Protein therapeutics for treatment of senescent cells

Abstract

To provide methods of generating conditionally active senolytic antibodies that target senescent cells and that are conditionally active in an extracellular environment of a senescent cell.SOLUTION: Provided is a method comprising: evolving a DNA encoding a parent antibody or antibody fragment that binds to a target associated with a senescent cell to create a mutant DNA; expressing the mutant DNA to obtain a mutant antibody or antibody fragment; subjecting the mutant antibody or antibody fragment to an assay for a binding activity to the target under an extracellular condition of the senescent cell and to an assay for a binding activity to the target under a normal physiological condition; and selecting a conditionally active senolytic antibody or antibody fragment, the ratio of the activity of the conditionally active senolytic antibody or antibody fragment in the assay under extracellular conditions to the activity in the assay under normal physiological conditions being at least 1.3:1.SELECTED DRAWING: Figure 18B

Description

[Technical Field]

[0001] Areas of disclosure This disclosure relates to the field of treating or purifying senescent cells, and / or the field of treating diseases or disorders related to senescent cells. More specifically, this disclosure relates to conditionally active proteins that target senescent cells, and to methods for producing such conditionally active proteins. [Background technology]

[0002] Background of Disclosure Senescent cells are metabolically active but are trapped in the G1 phase of the cell proliferation cycle, and their lifespan is controlled by multiple dominant genes (Stanulis-Praeger, Mech.Ageing Dev., vol.38, pp.1-48, 1987). Senescent cells differ from quiescent and terminally differentiated cells in several important aspects, and have characteristic morphological changes such as dilation, flattening, and increased granularity (Dimri et al., Proc. Nat. Acad. Sci. USA, vol. 92, pp.9363-9367, 1995). Senescent cells do not divide even when stimulated by mitotic factors (Campisi, Trends Cell Biol., vol. 11, pp. S27-S31, 2001). Aging involves the activation of p53 and / or Rb, as well as their regulators, such as p16INK4a, p21, and ARF. Aging is generally irreversible unless p53 or Rb is inactivated.

[0003] Senescent cells express high levels of plasminogen activator inhibitor (PAI) and stain for β-galactosidase activity at pH 6 (Sharpless et al., J. Clin. Invest., vol. 113, pp.160-168, 2004). Irreversible G1 arrest is mediated by the inactivation of cyclin-dependent kinase (CdK) complexes that phosphorylate Rb. P21 accumulates in senescent cells and inhibits CdK4-CdK6. P16 also inhibits CdK4-CdK6 and accumulates in senescent cells in proportion to β-galactosidase activity and cell volume (Stein et al., Mol. Cell. Biol., vol. 19, pp.2109-2117, 1999). Evidence suggests that p21 is expressed at the onset of aging but is not required to maintain aging, while p16 expression supports the maintenance of aging once it has begun.

[0004] In some cases, aging is related to the progression of telomere shortening with each cell division, and aging is induced when specific chromosomal telomeres reach a critical length (Mathon and Lloyd, Nat. Rev. Cancer, vol. 3, pp.203-213, 2001; Martins, UM Exp Cell Res., vol. 256, pp.291-299, 2000). Aging can be eliminated by the expression of telomerase, which elongates telomeres. For example, human fibroblasts, when transfected to express telomerase, undergo indefinite replication. Most cancer cells express telomerase to maintain telomere length and replicate indefinitely. A small number of cancer cells that do not express telomerase have an alternative mechanism (ALT) for telomere elongation.

[0005] There are other causes of aging. Collectively, these other causes are often referred to as stress-induced premature cellular senescence (SIPS). Oxidative stress can shorten telomeres, thereby inducing aging (von Zglinicki, Trends Biochem. Sci., vol. 27, pp.339-344, 2002). Hyperoxia has been shown to induce aging. Gamma irradiation of human fibroblasts in the early to mid-G1 phase induces p53-dependent aging (Di Leonardo et al., Genes Dev., vol. 8, pp.2540-2551, 1994). Ultraviolet irradiation also induces aging. Other substances that may induce aging include hydrogen peroxide (Krtolica et al., Proc. Nat. Acad. Sci. USA, vol. 98, pp.12072-12077, 2001), sodium butyrate, 5-azacitadine, and transfection of the Ras oncogene (Tominaga, Mech. Ageing Dev., vol. 123, pp.927-936, 2002). Chemotherapy agents including doxorubicin, cisplatin, and other hosts have been shown to induce aging in cancer cells (Roninson, Cancer Res., vol. 63, pp.2705-2715, 2003). Treatment with 5-bromodeoxyuridine induces senescence in both normal and malignant tumor cells (Michishita et al., J. Biochem., vol. 126, pp.1052-1059, 1999). Generally speaking, drugs that damage DNA can induce senescence.

[0006] Evidence suggests a link between aging and senescence. Cultured cells from older donors show aging after fewer proliferation cycles than cells from younger donors (Martin et al., Lab. Invest., vol. 23, pp.86-92, 1970; Schneider et al., Proc. Nat. Acad. Sci. USA, vol. 73, pp.3584-3588, 1976). Cells from short-lived species age after fewer proliferation cycles than cells from long-lived species (Rohme, D., Proc. Nat. Acad. Sci. USA, vol. 78, pp.5009-3320, 1981). Cultured cells from donors with genetic progeria syndromes such as Werner syndrome show aging after fewer proliferation cycles than cells from age-matched controls.

[0007] Aging confers functional changes to senescent cells associated with a variety of age-related diseases and disorders (Chang et al., Proc. Nat. Acad. Sci. USA, vol. 97, pp.4291-4296, 2000). Senescent cells accumulate in an individual's tissues and organs as they age and are found in areas of age-related pathology. Given that senescent cells may be causally linked to certain aspects of age-related health decline, contribute to specific diseases, and are also induced as a result of necessary life-sustaining chemotherapy and radiation therapy, the presence of senescent cells can have detrimental effects on millions of patients worldwide. In a broad sense, the selective elimination of senescent cells is thought to be able to prevent and treat age-related diseases and disorders.

[0008] Senescent cells can also promote tumorigenesis. Senescent stromal cells express tumorigenic factors that exert paracrine effects on neighboring epithelial cells. These effects include pro-mitotic and anti-apoptotic properties (Chang et al., Proc. Nat. Acad. Sci. USA, vol. 97, pp.4291-4296, 2000). Senescent fibroblasts have been shown to stimulate pre-malignant and malignant epithelial cells but not normal epithelial cells, leading to tumor formation in mice. This occurred when only 10% of fibroblasts senescent (Krtolica et al., Proc. Nat. Acad. Sci. USA, vol. 98, pp.12072-12077, 2001). Tumor-promoting factors secreted by senescent cells are partially mediated by P21waf1 / cip1 / sdi1 (Roninson, Cancer Res., vol. 63, pp.2705-2715, 2003). The threshold of senescent stromal cells appears to provide an environment that allows adjacent pre-malignant epithelial cells to survive, migrate, and divide (Campisi, Nat. Rev. Cancer, vol. 3, pp. 339-349, 2003).

[0009] As a result, therapies targeting senescent cells are a promising treatment option for age-related diseases and disorders. US2016 / 0038576 discloses immunogenic compositions for inducing adaptive immune responses specifically directed to senescent cells for the treatment and defense of age-related diseases and disorders, as well as other diseases and disorders associated with or exacerbated by the presence of senescent cells. The immunogenic composition comprises at least one or more of a senescent cell-associated antigen, a polynucleotide encoding the senescent cell-associated antigen, and a recombinant expression vector containing the polynucleotide for use when administered to a subject.

[0010] WO2015116740 discloses a method for administering a therapeutically effective dose of a small molecule senescent cell scavenger that selectively kills senescent cells compared to non-senescent cells for the treatment of senescent cell-related diseases and disorders. Senescent cell-related diseases and disorders that can be treated by this method include arteriosclerosis such as atherosclerosis, idiopathic pulmonary fibrosis, chronic obstructive pulmonary disease, osteoarthritis, age-related ophthalmic diseases and disorders, and cardiovascular diseases and disorders associated with or induced by age-related skin diseases and disorders.

[0011] US2015 / 0064137 discloses polypeptides useful for selective elimination of senescent cells, and viruses containing these polypeptides. These polypeptides and viruses can induce apoptosis in senescent cells. The polypeptides are selected from the products of pro-apoptotic genes. The viruses contain pro-apoptotic genes whose expression is regulated by a p16 promoter. The p16 promoter may be a classical p16 promoter or a non-classical p16 promoter. [Overview of the Initiative] [Problems that the invention aims to solve]

[0012] These therapeutics target one or more proteins in senescent cells to kill or eliminate them. However, these target proteins in senescent cells may also be present in other types of cells, potentially inducing undesirable side effects. Therefore, it would be advantageous to develop a classification of therapeutic proteins that preferentially and / or specifically bind to targets on senescent cells while minimizing or eliminating binding to the same targets on other types of cells. [Means for solving the problem]

[0013] Summary of Disclosure In one embodiment, the disclosure is a method for generating a conditionally active protein that binds to a target associated with senescent cells from a parent protein that binds to the target associated with senescent cells, (i) Using one or more evolutionary techniques to evolve the DNA encoding the parent protein to create mutant DNA; (ii) expressing the mutant DNA to obtain a mutant protein; (iii) subjecting the mutant protein to assays under extracellular conditions of the senescent cells and assays under normal physiological conditions; (iv) (a) a decrease in the same activity in the assay under normal physiological conditions compared to the activity of the parent protein in the assay, and an increase in the same activity in the assay under extracellular conditions of the senescent cells compared to the activity of the conditional active protein in the assay under normal physiological conditions; and (b) a decrease in the same activity in the assay under normal physiological conditions compared to the activity of the parent protein in the assay, and an increase in the same activity in the assay under extracellular conditions of the senescent cells compared to the activity of the parent protein in the assay under extracellular conditions of the senescent cells, selecting the conditional active protein from the mutant protein showing at least one of the above; A method is provided that includes the above steps.[[ID=(13)]]

[0014] In some embodiments, the parent protein may be selected from an enzyme, an antibody, a receptor, a ligand, an enzyme fragment, an antibody fragment, a receptor fragment, and a ligand fragment.

[0015] In each of the above embodiments, the activity may be a binding activity to the target.

[0016] In each of the above embodiments, the parent protein may be an enzyme, and the activity may be an enzyme activity using at least a part of the senescent cells as a substrate.

[0017] In each of the embodiments described above, the conditionally active protein may be a cyclic peptide. The cyclic peptide may have a length of about 5 to about 500 amino acids, or about 10 to about 50 amino acids.

[0018] In each of the embodiments described above, the target may be a surface molecule located on the outer surface of a senescent cell. In each of the embodiments described above, the surface molecule may be a cell membrane protein of a senescent cell. In each of the embodiments described above, the target may be APC, ARHGAP1, ARMCX-3, AXL, B2MG, BCL2L1, CAPNS2, CD261, CD39, CD54, CD73, CD95, CDC42, CDKN2C, CLYBL, COPG1, CRKL, DCR1, DCR2, DCR3, DEP1, DGKA, EBP, EBP50, FASL, FGF1, GBA3, GIT2, ICAM1, ICAM3, IGF1, ISG20, ITGAV, KITLG, Lamin B1, LANCL1, LCMT2, LPHN1, MADCAM1, MAG, MAP3K14 , MAPK, MEF2C, miR22, MMP3, MTHFD2, NAIP, NAPG, NCKAP1, Nectin4, NNMT, NOTCH3, NTAL, OPG, OSBPL3, p16, p16INK4a, p19, p21, p53, PAI1, PARK2, PFN1, PGM, PLD3, PMS2, POU5F1, PPP1A, PPP1CB, PRKRA, PRPF19, PRTG, RAC1, RAPGEF1, RET, Smurf2, STX4, VAMP3, VIT, VPS26A, WEE1, YAP1, YH2AX, and YWHAE may be selected. In addition, it should be recognized that the target may be any combination of the above.

[0019] In each of the embodiments described above, the ratio of the activity of the conditionally active protein in the assay under extracellular conditions of senescent cells to the activity of the conditionally active protein in the assay under normal physiological conditions is at least about 1.3:1, or 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, Alternatively, the ratio may be at least approximately 12:1, or at least approximately 13:1, or at least approximately 14:1, or at least approximately 15:1, or at least approximately 16:1, or at least approximately 17:1, or at least approximately 18:1, or at least approximately 19:1, or at least approximately 20:1, or at least approximately 30:1, or at least approximately 40:1, or at least approximately 50:1, or at least approximately 60:1, or at least approximately 70:1, or at least approximately 80:1, or at least approximately 90:1, or at least approximately 100:1.

[0020] In each of the embodiments described above, the extracellular conditions of the senescent cells may be a pH within the range of about 5.5 to about 7.0, or about 6.0 to about 7.0, or about 6.2 to about 6.8.

[0021] In each of the embodiments described above, the normal physiological conditions may be a pH within the range of about 7.2 to about 7.8, or about 7.2 to about 7.6, or about 7.4 to about 7.6.

[0022] In each of the embodiments described above, the extracellular conditions of the senescent cells may be such that the concentration of deoxynucleotide is lower than the normal physiological concentration of the deoxynucleotide.

[0023] In each of the embodiments described above, the extracellular conditions of the senescent cells may be oxygen at a lower concentration than the normal physiological concentration of oxygen.

[0024] In each of the embodiments described above, the extracellular conditions of the senescent cells may be NAD+ / NADH at a ratio lower than the normal physiological ratio.

[0025] In each of the embodiments described above, the extracellular conditions of the senescent cells may be at least one of the redox homeostasis metabolites selected from hypotaurine, cysteine ​​sulfinic acid, cysteine-glutathione disulfide, gamma-glutamylalanine, gamma-glutamylmethionine, pyridoxate, gamma-glutamylglutamine, and alanine, at a higher concentration than the normal physiological concentration of the said redox homeostasis metabolite.

[0026] In each of the embodiments described above, the extracellular conditions of the senescent cells may be a high concentration of at least one nucleotide metabolite selected from 3-ureidopropionate, urat, 7-methylguanine, and hypoxanthine compared to the normal physiological concentration of the same nucleotide metabolite.

[0027] In each of the embodiments described above, the extracellular conditions of the senescent cells may be a low concentration of thymidine compared to the normal physiological concentration of thymidine.

[0028] In each of the embodiments described above, the extracellular conditions of the senescent cells may be at a lower concentration of at least one dipeptide selected from glycylisoleucine, glycylvaline, glycylleucine, isoleucylglycine, and valylglycine compared to the normal physiological concentration of the same dipeptide.

[0029] In each of the embodiments described above, the extracellular conditions of the senescent cells may be a low concentration of at least one fatty acid selected from linoleate, dihomolinoleate, and 10-heptadecanoate compared to the normal physiological concentration of the fatty acid.

[0030] In each of the embodiments described above, the extracellular conditions of the senescent cells may be a high concentration of at least one phospholipid metabolite selected from 2-hydroxypalmitate, 2-hydroxysteart, 3-hydroxydecanoate, 3-hydroxyoctanoate, and glycerophosphorylcholine compared to the normal physiological concentration of said phospholipid metabolite.

[0031] In each of the embodiments described above, the extracellular conditions of the senescent cells may be a high concentration of at least one amino acid metabolite selected from alanine, C-glycosyltryptophan, kynurenine, dimethylarginine, and orthithine compared to the normal physiological concentration of said amino acid metabolite.

[0032] In each of the embodiments described above, the extracellular conditions of the senescent cells may be a lower concentration of phenylpilbert compared to a normal physiological concentration of phenylpilbert.

[0033] In each of the embodiments described above, the extracellular conditions of the senescent cells may be a high concentration of at least one metabolite selected from humarate, malonate, eicosapentaenoate, and citrate compared to the normal physiological concentration of said metabolite.

[0034] In each of the embodiments described above, the extracellular conditions of the senescent cell may be a ratio of glycerophosphocholine to phosphocholine that is higher than the normal physiological ratio of glycerophosphocholine to phosphocholine.

[0035] In each of the embodiments described above, the extracellular conditions of the senescent cells may be a high concentration of the protein secreted by the senescent cells compared to the normal physiological concentration of the protein, where the protein secreted by the senescent cells is GM-CSF, GROa, GRC-α, β, γ, IGFBP-7, IL-1α, IL-6, IL-7, IL-8, MCP-1, MCP-2, MIP-la, MMP-1, MMP-2, MMP-10, MMP-3, amphiregulin, ENA-78, eotaxin-3, GCP-2, GITR, HGF, ICAM-1, IGFBP-1, IGFBP-2, IGFBP-3, IGFBP-4, IGFBP-5, IGFBP-6, IL-13, IL-Iβ, MCP-4, MIF, MIP-3a, MMP-12, MMP-13, MMP-14, NAP2, Oncostatin M, Osteoprotegerin, PIGF, RANTES, sgpl30, TIMP-2, TRAIL-R3, Acrp30, Angiogenin, AXL, bFGF, BLC, BTC, CTACK, EGF-R, Fas, FGF-7, G-CSF, GDNF, HCC-4, I-309, IFN-γ, IL-1Rl, IL-11, IL-15, IL-2R-a, IL-6R, I-TAC, Leptin, LIF, MSP-a, PAI-1, PAI-2, PDGF-BB, SCF, SDF-1, sTNF Selected from at least one of the following: RI, sTNF-RII, thrombopoietin, TIMP-1, tPA, uPA, uPAR, VEGF, MCP-3, IGF-1, TGF-β3, MIP-1-delta, IL-4, IL-16, BMP-4, MDC, IL-10, Fit-3 ligand, CNTF, EGF, BMP-6, and any combination thereof.

[0036] In each of the embodiments described above, the assay under normal physiological conditions and the assay under extracellular conditions of senescent cells may be carried out in an assay solution containing at least one component selected from inorganic compounds, ions, and organic molecules. In this embodiment, the at least one component may be present in substantially the same concentration in the assay solution in both the assay under normal physiological conditions and the assay under extracellular conditions of senescent cells. In these embodiments, the at least one component may be an inorganic compound, selected from boric acid, calcium chloride, calcium nitrate, diammonium phosphate, magnesium sulfate, monoammonium phosphate, monopotassium phosphate, potassium chloride, potassium sulfate, copper sulfate, iron sulfate, manganese sulfate, zinc sulfate, magnesium sulfate, calcium nitrate, calcium chelate, copper chelate, iron chelate, iron chelate, manganese chelate, zinc chelate, 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, and urea. In these embodiments, the at least one component may be an ion, selected from phosphorus ions, sulfur ions, chloride ions, magnesium ions, sodium ions, potassium ions, ammonium ions, iron ions, zinc ions, and copper ions. In these embodiments, the at least one component may be selected from one or more of the following: uric acid in a concentration range of 2 to 7.0 mg / dL, calcium ions in a concentration range of 8.2 to 11.6 mg / dL, chloride ions in a concentration range of 355 to 381 mg / dL, iron ions in a concentration range of 0.028 to 0.210 mg / dL, potassium ions in a concentration range of 12.1 to 25.4 mg / dL, sodium ions in a concentration range of 300 to 330 mg / dL, and carbonic acid in a concentration range of 15 to 30 mM.In these embodiments, the at least one component may be an organic molecule and is an amino acid selected from histidine, alanine, isoleucine, arginine, leucine, asparagine, lysine, aspartic acid, methionine, cysteine, phenylalanine, glutamic acid, threonine, glutamine, tryptophan, glycine, valine, pyrrolicin, proline, selenocysteine, serine, and tyrosine. In these embodiments, the at least one component may be an organic acid selected from citric acid, α-ketoglutaric acid, succinic acid, malic acid, fumaric acid, acetoacetic acid, β-hydroxybutyric acid, lactic acid, pyruvic acid, α-ketonic acid, acetic acid, and volatile fatty acids. In these embodiments, the at least one component may be a sugar selected from glucose, pentose, hexose, xylose, ribose, mannose, galactose, lactose, GlcNAcβ1-3Gal, Galα1-4Gal, Manα1-2Man, GalNAcβ1-3Gal, and O-, N-, C-, and S-glycosides. In these embodiments, the at least one component may be selected from magnesium ions, sulfate ions, bisulfate ions, carbonate ions, bicarbonate ions, nitrate ions, nitrite ions, phosphate ions, hydrogen phosphate ions, dihydrogen phosphate ions, persulfate ions, monopersulfate ions, borate ions, and ammonium ions.

[0037] In each of the embodiments described above, the extracellular conditions of the senescent cells may be a first pH in the range of about 5.5 to about 7.0, the normal physiological conditions may be a second pH in the range of about 7.2 to about 7.8, and one or more assays may be carried out in an assay solution containing at least one molecular species having a molecular weight of less than 900 a.mu and a pKa at a maximum of 0.5, 1, 2, 3, or 4 pH units away from the first pH.

[0038] In each of the embodiments described above, the extracellular conditions of the senescent cells may be a first pH in the range of about 5.5 to about 7.0, the normal physiological conditions may be a second pH in the range of about 7.2 to about 7.8, and one or more assays may be carried out in an assay solution containing at least one molecular species having a molecular weight of less than 900 a.mu, the molecular species may have a pKa between the first pH and the second pH.

[0039] In each of the embodiments described above, the extracellular conditions of the senescent cells may be a first pH in the range of about 5.5 to about 7.0, the normal physiological conditions may be a second pH in the range of about 7.2 to about 7.8, and one or more assays may be carried out in an assay solution containing at least one molecular species selected from histidine, histamine, adenosine diphosphate, adenosine triphosphate, citrate, bicarbonate, acetate, lactate, disulfide, hydrogen sulfide, ammonium, and dihydrogen phosphate.

[0040] In each of the embodiments described above, step (iv) of selection may include selecting a conditionally active protein that exhibits (a) a decrease in the activity of the parent protein in the assay compared to the activity of the parent protein in the assay under normal physiological conditions, and an increase in the activity of the conditionally active protein in the assay under extracellular conditions of the senescent cell compared to the activity of the conditionally active protein in the assay under normal physiological conditions.

[0041] In each of the embodiments described above, the selection step (iv) may include selecting a conditionally active protein that exhibits (b) a decrease in the activity of the parent protein in the assay compared to the activity of the parent protein in the assay under normal physiological conditions, and an increase in the activity of the parent protein in the assay under extracellular conditions of senescent cells compared to the activity of the parent protein in the assay under extracellular conditions of senescent cells.

[0042] In another embodiment, the Disclosure provides a conditionally active protein produced by any of the methods described above. The conditionally active protein may be an antibody. The antibody may be a single-chain antibody or an antibody fragment. The antibody may be suitable for manipulation as part of a chimeric antigen receptor on a T cell. The antibody may be a humanized antibody, a bispecific antibody, or a multispecific antibody.

[0043] In each of the embodiments described above, the conditionally active protein may be selected from a receptor, a regulatory protein, a soluble protein, a cytokine, a receptor fragment, a regulatory protein fragment, a soluble protein fragment, and a cytokine fragment.

[0044] In each of the embodiments described above, the conditionally active protein may be a conditionally active antibody, which may be conjugated to a masking portion by a linker. The masking portion reduces the binding activity of the conditionally active antibody to the target by at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%.

[0045] In each of the embodiments described above, the linker may be covalently bound to the variable region of the conditionally active antibody.

[0046] In each of the embodiments described above, the masking portion may specifically bind to the variable region of the conditionally active antibody.

[0047] In each of the embodiments described above, the linker may include a flexible region and a cut portion.

[0048] In each of the embodiments described above, the cleavage site may be cleaved by a protease in the extracellular environment of the senescent cell.

[0049] In each of the embodiments described above, the conditionally active protein may be conjugated by a linker to a cytotoxic agent, a cell proliferation inhibitor, or an antiproliferative agent.

[0050] In each of the embodiments described above, the linker may contain a cleavage site for at least one protease in the extracellular environment of the senescent cell. The at least one protease is selected from ADAM10, ADAM12, ADAM17, ADAMTS, ADAMTS5, BACE, caspase 1-14, cathepsin A, cathepsin B, cathepsin D, cathepsin E, cathepsin K, cathepsin S, FAP, MT1-MMP, granzyme B, guanidinobenzoatase, hepsin, human neutrophil elastase, regmine, matryptase 2, meprine, MMP1-17, MT-SP1, neprilysin, NS3 / 4A, plasmin, PSA, PSMA, TRACE, TMPRSS 3, TMPRSS 4, and uPA.

[0051] In another embodiment, the Disclosure provides a pharmaceutical composition comprising an effective amount of any of the aforementioned conditionally active proteins and a pharmaceutically acceptable carrier.

[0052] In yet another embodiment, the Disclosure provides a method for treating aging or senescent cell-related diseases or disorders, comprising the step of administering one of the conditionally active proteins described above or one of the pharmaceutical compositions described above. In the embodiments described above, the senescent cell-related diseases or disorders may be selected from cognitive disorders, cardiovascular diseases, metabolic diseases and disorders, motor function disorders and disorders, cerebrovascular diseases, emphysema, osteoarthritis, lung diseases, inflammatory / autoimmune diseases and disorders, ophthalmic diseases or disorders, metastases, side effects of chemotherapy or radiotherapy, aging-related diseases and disorders, fibrous diseases and disorders.

[0053] In yet another embodiment, the Disclosure provides a method for producing conditionally active molecules having a molecular weight of less than approximately 3000 a.mu from a parent organic compound. The method includes the steps of: modifying the parent organic compound by introducing one or more partially charged or charged groups to the parent organic compound to produce one or more modified organic compounds; and selecting the modified organic compounds that exhibit higher activity in the assay under abnormal conditions compared to their activity in the assay under normal physiological conditions.

[0054] In yet another embodiment, the Disclosure provides a method for producing conditionally active molecules having a molecular weight of less than about 3000 a.mu from a parent organic compound, comprising the steps of: modifying the parent organic compound by removing one or more partially charged or charged groups from the parent organic compound to produce one or more modified organic compounds; and selecting the modified organic compound that exhibits higher activity in the assay under abnormal conditions compared to its activity in the assay under normal physiological conditions.

[0055] In yet another embodiment, the Disclosure provides a method for producing conditionally active molecules having a molecular weight of less than about 3000 a.mu from a parent organic compound, comprising the steps of: modifying the parent organic compound by substituting one or more groups of the parent organic compound with one or more partially charged or charged groups to produce one or more modified organic compounds; and selecting the modified organic compounds that exhibit higher activity in the assay under abnormal conditions compared to their activity in the assay under normal physiological conditions.

[0056] In each of the methods described above, the organic parent compound may have a molecular weight in the range of about 100 a.mu to about 3000 a.mu, or about 100 a.mu to about 1500 a.mu, or about 150 a.mu to about 1250 a.mu, or about 300 a.mu to about 1100 a.mu, or about 400 a.mu to about 1000 a.mu.

[0057] In each of the methods described above, the abnormal condition may be a value of the extracellular condition of senescent cells, and the normal physiological condition may be a different value of the same extracellular condition of normal cells.

[0058] In each of the methods described above, the abnormal condition may be a pH in the range of about 5.0 to about 7.0, or about 5.5 to about 7.0, or about 6.0 to about 7.0, or about 6.2 to about 6.8, and the normal physiological condition is a pH in the range of about 7.0 to about 7.8, or about 7.2 to about 7.8, or about 7.2 to about 7.6.

[0059] In each of the methods described above, the conditionally active protein may be conjugated with a drug selected from a toxic agent, a radiopharmaceutical, or a D-retroinverso peptide.

