Protein Therapies for the Treatment of Senescent Cells

Abstract

Methods for generating conditionally active proteins that target senescent cells and are conditionally active in the extracellular environment of senescent cells include heuristics using libraries of evolved proteins and assays using physiological concentrations of components of body fluids. Also disclosed are conditionally active proteins for killing or eliminating senescent cells, pharmaceutical compositions using these conditionally active proteins, and methods for treating age-related diseases, illnesses, or disorders using the same. The conditionally active proteins may be further evolved, conjugated to other molecules, masked, or have their activity reduced by the attachment of cleavable moieties. [Selection diagram] 18B

Description

[Technical Field]

[0001] Field of Disclosure The present disclosure relates to the field of treating or purging senescent cells and / or treating diseases or disorders associated with senescent cells. In particular, the present disclosure relates to conditionally active proteins that target senescent cells and to methods of making such conditionally active proteins. [Background technology]

[0002] Background to the disclosure Senescent cells are metabolically active but are arrested 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, exhibiting characteristic morphological changes such as enlargement, 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 mitogens (Campisi, Trends Cell Biol., vol. 11, pp. S27-S31, 2001). Senescence involves the activation of p53 and / or Rb and their regulators, such as p16INK4a, p21, and ARF. Senescence is generally irreversible unless p53 or Rb is inactivated.

[0003] Senescent cells express high levels of plasminogen activator inhibitor (PAI) and exhibit staining 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 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 senescence but is not required for the maintenance of senescence, and that expression of p16 helps maintain senescence once initiated.

[0004] In some cases, senescence is associated with the progressive shortening of telomeres with each cell division, triggering senescence when telomeres of specific chromosomes 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). Senescence can be prevented by expressing telomerase, which elongates telomeres. For example, human fibroblasts can be transfected to express telomerase, causing the fibroblasts to replicate indefinitely. 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 senescence (von Zglinicki, Trends Biochem. Sci., vol. 27, pp. 339-344, 2002). Hyperoxia has been shown to induce senescence. Gamma irradiation of human fibroblasts in early-mid G1 phase induces senescence in a p53-dependent manner (Di Leonardo et al., Genes Dev., vol. 8, pp. 2540-2551, 1994). UV irradiation also induces senescence. Other substances that can induce senescence include hydrogen peroxide (Krtolica et al., Proc. Nat. Acad. Sci. USA, vol. 98, pp. 12072-12077, 2001), sodium butyrate, 5-azacytadine, and transfection with the Ras oncogene (Tominaga, Mech. Ageing Dev., vol. 123, pp. 927-936, 2002). Chemotherapeutic agents, including doxorubicin, cisplatin, and a host of other agents, have been shown to induce senescence 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, agents that damage DNA can induce senescence.

[0006] Evidence suggests a link between senescence and aging. Cultured cells from older donors show senescence 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 senesce 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 Warner syndrome, show senescence after fewer proliferation cycles than cells from age-matched controls.

[0007] Aging confers functional changes to senescent cells, which are associated with various 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 the individual ages and are found at the site of age-related pathologies. Given that senescent cells are causally associated with certain aspects of age-related health decline and can contribute to certain diseases, as well as being induced as a result of necessary life-sustaining chemotherapy and radiation treatments, the presence of senescent cells may have a detrimental effect on millions of patients worldwide. It is widely believed that selective elimination of senescent cells can prevent and treat age-related diseases and disorders.

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

[0009] As a result, therapy targeting senescent cells is a promising treatment option for aging-related diseases and disorders. US2016 / 0038576 discloses an immunogenic composition for inducing an adaptive immune response specifically directed against senescent cells for the treatment and prevention 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 comprising the polynucleotide for use in administration to a subject.

[0010] WO2015116740 discloses a method for administering a therapeutically effective amount of a small molecule senolytic agent that selectively kills senescent cells relative to non-senescent cells for the treatment of senescent cell-associated diseases and disorders, including arteriosclerosis such as atherosclerosis, idiopathic pulmonary fibrosis, chronic obstructive pulmonary disease, osteoarthritis, aging-associated ophthalmologic diseases and disorders, and cardiovascular diseases and disorders associated with or induced by aging-associated skin diseases and disorders.

