the absence of the small molecule or ion.

9. The method of claim 1, wherein the ratio of the binding activity in the first assay to the binding activity in the second assay is at least 4.

0.

10. The method of claim 1, wherein the small molecule or ion is bicarbonate.

11. The method of claim 1, wherein the small molecule or ion is hydrogen sulfide.

12. The method of claim 1, wherein the pKa of the small molecule or ion differs from the first pH by at most 0.5, 1, 2, 3, or 4 pH units.

13. The method of claim 1, wherein the pKa of the small molecule or ion is between the first pH and the second pH.

14. The method of claim 1, wherein the pKa of the small molecule or ion is greater than 6.2 and less than 7.

2.

15. The method of claim 1, wherein the antibody, single chain antibody or antibody fragment is an antibody, single chain antibody or antibody fragment derived from a non-naturally occurring mutation of a parent antibody, single chain antibody or antibody fragment.

16. The method of claim 15, wherein the mutated antibody, single chain antibody or antibody fragment has a higher proportion of charged amino acid residues compared to the parent antibody, single chain antibody or antibody fragment.

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