[0060] In each of the embodiments described above, the D retroinverso peptide may include LTLRKEPASE IAQSILEAYS QNGWANRRSG GKRP (SEQ ID NO: 5), LTLRKEPASE IAQSILEAYS QNGWANRRSG GKRPPPRRRQ RRKKRG (SEQ ID NO: 6), or SEIAQSILEAYSQNGW (SEQ ID NO: 7). [Brief explanation of the drawing]

[0061] [Figure 1] This plot shows the selectivity of the conditionally activated antibody selected in Example 9 at pH 6.0, which is above pH 7.4. [Figure 2]This diagram illustrates the formation of salt crosslinks in deoxyhemoglobin, where three amino acid residues form two salt crosslinks, stabilizing the T quaternary structure of deoxyhemoglobin and leading to a lower affinity for oxygen. [Figure 3] This is a diagram illustrating the structure of a chimeric antigen receptor (CAR). [Figure 4] This demonstrates the binding activity of conditionally active antibodies to antigens assayed in different buffer solutions. [Figure 5] This study demonstrates the effect of changing the composition of Krebs buffer on the binding activity of conditionally active antibodies. [Figure 6] The binding activity of the three different conditionally active antibodies was shown to be dependent on the presence and concentration of bicarbonate at H7.4, as described in Example 12. [Figure 7] This paper illustrates the design principles for D-retroinverso (DRI) peptides derived from natural or wild-type peptides. [Figure 8] This diagram shows the signaling pathways that regulate the FOXO family, including FOXO4. "+p" indicates phosphorylation, "-p" indicates dephosphorylation, and "+m" indicates methylation. Arrows indicate activation, and lines with crossing bars at the end indicate inhibition. Each of these is associated with a target gene. [Figure 9A] Untreated MCF-7 cells are shown. [Figure 9B] This image shows MCF-7 cells treated with 1 μM palbociclib isethionate. [Figure 9C] This shows the separation of untreated and treated MCF-7 cells by fluorescence-activated cell sorting (FACS). [Figure 9D] The target expression profiles of untreated MCF-7 cells and MCF-7 cells treated with palbociclib isethionate are shown. [Figure 10A] Untreated MDA-MB231 cells are shown. [Figure 10B] This image shows MDA-MB231 cells treated with 1 μM palbociclib isethionate. [Figure 10C]This image shows the isolation of untreated MDA-MB231 cells and MDA-MB231 cells treated with palbociclib isethionate by FACS. [Figure 10D] The target expression profiles of untreated MDA-MB231 cells and MDA-MB231 cells treated with palbociclib isethionate are shown. [Figure 11A] Untreated MDA-MB468 cells are shown. [Figure 11B] This image shows MDA-MB468 cells treated with 1 μM palbociclib isethionate. [Figure 11C] This shows that untreated MDA-MB468 cells and MDA-MB468 cells treated with palbociclib isethionate could not be separated by FACS. [Figure 11D] Untreated MDA-MB468 cells and MDA-MB468 cells treated with palbociclib isethionate show similar target expression profiles. [Figure 12A] Untreated MDA-MB231 cells are shown. [Figure 12B] This image shows MDA-MB231 cells treated with palbociclib isethionate. [Figure 13A] Untreated MDA-MB468 cells are shown. [Figure 13B] This image shows MDA-MB468 cells treated with palbociclib isethionate. [Figure 14A] This shows FACS cell sorting of untreated MDA-MB231 cells that were negative for B-gal staining. [Figure 14B] This shows FACS cell sorting of MDA-MB231 cells treated with palbociclib isethionate, which were negative for B-gal staining. [Figure 14C] This shows FACS cell sorting of untreated MDA-MB231 cells that were positive for B-gal staining. [Figure 14D] This shows FACS cell sorting of MDA-MB231 cells treated with palbociclib isethionate, which were positive for B-gal staining. [Figure 15A] This shows the FACS sorting of untreated MDA-MB231 cells. [Figure 15B] This shows FACS sorting of MDA-MB231 cells treated with palbociclib isethionate. [Figure 16A] This shows FACS cell sorting of untreated MDA-MB468 cells that were negative for B-gal staining. [Figure 16B] This shows FACS sorting of MDA-MB468 cells treated with palbociclib isethionate, which were negative for B-gal staining. [Figure 16C] This shows FACS cell sorting of untreated MDA-MB468 cells that were positive for B-gal staining. [Figure 16D] This shows FACS cell sorting of MDA-MB468 cells treated with palbociclib isethionate, which were positive for B-gal staining. [Figure 17A] This shows the FACS sorting of untreated MDA-MB468 cells. [Figure 17B] This shows FACS sorting of MDA-MB468 cells treated with palbociclib isethionate. [Figure 18A] This shows the CD73 expression levels of MDA-MB231 and MDA-MB468 cells before and after treatment with palbociclib isethionate. [Figure 18B] This demonstrates senescent cell killing by an anti-CD73 conditionally activated antibody. [Modes for carrying out the invention]

[0062] definition To facilitate understanding of the examples provided herein, certain frequently occurring methods and / or terms are defined herein.

[0063] Terms such as "about", "activity", "drug", "ambiguous base requirement", "amino acid", "amplification", "chimeric properties", "homogeneous", "comparison window", "conservative amino acid substitution", "corresponding to", "effective degradation", "defined sequence framework", "digestion", "directed ligation", "DNA shuffle", "drug" or "drug molecule", "effective amount", "electrolyte", "epitope", "enzyme", "evolution" or "evolving", "fragment", "derivative", "analog", "full range of single amino acid substitution", "gene", "genetic instability", "heterogeneity", "homologous or homeologous", "industrial application", "identical" or "identity", "area of ​​identity", "isolated", "isolated nucleic acid", "ligand", "ligation", "linker" or "spacer", "microenvironment", "molecular properties to evolve", "mutation", "naturally occurring", "normal physiological conditions" or "wild-type operating conditions", "nucleic acid molecule", "nucleic acid molecule", "~ "coding nucleic acid sequence" or "DNA coding sequence of ~" or "nucleotide sequence encoding ~", "promoter sequence", "enzyme (protein) encoding nucleic acid" or "enzyme (protein) encoding DNA" or "enzyme (protein) encoding polynucleotide", "specific nucleic acid molecular species", "assembling a working nucleic acid sample into a nucleic acid library", "nucleic acid library", "nucleic acid construct" or "nucleotide construct" or "DNA construct", "construct", "oligonucleotide" or "oligo", "homogenetic", "operably linked", "operably linked to ~", "parent polynucleotide set", "patient" or "subject", "physiological conditions", "population", "substitute form", "pre-pro-form", "pseudo-random", "pseudo-repeating unit", "random peptide library", "random peptide sequence", "receptor", "recombinant", "synthetic", "related polynucleotide", "reductive recombinationThe definitions of "reassortment," "reference sequence," "comparison window," "sequence identity," "percentage of sequence identity," "substantially identical," "reference sequence," "repetition index (RI)," "restriction site," "selective polynucleotide," "sequence identity," "similarity," "specifically binding," "specific hybridization," "specific polynucleotide," "stringent hybridization conditions," "substantially identical," "substantially pure enzyme," "substantially pure," "to treat," "variable segment," "mutant," "wild type," "wild type protein" or "wild type biological protein," "parent molecule" or "target protein," "to work," "conditionally active antibody," "antibody-dependent cell-mediated cytotoxicity" or "ADCC," "cancer" and "oncogenic," "multispecific antibody," "full-length antibody," "library," "recombinant antibody," and "individual" or "subject" are the same as in WO2016 / 138071.

[0064] As used herein, the term “antibody” refers to intact immunoglobulin molecules capable of binding to an antigen’s epitope, and fragments of immunoglobulin molecules, e.g., Fab, Fab', (Fab')2, Fv, and SCA fragments. These antibody fragments, which retain some ability to selectively bind to the antigen (e.g., polypeptide antigen) of the antibody to be induced, can be prepared using methods well known in the art (see, for example, Harlow and Lane above), which are further described below. Antibodies useful in the practice of the claimed invention may be IgG1, IgG2, IgG3, IgG4, IgM, IgA1, IgA2, sIgA, IgD, or IgE. Using antibodies, the preparation of the antigen can be isolated by immunoaffinity chromatography. Various other uses of such antibodies include for diagnosing and / or staging diseases (e.g., neoplasms), as well as for therapeutic applications to treat diseases such as neoplasms, autoimmune diseases, AIDS, cardiovascular diseases, infectious diseases, and similar conditions. Chimeric antibodies, human-like antibodies, humanized antibodies, or fully human antibodies are particularly useful for administration to human patients.

[0065] Fab fragments consist of monovalent antigen-binding fragments of antibody molecules and can be generated by digesting the entire antibody molecule with the enzyme papain, which produces fragments consisting of the light and heavy chains of the intact antibody.

[0066] The Fab' fragment of an antibody molecule can be obtained by treating the entire antibody molecule with pepsin and then reducing it to produce a molecule consisting of parts of the intact light and heavy chains. Two Fab' fragments are obtained from antibody molecules treated using this method.

[0067] The (Fab')2 fragment of an antibody can be obtained by treating the entire antibody molecule with the enzyme pepsin without subsequent reduction. The (Fab')2 fragment is a dimer of two Fab' fragments held together by two disulfide bonds.

[0068] An Fv fragment is defined as a genetically engineered fragment containing a variable light chain region and a variable heavy chain region, expressed as two separate strands.

[0069] A single-chain antibody ("SCA" or scFv) is a genetically engineered single-chain molecule comprising a variable region of a light chain and a variable region of a heavy chain linked by a suitable flexible polypeptide liner, and potentially containing additional amino acid sequences at the amino and / or carboxyl terminals. For example, a single-chain antibody may include a tether segment for linkage to an encoding polynucleotide. A functional single-chain antibody generally comprises a sufficient portion of the variable region of the light chain and a sufficient portion of the variable region of the heavy chain to retain the properties of a full-length antibody for binding to a specific target molecule or epitope.

[0070] As used herein, the terms “antigen” or “Ag” are defined as molecules capable of eliciting an immune response. This immune response may include either antibody production or activation of specific immune-competent cells, or both. Those skilled in the art will understand that virtually any macromolecule, including proteins or peptides, can act as an antigen. It will be immediately apparent that antigens can be constructed, synthesized, or derived from biological samples. Such biological samples may include, but are not limited to, tissue samples, tumor samples, cells, or biological fluids.

[0071] As used herein, the term "apoptosis" refers to a single-cell-acting mechanism of cell death characterized by cellular atrophy, chromatin coagulation, and fragmentation of the cell into membrane junctions that are eliminated by phagocytosis. The term "apoptosis" is often used synonymously with the term "programmed cell death."

[0072] As used herein, the term “apoptosis-inducing activity” refers to the intrinsic property of a compound to selectively induce apoptosis in (i) specific cell types and / or (ii) cells at a particular stage of development or differentiation, in response to internal or external stimuli. Experienced practitioners are aware of the existence of in vitro standard assays for measuring the apoptosis-inducing activity of compounds in cell cultures, such as tests that assess the levels of cytoplasmic cytochrome C (a marker of apoptosis) and TUNEL (a marker of apoptosis). Using these standard assays, experienced practitioners can easily assess and compare the apoptosis-inducing activity of different compounds in relation to different cell types or cells at different stages of development, for example, in comparison of senescent and non-senescent cells. Other standard apoptosis assays include the annexin V assay and cleaved caspase-3 staining.

[0073] The terms “biosimilar” or “post-product” are used in a manner consistent with the practical definition published by the U.S. Food and Drug Administration (FDA), which defines a biosimilar as a product that is “highly similar” to a reference product (with only minor differences in clinically inactive components). In practice, there can be no clinically significant differences in safety, purity, and efficacy between a reference product and a biosimilar product (Public Health Service (PHS) Act §262). A biosimilar may also conform to one or more guidelines adopted by the European Medicines Agency's Committee for Medicinal Products for Human Use (CHMP) on May 30, 2012, and published by the European Union as “Guideline on similar biological medicinal products containing monoclonal antibodies—non-clinical and clinical issues” (Document Reference EMA / CHMP / BMWP / 403543 / 2010). For example, a “biosimilar antibody” typically refers to a subsequent version of an innovator’s antibody (reference antibody) produced by a different company. The differences between biosimilar antibodies and reference antibodies can include post-translational modifications, such as the attachment of other biochemical groups to the antibody (e.g., phosphates, various lipids and carbohydrates); post-translational proteolytic cleavage; alteration of the chemical properties of amino acids (e.g., formylation); or many other mechanisms. Other post-translational modifications may be the result of manufacturing process operations, such as glycation, which can occur upon exposure of the product to reducing sugars. In some cases, storage conditions may be acceptable for certain degradation pathways that occur, such as oxidation, deamidation, or aggregation, because all of these product-related variants can be present in biosimilar antibodies.

[0074] The terms “cancer” and “malignant” refer to or describe a physiological condition in mammals typically characterized by unregulated cell growth / proliferation. “Tumor” includes one or more cancerous cells. Examples of cancer include, but are not limited to, carcinomas, lymphomas, blastomas, sarcomas, and leukemia or lymphomas. More specific examples of such cancers include squamous cell carcinoma (e.g., squamous cell carcinoma), small cell lung cancer, non-small cell lung cancer ("NSCLC"), lung cancers such as adenocarcinoma and squamous cell carcinoma of the lung, peritoneal cancer, hepatocellular carcinoma, gastric 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 or uterine cancer, salivary gland cancer, kidney or renal cancer, prostate cancer, vulvar cancer, thyroid cancer, liver cancer, anal cancer, testicular cancer, and head and neck cancer.

[0075] The term “conditionally active protein” refers to a variant or mutant of a parent protein that exhibits greater or less activity under one or more abnormal conditions compared to the activity of a control or under normal physiological conditions. This conditionally active protein also exhibits activity in selected areas within the body and / or increased or decreased activity under abnormal or acceptable physiological conditions. Normal physiological conditions are conditions considered within the normal range at a location of the subject, such as the tissue or organ of the administration or site of action. Abnormal conditions are conditions that deviate from the normally acceptable range for that location. In one embodiment, the conditionally active protein is virtually inactive under normal physiological conditions but active under abnormal or acceptable conditions. For example, in one embodiment, an evolved conditionally active protein is virtually inactive at body temperature but active at lower or higher temperatures. In another embodiment, the conditionally active protein may be reversibly or irreversibly inactivated under normal physiological or control conditions. In a further embodiment, the conditionally active protein is a therapeutic protein. In another embodiment, the conditionally active protein is used as a drug or therapeutic agent. In yet another embodiment, the conditionally active protein is more or less active in highly oxygenated blood, such as in a lower pH environment found after passing through the lungs or in the kidneys. The conditionally active protein may also be a conditionally active biological protein.

[0076] As used herein, the term "cyclic peptide" refers to a polypeptide chain in which the amino and carboxyl terms themselves are linked to one another by peptide bonds that form a cyclic chain (i.e., between a carboxyl residue and another alpha-amine). For the purposes of this application, cyclic peptides may also include non-peptide linkages, such as non-alpha-amide linkages, and thioether linkages between Trp and Cys residues. The length of a cyclic peptide may be in the range of about 5 to about 500 amino acids, or about 8 to about 300 amino acids, or about 8 to about 200 amino acids, or about 10 to about 100 amino acids, or about 10 to about 50 amino acids. In addition, amino acids other than naturally occurring amino acids, such as β-alanine, phenylglycine, and homoarginine, may be included in the cyclic peptide.

[0077] As used herein, the abbreviation "DRI" refers to a D-retroinverso isoform of an L-peptide in which the amino acid sequence is reversed compared to a fragment or full-length version of the natural or wild-type protein, and at least some of the amino acid residues in the DRI peptide are D amino acid residues instead of L amino acid residues in the natural or wild-type protein (Figure 7). D-retroinverso peptides can be produced by identifying the amino acid sequence of a fragment or full-length version of the natural protein, reversing the sequence, and synthesizing D-retroinverso peptides using known methods to provide peptides that have the reversed amino acid sequence of a fragment or full-length version of the natural protein and contain a sufficient number of D amino acids to provide the desired function in the D-retroinverso peptide.

[0078] The terms “disease or illness for which removal of senescent cells is beneficial,” “disease or illness associated with the presence of senescent cells,” and “disorder for which removal of senescent cells is beneficial” are used interchangeably and refer to any such disease or illness in a mammalian subject, e.g., a human subject, for which removal, or cleansing or reduction of the viability of senescent cells, is beneficial to the disease or disease-affected subject. The terms encompass situations in which senescent cells are one or the sole cause of the disease, or contribute to its progression. The terms further relate to situations in which senescent cells may cause the disease or illness in the subject in the future. For example, a treatment for a disease or illness for which removal of senescent cells is beneficial is a disease or illness that is prevented, can be prevented, or improved by removing senescent cells. For example, chemotherapy and radiotherapy are known to induce cellular senescence. Removing these senescent cells to prevent the onset of a disease or illness associated with cellular senescence is advantageous. The terms further encompass diseases or illnesses for which removal of senescent cells alleviates or reduces the symptoms of the disease or illness.

[0079] The removal of senescent cells is particularly beneficial when a disease or illness can be cured or prevented, or when the symptoms of such a disease or illness can be reduced or alleviated. The removal of senescent cells may be achieved by inducing apoptosis in senescent cells. For example, diseases or conditions for which the removal of senescent cells is beneficial are selected from the group formed by chronic inflammatory diseases such as atherosclerosis, arthritis or arthropathy, cancer, osteoarthritis, diabetes, diabetic ulcers, kyphosis, sclerosis, liver failure, cirrhosis, Hutchinson-Gilford progeria syndrome (HGPS), disc disease, osteoporosis, dementia, (cardio)vascular disease, obesity, metabolic syndrome, acute myocardial infarction, emphysema, insulin sensitivity, Boutonuse fever, sarcopenia, Alzheimer's disease, neurodegenerative diseases such as Huntington's disease or Parkinson's disease, cataracts, anemia, hypertension, fibrosis, age-related macular degeneration, COPD, asthma, renal failure, incontinence, hearing loss such as hearing impairment, visual loss such as blindness, sleep disorders, pain such as joint pain or leg pain, balance disorders, phobias, depression, dyspnea, weight loss, alopecia, muscle loss, decreased bone density, frailty and / or poor health. Diseases or illnesses for which the removal of senescent cells is beneficial are diseases or illnesses related to or leading to inflammation, specifically chronic inflammation in mammals, such as humans, where the inflammation is induced or mediated by senescent cells. In some embodiments, the senescent cells induced or mediated by the inflammation are at least partially present in the same organ or tissue as the organ or tissue affected by the disease or illness, more preferably in the same tissue.

[0080] As used herein, “disease or illness associated with the presence of senescent cells” means any disease or illness in a mammal, such as a human subject, in which the presence of senescent cells or cellular senescence in the said subject leads to the said disease or illness in the said subject. In this context, “leading to” may mean, in particular, (i) as at least a partial cause of the disease or illness, or (ii) as at least a partial cause of the symptoms. In some embodiments, diseases or illnesses associated with the presence of senescent cells are selected from the group formed by chronic inflammatory diseases such as atherosclerosis, arthritis or arthropathy, cancer, osteoarthritis, diabetes, diabetic ulcers, kyphosis, sclerosis, liver failure, cirrhosis, Hutchinson-Gilford progeria syndrome (HGPS), disc disease, osteoporosis, dementia, (cardio)vascular disease, obesity, metabolic syndrome, acute myocardial infarction, emphysema, insulin sensitivity, Boutonuse fever, sarcopenia, neurodegenerative diseases such as Alzheimer's disease, Huntington's disease or Parkinson's disease, cataracts, anemia, hypertension, fibrosis, age-related macular degeneration, COPD, asthma, renal failure, incontinence, hearing loss such as hearing impairment, visual loss such as blindness, sleep disorders, pain such as joint pain or leg pain, balance disorders, phobias, depression, dyspnea, weight loss, alopecia, muscle loss, decreased bone density, frailty and / or poor health. Specific diseases or conditions for which the removal of senescent cells is advantageous include diseases or conditions related to or leading to inflammation, typically chronic inflammation in mammals such as humans, where the inflammation is induced or mediated by senescent cells. In some embodiments, the senescent cells induced or mediated by the inflammation are at least partially present in the same organ or tissue as the organ or tissue affected by the disease or condition.

[0081] As used herein, the term “extracellular conditions of senescent cells” refers to the extracellular environment conditions immediately surrounding one or more senescent cells, which differ from the same conditions surrounding non-senescent cells. The extracellular environment of senescent cells may include, for example, any extracellular matrix or fluid adjacent to the senescent cells.

[0082] As used herein, the terms “FOXO4 peptide” and “FOXO4 protein” refer to the protein translated from the transcript of the forkheadbox protein O4 (FOXO4) gene. FOXO4 has two variants (SEQ ID NO:1 and SEQ ID NO:2). The term “FOXO4 DRI peptide” refers to a D retroinverso peptide having the reverse amino acid sequence of at least one fragment of the FOXO4 protein, and containing several, for example, all D amino acid residues.

[0083] The term "full-length antibody" refers to the antigen-binding variable region (V H or V L This refers to an antibody that includes a light chain constant domain (CL) and heavy chain constant domains CH1, CH2, and CH3. The constant domains may be the constant domain of the native sequence (e.g., the human native sequence constant domain) or an amino acid sequence variant thereof. Depending on the amino acid sequence of the heavy chain constant domain, full-length antibodies can be assigned to different "classes". There are five main classes of full-length antibodies: IgA, IgD, IgE, IgG, and IgM, some of which can be further subdivided into "subclasses" (isotypes), e.g., IgG1, IgG2, IgG3, IgG4, IgA, and IgA2. The heavy chain constant domains corresponding to different classes of antibodies are called alpha, delta, epsilon, gamma, and mu, respectively.

[0084] The “individual” or “subject” is a mammal. Mammals include, but are not limited to, domesticated animals (e.g., cattle, sheep, cats, dogs, and horses), primates (e.g., humans and non-human primates, e.g., monkeys), rabbits, and rodents (e.g., mice and rats).

[0085] As used herein, the term "library" refers to an assembly of proteins in a single pool. Libraries may be prepared using recombinant DNA technology. For example, a protein library may be prepared by inserting an assembly of cDNA or any other protein-coding DNA into an expression vector. Similarly, a bacteriophage display library of wild-type proteins may be prepared by inserting a cDNA or protein-coding DNA assembly into a phage genome. The cDNA assembly may be generated from a selected cell population or tissue sample, such as by the method disclosed by Sambrook et al. (Molecular Cloning, Cold Spring Harbor Laboratory Press, 1989). cDNA assemblies from selected cell types are also commercially available from companies such as Stratagene®. The wild-type protein library as used herein is not an assembly of biological samples.

[0086] As used herein, the term “ligand” refers to a molecule that is recognized by a particular receptor and specifically binds to that receptor at one or more binding sites. Examples of ligands include, but are not limited to, cell membrane receptor agonists and antagonists, toxins and venoms, viral epitopes, hormones, hormone receptor peptides, enzymes, enzyme substrates, cofactors, drugs (e.g., opioids, steroids, etc.), lectins, sugars, polynucleotides, nucleic acids, oligosaccharides, proteins, and monoclonal antibodies. Typically, a ligand comprises two structural parts: a first part involved in ligand binding to the receptor and a second part not involved in such binding.

[0087] As used herein, the term “multispecific antibody” refers to an antibody that has binding specificity to at least two different epitopes. Exemplary multispecific antibodies may bind to both BBB-R and brain antibodies. Multispecific antibodies can be prepared as full-length antibodies or antibody fragments (e.g., F(ab')2 bispecific antibodies). Manipulated antibodies having two, three or more (e.g., four) functional antigen-binding sites are also conceivable (see, for example, US2002 / 0004587 A1).

[0088] As used herein, the term “non-natural amino acid” refers to any amino acid not found in nature. Examples of non-natural amino acids include D-amino acids, amino acids with side chains not found in nature, and peptidomimetics. Examples of peptidomimetics include, but are not limited to, β-peptides, γ-peptides, and d-peptides; oligomers with backbones that can employ helical or sheet conformations, such as compounds with backbones utilizing bipyridine segments, compounds with backbones utilizing solubility interactions, compounds with backbones utilizing side-chain interactions, compounds with backbones utilizing hydrogen bonding interactions, and compounds with backbones utilizing metal coordination. Non-natural amino acids also include residues with side chains resistant to nonspecific protein adsorption, which can be designed to increase the presentation of antimicrobial peptides in biological fluids, and / or residues with polymerizable side chains that enable the synthesis of polymer brushes by utilizing non-natural amino acid residues within peptides as monomer units.

[0089] As used herein, the term “parent protein” refers to a polypeptide or protein that can be evolved to produce a conditionally active polypeptide or protein using the method of the present invention. The parent protein may be a wild-type protein or a non-naturally derived protein. For example, a therapeutic polypeptide or protein, or a mutant or variant polypeptide or protein, may be used as the parent polypeptide or protein. The parent protein may also be a fragment of another naturally derived protein, a wild-type protein, a therapeutic protein, or a mutant protein. Examples of parent proteins include antibodies, antibody fragments, enzymes, enzyme fragments, cytokines and their fragments, hormones and their fragments, ligands and their fragments, receptors and their fragments, regulatory proteins and their fragments, and growth factors and their fragments.

[0090] As used herein, the term "polypeptide" refers to a polymer in which monomers are amino acids linked together through peptide or disulfide bonds. A polypeptide may be a full-length naturally occurring amino acid chain or a fragment thereof, a mutant or variant, for example, a selected region of the relevant amino acid chain within a binding interaction. A polypeptide may also be a synthetic amino acid chain, or a combination of a naturally occurring amino acid or a fragment thereof with a synthetic amino acid chain. A fragment is a part of a full-length protein and refers to an amino acid sequence typically between about 8 and about 500 amino acids in length, preferably about 8 to about 300 amino acids, more preferably about 8 to about 200 amino acids, and more preferably about 10 to about 50 or 100 amino acids. In addition, amino acids other than naturally occurring amino acids, such as β-alanine, phenylglycine, and homoarginine, may be contained in the polypeptide. Commonly encountered amino acids that are not genetically encoded may be contained in the polypeptide. These amino acids may be D- or L-optical isomers. D-isomers are preferred for use in the specific situations further described below. In addition, other peptidomimetics are also useful, for example, in the linker sequences of polypeptides (see Spatola, 1983, 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 include any significant difference from the term "polypeptide" other than that it includes structures containing two or more polypeptide chains held together by covalent or noncovalent bonds.

[0091] As used herein, the term "protein" refers to a polymer in which monomers are amino acids and are linked to one another through peptides or disulfide bonds. A protein may be an extended naturally occurring amino acid chain or a fragment thereof, a mutant or variant, for example, a selected region of the relevant amino acid chain within a binding interaction. A protein may be a cyclic peptide having an amino acid polymer that forms a cyclic structure using all or part of the polymer. A protein may be a synthetic amino acid chain, an amino acid chain containing non-natural amino acids, or a combination of a natural amino acid chain or fragment thereof with a synthetic amino acid chain. A fragment is a part of a full-length protein and refers to an amino acid sequence that is typically between about 8 and about 500 amino acids in length, preferably between about 8 and about 300 amino acids, more preferably between about 8 and about 200 amino acids, and more preferably between about 10 and about 50 or 100 amino acids. In addition, amino acids other than naturally occurring amino acids, such as β-alanine, phenylglycine, and homoarginine, may be contained in the polypeptide. Commonly encountered amino acids that are not genetically encoded may be contained in the polypeptide. The amino acids may be D- or L-optical isomers. D-isomers are preferred for use in specific situations further described below. In addition, other peptidomimetics are also useful, for example, in the linker sequences of polypeptides (see Spatola, 1983, 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 include any significant difference from the term "polypeptide" other than that it includes structures containing two or more polypeptide chains held together by covalent or noncovalent bonds.

[0092] As used herein, the term "receptor" refers to a molecule that has affinity for a given ligand. Receptors may be naturally occurring or synthetic molecules. Receptors may be used in their unmodified state or as aggregates with other molecular species. Receptors can be attached to binding membranes covalently or noncovalently, either directly or via specific binding agents. Examples of receptors include, but are not limited to, antibodies such as monoclonal antibodies and antisera that react with specific antigenic determinants (such as viruses, cells, or other materials), cell membrane receptors, complex carbohydrates and glycoproteins, enzymes, and hormone receptors. Ligand binding to a receptor represents a combination of ligand and receptor molecules through specific molecular recognition, forming a complex that can be detected by various ligand-receptor binding assays known to those skilled in the art.

[0093] As used herein, the terms “senescence” or “cellular senescence” refer to the transition from actively dividing cells to metabolically active, non-dividing cells. The term “senescence” refers to a state in which a cell enters after multiple divisions, in which future cell division is inhibited, even though the cell remains metabolically active.

[0094] As used herein, the term “senescent cells” refers to cells that are metabolically active but have permanently left the cell cycle (see, e.g., Campisi, Cell, vol. 120, pp. 513-522, 2005). Senescent cells do not replicate and have one or more of the following further characteristics attributable to senescence: cell cycle arrest in G1 phase; enlarged and flattened morphology; granular enlargement; staining of β-galactosidase activity at pH 6; senescence-related heterochromatin formation; and characteristic gene expression partially regulated by p16 and p21. Examples of senescent cells include senescent adipocytes, senescent endothelial cells, senescent fibroblasts, senescent neurons, senescent epithelial cells, senescent mesenchymal cells, senescent smooth muscle cells, senescent macrophages, and senescent chondrocytes.

[0095] As used herein, the term “senescent cell scavenger” refers to an agent that selectively (preferentially or to a greater degree) destroys, kills, removes, or facilitates the selective destruction of senescent cells. In other words, the senescent cell scavenger destroys or kills senescent cells in a manner that is biologically, clinically, and / or statistically significant compared to its ability to destroy or kill non-senescent cells. The senescent cell scavenger may be a small compound or a biological molecule such as a protein or polynucleotide. The senescent cell scavenger is used in an amount and time that is sufficient to selectively kill a defined number of senescent cells but insufficient to kill (destroy, cause death) a clinically significant or biologically significant number of non-senescent cells. In certain embodiments, the senescent cell scavenger described herein modifies at least one signaling pathway in a manner that induces (initiates, stimulates, triggers, activates, promotes) and brings about (i.e., induces, leads to) the death of senescent cells. The senescent cell scavenging agent may, for example, modify either or both of the cell survival signaling pathway (e.g., the Akt pathway) or the inflammatory pathway by antagonizing, for example, proteins in viable cells and / or the inflammatory pathway in senescent cells.

[0096] As used herein, the term "small molecule" refers to a molecule or ion having a molecular weight of less than 900 a.mu, more preferably 500 a.mu, more preferably less than 200 a.mu, or more preferably less than 100 a.mu. In the assays and environments of the present invention, small molecules may often exist as mixtures of molecules and deprotonated ions of molecules, depending primarily on the pH of the assay or environment.

[0097] As used herein, the term "targets associated with senescent cells" means molecules, such as proteins, that are located on the surface of senescent cells (e.g., cell membrane proteins), or are present within senescent cells, or are secreted by senescent cells into the extracellular environment of senescent cells.

[0098] As used herein, the term “therapeutic protein” refers to any protein and / or polypeptide that can be administered to a mammal, for example by an examiner or physician, to induce a biological or medical response in the tissue, system, animal, or human being being sought. A therapeutic protein may induce one or more biological or medical responses. Examples of therapeutic proteins include antibodies, enzymes, hormones, cytokines, regulatory proteins, and fragments thereof.

[0099] As used herein, the term “therapeutic effective dose” means any dose that, indefinitely, results in the cure, prevention, or improvement of a disease, disorder, or side effect, or slows the progression of a disease or disorder, compared to a corresponding subject not receiving such dose. The term also includes, within its scope, doses that are effective in enhancing normal physiological function, and doses that are effective in inducing physiological function in a patient to enhance or assist the therapeutic effect of a second medicinal agent.

[0100] As used herein, the terms “to treat” and “treatment” refer to the medical management of a disease, disorder, or illness in a subject (i.e., a patient) (e.g., Stedman's Medical Dictionary). Generally, an appropriate dose and treatment regimen provides a senescent agent in an amount sufficient to provide therapeutic and / or preventive benefits. Therapeutic benefits to a subject to whom the senescent agents described herein are administered include, for example, improved clinical outcomes in which the subject prevents, delays, or inhibits (reduces) undesirable physiological changes associated with the disease, or prevents, delays, or inhibits (reduces) the progression or severity of such disease.

[0101] As used herein, the term “tumor microenvironment” refers to the microenvironment within and around solid tumors that supports the growth and metastasis of tumor cells. The tumor microenvironment includes surrounding blood vessels, immune cells, fibroblasts, other cells, soluble factors, signaling molecules, extracellular matrix, and mechanical cues that enable the promotion of neoplasm transformation, support tumor growth and invasion, protect the tumor’s host immune response, cultivate treatment resistance, and provide a niche for dormant metastases to grow. The tumor and its surrounding microenvironment are closely related and constantly interacting. Tumors can influence the microenvironment by releasing extracellular signals, promoting tumor angiogenesis, and inducing peripheral immune resistance, while immune cells within the microenvironment can influence the growth and evolution of cancer cells. Swarts et al. “Tumor Microenvironment Complexity: Emerging Roles in Cancer Therapy,” Cancer Res, vol., 72, pages 2473-2480, 2012; Weber et al., “The tumor microenvironment,” Surgical Oncology, vol. 21, pages 172-177, 2012; Blagosklonny, “Antiangiogenic therapy and tumor progression,” Cancer Cell, vol. 5, pages 13-17, 2004; Siemann, “Tumor microenvironment,” Wiley, 2010; and Bagley, “The tumor microenvironment,” Springer, 2010.

[0102] Detailed description It should be noted that, as used herein and in the appended claims, the singular forms “a,” “an,” and “the” include multiple subjects unless otherwise clearly indicated. Furthermore, the terms “a” (or “an”), “one or more,” and “at least one” may be used interchangeably herein. The terms “including,” “incorporating,” “having,” and “constructed from” may also be used interchangeably.

[0103] Unless otherwise indicated, all figures used herein and in the claims to express quantities, properties, e.g., molecular weight, percentage, ratio, reaction conditions, etc., of raw materials, should be understood to be modified in all examples by the term "about," whether or not the term "about" is present. Thus, unless otherwise indicated, the numerical parameters expressed herein and in the claims are approximations that may vary depending on the desired properties sought as obtained by this disclosure. At a minimum, and not in an attempt to limit the application of the doctrine of the equivalent of the claims, each numerical parameter should be interpreted at least in light of the number of significant figures reported and by applying the usual rounding techniques. While the numerical ranges and parameters that represent the broad scope of this disclosure are approximations, the numerical values ​​shown in specific examples are reported as precisely as possible. However, any numerical value inherently contains certain errors that inevitably arise from the standard deviation found in each test measurement.

[0104] It should be understood that each component, compound, substituent, or parameter disclosed herein is to be interpreted as being disclosed for use alone or in combination with one or more other components, compounds, substituents, or parameters disclosed herein.

[0105] Similarly, it should be understood that each quantity / value or range of quantity / value for each component, compound, substituent or parameter disclosed herein is to be interpreted as being disclosed in combination with each quantity / value or range of quantity / value disclosed for any other component(s), compound(s), substituent(s), or parameter(s) disclosed herein, and that any combination or range of quantity / value for two or more components(s), compounds(s), substituent(s), or parameters disclosed herein is thus disclosed in combination with each other for the purposes of this specification.

[0106] It will be further understood that each range disclosed herein should be interpreted as a disclosure of each specific value within the disclosed range having the same number of significant figures. Thus, the range 1–4 should be interpreted as representing a disclosure of the values ​​1, 2, 3, and 4. Furthermore, it will be understood that each lower limit of each range disclosed herein must be interpreted as being disclosed in combination with each upper limit of each range disclosed herein and each specific value within each range for the same component, compound, substitution, or parameter. Thus, this disclosure should be interpreted as a disclosure of all ranges obtained by combining each lower limit of each range with each upper limit of each range or each specific value within each range, or by combining each upper limit of each range with each specific value within each range.

[0107] Furthermore, any specific amount / value of a component, compound, substituent, or parameter disclosed herein or in the examples shall be interpreted as either a lower or upper limit disclosure of a range, and thus can be combined with any other lower or upper limit of the range or specific amount / value of the same component, compound, substituent, or parameter elsewhere in this application to form the range of that component, compound, substituent, or parameter.

[0108] The present invention provides a method for generating a conditionally active protein having activity against senescent cells from a parent protein that binds to a target associated with senescent cells. The method is as follows: (i) The step of evolving the DNA encoding the parent protein using one or more evolutionary techniques to produce mutant DNA; (ii) The step of expressing the mutant DNA to obtain a mutant protein; (iii) The step of subjecting the mutant protein to an assay under extracellular conditions of the senescent cells and an assay under normal physiological conditions; (iv)(a) Decreased activity of the parent protein in the assay under normal physiological conditions compared to the activity of the parent protein in the assay, and increased activity of the conditionally active protein in the assay under extracellular conditions of senescent cells compared to the activity of the conditionally active protein in the assay under normal physiological conditions; and (b) a decrease in the activity of the parent protein in the assay compared to the activity of the parent protein in the assay under normal physiological conditions, and an increase in the activity of the parent protein in the assay under extracellular conditions of senescent cells compared to the activity of the parent protein in the assay under extracellular conditions of senescent cells, The steps include selecting the conditionally active protein from the mutant protein exhibiting at least one of the following: Includes.

[0109] The parent protein may be an antibody, ligand, receptor, or enzyme, or a fragment of any of the aforementioned. Examples of ligands include cytokines and their fragments, hormones and their fragments, regulatory proteins and their fragments, and growth factors and their fragments.

[0110] In the case of antibodies, ligands, or receptors, the parent protein binds to a target associated with senescent cells, and the activity may be binding activity to that target. In the case of enzymes, the parent protein can utilize at least a portion of senescent cells, since the substrate and activity are enzymatic activity that uses at least a portion of senescent cells as the substrate.

[0111] In some embodiments, the parent protein may be a therapeutic protein or a biosimilar.

[0112] Targets associated with senescent cells are typically proteins on senescent cells. In some examples, these targets are proteins on the cell membrane of senescent cells. In some embodiments, these targets are selected from DEP-1, NTAL, EBP50, STX4, VAMP3, ARMCX-3, LANCL1, B2MG, PLD3, and VPS26A. These proteins are recognized as biomarkers for senescent cells as described in WO2015 / 181526. In some embodiments, the target is selected from ITGAV, RAC1, ARHGAP1, RAPGEF1, CRKL, NCKAP1, CDC42, CAPNS2, EBP, FGF1, ISG20, KITLG, LPHN1, MAG, MEF2C, OSBPL3, PFN1, POU5F1, PPP1CB, pl6INK4a, PRKRA, APC, AXL, BCL2L1, CDKN2C, CLYBL, COPG1, DGKA, GBA3, GIT2, IGF1, LCMT2, MADCAM1, MAP3K14, MTHFD2, NAIP, NAPG, NNMT, PARK2, PMS2, PRPF19, PRTG, RAPGEF1, RET, VIT, WEE1, YAP1, and YWHAE.

[0113] In some embodiments, the target is the Fas protein or cell death receptor (DR). Fas is sometimes referred to as tumor necrosis factor receptor superfamily member 6A (TNFSF6). It is a membrane receptor that is easily accessible from the outside of senescent cells. DR is a TNF-associated apoptosis-inducing ligand (TRAIL); see Guicciardi et al., “Life and death by death receptors,” FASEB J. vol. 23, pp. 1625-1637, 2009. Examples of DRs include DR4 and DR5.

[0114] In some embodiments, the target associated with the senescent cells is selected from MDM2, AKT (AKT1, AKT2, and AKT3), NOTCH3, DcR2 (TNFRSF10D), and proteins of the BCL-2 anti-apoptotic protein family. The proteins in this family may have BH1-BH4 domains (BCL-2 (i.e., the BCL-2 protein member of the BCL-2 anti-apoptotic protein family), BCL-xL, BCL-w, Al, MCL-1, and BCL-B); or BH1, BH2, and BH3 domains (BAX, BAK, and BOK); or BH3 domain only (BIK, BAD, BID, BIM, BMF, HRK, NOXA, and PUMA) (see, for example, Cory et al., Nature Reviews Cancer, vol. 2, pp. 647-56, 2002; Cory et al., Cancer Cell, vol. 8, pp. 5-6, 2005; Adams et al, Oncogene, vol. 26, pp. 1324-1337, 2007). Further targets related to senescent cells suitable for use in the present invention are described in Althubiti et al, Cell Death and Disease, vol. 5, p. el528, 2014.

[0115] In some embodiments, the target associated with the senescent cells is selected from misfolded forms of proteins selected from prion proteins (PrP), CD38, Notch-1, CD44, CD59, Fas ligand, TNF receptor, and EGF receptor, as described in US2016 / 0115237. The target may also be p16INK4a, or a protein selected from Tables 1-3 of US 2016 / 0038576.

[0116] In some embodiments, once a target associated with the senescent cells has been selected, the parent protein that binds to the target may be selected to be an enzyme that binds to the selected target and uses at least a portion of the senescent cells as a substrate, or an antibody, ligand, or receptor that binds to the target. Some examples of suitable parent proteins for use in the present invention are described in the “Target Wild-type Proteins” section of WO 2016 / 138071.

[0117] The parent protein may be selected from a library as described in WO 2016 / 138071. In some embodiments, the parent protein is selected from a library by utilizing an assay under conditions such as pH less than 7.0, e.g., 5.0 to less than 7.0, or 5.5 to less than 7.0, or 6.0 to less than 7.0, or 6.2 to 6.8.

[0118] In some other embodiments, the parent protein is selected from a library using a screening solution that does not contain small molecules having pKas between, for example, 6 and 7.5, preferably between 6 and 7, and more preferably between 6.2 and 6.8. Examples of such small molecules are described in this application.

[0119] In some embodiments, the parent protein is an antibody. In some embodiments, the parent protein has one or more preferred characteristics based on what is selected for use as a parent antibody. For example, in certain embodiments, the parent antibody may be selected based on having good binding activity under one or more extracellular conditions of senescent cells, such as a pH of 5.0 to less than 7.0.

[0120] In some embodiments, the parent antibody is selected for its binding activity to a specific epitope. Selection based on binding activity to a specific epitope may be combined with one or more other selection criteria, such as selection for good binding activity under one or more extracellular conditions of senescent cells.

[0121] In other embodiments, the parent antibody is selected based on its internalization efficiency. Selection based on internalization efficiency may be combined with one or more other selection criteria, such as binding activity to a specific epitope or good binding activity under one or more extracellular conditions of senescent cells.

[0122] In other embodiments, the parent antibody may have similar binding activity and / or properties under both normal physiological conditions and extracellular conditions of senescent cells. In such embodiments, the parent antibody is selected based on having the most similar combination of binding activity and / or one or more properties under both normal physiological conditions and extracellular conditions of senescent cells. For example, if normal physiological conditions and extracellular conditions of senescent cells may be pH 7.4 and 6.4, respectively, an antibody having the most similar binding activity at pH 7.4 and 6.4 may be selected as the parent antibody rather than an antibody having less similar binding activity at pH 7.4 and 6.4.

[0123] In some embodiments, the parent protein may be a fragment of a naturally occurring protein. For example, the parent protein may be the catalytic domain of an enzyme, the binding domain of a ligand or receptor, or the variable region of an antibody. In some embodiments, the parent protein may be a peptide or cyclic peptide consisting of only 8 amino acid units.

[0124] After the parental protein has been selected, the DNA encoding the parental protein may evolve using appropriate evolutionary techniques to generate mutant DNA, which may then be expressed to produce mutant proteins for screening to identify conditionally active proteins. Appropriate techniques for evolving the DNA encoding the parental protein, for expressing the mutant DNA to generate mutant proteins, and for screening the mutant proteins are described in WO2016 / 138071.

[0125] If selected, the conditionally active protein may be synthesized in "mimetic" and "peptidomimetic" forms as described in WO2016 / 138071.

[0126] The selected conditionally active protein may also be generated using a polypeptide-expressing cell-generating host or organism. To make the generation process more efficient, the DNA encoding the conditionally active protein may be subjected to codon optimization for the cell-generating host or organism.Codon optimization is described in the following publications: Narum et al., “Codon optimization of gene fragments encoding Plasmodium falciparum merzoite proteins enhances DNA vaccine protein expression and immunogenicity in mice,” Infect. Immun., vol. 69, pp. 7250-3, 2001; Outchkourov et al., “Optimization of the expression of Equistatin in Pichia pastoris, protein expression and purification,” Protein Expr. Purif., vol. 24, pp. 18-24, 2002; and Feng et al., “High level expression and mutagenesis of recombinant human phosphatidylcholine transfer protein using a synthetic gene: evidence for a C-terminal membrane binding domain,” Biochemistry, vol. 39, pp. 15399-409, describing codon optimization in mice; Previous studies have described how codon usage affects protein secretion in Escherichia coli, such as Humphreys et al., "High-level periplasmic expression in Escherichia coli using a eukaryotic signal peptide: importance of codon usage at the 5' end of the coding sequence", Protein Expr. Purif., vol. 20, pp. 252-64, 2000.

[0127] The cell-generating host may be a mammalian cell-generating host selected from one of the following groups: CHO, HEK293, IM9, DS-I, THP-I, Hep G2, COS, NIH 3T3, C33a, A549, A375, SK-MEL-28, DU 145, PC-3, HCT 116, Mia PACA-2, ACHN, Jurkat, MML-I, Ovcar 3, HT 1080, Panc-1, U266, 769P, BT-474, Caco-2, HCC 1954, MDA-MB-468, LnCAP, NRK-49F, and SP2 / 0 cell lines; as well as mouse splenocytes and rabbit PBMCs. The mammalian cell-generating host may be selected from, for example, the CHO or HEK293 cell line. In one specific embodiment, the mammalian cell-generating host is the CHO-S cell line. In another embodiment, the mammalian cell-generating host is the HEK293 cell line.

[0128] In some embodiments, the cell-generating host is yeast cells, such as S. cerevisiae yeast cells or Pichia yeast cells. In some embodiments, the cell-generating 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). The conditionally active protein may also be produced in plant cells or plants (Firek et al., Plant Mol. Biol., vol. 23, p.861, 1993).

[0129] The conditionally active protein may be modified through natural processes or by utilizing the chemical modification techniques described in WO2016 / 138071. The conditionally active protein may also be synthesized by solid-phase chemical peptide synthesis, as also described in WO2016 / 138071.

[0130] The conditionally active protein may be selected using assays under extracellular conditions of senescent cells and / or under normal physiological conditions. The selected conditionally active protein is (a) a decrease in the activity of the parent protein in the assay compared to the activity of the parent protein in the assay under normal physiological conditions, and an increase in the activity of the conditionally active protein in the assay under extracellular conditions of senescent cells compared to the activity of the conditionally active protein in the assay under normal physiological conditions; and (b) a decrease in the activity of the parent protein in the assay compared to the activity of the parent protein in the assay under normal physiological conditions, and an increase in the activity of the parent protein in the assay under extracellular conditions of senescent cells compared to the activity of the parent protein in the assay under extracellular conditions of senescent cells, Show at least one of them.

[0131] The conditions in the assay under extracellular conditions for senescent cells are the same as those in the assay under normal physiological conditions, but have different values. For example, the condition may be pH, and the pH value under normal physiological conditions may be 7.2-7.8 or 7.2-7.6, while the pH value under extracellular conditions for senescent cells may be 6.0-7.0 or 6.2-6.8.

[0132] The activity may be any activity relating to the treatment of any senescent cells, such as the binding activity of a conditionally active antibody to the target, or a specific epitope, or the internalization efficiency of the protein, or, in the case of an enzyme, the activity may be, for example, the enzymatic activity of a conditionally active enzyme to at least a portion of the senescent cells as a substrate.

[0133] The extracellular conditions of senescent cells are selected from one or more of the differences induced in the extracellular environment immediately adjacent to the senescent cell, which are the result of specific characteristics of senescent cells compared to, for example, the characteristics of normal cells. A group of specific characteristics of senescent cells useful in this invention is the metabolic activity of senescent cells. For example, senescent cells may exhibit one or more of the following specific characteristics: (1) the cessation of senescent cell growth is essentially permanent and cannot be reversed by known physiological stimuli; (2) senescent cells increase in size, sometimes expanding to more than twice the size of their non-senescent counterparts; (3) senescent cells express senescence-associated β-galactosidase (SAP-gal), which partially reflects an increase in lysosome volume; (4) many senescent cells express pl6INK4a, which is not generally expressed by quiescent or terminally differentiated cells; (5) some senescent cells have persistent DNA damage response (DDR) signaling. (6) Senescent cells harbor persistent nuclear foci called DNA segments, chromatin changes that reinforce aging (DNA-scars such as dysfunctional telomeres or telomere dysfunction-induced foci (TIFs)), contain activated DDR proteins, and are distinguishable from transient damaged foci; (7) Senescent cells may express and secrete aging-related molecules, which in certain cases may be observed in the presence of persistent DDR signaling; (8) The nuclei of senescent cells lose structural proteins such as laminin B1, or chromatin-related proteins such as histones and HMGB1. For example, Freund et al, Mol. Biol. Cell, vol. 23, pp. 2066-75, 2012; Davalos et al, J. Cell Biol., vol. 201, pp. 613-29, 2013; Ivanov et al, J. Cell Biol., DOI:10.1083 / jcb.201212110, pp. 1-15, 2013; Funayama et al, J. Cell Biol., vol. 175, pp. 869-80, 2006.

[0134] In some embodiments, the extracellular conditions of senescent cells are low pH, induced by increased glycolysis in senescent cells (James et al., “Senescent human fibroblasts show increased glycolysis and redox homeostasis with extracellular metabolomes that overlap with those of irreparable DNA damage, aging, and disease,” J Proteome Res., vol. 14, pp. 1854-71, 2015). Glycolysis involves breaking down sugars to form two pyruvates and two ATP molecules, where pyruvates may be converted to lactates and excreted, thus lowering the pH in the extracellular environment of senescent cells (Wiley and Campisi, “From Ancient Pathways to Aging Cells-Connecting Metabolism and Cellular Senescence,” Cell Metab., vol. 23, pp. 1013-21, 2016). This is similar to the tumor microenvironment, where glycolysis in cancer cells lowers the pH in the tumor microenvironment. Therefore, the extracellular conditions of senescent cells can be acidic pH in the range of about 5.5 to about 7.2, or about 6.0 to about 7.0, or about 6.2 to about 7.0, or about 6.2 to about 6.8, or about 6.4 to about 6.8. The corresponding normal physiological conditions are normal physiological pH in the range of about 7.2 to about 7.8, preferably 7.2 to about 7.6, or more preferably about 7.4 to about 7.6.

[0135] In some embodiments, the extracellular conditions of senescent cells may be lower in concentration of deoxynucleotides compared to the normal physiological concentration of deoxynucleotides in the normal cellular environment (Wiley and Campisi, “From Ancient Pathways to Aging Cells-Connecting Metabolism and Cellular Senescence,” Cell Metab., vol. 23, pp. 1013-21, 2016). Some senescent cells may have lost the ability to synthesize deoxynucleotides, which can lead to lower concentrations of deoxynucleotides in the extracellular environment of senescent cells compared to the extracellular concentration of deoxynucleotides in the extracellular environment of normal cells. Therefore, the extracellular conditions of senescent cells may be selected to be lower in concentration of deoxynucleotides compared to the normal physiological concentration of deoxynucleotides in the extracellular environment of normal cells, where the corresponding normal physiological conditions are the concentration of deoxynucleotides in the extracellular environment of normal cells.

[0136] In some embodiments, the extracellular conditions of senescent cells may be lower in oxygen concentration compared to the physiological oxygen concentration in the extracellular environment of normal cells (Wiley and Campisi, “From Ancient Pathways to Aging Cells-Connecting Metabolism and Cellular Senescence,” Cell Metab., vol. 23, pp. 1013-21, 2016). Senescent cells have increased oxygen consumption compared to non-senescent cells and can have lower oxygen concentrations in their extracellular environment compared to that of normal cells. Therefore, the extracellular conditions of senescent cells may be selected to be lower in oxygen concentration compared to the normal physiological oxygen concentration in the extracellular environment of normal cells, where the corresponding normal physiological condition is the oxygen concentration in the extracellular environment of normal cells.

[0137] In some embodiments, the extracellular conditions of the senescent cells may be a lower NAD+ / NADH ratio than that of normal cells in the extracellular environment (Wiley and Campisi, “From Ancient Pathways to Aging Cells-Connecting Metabolism and Cellular Senescence,” Cell Metab., vol. 23, pp. 1013-21, 2016). Therefore, the extracellular conditions of the senescent cells may be selected to have a lower NAD+ / NADH ratio compared to the normal physiological NAD+ / NADH ratio in the extracellular environment of normal cells, the corresponding normal physiological condition being the normal NAD+ / NADH ratio in the extracellular environment of normal cells.

[0138] In some embodiments, the extracellular conditions of the senescent cells may be higher concentrations of redox homeostasis metabolites selected from hypotaurine, cysteine ​​sulfinate, cysteine-glutathione disulfide, gamma-glutamylalanine, gamma-glutamylmethionine, pyridoxate, gamma-glutamylglutamine, and alanine compared to the normal concentrations of these metabolites in the extracellular environment of normal growing, confluent, or quiescent cells (James et al., “Senescent human fibroblasts show increased glycolysis and redox homeostasis with extracellular metabolomes that overlap with those of irreparable DNA damage, aging, and disease,” J Proteome Res., vol. 14, pp. 1854-71, 2015). Therefore, the extracellular conditions of the senescent cells may be selected to result in higher concentrations of the redox homeostasis metabolite compared to the normal physiological concentration of the redox homeostasis metabolite in the extracellular environment of normal cells, which can be selected from growing cells, confluent cells, or quiescent cells, the corresponding normal physiological conditions being the concentration of the redox homeostasis metabolite in the extracellular environment of normal cells.

[0139] In some embodiments, the extracellular conditions of the senescent cells may be higher concentrations of nucleotide metabolites selected from 3-ureidopropionate, urat, 7-methylguanine, and hypoxanthine compared to the concentrations of these metabolites in the extracellular environment of normal growing, confluent, or quiescent cells (James et al., “Senescent human fibroblasts show increased glycolysis and redox homeostasis with extracellular metabolomes that overlap with those of irreparable DNA damage, aging, and disease,” J Proteome Res., vol. 14, pp. 1854-71, 2015). Thus, the extracellular conditions of the senescent cells may be selected to be higher concentrations of these nucleotide metabolites compared to the normal physiological concentrations of these metabolites in the extracellular environment of normal cells, which can be selected from growing, confluent, or quiescent cells, where the corresponding normal physiological conditions are the concentrations of nucleotide metabolites in the extracellular environment of normal cells.

[0140] In some embodiments, the extracellular conditions of the senescent cells may be low in thymidine compared to the concentration of thymidine in the extracellular environment of normal growing, confluent, or quiescent cells (James et al., “Senescent human fibroblasts show increased glycolysis and redox homeostasis with extracellular metabolomes that overlap with those of irreparable DNA damage, aging, and disease,” J Proteome Res., vol. 14, pp. 1854-71, 2015). Thus, the extracellular conditions of the senescent cells may be selected to be low in thymidine compared to the normal physiological concentration of thymidine in the extracellular environment of normal cells, which can be selected from growing, confluent, or quiescent cells, the corresponding normal physiological conditions being the concentration of thymidine in the extracellular environment of normal cells.

[0141] In some embodiments, the extracellular conditions of the senescent cells may be at a lower concentration of the dipeptide selected from glycylisoleucine, glycylvaline, glycylleucine, isoleucylglycine, and valylglycine compared to the concentration of the dipeptide in the extracellular environment of normal growing, confluent, or quiescent cells (James et al., “Senescent human fibroblasts show increased glycolysis and redox homeostasis with extracellular metabolomes that overlap with those of irreparable DNA damage, aging, and disease,” J Proteome Res., vol. 14, pp. 1854-71, 2015). Thus, the extracellular conditions of the senescent cells may be selected to be at a lower concentration of the dipeptide compared to the normal physiological concentration of the dipeptide in the extracellular environment of normal cells, which can be selected from growing, confluent, or quiescent cells, where the corresponding normal physiological condition is the concentration of the dipeptide in the extracellular environment of normal cells.

[0142] In some embodiments, the extracellular conditions of the senescent cells may be lower in concentration of a fatty acid selected from linoleate, dihomolinoleate, and 10-heptadecenoate compared to the concentration of the fatty acid in the extracellular environment of normal growing cells, confluent cells, or quiescent cells (James et al., “Senescent human fibroblasts show increased glycolysis and redox homeostasis with extracellular metabolomes that overlap with those of irreparable DNA damage, aging, and disease,” J Proteome Res., vol. 14, pp. 1854-71, 2015). Thus, the extracellular conditions of the senescent cells may be selected to be lower in concentration of the fatty acid selected from linoleate, dihomolinoleate, and 10-heptadecenoate compared to the normal physiological concentration of the fatty acid in the extracellular environment of normal cells, which can be selected from growing cells, confluent cells, or quiescent cells, where the corresponding normal physiological condition is the concentration of the fatty acid in the extracellular environment of normal cells.

[0143] In some embodiments, the extracellular conditions of the senescent cells may be higher concentrations of phospholipid metabolites selected from 2-hydroxypalmitate, 2-hydroxystearate, 3-hydroxydecanoate, 3-hydroxyoctanoate, and glycerophosphorylcholine compared to the concentrations of these metabolites in the extracellular environment of normal growing, confluent, or quiescent cells (James et al., “Senescent human fibroblasts show increased glycolysis and redox homeostasis with extracellular metabolomes that overlap with those of irreparable DNA damage, aging, and disease,” J Proteome Res., vol. 14, pp. 1854-71, 2015). Thus, the extracellular conditions of the senescent cells may be selected to be higher concentrations of these phospholipid metabolites compared to the normal physiological concentrations of these metabolites in the extracellular environment of normal cells, which can be selected from growing, confluent, or quiescent cells, with the corresponding normal physiological conditions being the concentrations of these phospholipid metabolites in the extracellular environment of normal cells.

[0144] In some embodiments, the extracellular conditions of the senescent cells may be higher concentrations of amino acid metabolites selected from alanine, C-glycosyltriprofen, kynurenine, dimethylarginine, and ortithine compared to the concentrations of these metabolites in the extracellular environment of normal growing, confluent, or quiescent cells (James et al., “Senescent human fibroblasts show increased glycolysis and redox homeostasis with extracellular metabolomes that overlap with those of irreparable DNA damage, aging, and disease,” J Proteome Res., vol. 14, pp. 1854-71, 2015). Therefore, the extracellular conditions of the senescent cells may be selected to be higher concentrations of these amino acid metabolites compared to the normal physiological concentrations of these metabolites in the extracellular environment of normal cells, which can be selected from growing, confluent, or quiescent cells, with the corresponding normal physiological conditions being the concentrations of amino acid metabolites in the extracellular environment of normal cells.

[0145] In some embodiments, the extracellular conditions of the senescent cells may be lower in concentration of phenylpilbert compared to the concentration of phenylpilbert in the extracellular environment of normal growing, confluent, or quiescent cells (James et al., “Senescent human fibroblasts show increased glycolysis and redox homeostasis with extracellular metabolomes that overlap with those of irreparable DNA damage, aging, and disease,” J Proteome Res., vol. 14, pp. 1854-71, 2015). Therefore, the extracellular conditions of the senescent cells may be selected to be lower in concentration of phenylpilbert compared to the normal physiological concentration of phenylpilbert in the extracellular environment of normal cells, the corresponding normal physiological condition being the concentration of phenylpilbert in the extracellular environment of normal cells.

[0146] In some embodiments, the extracellular conditions of the senescent cells may be higher concentrations of a metabolite selected from humarate, malonate, eicosapentaenoate, and citrate compared to the concentrations of such metabolites in the extracellular environment of normal growing, confluent, or quiescent cells (James et al., “Senescent human fibroblasts show increased glycolysis and redox homeostasis with extracellular metabolomes that overlap with those of irreparable DNA damage, aging, and disease,” J Proteome Res., vol. 14, pp. 1854-71, 2015). Therefore, the extracellular conditions of the senescent cells may be selected to be higher concentrations of a metabolite selected from humarate, malonate, eicosapentaenoate, and citrate compared to the normal physiological concentrations of such metabolites in the extracellular environment of normal cells, where the corresponding normal physiological conditions are the concentrations of such metabolites in the extracellular environment of normal cells.

[0147] In some embodiments, the extracellular conditions of senescent cells may be high equimratios compared to the phosphocholine-to-glycerophosphocholine ratio in the extracellular environment of normal non-quiescent cells (Gey and Seeger, “Metabolic changes during cellular senescence investigated by proton NMR-spectroscopy,” Mech Ageing Dev., vol. 134, pp. 130-8, 2013). The extracellular conditions of senescent cells may be selected to be high equimratios compared to the phosphocholine-to-glycerophosphocholine ratio in the extracellular environment of normal non-quiescent cells, the corresponding normal physiological condition being the phosphocholine-to-glycerophosphocholine ratio in the extracellular environment of normal non-quiescent cells.

[0148] Senescent cells secrete various different proteins collectively known as senescent cell-associated secretory phenotypes (SASPs). Examples of these secreted proteins include GM-CSF, GROa, GRC-α, β, γ, IGFBP-7, IL-1α, IL-6, IL-7, IL-8, MCP-1, MCP-2, MIP-a, MMP-1, MMP-10, MMP-3, amphiregulin, ENA-78, eotaxin-3, GCP-2, GITR, HGF, ICAM-1, IGFBP-2, IGFBP-4, IGFBP-5, IGFBP-6, IL-13, IL-Iβ, MCP-4, MIF, MIP-3a, MMP-12, MMP-13, MMP-14, NAP2, and oncostatin. M, Osteoprotegerin, PIGF, RANTES, sgpl30, TIMP-2, TRAIL-R3, Acrp30, Angiogenin, Axl, bFGF, BLC, BTC, CTACK, EGF-R, Fas, FGF-7, G-CSF, GDNF, HCC-4, I-309, IFN-γ, IGFBP-1, IGFBP-3, IL-1 Rl, IL-11, IL-15, IL-2R-a, IL-6 R, I-TAC, Leptin, LIF, MMP-2, MSP-a, PAI-1, PAI-2, PDGF-BB, SCF, SDF-1, sTNF RI, sTNF RII, Thrombopoietin, TIMP-1, tPA, uPA, uPAR, VEGF, MCP-3, IGF-1, TGF-β3, MIP-1 Examples include delta, IL-4, FGF-7, PDGF-BB, IL-16, BMP-4, MDC, MCP-4, IL-10, TIMP-1, Fit-3 ligand, ICAM-1, Axl, CNTF, INF-γ, EGF, and BMP-6.Further proteins secreted by senescent cells include IGF-2, IGF-2R, IGFBP-3, IGFBP-7, TGF-β, WNT2, CXCR2-bound chemokines, WNT16B, SFRP2, SPINK1, ENPP5, EREG, ANGPTL4, CSGALNACT, CCL26, AREG, ANGPT1, CCK, THBD, CXCL14, NOV, GAL, NPPC, FAM150B, CST1, MUCL1, NPTX2, TMEM155, EDN1, PSG9, ADAMTS3, CD24, PPBP, CXCL3, CST2, PSG8, PCOLCE2, PSG7, TNFSF15, C17orf67, CALCA, FGF18, BMP-2, MATN3, TFP1, SERPINI 1, TNFRSF25, and IL-23A. In some embodiments, the extracellular conditions of the senescent cells are either the presence of one or more of these secretory proteins at a high concentration compared to the concentration of these secretory proteins in the extracellular environment of normal cells, or the presence of one or more of these secretory proteins at a high concentration compared to the normal physiological conditions or normal physiological concentrations in the extracellular environment of normal cells in the absence of the said secretory proteins.

[0149] The conditionally active protein of the present invention may be used as a senescent cell scavenger to kill or remove senescent cells from a target. The interaction between the conditionally active protein and senescent cells may inhibit or kill senescent cells by inhibiting cell survival signaling pathways and / or inflammatory pathways that are activated during cellular senescence. Inhibition of cell survival signaling pathways and / or inflammatory pathways may induce cell death pathways, such as apoptosis in senescent cells, leading to the death of senescent cells (i.e., it may be initiated, induced, stimulated, or, in some way, eliminate or inhibit the suppression of cell death pathways).

[0150] Cell survival signaling pathways activated during aging include the src kinase signaling pathway, PI3K / Akt pathway, PBK / Akt / mTor pathway, p38 / MAPK pathway, ERK / MAPK pathway, mTOR pathway, insulin / IGF-1 signaling pathway, and TGF-β signaling pathway. Inflammatory pathways activated during aging include the p38 / MAPK signaling pathway, ERK / MAPK pathway, src kinase signaling pathway, and NF-κB pathway.

[0151] The src kinase signaling pathway is involved in the regulation of cell proliferation, differentiation, apoptosis, cell adhesion, and stress responses (see, e.g., Wang, Oncogene, vol. 19, pp. 5643-50, 2000 and Thomas et al, Annu. Rev. Cell Dev. Biol., vol. 13, pp. 513-609, 1997). The src kinase signaling pathway is also involved in inflammatory responses, including macrophage-mediated immune responses (see, e.g., Byeon et al, Mediators of Inflammation, vol. 2012, article ID 512926, 2012) and acute inflammatory responses (see, e.g., Okutani et al, Am. J. Physiol. Lung Cell Mol. Physiol., vol. 291, pp. L129-L141, 2006). Therefore, conditionally active proteins that modify the src kinase signaling pathway may modify both the signaling pathway and the inflammatory pathway.

[0152] Modifying cellular signaling pathways and / or inflammatory pathways may affect the function of one or more downstream proteins, or the interaction between one or more downstream proteins and other components of each cellular signaling or inflammatory pathway. For example, conditionally active proteins that modify the src kinase signaling pathway or the PBK / Akt pathway may modify the function of one or more downstream proteins in each pathway, or the interaction between one or more downstream proteins and other components of each pathway (see, e.g., Example 1; Figures 2B-2D). Exemplary proteins upregulated in senescent cells include P38 / MAPK, ERK1 / 2, and PBK (complex). In certain embodiments, the PBK / Akt pathway, a cellular signaling pathway, is activated during aging, and the conditionally active proteins described herein inhibit the PBK / Akt pathway to promote or induce apoptosis in senescent cells.

[0153] Assay solutions for assays of senescent cells under extracellular conditions and under normal physiological conditions include components selected from, for example, citrate buffers such as sodium citrate, phosphate buffers, bicarbonate buffers such as Krebs buffer, phosphate-buffered saline (PBS) buffer, Hanks buffer, Tris buffer, HEPES buffer, etc. Other buffers known to those skilled in the art and suitable for the assay may be used.

[0154] The assay solution of the present invention may contain at least one component selected from inorganic compounds, ions, and organic molecules, such as components commonly found in the bodily fluids of mammals or animals, including humans. These inorganic compounds, ions, and organic molecules are described in detail in WO2016 / 138071.

[0155] The conditionally active protein may interact with one or more inorganic compounds, ions, and organic molecules. Such interactions between the conditionally active protein and the components, which may be selected from inorganic compounds, ions, and organic molecules, include hydrogen bonding, hydrophobic interactions, and van der Waals interactions.

[0156] In some embodiments, the extracellular conditions of the senescent cells are a lower pH in the range of 5.5–7.2, or 6.0–7.0, or 6.2–6.8, while normal physiological conditions are a normal physiological pH, for example, in the range of 7.2–7.8. The assay solution for pH as extracellular conditions may contain a pKa component between the lower pH of the extracellular conditions and the normal physiological pH. The pKa is, for example, up to 0.5, 1, 1.5, 2, 2.5, or 3 units away from the lower pH of the extracellular conditions. In some embodiments, this component has a molecular weight of less than 900 a.mu and may be selected from, for example, histidine, histamine, hydrogenated adenosine diphosphate, hydrogenated adenosine triphosphate, citrate, bicarbonate, acetate, lactate, disulfide, hydrogen sulfide, ammonium, dihydrogen phosphate, and any combination thereof.

[0157] It has been observed that certain conditionally active proteins contain a large number (high proportion) of charged amino acid residues compared to the amino acid residues of the parent protein from which the conditionally active protein is induced. Three positively charged amino acid residues—lysine, arginine, and histidine—and two negatively charged amino acid residues—aspartic acid and glutamic acid—are present. These charged amino acid residues are over-represented in certain conditionally active proteins compared to the parent protein from which the conditionally active protein is induced. As a result, the increased number of charged amino acid residues in the conditionally active protein makes it more likely to interact with charged molecular species in the assay solution. This also affects the activity of the conditionally active protein.

[0158] It has also been observed that certain conditionally active proteins typically exhibit different activities in the presence of different molecular species in the assay solution. Molecular species having at least two ionization states: an uncharged or low-charged state at one value of the condition, such as pH, and a charged or higher-charged state at different values ​​of the same condition, can modify the activity of the conditionally active protein. The charged or higher-charged state of the molecular species can increase the interaction between the molecular species and charged amino acid residues present in the conditionally active protein. This mechanism may be used to increase the selectivity and / or pH-dependent activity of the conditionally active protein.

[0159] The nature of the charge(s) of the conditionally active protein may be one factor used to determine which molecular species are suitable for influencing the activity of the conditionally active protein. In some embodiments, the conditionally active protein may have more positively charged amino acid residues: lysine, arginine, and histidine, compared to the parent protein. Thus, the conditionally active protein may be selected to have a desired level of interaction with specific molecular species present in the extracellular environment of senescent cells where the activity is desirable, and / or to have a desired level of interaction with specific molecular species present under normal physiological conditions where low activity is desirable.

[0160] The position of charged amino acid residues in the conditionally active protein may have an effect on its activity. For example, the proximity of charged amino acid residues to the binding site of the conditionally active protein may be used to influence its activity.

[0161] In some embodiments, the interaction between the charged molecular species and the conditionally active protein may result in the formation of salt bridges between different regions of the protein, particularly charged or polarized regions. The formation of salt bridges is known to stabilize polypeptide structures (Donald, et al., “Salt Bridges: Geometrically Specific, Designable Interactions,” Proteins, 79(3): 898-915, 2011; Hendsch, et al., “Do salt bridges stabilize proteins? A continuum electrostatic analysis,” Protein Science, 3:211-226, 1994). These salt bridges can stabilize or fix protein structures that normally undergo a series of subtle structural changes known as “breathing” (Parak, “Proteins in action: the physics of structural fluctuations and conformational changes,” Curr Opin Struct Biol., 13(5):552-557, 2003). Structural variations are crucial for the efficient recognition and binding of the conditionally active protein to its partner; therefore, "bracing" of the protein structure is important for protein function and its binding to its partner (Karplus, et al., “Molecular dynamics and protein functions,” PNAS, vol. 102, pp. 6679-6685, 2015). It is possible that the salt crosslink can directly block the partner from approaching the binding site, thus reducing the accessibility of the binding site, particularly the binding pocket, to the partner on the conditionally active protein. Even salt crosslinks far from the binding site can exert an allosteric effect, altering the conformation of the binding site and inhibiting binding. Therefore, after the salt crosslink stabilizes (fixes) the structure of the conditionally active protein, the protein may become less active upon binding to its partner, potentially leading to decreased activity.

[0162] Hemoglobin is a known example of a protein whose structure is stabilized by salt crosslinking. Structural and chemical studies have revealed that at least two combinations of chemical groups, namely the amino terminus and side chains of histidine β146 and α122 with pKa values ​​around pH 7, are responsible for salt crosslinking. In deoxyhemoglobin, the terminal carboxylic acid group of β146 forms a salt crosslink with the lysine residue in the α subunit of the other αβ dimer. This interaction, assuming that the imidazole group of the histidine residue is protonated, fixes the side chain of histidine β146 to a position where it can participate in salt crosslinking with the negatively charged aspartic acid 94 within the same chain (Figure 2). At high pH, ​​the side chain of histidine β146 is not protonated, and no salt crosslinking is formed. However, as the pH decreases, the side chain of histidine β146 becomes protonated, and a salt crosslink is formed between histidine β146 and aspartate β94. This stabilizes the quaternary structure of deoxyhemoglobin, increasing the tendency for oxygen to be released (at lower pH) during active tissue metabolism. This hemoglobin exhibits pH-dependent oxygen binding activity; at low pH, oxygen binding activity decreases due to the formation of salt crosslinks. Conversely, at high pH, ​​oxygen binding activity increases because salt crosslinks are absent.

[0163] Similarly, small molecules such as bicarbonates may reduce the binding activity of the conditionally active protein to its partner by forming salt crosslinks in the conditionally active protein. For example, at pH less than pKa 6.4, the bicarbonate is protonated and therefore uncharged. Uncharged bicarbonates cannot form salt crosslinks and thus have little effect on the binding of the conditionally active protein to its partner. Therefore, the conditionally active protein has high binding activity to its partner at low pH. On the other hand, at high pH greater than the pKa of the bicarbonate, the bicarbonate ionizes by releasing protons and thus becomes negatively charged. Negatively charged bicarbonates will stabilize the structure of the conditionally active protein by forming salt crosslinks between the positively charged or polarized portions on the conditionally active protein. This will block or reduce the binding of the conditionally active protein to its partner. Therefore, the conditionally active protein has low activity at high pH. Thus, the conditionally active protein has pH-dependent activity in the presence of bicarbonate and has higher binding activity at low pH than at high pH.

[0164] If molecular species such as bicarbonates are not present in the assay solution, the conditionally active protein may lose its conditional activity. This may be due to the lack of salt crosslinks on the conditionally active protein that stabilize (fix) the protein's structure. Therefore, the partner will have similar accessibility to the binding site on the conditionally active protein at any pH and will produce similar activity at the first and second pH levels.

[0165] While salt crosslinking (ionic bonding) is the most powerful and common method by which molecular species influence the activity of a conditionally active protein, it must be understood that other interactions between such molecular species and the conditionally active protein can also contribute to the stabilization (fixation) of the protein's structure. Other interactions include hydrogen bonding, hydrophobic interactions, and van der Waals interactions.

[0166] In some embodiments, the conditionally active protein is compared to a parent protein to select a suitable compound or ion as the molecular species, and is evolved to determine whether the conditionally active protein has a higher proportion of negatively charged or positively charged amino acid residues. A compound with the appropriate charge at a normal physiological pH may then be selected to influence the activity of the conditionally active protein. For example, if the conditionally active protein has a higher proportion of positively charged amino acid residues than the parent protein, the suitable compound should typically be negatively charged at a normal physiological pH in order to interact with the conditionally active protein. Conversely, if the conditionally active protein has a higher proportion of negatively charged amino acid residues than the parent protein, the suitable small molecule should typically be positively charged at a normal physiological pH in order to interact with the conditionally active protein.

[0167] Therefore, suitable molecular species may be inorganic or organic molecules that transition from an uncharged or low-charged state at lower pH extracellular conditions of senescent cells to a charged or higher-charged state at normal physiological pH. Such molecular species should typically have a pKa between the lower pH and normal physiological pH. For example, bicarbonate has a pKa of 6.4. Therefore, at higher pH levels such as pH 7.4, negatively charged bicarbonate will bind to charged amino acid residues in the conditionally active protein, reducing its activity. On the other hand, at lower pH levels such as pH 6.0–6.2, less charged bicarbonate will not bind to the conditionally active protein in the same amount, thus allowing for higher activity of the conditionally active protein.

[0168] The disulfide has a pKa of 7.05. Therefore, at higher pH levels, such as pH 7.4, the more negatively charged disulfide will bind to the positively charged amino acid residues of the conditionally active protein, reducing its activity. On the other hand, at lower pH levels, such as pH 6.0-6.8, the less charged hydrogen sulfide / disulfide will not bind to the same level of the conditionally active protein, thus allowing for higher activity of the conditionally active protein.

[0169] Some molecular species are selected from disulfides, hydrogen sulfide, histidine, histamine, citrate, bicarbonate, acetate, and lactate. Each of these small molecules has a pKa between 6.2 and 7.0. Other suitable small molecules may be found in textbooks utilizing the principles of this application, such as the CRC Handbook of Chemistry and Physics, 96th Edition, 2015 by CRC press; and the Chemical Properties Handbook, McGraw-Hill Education, 1998.

[0170] For example, these molecular species have low molecular weight and / or relatively small conformations, ensuring maximum access to small pockets on conditionally active proteins by minimizing steric hindrance. For this reason, small molecules typically have molecular weights of less than 900 a.mu, more preferably less than 500 a.mu, more preferably less than 200 a.mu, and more preferably 100 a.mu. For example, hydrogen sulfide, disulfides, and bicarbonates all have low molecular weight and small structures, providing access to pockets on conditionally active proteins.

[0171] The concentration of the molecular species in the assay solution is, for example, the physiological concentration of the molecular species in the subject, or close to it. For example, the physiological concentration of bicarbonate (in human serum) is in the range of 15 to 30 mM. Therefore, the concentration of bicarbonate in the assay solution may be 10 mM to 40 mM, or 15 mM to 30 mM, or 20 mM to 25 mM, or about 20 mM. The physiological concentration of disulfide is also low. The concentration of disulfide in the assay solution may be 3 to 500 nM, or 5 to 200 nM, or 10 to 100 nM, or 10 to 50 nM.

[0172] The molecular species may be present in the assay solution for extracellular conditions of senescent cells and in the assay solution for normal physiological conditions at substantially the same concentration, for example, about 20 μM for bicarbonate.

[0173] In some embodiments, the conditionally active protein is pH-dependent when two or more different small molecules, such as a combination of bicarbonate and histidine, are present. Therefore, these two or more small molecules are present in the assay solution.

[0174] The molecular species in the assay solution may be formed in situ from the components of the assay solution, or they may be directly contained in the assay solution. For example, CO2 from the air may be dissolved in the assay solution to provide bicarbonate as a molecular species in the assay solution. As another example, sodium dihydrogen phosphate may be added to the assay solution to provide hydrogen phosphate as a molecular species in the assay solution.

[0175] In the absence of the molecular species, the conditionally active protein may lose its pH dependence. Therefore, in the absence of the molecular species, the conditionally active protein may have similar activity between the lower pH of the extracellular conditions of senescent cells and the normal physiological pH in the absence of the molecular species. This same result can be achieved based on any extracellular conditions of senescent cells that differ from normal physiological conditions.

[0176] In some embodiments, the conditionally active protein exhibits higher activity at lower pH levels of extracellular conditions in senescent cells compared to its activity at normal physiological pH, in the presence of an auxiliary protein. The auxiliary protein may be a protein present in blood or human serum. One suitable protein may be albumin, particularly mammalian albumin such as bovine albumin or human albumin.

[0177] In one embodiment, the auxiliary protein, such as albumin, is present in an assay solution used to screen and select the conditionally active protein from mutant proteins produced by the evolutionary step. In another embodiment, the assay solution containing the auxiliary protein, such as albumin, is used to test the activity of the selected conditionally active protein under the same or different conditions.

[0178] In some embodiments, two or more of these inorganic compounds, ions, and organic molecules discussed in this application are added to both assay solutions at substantially the same concentrations for normal physiological conditions and for extracellular conditions of senescent cells. For example, both bicarbonate and histidine are added to both assay solutions.

[0179] In one embodiment, human serum may be added to both assay solutions at substantially the same concentration for normal physiological conditions and for extracellular conditions of senescent cells. Since human serum contains numerous inorganic compounds, ions, and organic molecules (such as proteins), the assay solution will have multiple components selected from inorganic compounds, ions, and organic molecules that are present at substantially the same concentration between the two assay solutions.

[0180] In some other embodiments, at least one of two or more components is added to the assay solution at different concentrations for normal physiological conditions and for extracellular conditions of senescent cells. For example, both bicarbonate and histidine are added to the assay solution. The bicarbonate concentration may differ between the assay solutions, but the histidine may be at the same concentration in both assay solutions.

[0181] In some embodiments, the assay solution may be designed to select a conditionally active bioprotein having activity dependent on two or more conditions. In one exemplary embodiment, the conditionally active protein may have activity dependent on both pH and bicarbonate. The assay solution for selecting such a conditionally active protein may be an assay solution for normal physiological conditions having a pH of 7.2–7.6 and a bicarbonate concentration in the range of 25–30 mM. The assay solution for extracellular conditions of senescent cells may have a pH of 6.4–6.8 and a bicarbonate concentration in the range of 10–20 mM. Optionally, both the assay solutions for normal physiological conditions and extracellular conditions of senescent cells may also contain ions to facilitate binding between the mutant protein and its binding partner, thus increasing the number of hits to the conditionally active protein.

[0182] In some embodiments, specific components of serum may be minimized or excluded from the assay solution for a particular purpose. For example, when screening antibodies, serum components that bind to or adsorb antibodies may be minimized or excluded from the assay solution. Such binding antibodies, such as conjugated mutant antibodies that bind to components present in serum rather than conditionally active under various different conditions, may thereby give false positives. Therefore, careful selection of assay components to minimize or exclude components that may potentially bind to mutant proteins in the assay can reduce the number of false-positive mutant proteins that may be inadvertently identified as positive for conditional activity by binding to components in the assay other than the desired binding partner. For example, in some embodiments where mutant proteins that tend to bind to components in human serum are screened, bovine serum albumin may be used in the assay solution to reduce or eliminate the possibility of false positives induced by mutant proteins that bind to components in human serum. Other similar substitutions may also be made in specific examples to achieve similar objectives that are readily apparent to those skilled in the art.

[0183] In some embodiments, the evolutionary step may generate mutant proteins that simultaneously possess other desired properties of the conditional activity features discussed earlier. Suitable other desired properties that can be evolved include binding affinity, expression, and humanization. Therefore, the present invention may also be used to generate conditional activity proteins that also have improvements of at least one of these other desired properties.

[0184] In some embodiments, the conditionally active protein may be further mutated, for example in a second evolutionary step, using one of the mutagenicity techniques disclosed herein, to improve other properties of the conditionally active protein, such as binding affinity, expression, or humanization. After the second evolutionary step, the mutant protein may be screened for both the conditional activity and the improved properties.

[0185] In some embodiments, after evolving the parent protein to generate a mutant protein, (a) a decrease in the activity of the parent protein in the assay compared to the activity of the parent protein in the assay under normal physiological conditions, and an increase in the activity of the conditionally active protein in the assay under extracellular conditions of senescent cells compared to the activity of the conditionally active protein in the assay under normal physiological conditions; and (b) a decrease in the activity of the parent protein in the assay compared to the activity of the parent protein in the assay under normal physiological conditions, and an increase in the activity of the parent protein in the assay under extracellular conditions of senescent cells compared to the activity of the parent protein in the assay under extracellular conditions of senescent cells, A first conditionally active protein is selected that exhibits at least one of the following characteristics.

[0186] The first selected conditionally active protein is then subjected to one or more additional evolution, expression, and selection steps, as well as (a) a decrease in the activity of the parent protein in the assay compared to the activity of the parent protein in the assay under normal physiological conditions, and an increase in the activity of the conditionally active protein in the assay under extracellular conditions of senescent cells compared to the activity of the conditionally active protein in the assay under normal physiological conditions; and (b) a decrease in the activity of the parent protein in the assay compared to the activity of the parent protein in the assay under normal physiological conditions, and an increase in the activity of the parent protein in the assay under extracellular conditions of senescent cells compared to the activity of the parent protein in the assay under extracellular conditions of senescent cells, At least one second conditionally active protein exhibiting at least one of the following can be selected. The second activity may be similar to the first activity, in which case it is desirable that the second conditionally active protein has a larger ratio between activity under extracellular conditions and activity under normal physiological conditions compared to the first conditionally active protein. In some embodiments, the second activity may be different from the first activity, in which case the second activity may be an activity such as internalization efficiency or binding to a specific epitope.

[0187] In certain embodiments, the present invention aims to generate conditionally active proteins having a ratio of activity under extracellular conditions of senescent cells to activity under normal physiological conditions greater than 1.0 (e.g., high selectivity between the two conditions). The ratio or selectivity of activity under extracellular conditions of senescent cells to activity under normal physiological conditions may be at least about 1.3:1, or 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.

[0188] In one embodiment, the conditionally active protein is an antibody which may have a ratio of activity under extracellular conditions of senescent cells 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 20:1, or at least about 40:1, or at least about 70:1, or at least about 100:1.

[0189] In some embodiments, the conditionally active protein is a probody containing an antibody or antibody fragment (collectively referred to as "antibody") conjugated to a masking moiety (MM) via a linker (L). The probody is more active in the extracellular environment of senescent cells than in the extracellular environment of normal cells. In particular, in the extracellular environment of normal cells, the masking moiety of the probody masks the activity of the antibody, resulting in lower binding activity to target senescent cells. The masking moiety is cleaved from the antibody by proteases present in the extracellular environment of senescent cells. The antibody is then exposed and can freely bind to target senescent cells. Therefore, the probody has higher binding activity to target senescent cells in the extracellular environment of senescent cells than to the same target in the extracellular environment of normal cells.

[0190] Antibody fragments that may be included in a probody include antibodies (V L , V H Examples include variable or hypervariable regions of the light and / or heavy chains, variable fragments (Fv), Fab' fragments, F(ab')2 fragments, Fab fragments, single-chain antibodies (scAb), single-chain variable regions (scFv), complementarity-determining regions (CDR), domain antibodies (dAb), single-domain heavy-chain immunoglobulins of the BHH or BNAR type, and single-domain light-chain immunoglobulins.

[0191] The masking portion functions to reduce the binding activity of the antibody in the probody to target senescent cells compared to the binding activity of the antibody without the masking portion (e.g., after the masking portion is cleaved from the probody). The binding activity of the antibody to target senescent cells may be reduced by the masking portion by at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%. The reduction in binding activity may occur over a period of, for example, at least 2, 4, 6, 8, 12, 28, 24, 30, 36, 48, 60, 72, 84, or 96 hours.

[0192] In one embodiment, the masking moiety (MM) is conjugated to one or more variable regions of an antibody (Ab) via a linker (L) to create a barrier between the antibody and target senescent cells. For example, the masking moiety may be conjugated to the N-terminus of one or more variable regions. The masking moiety and linker form a single chain conjugated to the N-terminus of one or more variable regions. In another example, the masking moiety may be conjugated to the side chain of an amino acid in one or more variable regions, in which case the masking moiety and linker form a single chain conjugated to the side chain of an amino acid in one or more variable regions. In yet another example, the masking moiety is conjugated to the C-terminus of one or more variable regions when the probody contains only a fragment of the antibody (e.g., only a variable region). In some embodiments, the probody has an MM-L-Ab structure from the N-terminus to the C-terminus. In another embodiment, the probody has an Ab-L-MM structure from the N-terminus to the C-terminus.

[0193] In some embodiments, the masking moiety may be identified by screening a diverse library of peptides for peptides that bind to one or more variable regions of the antibody (Desnoyers et al., “Tumor-specific activation of an EGFR-targeting probody enhances therapeutic index,” Sci Transl Med., vol. 5, 207ra144, 2013). A peptide that, when conjugated to the antibody via a linker, specifically binds to the antibody and can block the antibody's binding to target senescent cells is selected as the masking moiety. This screening may be carried out using known techniques, including, but not limited to, panning, fluorescence-activated cell sorting, and magnetic sorting using streptavidin-coated magnetic beads (Rice et al., “Bacterial display using circularly permuted outer membrane protein OmpX yields high affinity peptide ligands,” Protein Sciences, vol. 15, pp. 825-36, 2006).

[0194] In some embodiments, a random peptide library (e.g., peptides having about 2 to about 40 amino acids, or about 5 to about 30 amino acids, or about 8 to about 20 amino acids, or greater than 40 amino acids) may be used in a screening method to identify suitable masking moieties. For example, masking moieties having specific binding affinity to an antibody can be identified through a screening procedure that includes providing a library of peptide scaffolds, each of which consists of a transmembrane protein and a candidate substance. The library is then contacted with an antibody to identify one or more suitable masking moieties having detectable binding activity to the antibody. The screening may include another magnetically activated sorting or fluorescence-activated cell sorting.

[0195] Therefore, the present invention intends that the masking moiety may be specific to the antibody in the probody. One masking moiety that acts well on a particular antibody may have lower optimality for another antibody. Thus, screening a diverse peptide library using the antibody in the probody for the masking moiety best suited to the antibody may be important in some embodiments of the present invention.

[0196] In some embodiments, the masking moiety is screened from a diverse library of synthetic peptides. This type of masking moiety may have a specific level of similarity to target senescent cells (the antibody's natural binding partner). In certain embodiments, the masking moiety may be modeled after the antibody's natural binding partner. For example, the natural binding partner may be modified by altering one or more amino acid residues to slightly reduce its binding activity to the antibody. In other embodiments, the masking moiety has sequence identity of 5% or less, 7% or less, 10% or less, 15% or less, 20% or less, 25% or less, 30% or less, 35% or less, 40% or less, 45% or less, 50% or less, 55% or less, 60% or less, 65% or less, 70% or less, 75% or less, or 80% or less with respect to the antibody's natural binding partner.

[0197] The structural characteristics of the masking moiety depend on several factors, including the minimum amino acid sequence required to inhibit antibody binding to target senescent cells, the size of the antibody (whole antibody or fragment), and the length of the linker. In some embodiments, the masking moiety is covalently linked to the antibody. In one example, the antibody is linked to the masking moiety by a cysteine-cysteine ​​disulfide bridge between the linker and the antibody. In another example, the antibody is linked to the masking moiety by a peptide bond between the linker and the antibody.

[0198] In some embodiments, the masking portion is unable to specifically bind to the antibody, but rather interferes with the binding of the antibody to target senescent cells through one or more nonspecific interactions, such as steric hindrance. For example, the structure of the probody may be such that the masking portion is positioned within the probody so that it masks the antibody through charge-based interactions, thereby holding the masking portion in place and preventing it from approaching the antibody binding site.

[0199] The linker of the probody is located between the masking portion and the antibody. The linker includes a cleavage site (CS), where a protease present in the extracellular environment of the senescent cell cleaves the linker, releasing the masking portion from the probody. The antibody is then exposed and available for binding to the target senescent cell. The linker may further include one or more flexible regions (FRs) adjacent to one or both sides of the cleavage site. For example, the linker may have the structure -FR-CS-FR-, -FR-CS-, -CS-FR-, -FR-FR-CS-, -CS-FR-FR-, -FR-FR-CS-FR-FR-, -FR-FR-CS-FR-FR-.

[0200] The flexible region provides flexibility to the conformation of the masking portion, allowing it to reach the antibody binding site and thus hindering binding. The flexible region essentially consists of small amino acids such as glycine, serine, and alanine, which have small side chains to provide maximum flexibility. Glycine and glycine-serine polymers are relatively structurally undefined and therefore can act as neutral tethers between components. Glycine approaches the phi-psi space significantly more closely than alanine and is considerably less constrained than residues with longer side chains (see Scheraga, Rev. Computational Chem., pp. 11173-11142, 1992).

[0201] The appropriate flexible region may have different lengths, such as 1-20 amino acids, 2-15 amino acids, 3-12 amino acids, 4-10 amino acids, 5-9 amino acids, 6-8 amino acids, or 7-8 amino acids, and may be 1, 2, 3, 4, 5, 6, or 7 amino acids long.

[0202] Exemplary flexible regions include glycine polymers (G)n, glycine-serine polymers (e.g., (GS)n (SEQ ID NO: 14), (GGS)n (SEQ ID NO: 15), (GSGGS)n (SEQ ID NO: 16), (GSGGS)n (SEQ ID NO: 17), and (GGGS)n (SEQ ID NO: 18) (where n is an integer of at least 1)), glycine-alanine polymers, alanine-serine polymers, and other flexible regions known in the art. Further examples of flexible regions include GGSG (SEQ ID NO: 19), GGSGG (SEQ ID NO: 20), GGSSG (SEQ ID NO: 21), GGGGG (SEQ ID NO: 22), GGGSG (SEQ ID NO: 23), GSSSG (SEQ ID NO: 24), GSSGGSGGSGGSG (SEQ ID NO: 25), GSSGGSGGSGG (SEQ ID NO: 26), GSSGGSGGSGGS (SEQ ID NO: 27), GSSGGSGGSGGSGGGS (SEQ ID NO: 28), GSSGGSGGSG (SEQ ID NO: 29), GSSGGSGGSGS (SEQ ID NO: 30), GSSGT (SEQ ID NO: 31), or GSSG (SEQ ID NO: 32).

[0203] The cleavage site is a substrate for proteases in the extracellular environment of senescent cells. Generally, the cleavage site is included as part of the linker. However, in some cases, the cleavage site may be part of a masking region, so that all or part of the cleavage site facilitates antibody masking when the probody is inhibited, uncleaved, or masked.

[0204] The cleavage site may be selected based on proteases in the extracellular environment of senescent cells. Senescent cells are known to secrete proteases into the extracellular environment, such as matrix metalloproteinases (MMPs). Examples of MMP family members include stromelycin-1 and -2 (MMP-3 and -10, respectively) and collagenase-1 (MMP-1). Other MMPs include MMP1, MMP2, MMP7, MMP8, MMP9, MMP13, and MMP14. The native substrates of these proteases that can assist in the design of cleavage sites used in probodies are also known. For example, these MMPs can cleave MCP-1, -2, and -4, as well as IL-8. Various other CXCL / CCL family members can also be cleaved by MMP-9, -2, or -7. Serine proteases are also present in the extracellular environment of senescent cells. Examples of serine proteases include urokinase-type or tissue-type plasminogen activators (uPA or tPA, respectively). See Coppe et al., “The Senescence-Associated Secretory Phenotype: The Dark Side of Tumor Suppression,” Annu Rev Pathol., vol. 5, pp. 99-118, 2010.

[0205] In one exemplary embodiment, the cleavage site is a substrate of a matrix metalloproteinase and is therefore cleavable by an MMP to release the making moiety. In another embodiment, the cleavage site is a substrate of serine uPA or PSA. In some embodiments, the probody may contain more than one cleavage site, each of which may be a substrate of a different protease. Exemplary cleavage sites that can be substrates for proteases include ADAM10, ADAM12, ADAM17, ADAMTS, ADAMTS5, BACE, caspase 1-14, cathepsin A, cathepsin B, cathepsin D, cathepsin E, cathepsin K, cathepsin S, FAP, MT1-MMP, granzyme B, guanidinobenzoatase, hepsin, human neutrophil elastase, regmine, matryptase 2, meprine, MMP1-17, MT-SP1, neprilysin, NS3 / 4A, plasmin, PSA, PSMA, TRACE, TMPRSS 3, TMPRSS 4, and uPA. Some exemplary cleavage sites include PLGLWA (SEQ ID NO: 33), which can be cleaved by MMP, and GPQGIAGQ (SEQ ID NO: 34), which can be cleaved by collagenase. Other examples of transection sites include YGLLGIAGPPGP (SEQ ID NO: 35), SPGRVVRG (SEQ ID NO: 36), and VRG (SEQ ID NO: 37).

[0206] In some embodiments, the antibody in the probody has conditional activity of its own. In particular, the antibody itself may have higher binding activity to the target under the conditions of the extracellular environment of senescent cells compared to its binding activity to the target under normal physiological conditions. Such a probody provides twofold amplification once it reaches the extracellular environment of senescent cells by (1) cleaving the masking portion to dissociate the antibody binding site from the masking portion, and (2) the antibody having higher binding activity to the target under the conditions of the extracellular environment of senescent cells compared to its binding activity under normal physiological conditions.

[0207] In one embodiment, the conditionally active protein is an antibody intended to be conjugated with another drug. The conditionally active antibody may have a high ratio of activity under extracellular conditions of senescent cells to activity under normal physiological conditions of at least about 10:1, or at least about 11:1, or at least about 12:1, or at least about 13:1, or at least about 14:1, or at least about 15:1, or at least about 16:1, or at least about 17:1, or at least about 18:1, or at least about 19:1, or at least about 20:1, or at least about 40:1, or at least about 60:1, or at least about 80:1, or at least about 100:1. The above may be particularly important if the conjugated drug is, for example, toxic or radioactive, as such a conjugated drug will be concentrated at the disease or treatment site if desired.

[0208] In some embodiments, the conjugate is a D-retroinverso peptide ("DRI peptide"). Because the D amino acid is in reverse order, the DRI peptide can maintain a similar amino acid side-chain topology to the induced native protein. In addition, the DRI peptide is more resistant to proteolysis and therefore tends to have a considerably longer half-life than the induced native protein. Furthermore, the DRI peptide has a structure similar to that of the induced native protein. Finally, the DRI peptide has comparable bioavailability to the induced native protein. Therefore, the DRI peptide can be a functional substitute for the induced native protein and can compete with it. Thus, the DRI peptide is considered a promising pharmaceutical agent.

[0209] FOXO4 is a molecular pivot that determines whether damaged cells undergo senescence or apoptosis. The FOXO protein family, including FOXO1, 3, and 4, is negatively regulated by growth factor signaling but can also be activated by oxidative stress (Brunet, A. et al., Science, vol. 303, pp. 2011-2015 (2004); de Keizer, PL et al., Cancer Res, vol. 70, pp. 8526-8536 (2010); Essers, MA et al., EMBO J., vol. 23, pp. 4802-4812 (2004)). Constitutive foxo1- / - mice are embryonically lethal, and foxo3- / - mice exhibit genital defects, but foxo4- / - mice do not carry a significantly deficient phenotype (Hosaka, T. et al., Proc. Natl. Acad. Sci. USA, vol. 101, pp. 2975-2980 (2004); Castrillon, DH et al., Science, vol. 301, pp. 215-218 (2003)). Individual conditionally somatic foxo3- / - mice exhibit a significantly shortened lifespan, but conditionally somatic foxo1- / - and foxo4- / - do not (Paik, JH et al., Cell, vol. 128, pp. 309-323 (2007)). Somatic triple 1, 3, and 4- / - mice show increased lymphoma, indicating a functional surplus of each of these FOXO proteins (ibid.). Notably, however, single somatic foxo4- / - mice show neither shortened lifespan nor any change in tumor-free survival. Unlike its counterparts FOXO1 and FOXO3, mRNA and protein expression of FOXO4 significantly increases in response to aging-induced DNA damage levels.

[0210] Ionizing radiation (XRAY)-induced DNA damage-induced senescence is characterized by the formation of persistent nuclear foci called DNA-SCARS (or DNA Segments with Chromatin Alterations Reinforcing Senescence), which are necessary for growth arrest (Rodier, F. et al., J Cell Sci, vol. 124, pp. 68-81 (2011)). Under these DNA-damaging conditions, loss of FOXO4 expression using stable short hairpin RNA interference (shRNA) induced apoptosis instead of senescence. This suggests that FOXO4 is a pivot factor in the intramolecular determination of whether cellular senescence or apoptosis occurs in response to genotoxic stress.

[0211] The mechanism by which FOXO4 selectively selects senescence and limits apoptosis involves a physical relationship with the p53 tumor suppressor protein. p53 is well known to regulate cell fate after DNA damage (Rodier, F. et al., Nucleic Acids Res, vol. 35, pp. 7475-7484 (2007)) and is a major component of DNA-scars (Rodier, F. et al., Nat. Cell Biol., vol. 11, pp. 973-979 (2009)). Depending on post-translational modifications and their interaction partners, p53 can induce senescence and apoptosis (Vousden, KH et al., Nat. Rev. Mol. Cell Biol., vol. 8, pp. 275-283 (2007)). When p53 is phosphorylated on Ser46, it strongly influences apoptosis rather than cell cycle arrest (Bulavin, DV et al., EMBO J., vol. 18, pp. 6845-6854 (1999)). However, Ser46 is phosphorylated in response to several senescence-inducing stimuli, including activated oncogenes (Feng, L. et al., Cell Cycle, vol. 5, pp. 2812-2819 (2006); Bischof, O. et al., EMBO J., vol. 21, pp. 3358-3369 (2002)). Under DNA damage conditions, Ser46 phosphorylation of p53 increases, and interference with the HIPK2 kinase responsible for Ser46 phosphorylation (Dauth, I. et al., Cancer Res, vol. 67, pp. 2274-2279 (2007)) impairs the apoptotic response induced by FOXO4 deletion. Thus, FOXO4 suppresses apoptosis in senescent cells by stimulating senescence and suppressing the apoptotic function of p53 signaling. Inhibition of FOXO4, particularly its interaction with p53, will likely switch senescent cells to apoptosis.

[0212] The human foxo4 protein has two variants (seq ID no: 1 and 2). In some embodiments, any fragment of the foxo4 protein may be used as a basis for designing foxo4 DRI peptides. In one embodiment, the foxo4 fragment includes at least a portion of the functional domains of the foxo4 protein, such as a DNA-binding domain (seq ID no: 3) or a p53 interaction domain (seq ID no: 4).

[0213] Any FOXO4 DRI peptide capable of inhibiting the function of FOXO4 and / or interfering with its interaction with p53 may be used as a conjugate to a conditionally active antibody. In particular, three FOXO4 DRI peptides: LTLRKEPASE IAQSILEAYS QNGWANRRSG GKRP (SEQ ID NO: 5), LTLRKEPASE IAQSILEAYS QNGWANRRSG GKRPPPRRRQ RRKKRG (SEQ ID NO: 6), and SEIAQSILEAYSQNGW (SEQ ID NO: 7) are preferred for effectively interfering with the interaction between FOXO4 and p53. All three of these FOXO4 DRI peptides consist of D amino acid residues. At least some of the D amino acid residues in these FOXO4 DRI peptides may be replaced with L amino acid residues without significantly reducing their ability to induce apoptosis in senescent cells. These FOXO4 DRI peptides disrupt the interaction between FOXO4 and p53, thereby inhibiting FOXO4's function of suppressing p53 and inducing apoptosis in senescent cells.

[0214] FOXO4 is itself regulated by other proteins. Referring to Figure 8, members of the FOXO family, including FOXO4, are activated by other proteins through phosphorylation or methylation, specifically by AMPK, JNK, MST1, CK1, STAT3, and p38 through phosphorylation, and by PRMT1 through methylation. Stress-activated c-Jun N-terminal kinase (JNK) and energy-sensing AMP-activated protein kinase (AMPK) phosphorylate and activate FOXO upon exposure to oxidative and nutritional stress stimuli. Any protein that activates FOXO4 can form the basis for designing DRI peptides significant to the present invention (i.e., native or wild-type proteins). In some embodiments, the native protein is selected from the group consisting of AMPK, JNK, MST1, CK1, STAT3, p38, and PRMT1.

[0215] Let's take the JNK protein as an example. JNK is a c-Jun N-terminal kinase capable of phosphorylating and activating FOXO4. Human JNK has the amino acid sequence SEQ ID NO:8. DRI peptides based on the JNK protein can allosterically and selectively modulate JNK by blocking access to the substrate using a competitive mechanism (Bonny, C. et al. Diabetes, vol. 50, pp. 77-82 (2001); Borsello, T. et al. Trends Mol Med, vol. 10, pp. 239-244, (2004); and Borsello, T. et al. Nat Med, vol. 9, pp. 1180-1186, (2003)). One exemplary JNK DRI peptide is DQSRPVQPFLQLTTPRKP (SEQ ID NO:9).

[0216] Furthermore, activators of AMPK, JNK, MST1, CK1, STAT3, p38, and PRMT1 may also be used as native proteins for the design of the DRI peptide. For example, ASK1 is an apoptosis signal-regulated kinase 1 that activates JNK. Human ASK1 has GenBank accession number No. NP_005914. The ASK1 protein may also be a native protein for the design of the DRI peptide. Such a DRI peptide may inhibit ASK1, thereby suppressing JNK activity and leading to inhibition of FOXO4.

[0217] In some embodiments, the native proteins for designing the DRI peptides of the present invention are human proteins such as human FOXO4, AMPK, JNK, MST1, CK1, STAT3, p38, PRMT1, and ASK1. In some other embodiments, the native proteins for designing the DRI peptides of the present invention are mammalian proteins such as primate or mouse proteins of FOXO4, AMPK, JNK, MST1, CK1, STAT3, p38, PRMT1, and ASK1. It is generally understood that orthologous proteins can function in other molecular species, which means that DRI peptides designed based on orthologs can function in other molecular species. For example, a DRI peptide designed based on mouse FOXO4 may function on human FOXO4, and thus may be used as a conjugate of the present invention to induce apoptosis in senescent cells in humans.

[0218] In one embodiment, a DRI peptide is designed using a fragment of a native protein. In another embodiment, the DRI peptide is designed using the full-length of a native protein. In these embodiments, the amino acid sequence of the DRI peptide is the exact reverse of the amino acid sequence of a fragment or full-length native protein of FOXO4, AMPK, JNK, MST1, CK1, STAT3, p38, PRMT1, and ASK1.

[0219] In some other embodiments, the amino acid sequence of the DRI peptide is not an exact inverse of the amino acid sequence of the fragment or full-length native protein of FOXO4, AMPK, JNK, MST1, CK1, STAT3, p38, PRMT1, and ASK1. In such embodiments, the amino acid sequence of the DRI peptide may have at least 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% sequence identity with the reverse sequence of the fragment or full-length native protein.

[0220] The DRI peptide may be a small peptide, for example, to enable the entry of the DRI peptide into senescent cells. In some embodiments, the DRI peptide is 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 5 Contains 3, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100 or more amino acid residues.

[0221] In some embodiments, the DRI peptide consists of all D amino acid residues, while some functional DRI peptides may contain combinations of L amino acid residues and D amino acid residues. In some embodiments, the DRI peptide has amino acid residues that are up to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 35, 40, 45, 50, 55, or 60% L amino acid residues.

[0222] In some embodiments, the DRI peptide may further comprise one or more functional domains that are not part of a native protein that serves as the basis for designing the DRI peptide. In one embodiment, the DRI peptide comprises the sequence “PPRRRQRRKKRG” (SEQ ID NO: 10) which facilitates the entry of the DRI peptide into senescent cells and induces apoptosis. Those skilled in the art will understand that this functional domain may be replaced by any other protein domain that facilitates the entry of the DRI peptide into senescent cells.

[0223] Some other functional domains that may be included in the DRI peptide include primary amphiphilic peptides MPG (GALFLGFLGA AGSTMGAWSQ PKKKRKV, SEQ ID NO:11), Pep-1 (KETWWETWWT EWSQPKKKRKV, SEQ ID NO:12), secondary amphiphilic peptide CADY (Ac-GLWRALWRLLRSLWRLLWRA-Cya, SEQ ID NO:13), or cell-permeable peptides ("CPP") such as octaarginine (R(8)).

[0224] The functional domain within the DRI peptide does not possess any arbitrary apoptosis-inducing activity itself, but may function to increase the apoptosis-inducing activity of another portion of the DRI peptide. The functional domain comprises at least 1, 2, 3, 4, 5, 6, 7, or 10 amino acid residues, and more preferably all amino acid residues in the functional domain are D amino acid residues.

[0225] The DRI peptide according to the present invention has apoptosis-inducing activity in senescent cells when it kills, purifies, removes, inactivates, or reduces the viability of senescent cells. In some embodiments, the DRI peptide can kill, purify, remove, inactivate, or reduce the viability of at least 5, 10, 15, 20, 25, 30, 40, 50, 60, 70, 80, 90, or 95% of cells in a senescent cell culture.

[0226] In some embodiments, the DRI peptide selectively exhibits apoptosis-inducing activity in senescent cells, i.e., has little to no apoptosis-inducing activity in non-senescent cells. The DRI peptide can induce apoptosis in senescent cells more than in non-senescent cells at a ratio of at least 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.5, 3, 4, 5, 6, 7, 8, 9, 10, or higher.

[0227] Those skilled in the art can use common general knowledge to determine whether the DRI peptide according to the present invention exhibits apoptosis-inducing activity in senescent cells using standard in vitro tests. For example, a cell culture of senescent cells can be obtained by subjecting the cell culture to ionizing radiation or a chemotherapeutic agent, and then mixed with non-senescent cells. Other methods for providing senescent cells include (i) continuous passage until repeated senescence occurs (= telomere shortening), (ii) by the use of oxidative stressors such as H2O2 and rotenone, (iii) chromatin remodeler as sodium dibutyrate, or (iv) expression of hyperactivated oncogenes such as RASG12V or BRAFV600E. The presence of senescent cells can be confirmed by testing for SA-B-GAL.

[0228] The second step is to administer the peptide according to the present invention to a cell culture and measure one or more markers of apoptosis, such as (i) staining of cytoplasmic cytochrome C, or (ii) staining with TUNEL. Cytochrome C data can be quantified by counting the number of cells from which cytochrome C has been released from the mitochondria into the cytosol (cells may be indicated using DAPI), or (in later stages) the number of cells that have completely disappeared. Since caspases are required for cell death, this assay can be performed in the presence of a caspase inhibitor so that cells undergoing apoptosis (indicated by the release of cytochrome C into the cytosol) cannot actually die. An advantage of this assay is that it is possible to obtain a cumulative number of senescences over several days (e.g., 5 days). In TUNEL staining, the percentage of nuclei stained positively with TUNEL (DAPI positive) is counted. This can be easily done with the naked eye, but a software tool called Cellprofiler (freeware) can also be used.

[0229] In some embodiments, the conditionally active protein is covalently bound to a peptide linker, which also contains a prodrug conjugated to the conditionally active protein. The prodrug is a drug conjugated to the peptide linker. Due to the presence of the covalently bound peptide linker, the drug is not in an active form. The peptide linker is cleaved by a protease in the extracellular environment of senescent cells, thus releasing the covalently bound drug from the conditionally active protein in its active form.

[0230] The peptide linker between the drug and the conditionally active protein may contain the same cleavage sites used in the probody described in this application (e.g., the cleavage sites of SEQ ID NO: 33-37). The same protease in the extracellular environment of senescent cells that can release antibodies in the probody cleaves the peptide linker, releasing the active form of the prodrug from the conditionally active protein in the extracellular environment of senescent cells.

[0231] In some embodiments, the peptide linker may be cleaved by the enzyme legmine. Such a peptide linker includes a cleavage site for legmine. Some exemplary cutting sites are: PTN (SEQ ID NO:38); PNN (SEQ ID NO:39); PAN (SEQ ID NO:40); PPN (SEQ ID NO:41); TTN (SEQ ID NO:42); TNN (SEQ ID NO:43); TAN (SEQ ID NO:44); TPN (SEQ ID NO:45); NTN (SEQ ID NO:46); NNN (SEQ ID NO:47); NAN (SEQ ID NO:48); NPN (SEQ ID NO:49); ATN (SEQ ID NO:50); ANN (SEQ ID NO:51); AAN (SEQ ID NO:52); APN (SEQ ID NO:53); TTNL (SEQ ID NO:54); TTNA (SEQ ID NO:55); PTNL (SEQ ID NO:56); PTNA (SEQ ID NO:57); PNNL (SEQ ID NO:58); PNNA (SEQ ID NO:59);TNNL(SEQ ID NO:60);TNNA (SEQ ID NO:61);NK(SEQ ID NO:62);NL(SEQ ID NO:63);NA(SEQ ID NO:64);NE(SEQ ID NO:65);ND(SEQ ID NO:66);andNN(SEQ ID NO:67).

[0232] The drug covalently bound to the peptide linker in the prodrug may be a cytotoxic agent, a cell division inhibitor, or an antiproliferative agent. These drugs Alkaloids: Docetaxel, etoposide, irinotecan, paclitaxel, teniposide, tepotecan, vinblastine, vincristine, vindesine, Alkylating agents: Busulfan, Improsulfan, Piposulfan, Benzodepa, Carboquan, Metsuredepa, Uredepa, Altretamine, Triethylenemelamine, Triethylenephosphoramide, Triethylenethiophosphoramide, Chlorambucil, Chloranaphazine, Cyclophosphamide, Estramustine, Ifosfamide, Mechloretamine, Mechloretamine oxide hydrochloride, Melphalan, Novemevichin, Perphosphamide, Fenesterine, Prednimustine, Trophosphamide, Uracil mustard, Carmustine, Chlorozotosine, Fotemustine, Lomustine, Nimustine, Semustine, Ranimustine, Dacarbazine, Manomustine, Mitobronitol, Mitractol, Pipobroman, Temozolomide Antibiotics and their analogues: acrasinomycin, actinomycin, anthramycin, azaserin, bleomycin, kactinomycin, carbicin, cardinophilin, chromomycin, dactinomycin, daunorubicin, 6-diazo-5-oxo-L-norleucine, doxorubicin, epirubicin, idarubicin, menogalil, mitomycin, mycophenolic acid, nogaramycin, olibomycin, peplomycin, pirarubicin, plicamycin, porphyromycin, puromycin, streptonigrin, streptozocin, tubercidine, dinostatin, zolubicin, Antimetabolites: Denopterin, edatrexate, methotrexate, pyritrexime, pteropterin, tomdex, trimethrexate, cladridine, fludarabine, 6-mercaptopurine, pentostatin, thiamiprine, thioguanine, ancitabine, azacitidine, 6-azauridine, carmoftir, cytarabine, doxifluridine, emitefur, floxuridine, fluorouracil, gemcitabine, tegafur, Platinum complexes: Caloplatin, cisplatin, miboplatin, oxaliplatin, Others: Acetollan, Amsacrin, Bisanthren, Dephosphamide, Demecolsin, Diadiquan, Eflornithine, Elliptinium acetate, Etoglucide, Etopside, Fenretinide, Gallium nitrate, Hydroxyurea, Ronidamin, Miltefosin, Mitoguazone, Mitoxantrone, Mopidamol, Nitracrine, Pentostatin, Fenamet, Podophyllic acid, 2-Ethylhydrazide, Procarbazine, Lazoxane, Sobuzoxane, Spirogermanium, Teniposide, Tenuazonic acid, Triadiquan, 2,2',2''-Trichlorotriethylamine, Urethane, This is then made concrete.

[0233] The drug covalently bound to the peptide linker in the prodrug may be a chemotherapeutic agent. Chemotherapeutic agents may inhibit senescent cells in different ways. Chemotherapeutic agents may damage the DNA template by alkylation, crosslinking, or by double-strand breaks of DNA. Other chemotherapeutic agents may block RNA synthesis by intercalation. Some chemotherapeutic agents are spindle inhibitors, or antimetabolites that inhibit enzyme activity, or hormones and antihormones. Chemotherapeutic agents may be selected from a wide range of drugs, including but not limited to alkylating agents, antimetabolites, antitumor antibiotics, vinca alkaloids, epipodophyllotoxin, nitrosourea, hormones and antihormones, and toxins. Some examples are as follows: Examples of alkylating agents include cyclophosphamide, chlorambucil, busulfan, melphalan, thiotepa, ifosphamide, and nitrogen mustard. Examples of antimetabolites include methotrexate, 5-fluorouracil, cytosine arabinoside, 6-thioguanine, and 6-mercaptopurine. Examples of antitumor antibiotics include doxorubicin, daunorubicin, idorubicin, nimitoxantrone, dactinomycin, bleomycin, mitomycin, and plicamycin. Examples of vinca alkaloids and epipodophyllotoxins include vincristine, vinblastine, vindestine, etoposide, and teniposide. Examples of nitrosoureas include carmustine, lomustine, semustine, and streptozocin. Examples of hormones and antihormone agents include adrenocorticorticoids, estrogens, antiestrogens, progestins, aromatase inhibitors, androgens, and antiandrogens. Examples of randomly selected synthetic drugs include dacarbazine, hexamethylmelamine, hydroxyurea, mitotane, procarbazide, cisplatin, and carboplatin.

[0234] In another embodiment, the present invention provides conditionally active molecules or conditionally active drugs (CAMs) that are active under abnormal conditions rather than normal physiological conditions. The conditionally active molecules are organic compounds and / or salts thereof obtained from parent organic compounds having a molecular weight of less than about 3000 a.mu. The parent organic compounds may be therapeutically active compounds having molecular weights in the range of about 100 a.mu to about 1500 a.mu, or about 150 a.mu to about 1250 a.mu, or about 300 a.mu to about 1100 a.mu, or about 400 a.mu to about 1000 a.mu.

[0235] The parent organic compound may be selected from the group of drugs consisting of anticancer agents, antibacterial agents, immunomodulators, anti-obesity drugs, antidiabetic drugs, antifungal agents, antiviral agents, contraceptives, analgesics, anti-inflammatory agents (e.g., steroidal or non-steroidal anti-inflammatory drugs (NSAIDs)), antiemetics, vasodilators, vasoconstrictors, and cardiovascular agents. In particular, the parent compounds include anticancer agents such as azacitidine, bendamustine, bortezomib, cisplatin, carboplatin, cyclophosphamide, carmustine, daunorubicin, doxorubicin, etoposide, fludarabine, gemcitabine, melphalan, mitomycin, oxaliplatin, pemetrexed, pentostatin, streptozocin, thiotepa, topotecan, or vinblastine; cytoprotective agents such as amifostine; antibacterial agents such as tigecycline, doxycycline, chloramphenicol, azithromycin, or cefazolin; antifungal agents such as caspofungin, micafungin, anidurafungin, or voriconazole; antiviral agents such as acyclovir or ganciclovir; and thiothixene or midazo Antipsychotics such as lamb; anti-ulcer drugs such as esomeprazole, lansoprazole, or pantoprazole; analgesics such as metamizole, hydromorphone, or remifentanil; anti-inflammatory drugs such as hydrocortisone, methylprednisolone, indomethacin, ketoprofen, or parecoxib; immunomodulators such as methotrexate; antiemetics such as aprepitant, drasetron, fosaprepitant, granisetron, ondansetron, metoclopramide, hycosine, or promethazine; cardiovascular agents such as atenolol, dobutamine, or epoprostenol; anesthetics such as methexital; and pharmaceutically acceptable salts thereof or combinations thereof.

[0236] In some embodiments, the present invention provides a method for creating a conditionally active molecule from a parent organic compound. The method includes modifying the parent organic compound by introducing one or more charged groups to produce a modified organic compound; subjecting the modified organic compound to assays under normal physiological conditions and under abnormal conditions; and selecting, from the modified organic compound, a conditionally active molecule that exhibits higher activity under abnormal conditions as compared to normal physiological conditions.

[0237] The modification of the parent organic compound may be achieved by substituting one or more uncharged and / or partially charged groups in the parent organic compound form with one or more partially charged or charged groups, or by the addition of one or more partially charged or charged groups. The addition of one or more partially charged or charged groups to the parent organic compound may be modified by substituting one or more atoms or neutral groups, such as hydrogen atoms on the parent organic compound, with one or more partially charged or charged groups. The partially charged or charged group may be positively or negatively charged. Examples of suitable charged groups include, but are not limited to, -COO - -SO3 - -PO4 - -PO3 - -PO2 - -BO3 - -NH2 + -NH3 + and other charged groups. Examples of suitable partially charged groups include polar groups or polar side chains.

[0238] In other embodiments, the parent organic compound may be modified by removing one or more partially charged or charged groups from the parent organic compound.

[0239] The generated and modified organic compounds are subjected to assays under normal physiological conditions and under abnormal conditions. In some embodiments, the abnormal conditions are values ​​of extracellular conditions of senescent cells, such as pH in the range of about 5.0 to less than about 7.0, or about 5.5 to less than about 7.0, or about 6.0 to less than about 7.0, or about 6.2 to about 6.8. The normal physiological conditions are different values ​​of conditions in the extracellular environment of normal cells, such as pH in the range of about 7.0 to less than about 7.8, or about 7.2 to less than about 7.8, or about 7.2 to less than about 7.6.

[0240] The activity of the modified organic compound is measured in both assays. The conditionally active molecule is (a) a decrease in the activity of the parent protein in the assay compared to the activity of the parent protein in the assay under normal physiological conditions, and an increase in the activity of the conditionally active protein in the assay compared to the activity of the conditionally active protein in the assay under normal physiological conditions; and (b) a decrease in the activity of the parent protein in the assay under normal physiological conditions compared to the activity of the parent protein in the assay under abnormal conditions, and an increase in the activity of the parent protein in the assay under abnormal conditions compared to the activity of the parent protein in the assay under abnormal conditions. A modified organic compound exhibiting at least one of the following may be selected. The assay solution used in assays under abnormal conditions and assays under normal physiological conditions may contain the small molecules and / or molecular species discussed earlier.

[0241] The activity measured in assays under both abnormal and normal physiological conditions may be an assay for the binding of the molecule to the target.

[0242] In certain embodiments, the conditionally active molecule has a ratio of activity under abnormal conditions to activity under normal physiological conditions (e.g., high selectivity between the two conditions) greater than 1.0. The activity ratio may be at least about 1.3:1, or 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.

[0243] The conditionally active protein may be further manipulated as described in WO2016 / 138071. The conditionally active protein may be manipulated through antibody conjugation, to generate a multispecific antibody, to generate a bispecific conditionally active antibody against an immunoeffector cell surface antigen, to generate a masked conditionally active protein, and / or the Fc region of the antibody may be manipulated, each as described in WO2016 / 138071. The conditionally active protein may be used to manipulate conditionally active viral particles as described in WO2015 / 175375.

[0244] T cells are used by the mammalian immune system to fight substances or cells that contain foreign antigens. CAR-T technology utilizes genetic engineering to reprogram naturally circulating T cells by inserting chimeric antigen receptors (CARs) into T cells to generate highly specific CAR-T cells, where the CARs specifically bind to antigens on the surface of target tissues, thereby directing the engineered CAR-T cells toward the target tissue. Thus, these CAR-T cells can specifically target tumor cells, making them considerably more effective than naturally circulating T cells. These CAR-T cells may also be engineered to target senescent cells.

[0245] The CAR of the present invention includes 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 3 and Jensen et al., “Design and implementation of adoptive therapy with chimeric antigen receptor-modified T cells,” Immunol Rev., vol. 257, pp. 127-144, 2014. After the ASTR specifically binds to the target antigen, the ISD activates intracellular signaling within the CAR-T cell. For example, the ISD can rewrite CAR-T cell specificity and reactivity to target selected by a non-MHC-restricted approach to mimic the antigen-binding properties of an antibody. Non-MHC-restricted antigen recognition confers on CAR-T cells the ability to recognize senescent cells and initiate antigen processing. In one embodiment, the ESD and / or CSD are optional. In another embodiment, the ASTR has bispecificity and specifically binds to two different antigens or epitopes. The conditional active protein of the present invention may be engineered as an ASTR or a part thereof to make the CAR more active in the extracellular environment of senescent cells. Such a CAR can preferentially deliver T cells to senescent cells, thereby dramatically reducing side effects induced by T cell attack on normal tissues. Thereby, higher doses of T cells can be used to increase treatment efficacy and improve the subject's tolerance to the treatment.

[0246] The ASTR may include a conditional active protein such as an antibody that specifically binds to an antigen on senescent cells, particularly a single-chain antibody, or a fragment thereof. Some examples of proteins suitable for the ASTR include linked cytokines (leading to the recognition of cells bearing cytokine receptors), affibodies, ligand-binding domains from naturally derived receptors, and soluble protein / peptide ligands for receptors on senescent cells.

[0247] In some embodiments, the CAR of the present invention comprises at least two ASTRs that target at least two different antigens or two epitopes on the same antigen. In one embodiment, the CAR comprises three or more ASTRs that target three or more different antigens or epitopes. When multiple ASTRs are present within the CAR, the ASTRs may be arranged in series or separated by linker peptides (Figure 3).

[0248] In yet another embodiment, the ASTR comprises a diabody. In the diabody, the scFv is created by a linker peptide that is too short to fold the two variable regions together, inducing the scFv to dimerize. Even shorter linkers (one or two amino acids) lead to the formation of trimers, so-called triabodies or tribodies. Tetrabodies may be used in the ASTR.

[0249] Target antigens include surface proteins found on senescent cells, such as the surface proteins discussed earlier.

[0250] In some embodiments, the extracellular spacer domain and transmembrane domain may be resistant to ubiquitination, enhancing CAR-T cell signaling, i.e., enhancing their activity (Kunii et al., “Enhanced function of redirected human t cells expressing linker for activation of t cells that is resistant to ubiquitylation,” Human Gene Therapy, vol. 24, pp. 27-37, 2013). Within this region, the extracellular spacer domain is outside the CAR-T cell, i.e., exposed to different conditions and potentially generating conditional ubiquitination resistance.

[0251] The conditionally active proteins of the present invention may be included in pharmaceutical compositions, pharmaceutical devices, kits, or products for human pharmaceutical or diagnostic use, as described in detail in WO2016 / 138071.

[0252] The conditionally active proteins and pharmaceutical compositions of the present invention may be used to treat senescent cell-related diseases and disorders, such as age-related diseases and disorders, in subjects requiring treatment. Examples of senescent cell-related diseases, disorders, or illnesses that can be treated by administering the conditionally active proteins or pharmaceutical compositions described herein include: cognitive disorders (e.g., mild cognitive impairment (MCI), Alzheimer's disease and other dementias; Huntington's disease); cardiovascular diseases (e.g., atherosclerosis, cardiac diastolic dysfunction, aortic aneurysm, angina pectoris, arrhythmia, cardiomyopathy, congestive heart failure, coronary artery disease, myocardial infarction, endocarditis, hypertension, mild arterial disease, peripheral vascular disease, cardiac stress resistance, cardiofibrosis); metabolic diseases and disorders (e.g., obesity, diabetes, metabolic syndrome); neurological diseases and disorders, including neurodegenerative diseases and disorders (e.g., Parkinson's disease, motor neuron dysfunction (MND)); cerebrovascular diseases; emphysema; benign prostatic hyperplasia; lung diseases (e.g., idiopathic pulmonary fibrosis, chronic obstructive pulmonary disease (COPD), etc.) Examples include: tumors, bronchiolitis obliterans, asthma; pulmonary failure; inflammatory / autoimmune diseases and disorders (e.g., osteoarthritis, eczema, psoriasis, osteoporosis, mucositis, transplant-related diseases and disorders); ophthalmic diseases or disorders (e.g., age-related macular degeneration, cataracts, glaucoma, vision loss, presbyopia); diabetic ulcers; metastases; side effects of chemotherapy, side effects of radiotherapy; age-related diseases and disorders (e.g., kyphosis, renal failure or impaired renal function, frailty, alopecia, hearing loss, muscle fatigue, skin diseases, sarcopenia, and herniated discs) and other age-related diseases induced by aging (e.g., diseases / disorders resulting from radiation, chemical exposure, smoking, high-fat / high-sugar diets, and environmental factors); wound healing; skin nevi; and fibrous diseases and disorders (e.g., cystic fibrosis, renal fibrosis, hepatic fibrosis, pulmonary fibrosis, oral submucosal fibrosis, cardiac fibrosis, and pancreatic fibrosis).

[0253] In a more specific embodiment, a method is provided for treating senescence-related diseases or disorders by administering the conditionally active protein or pharmaceutical composition to kill or eliminate disease- or disorder-related senescent cells (i.e., confirmed senescent cells) in a subject having the disease or disorder. In a particular exemplary embodiment, the present invention is used to treat osteoarthritis; idiopathic pulmonary fibrosis; chronic obstructive pulmonary disease (COPD); or atherosclerosis.

[0254] Subjects who may benefit from the use of the methods described herein, including the administration of the conditionally active protein or pharmaceutical composition (i.e., patients, individuals (human or non-human animals)), also include subjects who may have cancer. Subjects treated by these methods may be considered to be in partial or complete remission (also known as cancer remission). As discussed in detail herein, the conditionally active protein or pharmaceutical composition for use in methods for the selective killing or removal of senescent cells is not intended to be used as a treatment for cancer, i.e., in a method that kills or destroys cancer cells in a statistically significant manner. Therefore, the methods disclosed herein do not include the use of the conditionally active protein or pharmaceutical composition in a manner that would be considered a first-line treatment for cancer. Even if the conditionally active protein is not used alone or in combination with other chemotherapeutic or radiotherapeutic agents in a manner sufficient to be considered a first-line cancer treatment, the conditionally active protein or pharmaceutical composition described herein may be used in a manner useful for inhibiting metastasis (e.g., a short course of treatment). In certain embodiments, the subject treated with the conditionally active protein or pharmaceutical composition does not have cancer (i.e., the subject has not been diagnosed with cancer by a person skilled in the art of medicine).

[0255] Cardiovascular diseases and disorders The senescent cell-related disease or disorder treated with the conditionally active protein or pharmaceutical composition may be a cardiovascular disease. The cardiovascular disease may be any one or more of the following: angina pectoris, arrhythmia, atherosclerosis, cardiomyopathy, congestive heart failure, coronary artery disease, carotid artery disease, endocarditis, heart attack (coronary thrombosis, myocardial infarction), hypertension / hypertension, aortic aneurysm, cerebral aneurysm, cardiac fibrosis, cardiac diastolic dysfunction, hypercholesterolemia / hyperlipidemia, mitral valve prolapse, peripheral vascular disease (e.g., peripheral artery disease), cardiac stress resistance, and stroke.

[0256] In certain embodiments, methods are provided for treating senescent cell-related cardiovascular diseases associated with or induced by arteriosclerosis (i.e., hardening of arteries). These cardiovascular diseases may be any one or more of the following: atherosclerosis (e.g., coronary artery disease (CAD) and carotid artery disease); angina pectoris, congestive heart failure, and peripheral vascular diseases (e.g., peripheral artery disease (PAD)). Methods for treating cardiovascular diseases associated with or induced by arteriosclerosis may reduce the likelihood of developing hypertension, angina pectoris, stroke, and heart attacks (i.e., coronary thrombosis, myocardial infarction (MI)). In certain embodiments, methods are provided for stabilizing atherosclerotic plaques (may be multiple) in a target vessel (e.g., artery) to reduce or delay the likelihood of developing thrombotic events such as stroke or MI. In certain embodiments, these methods, including the administration of a conditionally active protein, reduce the lipid content of atherosclerotic plaques in target blood vessels (e.g., arteries) (i.e., induce a reduction in lipid content) and / or increase the thickness of the fibrous capsule (i.e., induce an increase in the thickness of the fibrous capsule, promote or accelerate thickening).

[0257] In one embodiment, a method is provided for inhibiting the formation of atherosclerotic plaques (or reducing, eliminating, or inducing a decrease in their formation) by administering the conditionally active protein or pharmaceutical composition. In another embodiment, a method is provided for reducing (reducing, eliminating) the amount (i.e., level) of atherosclerotic plaques. The reduction in the amount of atherosclerotic plaques in a blood vessel (e.g., artery) may be determined, for example, by a reduction in the surface area of ​​the plaques, or by a reduction in the degree or extent (e.g., percentage) of embolism in the blood vessel (e.g., artery), which can be measured by angiography or other visualization methods used in the cardiovascular art. Also provided herein is a method for increasing (or improving, promoting, or enhancing) the stability of atherosclerotic plaques present in one or more blood vessels (e.g., one or more arteries) of a subject, the method comprising administering the conditionally active protein or pharmaceutical composition to the subject.

[0258] The efficacy of the conditionally active protein or pharmaceutical composition for treating or preventing (i.e., reducing or decreasing the likelihood of developing or occurring) cardiovascular disease (e.g., atherosclerosis) can be immediately determined by those skilled in the art in the medical and clinical industries. One or any combination of diagnostic methods, including physical examination, assessment and monitoring of clinical symptoms, and the performance of analytical tests and methods described herein and practiced in the art (e.g., angiography, electrocardiography, stress tests, non-stress tests), may be used to monitor the health status of the subject. The effect of treatment with the conditionally active protein or pharmaceutical composition can be analyzed using techniques known in the art, such as comparing the symptoms of patients with or at risk of cardiovascular disease who received the treatment with the symptoms of patients who did not receive such treatment or who received a placebo.

[0259] Inflammatory and autoimmune diseases and disorders In certain embodiments, senescent cell-related diseases or disorders, including, but not limited to, inflammatory diseases or disorders such as osteoarthritis, may be treated or prevented (i.e., reduced in likelihood of occurrence) according to the methods described herein, including the administration of the conditionally active protein or pharmaceutical composition. Other inflammatory or autoimmune diseases or disorders include osteoporosis, psoriasis, oral mucositis, rheumatoid arthritis, inflammatory bowel disease, eczema, kyphosis, herniated discs, and lung diseases, COPD, and idiopathic pulmonary fibrosis.

[0260] Unexpectedly, by selectively killing senescent cells, the conditionally active protein or pharmaceutical composition reduces the likelihood of development, reduces or inhibits the loss or erosion of the proteoglycan layer in the joint, reduces inflammation in the affected joint, and promotes (i.e., stimulates, enhances, induces) the production of collagen (e.g., type II collagen). The removal of senescent cells may induce a reduction in the amount (i.e., levels) of inflammatory cytokines such as IL-6 produced in the joint, thereby reducing inflammation. A method for treating osteoarthritis is provided herein, by administering at least one conditionally active protein to the subject, which may selectively kill or remove senescent cells present in the osteoarthritis joint of the subject, and / or induce the production of collagen (such as type II collagen) in the joint of the subject as needed. The conditionally active protein may also be used to reduce (inhibit, reduce) the production of metalloproteinase 13 (MMP-13), which breaks down collagen in the joints, and to restore the proteoglycan layer or inhibit the loss and / or degradation of the proteoglycan layer. Treatment with the conditionally active protein or pharmaceutical composition may prevent or reduce the likelihood of bone erosion, inhibit or reduce such erosion, or slow such erosion. As described in detail herein, in certain embodiments, the conditionally active protein or pharmaceutical composition is administered directly to the joints of osteoarthritis (e.g., by intra-articular, topical, transdermal, intradermal, or subcutaneous delivery). Treatment with the conditionally active protein or pharmaceutical composition may restore, improve, or inhibit the exacerbation of joint strength. In addition, methods comprising administering the conditionally active protein or pharmaceutical composition may reduce joint pain and are therefore useful for pain management in osteoarthritis joints.

[0261] The effectiveness of one or more conditionally active proteins for the treatment or prevention of osteoarthritis in subjects, and for monitoring subjects receiving one or more senescent agents, can be immediately determined by those skilled in the art of medicine and clinical technology. One or any combination of diagnostic methods, including physical examination (e.g., measuring flexibility, swelling, or redness of the affected joint), assessment and monitoring of clinical symptoms (e.g., pain, stiffness, mobility), and the performance of analytical tests and methods described herein and practiced in the art (e.g., levels of inflammatory cytokines or chemokines; X-ray images measuring cartilage loss indicated by narrowing of the space between bones in the joint; and magnetic resonance imaging (MRI) providing detailed images of bone and soft tissue, including cartilage), may be used to monitor the health status of subjects. The effect of treatment with one or more senescent agents can be analyzed by comparing the symptoms of patients who have, or are at risk of having, an inflammatory disease or disorder such as osteoarthritis, and who have received the treatment, with the symptoms of patients who have not received such treatment or who have received a placebo.

[0262] In certain embodiments, the conditionally active protein or pharmaceutical composition may be used to treat and / or prevent rheumatoid arthritis (RA) (i.e., to reduce or decrease the likelihood of its occurrence).

[0263] Chronic inflammation can also contribute to other age-related or aging-associated diseases and disorders, such as kyphosis and osteoporosis. Kyphosis is associated with cellular senescence. The ability of senescent agents to treat kyphosis may be measured in preclinical animal models used in the art. For example, TTD mice develop kyphosis (see, e.g., de Boer et al. Science, vol. 296, pp. 1276-1279, 2002); another mouse that may be used is BubRl, which is also known to develop kyphosis. H / HMice are one example (see, for example, Baker et al. Nature, vol. 479, pp. 232-36, 2011). The development of kyphosis is measured visually over time. The level of senescent cells reduced by treatment with senescent cell deconjugates can be measured by detecting the presence of one or more senescent cell-related markers, such as SA-P-Gal staining.

[0264] In yet another embodiment, inflammatory / autoimmune disorders that can be treated or prevented (i.e., whose likelihood of occurrence is reduced) with the conditionally active proteins or pharmaceutical compositions described herein include irritable bowel syndrome (IBS) and inflammatory bowel diseases such as ulcerative colitis and Crohn's disease. Diagnosis and monitoring of these disorders are carried out according to methods and diagnostic tests routinely practiced in the art, including blood tests, colonoscopy, flexible sigmoidoscopy, barium enema, CT scans, MRI, endoscopy, and small bowel contrast radiography.

[0265] In other embodiments, the methods described herein may be useful for treating subjects with herniated discs. These subjects with herniated discs exhibit elevated rates of cellular senescence in the blood and vascular walls (see, e.g., Roberts et al. Eur. Spine J., 15 Suppl 3: S312-316, 2006). High levels of pro-inflammatory molecules and matrix metalloproteinases are also found in age-related degenerated disc tissue and are suggested to play a role in senescent cells (see, e.g., Chang-Qing et al. Ageing Res. Rev., vol. 6, pp. 247-61, 2007). Animal models may be used to characterize the effectiveness of senescent agents in treating intervertebral disc herniation; intervertebral disc degeneration can be induced in mice by assessment of compression and pelvic strength (see, e.g., Lotz et al. Spine, vol. 23, pp. 2493-506, 1998).

[0266] Other inflammatory or autoimmune diseases that can be treated or prevented (i.e., whose likelihood of occurrence is reduced) by using the conditionally active protein or pharmaceutical composition include eczema, psoriasis, osteoporosis, and lung diseases (e.g., chronic obstructive pulmonary disease (COPD), idiopathic pulmonary fibrosis (IPF), asthma), inflammatory bowel disease, and mucositis (in some cases, such as radiation-induced oral mucositis). Certain fibrosis or fibrotic conditions of organs, such as renal fibrosis, hepatic fibrosis, pancreatic fibrosis, cardiac fibrosis, skin wound healing, and oral submucosal fibrosis, may also be treated with the conditionally active protein or pharmaceutical composition.

[0267] In certain embodiments, the senescent cell-related disorders are, in non-limiting examples, inflammatory disorders of the skin such as psoriasis and eczema, which can be treated or prevented (i.e., the likelihood of occurrence is reduced) by methods described herein, including the administration of the conditionally active protein or pharmaceutical composition. The effectiveness of the conditionally active protein or pharmaceutical composition for the treatment of psoriasis and eczema, and for monitoring subjects receiving such treatment, can be immediately measured by those skilled in the art of medicine or clinical practice. This may include one or any combination of diagnostic methods, including physical examination (e.g., skin appearance), assessment and / or monitoring of clinical symptoms (e.g., itching, swelling, and pain), and the performance of analytical tests and methods described herein and practiced in the art (i.e., measurement of levels of pro-inflammatory cytokines).

[0268] Lung diseases and disorders In one embodiment, a method is provided for treating or preventing (i.e., reducing the likelihood of occurrence of) an aging cell-related disease or disorder, which is the disease or disorder itself, by killing or removing senescent cells (i.e., established senescent cells) associated with the disease or disorder in a subject having a lung disease or disorder by administering the conditional active protein or pharmaceutical composition. Aging-related lung diseases and disorders include, for example, idiopathic pulmonary fibrosis (IPF), chronic obstructive pulmonary disease (COPD), asthma, cystic fibrosis, bronchiectasis, and emphysema. The involvement of cellular senescence in IPF is suggested by the observations that the incidence of the disease increases with age and that lung tissue in IPF patients is rich in SA-P-Gal-positive cells and contains high levels of the senescence marker p21 (see, for example, Minagawa et al, Am. J. Physiol. Lung Cell. Mol. Physiol., vol. 300, pp. L391-L401, 2011). Short telomeres are a risk factor common to both IPF and cellular senescence (see, for example, Alder et al, Proc. Natl. Acad. Sci. USA, vol. 105, pp. 13051-56, 2008). Without wishing to be bound by theory, the contribution of cellular senescence to IPF is reported to be suggested by the fact that SASP components of senescent cells such as IL-6, IL-8, and IL-1β promote the differentiation of fibroblasts to myofibroblasts and the epithelial-to-mesenchymal transition, resulting in large-scale remodeling of the extracellular matrix in the alveoli and interstitial space (see, for example, Minagawa et al cited above).

[0269] Other lung diseases or disorders that can be treated by using the conditional active protein or pharmaceutical composition include, for example, emphysema, asthma, bronchiectasis, and cystic fibrosis (see, for example, Fischer et al, Am J Physiol Lung Cell Mol Physiol., vol. 304, pp. L394-400, 2013).

[0270] Methods described herein for treating or preventing age-related lung diseases or disorders (i.e., reducing the likelihood of their occurrence) may be used to treat aging subjects, as well as subjects with loss (or degeneration) of lung function (i.e., reduced or impaired lung function compared to younger subjects) and / or degeneration of lung tissue. Senescent cells may be administered to aging subjects (including asymptomatic middle-aged adults) to kill and remove senescent cells from the respiratory tract, thereby slowing or inhibiting the decline of lung function. The effects of treatment with such conditionally active proteins or pharmaceutical compositions may be analyzed using techniques known in the art, such as comparing the symptoms of patients with or at risk of the treated lung disease to the symptoms of patients who have not received such treatment or who received a placebo. In addition, methods and techniques for evaluating the mechanical function of the lungs, such as techniques for measuring lung volume, elastance, and airway hyperresponsiveness, may be employed. To measure and monitor lung function throughout the procedure, numerous measurements may be obtained, namely expiratory reserve volume (ERV), forced vital capacity (FVC), fetal vital capacity (FEV) (e.g., FEV1 per second), FEV1 / FEV ratio, forced expiratory flow rate 25%–75%, and one of the following: maximal ventilation (MVV), peak respiratory flow (PEF), or vital capacity (SVC). Overall lung volume includes total lung volume (TLC), vital capacity (VC), residual volume (RV), and functional residual volume (FRC). Gas exchange across the alveolar capillary membrane can be measured using carbon monoxide diffusion capacity (DLCO). Peripheral capillary oxygen saturation (SpO2) may also be measured.

[0271] Neurological diseases and disorders Senescent cell-related diseases or disorders that can be treated by administering the conditionally active protein or pharmaceutical composition include neurological diseases or disorders. Such senescent cell-related diseases and disorders include Parkinson's disease, Alzheimer's disease (and other dementias), motor neuron dysfunction (MND), mild cognitive impairment (MCI), Huntington's disease, and eye diseases and disorders such as age-related macular degeneration. Other eye diseases associated with increasing age include glaucoma, vision loss, presbyopia, and cataracts.

[0272] The aging of dopamine-producing neurons is thought to contribute to the cell death observed in PD through the generation of reactive oxygen species (see, for example, Cohen et al, J. Neural Transm. Suppl. 19:89-103 (1983)), and therefore, the conditionally active proteins or pharmaceutical compositions described herein are useful for the treatment and defense of Parkinson's disease.

[0273] Methods for detecting, monitoring, or quantifying neurodegenerative defects and / or spontaneous movement deficits associated with Parkinson's disease are publicly known in the art, including histological, biochemical, and behavioral assessments (see, for example, US2012 / 0005765). Symptoms of Parkinson's disease are publicly known in the art and include, but are not limited to, difficulty initiating or ending voluntary movements, seizures, rigid movements, muscle atrophy, tremors, changes in heart rate, but normal reflexes, bradykinesia, and postural sway.

[0274] The efficacy of the conditional proteins or pharmaceutical compositions described herein in subjects who have received one or more senescent cell desorbents can be immediately determined by those skilled in the art in the medical and clinical industries. Physical examination, assessment and monitoring of clinical symptoms, and one or any combination of diagnostic methods, including the performance of analytical tests and methods described herein, may be used to monitor the health status of the subjects. The effects of administering the conditional active proteins or pharmaceutical compositions may be analyzed using techniques known in the art, such as comparing the symptoms of patients with or at risk of Alzheimer's disease who have received the treatment with the symptoms of patients who have not received such treatment or who have received a placebo.

[0275] Mild cognitive impairment (MCI) MCI is a neurological syndrome characterized by the onset and development of cognitive impairment that exceeds what is predicted based on the individual's age and education, but is not significant enough to interfere with the individual's daily activities. Administration of the conditionally active protein may reduce or inhibit MCI by killing or removing senescent cells. Methods for detecting, monitoring, quantifying or assessing neuropathological defects associated with MCI are known in the art, such as morphological analysis of astrocytes, acetylcholine release, silver staining for assessing neurodegeneration, and PiB PET imaging for detecting beta-amyloid deposition (see, for example, US2012 / 0071468). Methods for detecting, monitoring, quantifying or assessing behavioral abnormalities associated with MCI are also known in the art, such as the eight-direction radial maze paradigm, non-matching task, heterocentral spatial determination task in a water maze, Maurice maze test, visuospatial task, and delayed-response spatial memory task, and the olfactory novelty test (see the same book).

[0276] Motor neuron dysfunction (MND) MNDs are a group of progressive neurological disorders that destroy motor neurons, which are cells that control essential voluntary muscle activities such as speech, walking, breathing, and swallowing. Examples of MNDs include, but are not limited to, amyotrophic lateral sclerosis (ALS), also known as Lou Gehrig's disease; progressive bulbar palsy; pseudobulbar palsy; primary lateral sclerosis; progressive muscular atrophy; lower motor neuron disease; and spinal muscular atrophy (SMA) (e.g., SMA1, also known as Werdnig-Hoffmann disease; Kugelberg-Verander disease; and SMA2, SMA3, also known as Kennedy disease); post-polio syndrome; and hereditary spastic paraplegia. Administration of the conditionally active protein may reduce or inhibit MNDs by killing or removing senescent cells. Methods for detecting, monitoring, or quantifying spontaneous motor deficits and / or other deficiencies associated with Parkinson's disease, such as MNDs, are known in the art (see, for example, US20120005765). Methods for detecting, monitoring, quantifying, or assessing MND-related exercise deficiency and histopathological deficiencies, including histopathological, biochemical, and electrophysiological examinations and exercise activity analysis, are known in the art (see, for example, Rich et al., J Neurophysiol, vol. 88, pp. 3293-3304, 2002; Appel et al, Proc. Natl. Acad. Sci. USA, vol. 88, pp. 647-51, 1991).

[0277] Ophthalmic diseases and disorders In certain embodiments, senescent cell-related diseases or disorders are eye diseases, disorders, or illnesses, such as presbyopia, macular degeneration, or cataracts. In other specific embodiments, senescent cell-related diseases or disorders are glaucoma. Macular degeneration is a neurodegenerative disease that causes the loss of photoreceptor cells in the central part of the retina called the macula. The exact cause of age-related macular degeneration is still unknown, but the number of senescent retinal pigment epithelial (RPE) cells increases with age. Age and certain genetic and environmental factors are risk factors for developing ARMD (see, for example, Lyengar et al, Am. J. Hum. Genet., vol. 74, pp. 20-39, 2004; Kenealy et al, Mol. Vis., vol. 10, pp. 57-61, 2004; Gorin et al, Mol. Vis., vol. 5, p. 29, 1999). A reduction in microRNAs contributes to a senescent cell profile; DICER1 excision induces premature aging. The diagnosis and monitoring of subjects with macular degeneration can be achieved by those skilled in the art of ophthalmology, following the procedures of routine eye examinations accepted in the art and the reporting of symptoms by the subjects.

[0278] Age-related changes in the mechanical properties of the anterior and posterior lens capsules suggest that the mechanical strength of the posterior lens capsule decreases significantly with age (see, e.g., Krag et al, Invest. Ophthalmol. Vis. Sci., vol. 44, pp. 691-96, 2003; Krag et al, Invest. Ophthalmol. Vis. Sci., vol. 38, pp. 357-63, 1997). The layered structure of the lens also changes, which may be at least partly due to changes in tissue composition.

[0279] Studies suggest that type IV collagen influences cellular function, inferred from its placement in the basement membrane beneath the epithelial layer, and the role of type IV collagen in tissue stabilization is supported by data. Posterior capsular opacity (PCO) develops as a complication in approximately 20–40% of patients within several years after cataract surgery (see, e.g., Awasthi et al, Arch Ophthalmol., vol. 127, pp. 555-62, 2009). PCO arises from the proliferation and activation of residual lens epithelial cells along the posterior capsule in a response similar to wound healing. Growth factors such as fibroblast growth factor, transforming growth factor β, epidermal growth factor, hepatocyte growth factor, insulin-like growth factor, and interleukins IL-1 and IL-6 can also promote epithelial cell migration. As discussed herein, the production of these factors and cytokines by senescent cells contributes to SASP. In contrast, in vitro studies have shown that type IV collagen promotes the adhesion of lens epithelial cells (see, e.g., Olivero et al, Invest. Ophthalmol. Vis. Sci., vol. 34, pp. 2825-34, 1993). The adhesion of type IV collagen, fibronectin, and laminin to intraocular lenses may inhibit cell migration and reduce the risk of PCOS (see, e.g., Raj et al, Int. J. Biomed. Sci., vol. 3, pp. 237-50, 2007).

[0280] While we do not wish to tie this to any particular theory, the selective killing or removal of the conditionally active proteins described herein may slow or interfere with (delay, inhibit, or block) the division of type IV collagen networks. The removal of senescent cells and the resulting elimination of the inflammatory effects of SASP may reduce or inhibit epithelial cell migration, delay (suppress) the onset of presbyopia, or slow or slow the progression of disease severity (e.g., slowing the progression from mild to moderate or moderate to severe). The conditionally active proteins and pharmaceutical compositions described herein may also be useful in reducing the likelihood of PCO development after cataract surgery.

[0281] BubR1 hypoplasia mice develop bilateral posterior subcapsular cataracts early in life, suggesting that aging may play a role (see, e.g., Baker et al, Nat. Cell Biol., vol. 10, pp. 825-36, 2008). The presence and severity of cataracts can be monitored by ophthalmic examinations using methods routinely performed by those skilled in the art of ophthalmology.

[0282] In certain embodiments, at least one conditionally active protein that selectively kills senescent cells may be administered to subjects at risk of developing presbyopia, cataracts, or macular degeneration. Treatment with the conditionally active protein may be initiated when the human subject is at least 40 years of age to delay or inhibit the onset or development of cataracts, presbyopia, and macular degeneration. Since almost all humans develop presbyopia, in certain embodiments, a senescent cell scavenging agent may be administered to a human subject after the subject has reached 40 years of age, using the method described herein, to delay or inhibit the onset or development of presbyopia.

[0283] In certain embodiments, the age-related disease or disorder is glaucoma. Glaucoma is a broad term used to describe a group of diseases that cause visual field defects, often without other major symptoms. When the cellular network required for fluid outflow was subjected to SA-P-Gal staining, a four-fold increase in aging was observed in glaucoma patients (see, e.g., Liton et al, Exp. Gerontol., vol. 40, pp. 745-748, 2005).

[0284] Standard automated perimetry (visual field testing) is the most widely used technique for monitoring the effects of treatment on inhibiting the progression of glaucoma. In addition, several algorithms for detecting progression have been developed (see, e.g., Wesselink et al, Arch Ophthalmol., vol. 127, pp. 270-274, 2009 and its references). Additional methods include gonioscopy (examining the trabecular network and angles from which fluid drains from the eye); imaging techniques (e.g., scanning laser tomography (e.g., HRT3), laser polarization measurement (e.g., GDX), optical coherence tomography); ophthalmoscopic examination; and thickness measurement to measure the thickness of the central cornea.

[0285] Metabolic diseases or disorders Age-related diseases or disorders that can be treated by administering the conditionally active protein or pharmaceutical composition include metabolic diseases or disorders. Such senescent cell-related diseases and disorders include diabetes mellitus, metabolic syndrome, diabetic ulcers, and obesity. The conditionally active proteins described herein may be used to treat type 2 diabetes mellitus, particularly type 2 diabetes mellitus associated with age, diet, and obesity.

[0286] The involvement of senescent cells in metabolic diseases such as obesity and type 2 diabetes has been suggested as a response to injury or metabolic dysfunction (see, e.g., Tchkonia et al, Aging Cell, vol. 9, pp. 667-684, 2010). Adipose tissue derived from obese mice showed induction of aging markers SA-P-Gal, p53, and p21 (see, e.g., Minamino et al, Nat. Med., vol. 15, pp. 1082-1087, 2009). Simultaneous upregulation of pro-inflammatory cytokines such as tumor necrosis factor-alpha and Ccl2 / MCP1 was observed in the same adipose tissue (see, e.g., Minamino et al. above). Since pro-inflammatory SASP components have also been suggested to contribute to type 2 diabetes (see, e.g., Tchkonia et al. above), the induction of senescent cells in obesity has potential clinical implications. Similar patterns of upregulation of aging markers and SASP components are associated with diabetes in both mice and humans (see, e.g., Minamino et al. cited above). Therefore, the methods described herein, including the administration of senescent cells, may be useful in treating or preventing obesity and metabolic syndrome, in addition to type 2 diabetes. While not intended to be theoretical, it is possible that contact between presenting adipocytes and senescent cells, thereby killing them, could result in clinical and health benefits for individuals with diabetes, obesity, or metabolic syndrome.

[0287] A disease or disorder associated with diabetes and aging is diabetic ulcer (i.e., diabetic wound). An ulcer is a destruction of the skin, which can extend to include subcutaneous tissue, muscle, or even bone. These lesions occur particularly in the lower extremities. Patients with diabetic venous ulcers show increased presence of cellular senescence at the site of chronic wounds (see, e.g., Stanley et al. J. Vas. Surg., vol. 33, pp. 1206-1211, 2001). Chronic inflammation, such as diabetic ulcers, is also observed at the site of chronic wounds (see, e.g., Goren et al. Am. J. Pathol., vol. 168, pp. 65-77), suggesting that the phenotype of pro-inflammatory cytokines in senescent cells plays a role in the pathogenesis.

[0288] The efficacy of the conditionally active protein can be immediately determined by those skilled in the art of medicine and clinical technology. One or any combination of diagnostic methods, including physical examination, assessment and monitoring of clinical symptoms, and the performance of analytical tests and methods such as those described herein, may be used to monitor the health status of the subject. Subjects receiving one or more senescent cytophagectomy agents described herein for the treatment or defense of diabetes can be monitored, for example, by assaying glucose and insulin resistance, energy expenditure, body composition, adipose tissue, skeletal muscle, and hepatic inflammation, and / or lipid toxicity (muscle and hepatic lipids by in vivo imaging, as well as muscle, liver, bone marrow, and pancreatic β-cell lipid accumulation, and inflammation by histological examination). Other characteristic features or phenotypes of type 2 diabetes are known and can be assayed as described herein and by utilizing other methods and techniques known and routinely practiced in the art.

[0289] Individuals with type 2 diabetes or at risk of developing type 2 diabetes may have metabolic syndrome. Metabolic syndrome in humans is typically associated with obesity and is characterized by one or more of the following: cardiovascular disease, hepatic steatohepatosis, hyperlipidemia, diabetes, and insulin resistance. Individuals with metabolic syndrome may represent a group of metabolic disorders or abnormalities, which may include, for example, hypertension, type 2 diabetes, hyperlipidemia, dyslipidemia (e.g., hypertriglyceridemia, hypercholesterolemia), insulin resistance, hepatic steatohepatosis (steatohepatitis), hypertension, atherosclerosis, and one or more of the following other metabolic disorders.

[0290] Skin disease or disorder Senescent cell-related diseases or disorders that can be treated by administering the conditionally active proteins or pharmaceutical compositions described herein include skin diseases or disorders. Such senescent cell-related diseases and disorders include psoriasis and eczema, which are also inflammatory diseases and have been discussed in more detail previously. Other skin diseases and disorders associated with aging include fine wrinkles (wrinkles due to aging); psychogenic pruritus (associated with diabetes and aging); dysesthesia (as a side effect of chemotherapy associated with diabetes and multiple sclerosis); psoriasis (as described) and other papular-scalding disorders, e.g., erythroderma, lichen planus, and lichenoid dermatitis; atopic dermatitis (associated with the form and inflammation of eczema); and eczematous rash (which is more commonly observed in elderly patients and is associated with the side effects of certain drugs). Other skin diseases and disorders associated with aging include eosinophilic dermatosis (associated with certain types of hematological cancers); reactive neutrophilic dermatosis (associated with underlying conditions such as inflammatory bowel syndrome); pemphigus (an autoimmune disease in which autoantibodies form against desmoglein); pemphigus-like and other immunobullous dermatosis (autoimmune blister formation of the skin); age-related fibrous histiocytic proliferation of the skin; and cutaneous lymphoma, which is more common in older populations. Another skin disease that may be treatable according to the methods described herein is cutaneous lupus, a symptom of lupus erythematosus. Late-onset lupus may be associated with decreased T-cell and B-cell function (i.e., decline), as well as age-related cytokines (immunoseniation).

[0291] metastasis In certain embodiments, the conditionally active protein or pharmaceutical composition may be used to treat or prevent metastasis (i.e., the spread and proliferation of cancer or tumor cells) from one organ or tissue to another in the body. A subject with cancer may benefit from the administration of the conditionally active protein or pharmaceutical composition to inhibit metastasis. Such a conditionally active protein or pharmaceutical composition may inhibit tumor growth. Cancer metastasis occurs when cancer cells (i.e., tumor cells) spread beyond the anatomical site of origin and initial colonization to other areas of the subject's body. Tumor growth can be measured by tumor size, which can be measured by various methods familiar to those skilled in the art, such as PET scans, MRI, CAT scans, and biopsies. The effect of a therapeutic agent on tumor growth can also be evaluated by examining the differentiation of tumor cells.

[0292] As used herein and in the art, the terms cancer or tumor are clinical descriptive terms encompassing diseases typically characterized by cells exhibiting abnormal cell proliferation. The term cancer is commonly used to describe malignant tumors or disease conditions resulting from malignant tumors. Alternatively, abnormal growth may be referred to in the art as neoplasm. The term tumor, such as tissue-related tumors, generally refers to any abnormal tissue growth characterized by at least partially excessive and abnormal cell proliferation. Tumors can be metastatic and can spread beyond their anatomical site of origin and initial colonization to other areas of the body in question. Cancer may include solid tumors or “liquid” tumors (e.g., leukemia and other blood cancers).

[0293] Cells are induced to age by cancer treatments such as radiation and certain chemotherapeutic agents. The presence of senescent cells increases the secretion of inflammatory molecules (see the description herein of senescent cells), promoting tumor progression, which can accelerate tumor growth, increase tumor size, promote metastasis, and alter differentiation. When senescent cells are destroyed, tumor progression is significantly inhibited, resulting in smaller tumors with little or no metastatic growth observed (see, e.g., WO2013 / 090645). Therefore, the conditionally active protein or pharmaceutical composition may be administered after chemotherapy or radiotherapy to kill or eliminate these senescent cells. As discussed herein and understood in the art, the confirmation of senescence, such as by the presence of a senescent cell-associated secretory phenotype (SASP), takes place over several days; therefore, once senescence is confirmed, administration of senescent agents is initiated to kill the senescent cells, thereby reducing the likelihood of metastasis or the extent of metastasis.

[0294] In certain embodiments, when chemotherapy or radiotherapy is administered in a treatment cycle of at least one day of on-therapy (i.e., chemotherapy or radiotherapy) followed by at least one week of off-therapy, the conditionally active protein or pharmaceutical composition is administered for at least one day during the off-therapy interval, starting on or after the second day of the off-therapy interval and ending on or before the last day of the off-therapy interval. In more specific embodiments, when chemotherapy or radiotherapy is administered in a treatment cycle of at least one day of on-therapy (i.e., chemotherapy or radiotherapy) followed by at least one week of off-therapy, the conditionally active protein or pharmaceutical composition is administered on the sixth day of the off-therapy interval. In other specific embodiments, when chemotherapy or radiotherapy is administered in a treatment cycle of at least one day of on-therapy (i.e., chemotherapy or radiotherapy) followed by at least two weeks of off-therapy, the conditionally active protein or pharmaceutical composition is administered starting on the sixth day of the off-therapy interval and ending at least one day or at least two days before the first day of the subsequent course of chemotherapy or radiotherapy.

[0295] In another embodiment for treating metastasis, the conditionally active protein or pharmaceutical composition may be administered after the completion of a chemotherapy or radiotherapy treatment regimen. In a particular embodiment, the conditionally active protein or pharmaceutical composition is administered for at least one day within a treatment window (i.e., a treatment cycle of senescent agents) of 14 days or less after the completion of chemotherapy or radiotherapy.

[0296] The methods described herein are also useful for inhibiting, halting, or slowing the progression of metastatic cancer of any one of the tumor types described in the field of medical technology. These cancer (tumor) types include: adrenocortical carcinoma, childhood adrenocortical carcinoma, AIDS-related cancer, anal cancer, appendiceal cancer, basal cell carcinoma, childhood basal cell carcinoma, bladder cancer, childhood bladder cancer, bone cancer, brain tumor, childhood astrocytoma, childhood brainstem glioma, childhood atypical teratomas / rhabdomyosarcomas, childhood germ cell tumors, childhood craniopharyngiomas, childhood ependymomas, breast cancer, Childhood bronchial tumors, carcinoid tumors, childhood carcinoid tumors, gastrointestinal carcinoid tumors, cancer of unknown primary origin, childhood cancer of unknown primary origin, childhood cardiac tumors, cervical cancer, childhood cervical cancer, childhood chordoma, chronic myeloproliferative disorders, colon cancer, colorectal cancer, childhood colorectal cancer, extrahepatic bile duct cancer, ductal carcinoma in situ (DCIS), endometrial cancer, esophageal cancer, childhood esophageal cancer, childhood nasal neuroblastoma, eye cancer, malignant fibrous histiocytoma of bone, gallbladder cancer, gastric cancer cancer), childhood gastric cancer, gastrointestinal stromal tumor (GIST), childhood gastrointestinal stromal tumor (GIST), childhood extracranial germ cell tumor, extragonadal germ cell tumor, gestational choriocarcinoma, glioma, head and neck cancer, childhood head and neck cancer, hepatocellular carcinoma (liver) cancer, hypopharyngeal cancer, kidney cancer, renal cell carcinoma, Wilms' tumor, childhood renal tumor, Langerhans cell histiocytosis, laryngeal cancer, childhood laryngeal cancer, leukemia, acute lymphoblastic leukemia (ALL), acute myeloid leukemia (AML), chronic lymphocytic leukemia (CLL), chronic myeloid leukemia (CML), Hairy cell leukemia, lip cancer, primary liver cancer, primary childhood liver cancer, lobular carcinoma in situ (LCIS), lung cancer, non-small cell lung cancer, small cell lung cancer, lymphoma, AIDS-related lymphoma, Burkitt lymphoma, cutaneous T-cell lymphoma, Hodgkin lymphoma, non-Hodgkin lymphoma, primary central nervous system lymphoma (CNS), melanoma, childhood melanoma, intraocular melanoma, Merkel cell carcinoma, malignant mesothelioma, childhood malignant mesothelioma, metastatic squamous cell carcinoma of the neck with occult origin, midline tract carcinoma related to the NUT geneCarcinoma, oral cancer, childhood multiple endocrine neoplasia, mycosis fungoides, myelodysplastic syndrome, myelodysplastic neoplasm, spinal proliferative neoplasm, multiple myeloma, nasal cavity cancer, nasopharyngeal cancer, childhood nasopharyngeal cancer, neuroblastoma, oral cancer, childhood oral cancer, oropharyngeal cancer, ovarian cancer, childhood ovarian cancer, epithelial ovarian cancer, low-grade ovarian cancer, pancreatic cancer, childhood pancreatic cancer, pancreatic endocrine tumor (island cell tumor), childhood papillomatosis, paraganglioma, paranasal sinus cancer, parathyroid cancer, penile cancer, pharyngeal cancer, pheochromocytoma, pituitary tumor, plasmacytoma, childhood pleuropulmonary blastoma, prostate Cancer, rectal cancer, transitional cell carcinoma of the renal pelvis, retinoblastoma, salivary gland cancer, childhood salivary gland cancer, Ewing's sarcoma family of tumors, Kaposi's sarcoma, osteosarcoma, rhabdomyosarcoma, childhood rhabdomyosarcoma, soft tissue sarcoma, uterine sarcoma, Sézary syndrome, childhood skin cancer, non-melanoma skin cancer, small intestine cancer, squamous cell carcinoma, childhood squamous cell carcinoma, testicular cancer, childhood testicular cancer, laryngeal cancer, thymoma and thymic carcinoma, childhood thymoma and thymic carcinoma, thyroid cancer, childhood thyroid cancer, transitional cell carcinoma of the ureter, urethral cancer, uterine cancer of the endometrium, vaginal cancer, vulvar cancer, Waldenström macroglobulinemia.

[0297] Side effects of chemotherapy and radiotherapy In another embodiment, the senescent cell-related disorder or disease is an adverse effect of chemotherapy or radiotherapy. Examples of chemotherapeutic agents that induce senescence in non-cancer cells include anthracyclines (e.g., doxorubicin, daunorubicin); taxol (e.g., paclitaxel); gemcitabine; pomalidomide; and lenalidomide. One or more senescent cell scavenging agents administered as described herein may be used to treat and / or prevent (i.e., reduce the likelihood of their occurrence) adverse effects of chemotherapy or radiotherapy. The removal or destruction of senescent cells may improve acute toxicity, such as acute toxicity including energy imbalance of chemotherapy or radiotherapy. Acute toxic side effects include, but are not limited to, gastrointestinal toxicity (e.g., nausea, vomiting, constipation, loss of appetite, diarrhea), peripheral neuropathy, fatigue, malaise, low physical activity, hematological toxicity (e.g., anemia), hepatotoxicity, alopecia (hair loss), pain, infection, mucositis, fluid retention, skin toxicity (e.g., rash, dermatitis, hyperpigmentation, urticaria, photosensitivity, nail changes), oral problems (e.g., oral mucositis), gingival or pharyngeal problems, or any toxic side effects caused by chemotherapy or radiotherapy. For example, toxic side effects caused by radiotherapy or chemotherapy (see, for example, the National Cancer Institute website) may be mitigated by the methods described herein. Accordingly, in certain embodiments, methods are provided herein for improving (reducing, inhibiting, or preventing the occurrence of) acute toxicity of chemotherapy or radiotherapy or both in a subject receiving treatment, or for reducing the severity of toxic side effects (i.e., adverse side effects), wherein the methods include administering to the subject an agent that selectively kills, removes, or destroys senescent cells, or facilitates such selective destruction.

[0298] Administration of the conditionally active protein or pharmaceutical composition to treat, reduce the likelihood of, or reduce the severity of side effects of chemotherapy or radiotherapy may be achieved by the same treatment cycle described above for the treatment / prevention of metastasis. As described for treating or preventing metastasis (i.e., reducing the likelihood of its occurrence), the conditionally active protein or pharmaceutical composition is administered during the time interval between chemotherapy or radiotherapy treatments, or after the completion of the chemotherapy or radiotherapy treatment regimen.

[0299] In more specific embodiments, acute toxicity is acute toxicity involving energy imbalance and may include one or more of the following: weight loss, endocrine changes (e.g., hormonal imbalance, altered hormonal signaling), and changes in body composition (may include multiple). In certain embodiments, acute toxicity involving energy imbalance relates to a decrease or reduction in the subject's ability to be physically active, as indicated by lower or eliminated energy expenditure than observed in subjects who did not receive medical treatment. In a non-limiting example, such acute toxic effects involving energy imbalance include low physical activity. In other specific embodiments, energy imbalance includes fatigue or malaise.

[0300] In one embodiment, the side effect of chemotherapy treated or prevented (i.e., the likelihood of occurrence is reduced) by the conditionally active protein or pharmaceutical composition is cardiotoxicity. A subject with cancer being treated with anthracyclines (such as doxorubicin or daunorubicin) may be treated with one or more senescent agents described herein that reduce, improve, or decrease the cardiotoxicity of anthracyclines. As is well understood in the medical art, due to the cardiotoxicity associated with anthracyclines, even if the cancer responds to the drug, the maximum lifetime dose a subject can receive is limited. By administering one or more of the conditionally active proteins, cardiotoxicity may be reduced, thereby allowing the subject to receive further doses of anthracyclines, resulting in an improved prognosis related to cancer. In one embodiment, cardiotoxicity occurs with the administration of anthracyclines such as doxorubicin. Doxorubicin is an anthracycline poisomerase approved for the treatment of patients with ovarian cancer after platinum-based therapy has been unresponsive; Kaposi's sarcoma after primary systemic chemotherapy has been unresponsive or intolerant to such therapy; or multiple myeloma in combination with bortezomib in patients who have not previously received bortezomib or have received at least one prior treatment. The total lifetime dose of doxorubicin to patients is 550 mg / m². 2 If the dose exceeds this limit, it may cause myocardial damage that can lead to congestive heart failure. Cardiotoxicity can occur even at low doses if the patient also receives mediastinal radiation or another cardiotoxic drug. See drug product inserts (e.g., Doxil, Adriamycin).

[0301] In other embodiments, the conditionally active proteins or pharmaceutical compositions described herein may be used in the manner provided herein to improve chronic or long-term adverse events. Chronic toxic adverse events typically result from multiple exposures or administration of long-term chemotherapy or radiotherapy. Certain toxic effects appear considerably later than the treatment (also known as delayed toxic effects) and result from damage to organs or systems caused by the treatment. Organ dysfunction (e.g., nerve, pulmonary, cardiovascular, and endocrine gland dysfunction) has been observed in patients treated for cancer in childhood (see, e.g., Hudson et al, JAMA, vol. 309, pp. 2371-81, 2013). While we do not wish to tie to any particular theory, destroying senescent cells, particularly certain normal cells induced to senescence by chemotherapy or radiotherapy, may reduce the likelihood of developing chronic adverse events, reduce or eliminate the severity of chronic adverse events, or delay the onset of chronic adverse events. Chronic and / or late-stage toxic side effects that may occur in patients receiving chemotherapy or radiotherapy include, but are not limited to, cardiomyopathy, congestive heart disease, inflammation, premature menopause, osteoporosis, infertility, cognitive impairment, peripheral neuropathy, secondary cancer, cataracts and other vision problems, hearing loss, chronic fatigue, decreased lung capacity, and lung disease.

[0302] In addition, by administering the conditionally active protein or pharmaceutical composition, sensitivity to chemotherapy or radiotherapy may be clinically or statistically significantly enhanced in subjects with cancer by killing or eliminating senescent cells compared to cases where the conditionally active protein or pharmaceutical composition was not administered. In other words, the development of resistance to chemotherapy or radiotherapy may be inhibited when the conditionally active protein or pharmaceutical composition is administered to subjects treated with each chemotherapy or radiotherapy.

[0303] Age-related diseases and disorders The conditionally active protein or pharmaceutical composition may be useful in treating or preventing (i.e., reducing the likelihood of onset) age-related disorders or impairments that occur as part of the natural aging process or when a subject is exposed to aging inducers or factors (e.g., radiation, chemotherapy, smoking, high-fat / high-sugar diet, other environmental factors). Age-related disorders or disorders, or age-sensitive characteristics, may be associated with aging-inducing stimuli. The efficacy of the treatment methods described herein may be achieved by reducing the number of symptoms of age-related disorders or age-sensitive characteristics associated with aging-inducing stimuli, by reducing the severity of one or more symptoms, or by delaying the progression of age-related disorders or age-sensitive characteristics associated with aging-inducing stimuli. In other specific embodiments, preventing age-related disorders or age-sensitive characteristics associated with aging-inducible stimuli means preventing (i.e., reducing the likelihood of onset) or delaying the onset of age-related disorders or age-sensitive characteristics associated with aging-inducible stimuli, or the recurrence of one or more age-related disorders or age-sensitive characteristics associated with aging-inducible stimuli.

[0304] Age-related diseases or conditions include, for example, renal dysfunction, kyphosis, herniated discs, frailty, alopecia, hearing loss, visual loss (blindness or visual impairment), muscle fatigue, skin disorders, skin nevi, diabetes, metabolic syndrome, and sarcopenia. Visual loss refers to a lack of vision that the subject has had in the past. Various scales have been developed to describe the extent of vision and visual loss based on visual acuity. Age-related diseases and conditions also include skin diseases, for example, treating one or more of the following conditions: wrinkles such as superficial fine lines; hyperpigmentation; scarring; keloids; dermatitis; psoriasis; eczema (such as seborrheic dermatitis); rosacea; vitiligo; ichthyosis vulgaris; dermatomyositis; and ultraviolet keratosis.

[0305] Frailty is defined as a clinically recognizable state of increased vulnerability resulting from an age-related decline in reserve and function across numerous physiological systems, impairing the subject's ability to cope with daily or acute stressors. In certain embodiments, aging, and age-related diseases and disorders, may be treated or prevented (i.e., the likelihood of their occurrence is reduced) by administering the conditionally active protein or pharmaceutical composition. The conditionally active protein or pharmaceutical composition may inhibit the aging of adult stem cells, inhibit the accumulation of aged adult stem cells, kill adult stem cells, or promote their removal. See, for example, Park et al, J. Clin. Invest., vol. 113, pp. 175-79, 2004, and Sousa-Victor, Nature, vol. 506, pp. 316-21, 2014, for their discussion of the importance of preventing aging in stem cells to maintain tissue regenerative capacity.

[0306] The efficacy of the conditionally active protein or pharmaceutical composition for the treatment of senescent cell-related diseases or disorders described herein can be readily determined by those skilled in the art in the medical and clinical fields. One or any combination of diagnostic methods appropriate for a particular disease or disorder, which are well known to those skilled in the art, including physical examination, patient self-assessment, assessment and monitoring of clinical symptoms, clinical tests, physical fitness tests, and analytical tests and methods including diagnostic surgery, may be used, for example, to monitor the health status of the subject and the efficacy of the senescent cell scavenging agent. The effects of the treatment methods described herein may be analyzed using techniques known in the art, such as comparing the symptoms of patients who have or are at risk of having a particular disease or disorder and have received the conditionally active protein or pharmaceutical composition with the symptoms of patients who have not been treated with the conditionally active protein or pharmaceutical composition or who have received a placebo.

[0307] The efficacy of the conditionally active protein or pharmaceutical composition may, without limitation, include: reduction, decrease, or alleviation of symptoms arising from or associated with the treated disease; reduction of symptom occurrence; improvement of quality of life; longer disease-free status (i.e., a reduction in the subject's likelihood or tendency to exhibit symptoms based on the diagnosis of the disease); reduction of disease scope; stable (i.e., non-exacerbating) state of the disease; delay or slowing of disease progression; improvement or alleviation of the disease state; and remission (whether partial or complete), whether detectable or undetectable; and / or beneficial or desired clinical outcomes, including overall survival. The efficacy of the conditionally active protein or pharmaceutical composition may also mean an extension of survival compared to the survival predicted if the subject had not received the conditionally active protein or pharmaceutical composition.

[0308] Subjects, patients, or individuals requiring treatment with the conditionally active proteins or pharmaceutical compositions described herein may be humans who have developed symptoms of senescent cell-associated disease or disorder, or who are at risk of developing senescent cell-associated disease or disorder, or non-human primates or other animals (i.e., veterinary use). Non-human animals that can be treated include mammals, such as non-human primates (e.g., monkeys, chimpanzees, gorillas, etc.), rodents (e.g., rats, mice, gerbils, hamsters, ferrets, rabbits), lagomorphs, domesticated pigs (e.g., pigs, miniature pigs), horses, dogs, cats, cattle, elephants, bears, and other domesticated animals, livestock, and zoo animals.

[0309] Examples Examples 1-9 for producing conditionally active proteins are described in WO2016 / 138071.

[0310] Example 10: Activity of conditionally active antibodies in different buffers

[0311] The activity of conditionally active antibodies evolved from two monoclonal antibodies (mAb 048-01 and mAb 048-02 as parent antibodies) was measured in two different buffers (Figure 4). The two buffers were phosphate buffer (condition IV) and Krebs buffer (condition I). Six conditionally active antibodies: CAB Hit 048-01, CAB Hit 048-02, CAB Hit 048-03, CAB Hit 048-04, CAB Hit 048-05, and CAB Hit 048-06 were evolved from mAb 048-01. Three conditionally active antibodies: CAB Hit 048-07, CAB Hit 048-08, and CAB Hit 048-09 were evolved from mAb 048-02.

[0312] This study demonstrated that the selectivity of the conditionally active antibody (the ratio of activity in the pH 6.0 assay to activity in the pH 7.4 assay) was affected by the buffer used in the assay. The conditionally active antibody evolved from wild-type mAb 048-02 showed significantly higher selectivity in Krebs buffer than in phosphate buffer (Figure 4).

[0313] Example 11: Selectivity of conditionally active antibodies and bicarbonates

[0314] In Example 10, higher selectivity of the conditionally active antibody was observed in Krebs buffer (Condition I) than in phosphate buffer (Condition IV). This led to the identification of the component in the Krebs buffer that most significantly contributed to the higher selectivity observed in Example 10. The selectivity of one conditionally active antibody was retested with buffers obtained from Krebs buffers in which one component was subtracted from various components at a time (Figure 5, left bar group). When complete Krebs buffer was used, the selectivity of the conditionally active antibody was high, with an activity ratio of approximately 8 at pH 6.0 / 7.4. When components A to F were each subtracted from the Krebs buffer, the selectivity of the conditionally active antibody was not lost, but the selectivity of the conditionally active antibody decreased when components C and D were each subtracted. However, when component G (bicarbonate) was subtracted from the Krebs buffer, the selectivity of the conditionally active antibody was completely lost. See Figure 5. This indicates that bicarbonate contributes, at least partially, to the high selectivity of the conditionally active antibody in Krebs buffer.

[0315] Subsequently, the selectivity of the same conditionally active antibody was measured in phosphate buffer without bicarbonate (Condition IV), and it was observed that the selectivity of the conditionally active antibody was completely lost in the phosphate buffer. When bicarbonate was added to the phosphate buffer, the selectivity of the conditionally active antibody was restored to the level observed in Krebs buffer. This confirmed that bicarbonate is necessary for the selectivity of this conditionally active antibody.

[0316] Example 12: Bicarbonate inhibits binding at pH 7.4.

[0317] In this example, the binding activity of three conditionally active antibodies (CAB Hit A, CAB Hit B, and CAB Hit C) at pH 7.4 was measured in buffers with different bicarbonate concentrations ranging from 0 to the physiological concentration of bicarbonate (approximately 20 mM) (Figure 6). It was observed that the binding activity of all three conditionally active antibodies at pH 7.4 decreased in a dose-dependent manner as the bicarbonate concentration increased from 0 to the physiological concentration (Figure 6). On the other hand, the binding activity of the wild-type antibody was not affected by bicarbonate. This test suggests that the selectivity of the conditionally active antibodies in the presence of bicarbonate may be at least partly due to the loss of binding activity to the conditionally active antibodies at pH 7.4 due to interaction with bicarbonate.

[0318] Example 13: Induction of senescent cells

[0319] Cell seeding: In a 6-well plate, seed 1.0 × 10 cells per well in 2 mL of medium for blank and treatment purposes. 5 Cellular MDA-MB468(P10), MDA-MB231(Px), and 2.0 × 10 5 The cells were seeded as MCF-7(Px). The cells were cultured overnight. MCF-7 is an ERa+ cell line. Palbociclib exhibits antiproliferative activity in this cell line, halting cell proliferation and inducing senescent cells. MDA-MB231 is an ERa-cell line. Palbociclib exhibits antiproliferative activity in this cell line, halting cell proliferation and inducing senescent cells. MDA-MB468 is another ERa-cell line. Palbociclib does not have an antiproliferative effect in this cell line and therefore does not halt growth or induce senescent cells.

[0320] Preparation of palbociclib solution: 25 mg of palbociclib isethionate (PD-0332991, Selechchem, catalog number S1579, batch 4, 25 mg) was added to 0.5 mL of H2O to prepare a stock solution of palbociclib at a concentration of 87.15 mM. 2.3 μL of the stock solution was mixed with 198 μL of H2O to prepare a 1 mM palbociclib solution.

[0321] Induction of senescent cells: 2 μL of 1 mM palbociclib solution was added to 2 mL of culture medium to obtain a final concentration of 1 μM palbociclib for treatment of cultured cells (MCF-7, MDA-MB231, and MDA-MB468). The cultured cells were treated in this medium for 7 days to attempt induction of senescent cells.

[0322] Detection of senescent cells by FAC (co-staining with B-gal and antibodies): After 7 days of treatment with palbociclib, the cells were co-stained with SA-B-Gal fluorescent substrate (C12FDG) and a series of antibodies, and then Zombie NIR live-death dye was applied. 1. Wash the cells twice with PBS and detach them with Detachin® cell detachment solution. 2. Stop the reaction between Detachin (trademark) and DMEM, and count the cells. 3. 2 mM C12FDG in PBS (final concentration 33 μM), antibody (5 μL, 1 × 10⁶) 6 Stain the cells with Zombie NIR dye (1:1000) and on ice for 1 hour. 4. Wash the cells twice with PBS and fix them with 4% PFA at room temperature for 10 minutes. 5. Wash with PBS and recover the FAC in 100 μL of PBS. 6. Apply FITC-PE-APC / Cy7. 7. Cells are co-stained with the following antibodies to detect the expression of the corresponding antigen: a) PE anti-human CD54 clone HCD54, 200 μg / mL, isotype: Ms IgG1. Biolegend, catalog number 322707, lot number B232865, 5 μl / 10 6 cell b) PE anti-human CD73 clone AD2, isotype: Ms IgG1. Biolegend, catalog number 344004, lot number B216193, 5 μl / 10 6 cell c) PE anti-human CD261 (DR4, TRAIL-R1) clone DJR1, 200 μg / mL, isotype: Ms IgG1. Biolegend, catalog number 307205, lot number B189821, 5 μl / 10 6 cell d) PE anti-human CD95 (Fas) clone DX2, 100 μg / mL, isotype: Ms IgG1. Biolegend, catalog number 305607, lot number B203942, 5 μl / 10 6 cell e) PE anti-human CD39 clone A1, 50 μg / mL, isotype: Ms IgG1. Biolegend, catalog number 328208, lot number B199643, 5 μl / 10 6 cell f) PE anti-human Nectin 4, isotype: Ms IgG1. R&D systems, catalog number FAB2659P, lot number AAAA0217031, 5 μl / 10 6 cell g) PE-isotype mouse anti-IgG1, k: clone MOPC-21, 0.2 mg / mL. Biolegend, catalog number 400112, lot number B220359, 5 μl / 10 6 cell.

[0323] Induced senescent cells were detected by FACS. Stained cells were washed with PBS, fixed with 4% paraformaldehyde (PFA) at room temperature for 10 minutes, and used for FACS analysis. SA-B-gal (Senescence Associated B-Gal) staining using the Cell Biolabs CBA-230 kit was also performed as a control.

[0324] Cell lines (MCF-7, MDA-MB231, and MDA-MB468 cells) were observed under a microscope after palbociclib treatment. Furthermore, the target profiles expressed in these cell lines after palbociclib treatment were analyzed by staining with the corresponding antibodies. The profiled targets were target 1 (CD54), target 2 (CD73), target 3 (CD261), target 4 (CD95), target 5 (CD39), and target 6 (nectin 4).

[0325] MCF-7 cells were induced to become senescent cells in response to palbociclib treatment (Figure 9A-9B). MCF-7 cells formed clusters with an extracellular environment suitable for senescence (Figure 9B). FACS analysis clearly showed that palbociclib-treated cells (senescent cells) differed from untreated cells (non-senescent cells) (Figure 9C). The target profile of palbociclib-treated cells (senescent cells) was found to differ from that of untreated cells (non-senescent cells) (Figure 9D). Specifically, targets 1, 2, and 6 were more highly expressed in senescent cells, and target 2 showed the highest increase in expression level.

[0326] Similarly, MDA-MB231 cells also induced senescence in response to palbociclib treatment (Figures 10A-10B). MCF-MB231 cells also formed clusters with an extracellular environment (Figure 10B). FACS analysis clearly showed that palbociclib-treated cells (senescent cells) differed from untreated cells (non-senescent cells) (Figure 10C). The target profile of palbociclib-treated cells (senescent cells) was found to differ from that of untreated cells (non-senescent cells) (Figure 10D). Specifically, targets 1 and 2 showed significantly higher expression levels in senescent cells compared to untreated non-senescent cells.

[0327] Control MDA-MB468 cells did not respond to palbociclib treatment, and therefore this treatment did not induce control cells to become senescent cells (Figures 11A-11B). FACS and target profile analysis showed no significant differences between treated and untreated cells.

[0328] Example 14: Palbociclib treatment and beta-galactosidase staining of MDA-MB231 cells

[0329] MDA-MB231 cells were placed in a 6-well plate at a rate of 1 × 10⁶ 5 Cells were seeded in wells and cultured overnight. The cultured cells were separated into two batches; one batch was treated with 1 μM palbociclib isethionate for 7 days, and the other batch was left untreated. Both batches were collected by detaching the cells from the wells.

[0330] The harvested cells were stained on ice for 1 hour with beta-galactosidase (B-gal) substrate (FITC), a target antibody (anti-CD73 antibody), and a biodegradable dye (APC / Cy7) in PBS buffer. B-gal staining was performed using the Cell Signaling Technologies, catalog number 9860S kit. The stained MDA-MB231 cells were observed under a microscope. Figure 12A shows that untreated MDA-MB231 cells had almost no senescent cells, as no cell clusters were observed. Figure 12B shows MDA-MB231 cells treated with palbociclib. Some cells were induced to form clustered senescent cells, and an extracellular environment was also present.

[0331] Both untreated and treated stained cells were washed with PBS and fixed with 4% paraformaldehyde at room temperature for 10 minutes. The fixed cells were then used for FACS cell sorting.

[0332] Most untreated cells were B-gal negative, but they were separated by CD73 activity by FACS sorting (Figure 14A). Significantly fewer B-gal positive cells were present, but these were also separated by CD73 activity by FACS sorting (Figure 14C). In contrast, palbociclib-treated cells had nearly the same number of B-gal negative and B-gal positive cells (Figures 14B and 14D). Similarly, treated cells, whether B-gal negative or B-gal positive, were separated by CD73 activity by FACS sorting (Figures 14B and 14D).

[0333] The results of FACS sorting of MDA-MB231 cells are summarized in Figures 15A-15B. Figure 15A shows that the number of senescent cells was considerably lower and the CD73 activity of these cells was considerably lower compared to treated cells containing a larger number of senescent cells and higher CD73 activity (Figure 15B).

[0334] Example 15: Palbociclib treatment and beta-galactosidase staining of MDA-MB468 cells

[0335] MDA-MB468 cells were cultured, stained, and harvested as described for MDA-MB231 cells in Example 14. Stained MDA-MB468 cells were observed under a microscope. Figure 13A shows untreated MDA-MB468 cells. Figure 13B shows MDA-MB468 cells treated with palbociclib. No significant senescent cells (cell clusters) were observed after treatment. Morphologically, the untreated and treated cells appeared similar under a microscope.

[0336] Both untreated and palbociclib-treated stained cells were washed with PBS and fixed with 4% paraformaldehyde at room temperature for 10 minutes. The fixed cells were used for FACS cell sorting.

[0337] Most untreated cells were B-gal negative, but they were separated by CD73 activity by FACS sorting (Figure 16A). The separation was not as clear as that of the MDA-MB231 cells in Example 14. A significantly smaller number of B-gal positive cells were present, but these were also separated by CD73 activity by FACS sorting (Figure 16C). Similarly, most treated cells were B-gal negative (Figures 16B and 16D). Likewise, treated cells, whether B-gal negative or B-gal positive, were separated by CD73 activity by FACS sorting, but this was not as clear as that of the MDA-MB231 cells in Example 14 (Figures 14B and 14D).

[0338] The results of FACS sorting of MDA-MB468 cells are summarized in Figures 17A-17B. Treated and untreated cells had similar senescent cell counts and CD73 activity levels. These results indicate that palbociclib treatment did not induce a significant increase in senescent cell counts.

[0339] Example 16: CD73 expression in MDA-MB231 and MDA-MB468 cells

[0340] CD73 expression levels were measured in MDA-MB231 and MDA-MB468 cells after palbociclib treatment (Figure 18A). In MDA-MB231 cells, palbociclib treatment significantly increased CD73 expression levels (the two left bars in Figure 18A). Untreated MDA-MB468 cells had lower CD73 expression levels than untreated MDA-MB231 cells (the first and third bars in Figure 18A). Furthermore, palbociclib treatment did not significantly increase CD73 expression levels in MDA-MB468 cells (the two right bars in Figure 18A).

[0341] Example 17: Senescent cell killing measured by ZAP assay

[0342] The ZAP assay was performed according to the protocol recommended by the manufacturer of the ZAP assay kit from Advanced Targeting Systems.

[0343] Since palbociclib treatment was observed to induce senescent cells with increased CD73 expression in MDA-MB231 cells, a cell toxicization assay (ZAP assay) was performed on MDA-MB231 cells. Briefly, cells were placed in 4 × 10⁶ well plates. 3 Cells were seeded in wells and cultured overnight. The cultured cells were separated into two batches: one batch was treated with 1 μM palbociclibissethionate for 7 days, and the other batch was not treated with palbociclibissethionate.

[0344] Both types of MDA-MB231 cells were used in the ZAP assay. Each cell type was assayed in four groups: BAP147-CD73 (conditionally active anti-CD73 antibody), B12 (isotype-negative control), saporin (negative control), and culture medium only (negative control). The ZAP assay was performed for 72 hours.

[0345] The results of cell toxication using the conditionally activated anti-CD73 antibody and negative control are shown in Figure 18B. Y-axis OD 450nm The value represents the total number of viable cells. The culture medium had a similar effect on palbociclib-treated and untreated cells, indicating that the medium does not have cytotoxic activity against senescent cells. The conditionally activated anti-CD73 antibody induced a significant reduction in the number of cells in palbociclib-treated cells compared to untreated cells, indicating that the conditionally activated anti-CD73 antibody has significant cytotoxic activity against senescent cells.

[0346] B12 appeared to have a small effect on palbociclib-treated cells compared to untreated cells, indicating that B12 has a small and insignificant cytotoxicity to senescent cells. Interestingly, saporins also induced a similarly small reduction in senescent cell counts compared to B12. See Figure 18B.

[0347] This example demonstrates that the conditionally active anti-CD73 antibody can target CD73 overexpressed in palbociclib-induced senescent cells, thereby killing a significant number of these senescent cells.

[0348] All documents referenced herein, either in whole or by reference providing specific disclosures, are incorporated herein by reference. The applicant(s) hereby have no intention of publicly presenting any disclosed embodiments, and any disclosed improvements or modifications are considered part thereof under the doctrine of equivalents, in that they cannot be literally included in the claims.

[0349] However, even though many of the features and advantages of the present invention are shown in the foregoing description along with details of its structure and function, this disclosure is merely illustrative, and it should be understood that modifications can be made in detail, particularly with respect to the shape, dimensions, and arrangement of parts, to the extent indicated in the broad sense of the terms expressed in the appended claims, especially within the principles of the present invention.

Claims

1. 1. A method for generating a conditionally active senolytic antibody or antibody fragment that binds to a target associated with senescent cells from a parent antibody or antibody fragment that binds to said target associated with said senescent cells, comprising: (i) evolving DNA encoding said parent antibody or antibody fragment using one or more evolutionary techniques to generate mutant DNA; (ii) expressing the mutant DNA to obtain a mutant antibody or antibody fragment; (iii) subjecting the mutant antibody or antibody fragment to an assay for binding activity to a target under the extracellular conditions of the senescent cell and under normal physiological conditions; (iv) from the mutant antibody or antibody fragment assayed in step (iii), a reduction in the binding activity to the target in the assay compared to the binding activity of the parent antibody or antibody fragment to the target in the assay under normal physiological conditions; and an increase in the binding activity of the senescent cell to the target in the assay under the extracellular conditions compared to the binding activity of the parent antibody or antibody fragment to the target in the assay under the extracellular conditions of the senescent cell. selecting said conditionally active senolytic antibody or antibody fragment that exhibits: wherein the target is selected from at least one of BCL2L1, p16, p16INK4a, p21, and p53; the extracellular conditions of the senescent cell are at a first pH in the range of 5.5 to 7.0, and the normal physiological conditions are at a second pH in the range of 7.2 to 7.8; the assay under normal physiological conditions and the assay under the extracellular conditions of the senescent cells are carried out in an assay solution having a bicarbonate concentration of at least 10 mM; the ratio of the activity of the conditionally active senolytic antibody or antibody fragment in the assay under the extracellular conditions of the senescent cells to the activity of the conditionally active senolytic antibody or antibody fragment in the assay under the normal physiological conditions is at least 1.3:

1.

2. The method of claim 1 , wherein the parent antibody or antibody fragment is an antibody.

3. 2. The method of claim 1, wherein the ratio of the activity of the conditionally active senolytic antibody or antibody fragment in the assay under the extracellular conditions of the senescent cells to the activity of the conditionally active senolytic antibody or antibody fragment in the assay under the normal physiological conditions is at least 2:1, or at least 3:1, or at least 4:1, or at least 5:1, or at least 6:1, or at least 7:1, or at least 8:1, or at least 9:1, or at least 10:

1.

4. The method of any one of claims 1 to 3, wherein the first pH is in the range of 6.0 to 7.0, or 6.2 to 6.

8.

5. The method of any one of claims 1 to 4, wherein the second pH is in the range of 7.2 to 7.6, or 7.4 to 7.

6.

6. 2. The method of claim 1 , wherein said selecting step (iv) comprises: (a) selecting a conditionally active senolytic antibody or antibody fragment that exhibits a decreased activity in the assay compared to the activity of the parent antibody or antibody fragment in the assay under normal physiological conditions, and an increased activity in the assay under the extracellular conditions of the senescent cells compared to the activity of the conditionally active senolytic antibody or antibody fragment in the assay under normal physiological conditions.

7. 2. The method of claim 1 , wherein said selecting step (iv) comprises (b) selecting a conditionally active senolytic antibody or antibody fragment that exhibits a decreased activity in the assay compared to the activity of the parent antibody or antibody fragment in the assay under normal physiological conditions, and an increased activity in the assay under the extracellular conditions of senescent cells compared to the activity of the parent antibody or antibody fragment in the assay under the extracellular conditions of senescent cells.

8. The method of any one of claims 1 to 7, further comprising the step of conjugating the conditionally active antibody to a masking moiety by a linker.

9. 9. The method of claim 8, wherein the masking moiety reduces the activity of the conditionally active antibody in binding to the target by at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%.

10. 10. The method of claim 8 or 9, wherein the linker is covalently attached to the variable region of the conditionally active antibody.

11. The method of any one of claims 8 to 10, wherein the masking moiety specifically binds to the variable region of the conditionally active antibody.

12. 12. The method of claim 11 , wherein the masking moiety has 5% or less, 7% or less, 10% or less, 15% or less, 20% or less, 25% or less, 30% or less, 35% or less, 40% or less, 45% or less, 50% or less, 55% or less, 60% or less, 65% or less, 70% or less, 75% or less, or 80% or less sequence identity to the target.

13. The method of claim 11 or 12, wherein the linker comprises a flexible region and a cleavage site.

14. 14. The method of claim 13, wherein the flexible region consists essentially of at least one amino acid selected from glycine, alanine, and serine.

15. 15. The method of claim 13 or 14, wherein the flexible region has a length of 1 to 20 amino acids, 2 to 15 amino acids, 3 to 12 amino acids, 4 to 10 amino acids, 5 to 9 amino acids, or 6 to 8 amino acids.

16. The method of any one of claims 13 to 15, wherein the cleavage site can be cleaved by a protease in the extracellular environment of the senescent cell.

17. The method of claim 16, wherein the protease is selected from at least one of ADAM10, ADAM12, ADAM17, ADAMTS, ADAMTS5, BACE, caspases 1 to 14, cathepsin A, cathepsin B, cathepsin D, cathepsin E, cathepsin K, cathepsin S, FAP, MT1-MMP, granzyme B, guanidinobenzoatase, hepsin, human neutrophil elastase, legumain, matriptase 2, meprin, MMPs 1 to 17, MT-SP1, neprilysin, NS3 / 4A, plasmin, PSA, PSMA, TRACE, TMPRSS 3, TMPRSS 4, and uPA.

18. 6. The method of any one of claims 1 to 5, further comprising the step of conjugating the conditionally active senolytic antibody or antibody fragment to a cytotoxic, cytostatic, or antiproliferative agent via a linker.

19. 19. The method of claim 18, wherein the linker comprises a cleavage site that can be cleaved by a protease in the extracellular environment of the senescent cell.

20. 20. The method of claim 19, wherein the protease is selected from at least one of ADAM10, ADAM12, ADAM17, ADAMTS, ADAMTS5, BACE, caspases 1 to 14, cathepsin A, cathepsin B, cathepsin D, cathepsin E, cathepsin K, cathepsin S, FAP, MT1-MMP, granzyme B, guanidinobenzoatase, hepsin, human neutrophil elastase, legumain, matriptase 2, meprin, MMPs 1 to 17, MT-SP1, neprilysin, NS3 / 4A, plasmin, PSA, PSMA, TRACE, TMPRSS 3, TMPRSS 4, and uPA.

21. 19. The method of claim 18, wherein the antiproliferative agent is a chemotherapeutic agent.

22. 6. The method of any one of claims 1 to 5, further comprising the step of conjugating the conditionally active senolytic antibody or antibody fragment to an agent selected from a toxic agent, a radioactive agent, or a D retro-inverso peptide.

23. 23. The method of claim 22, wherein the conditionally active senolytic antibody or antibody fragment is a conditionally active antibody.

24. 24. The method of claim 22 or 23, wherein the D retro-inverso peptide has an amino acid sequence that has at least 70%, or at least 80%, or at least 90%, or at least 95%, or at least 98%, or 100% amino acid sequence identity with a fragment or full-length reverse sequence of a native protein.

25. 25. The method of claim 24, wherein the native protein is selected from at least one of FOXO4, AMPK, JNK, MST1, CK1, STAT3, p38, PRMT1, and ASK1.

26. 26. The method of any one of claims 22-25, wherein the D retro-inverso peptide has a length of 5 or less, 10 or less, 15 or less, 20 or less, 25 or less, 30 or less, 35 or less, 40 or less, 45 or less, 50 or less, 60 or less, 70 or less, 80 or less, 90 or less, or 100 or less amino acid residues.

27. 27. The method of any one of claims 22 to 26, wherein the D retro-inverso peptide has at most 0, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55 or 60% of the amino acid residues that are L amino acid residues.

28. 27. The method of any one of claims 22 to 26, wherein the D retro-inverso peptide has 100% D amino acid residues.

29. 29. The method of any one of claims 22 to 28, wherein the D retro-inverso peptide comprises one or more functional domains selected from PPRRRQRRKKRG (SEQ ID NO: 10), GALFLGFLGA AGSTMGAWSQ PKKKRKV (SEQ ID NO: 11), KETWETWWT EWSQPKKKRKV (SEQ ID NO: 12), Ac-GLWRALWRLLRSLWRLLWRA-Cya (SEQ ID NO: 13), and octaarginine.

30. 30. The method of any one of claims 23 to 29, wherein the D retro-inverso peptide comprises LTLRKEPASE IAQSILEAYS QNGWANRRSG GKRP (SEQ ID NO: 5), LTLRKEPASE IAQSILEAYS QNGWANRRSG GKRPPPPRRRQ RRKKRG (SEQ ID NO: 6), or SEIAQSILEAYSQNGW (SEQ ID NO: 7).

31. 1. A method for generating a target-binding, conditionally active, senolytic antibody or antibody fragment having a molecular weight of less than 3000 a.m.u. from a target-binding parent antibody or antibody fragment, comprising: modifying the parent antibody or antibody fragment by introducing one or more partial charges or charged groups into the parent antibody or antibody fragment to produce one or more modified antibodies or antibody fragments; subjecting the one or more modified antibodies or antibody fragments to an assay for binding activity to a target under the extracellular conditions of the senescent cells and under normal physiological conditions; (a) an increase in the binding activity of the conditionally active senolytic antibody or antibody fragment to the target in the assay under the extracellular conditions of the senescent cell compared to the binding activity of the conditionally active senolytic antibody or antibody fragment to the target in the assay under normal physiological conditions; and (b) a decrease in the binding activity of the parent antibody or antibody fragment to the target in the assay compared to the binding activity of the parent antibody or antibody fragment to the target in the assay under normal physiological conditions. selecting said conditionally active senolytic antibody or antibody fragment from said modified antibodies or antibody fragments which exhibit wherein the target is selected from at least one of BCL2L1, p16, p16INK4a, p21, and p53; the extracellular conditions of the senescent cell are at a first pH in the range of 5.5 to 7.0, and the normal physiological conditions are at a second pH in the range of 7.2 to 7.8; The method, wherein the assay under normal physiological conditions and the assay under the extracellular conditions of the senescent cells are performed in an assay solution having a bicarbonate concentration of at least 10 mM.

32. 32. The method of claim 31 , wherein the parent organic compound has a molecular weight in the range of 100 a.m.u to 3000 a.m.u, or 100 a.m.u to 1500 a.m.u, or 150 a.m.u to 1250 a.m.u, or 300 a.m.u to 1100 a.m.u, or 400 a.m.u to 1000 a.m.u.

33. 32. The method of claim 31, wherein the first pH is in the range of 6.0 to 7.0, or 6.2 to 6.8, and the second pH is in the range of 7.2 to 7.8, or 7.2 to 7.6.

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