[0011] US2015 / 0064137 discloses a polypeptide useful for selectively eliminating senescent cells, and a virus containing the polypeptide. The polypeptide and virus can induce apoptosis in senescent cells. The polypeptide is selected from the products of pro-apoptotic genes. The virus contains a pro-apoptotic gene whose expression is regulated by the p16 promoter. The p16 promoter can be a classical p16 promoter or a non-classical p16 promoter. Summary of the Invention [Problem to be solved by the invention]

[0012] These therapeutic agents target one or more proteins in senescent cells to kill or eliminate them. However, these target proteins in senescent cells may also be present on other types of cells, which may induce undesirable side effects. Therefore, it would be advantageous to develop a class 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] Disclosure Overview In one embodiment, the disclosure provides a method for generating a conditionally active protein that binds to a target associated with a senescent cell from a parent protein that binds to said target associated with said senescent cell, the method comprising: (i) evolving DNA encoding the parent protein using one or more evolutionary techniques to generate mutant DNA; (ii) expressing the mutant DNA to obtain a mutant protein; (iii) subjecting the mutant protein to assay under extracellular conditions of the senescent cells and under normal physiological conditions; (iv) (a) a decrease in 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 the extracellular conditions of the senescent cell, compared to the activity of the parent 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 the normal physiological conditions, and an increase in the activity of the parent protein in the assay under the extracellular conditions of the senescent cells compared to the activity of the parent protein in the assay under the extracellular conditions of the senescent cells; selecting the conditionally active protein from the mutant proteins that exhibit at least one of: The present invention provides a method comprising:

[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 foregoing embodiments, the activity may be binding activity to the target.

[0016] In each of the foregoing embodiments, the parent protein may be an enzyme and the activity is an enzymatic activity that uses at least a portion of the senescent cell as a substrate.

[0017] In each of the foregoing embodiments, the conditionally active protein may be a cyclic peptide, which 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 foregoing embodiments, the target may be a surface molecule located on the exterior surface of the senescent cell. In each of the foregoing embodiments, the surface molecule may be a plasma membrane protein of the senescent cell. In each of the foregoing embodiments, 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. In addition, it should be recognized that the target may be any combination of the foregoing.

[0019] In each of the foregoing embodiments, the ratio of the activity of the conditionally active protein in the assay under the extracellular conditions of a senescent cell 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, or 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.

[0020] In each of the foregoing embodiments, the extracellular conditions of the senescent cells may be at 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 foregoing embodiments, 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 foregoing embodiments, the extracellular conditions of the senescent cell may be a lower concentration of deoxynucleotides than the normal physiological concentration of deoxynucleotides.

[0023] In each of the foregoing embodiments, the extracellular conditions of the senescent cells may be a concentration of oxygen that is lower than the normal physiological concentration of oxygen.

[0024] In each of the foregoing embodiments, the extracellular condition of the senescent cell may be a ratio of NAD+ / NADH that is lower than the normal physiological ratio of NAD+ / NADH.

[0025] In each of the foregoing embodiments, the extracellular condition of the senescent cell may be an elevated concentration of at least one redox homeostasis metabolite selected from hypotaurine, cysteine ​​sulfinic acid, cysteine-glutathione disulfide, gamma-glutamylalanine, gamma-glutamylmethionine, pyridoxate, gamma-glutamylglutamine, and alanine relative to a normal physiological concentration of the redox homeostasis metabolite.

[0026] In each of the foregoing embodiments, the extracellular condition of the senescent cell may be an elevated concentration of at least one nucleotide metabolite selected from 3-ureidopropionate, urate, 7-methylguanine, and hypoxanthine relative to normal physiological concentrations of the nucleotide metabolite.

[0027] In each of the foregoing embodiments, the extracellular condition of the senescent cell may be a low concentration of thymidine compared to normal physiological concentrations of thymidine.

[0028] In each of the foregoing embodiments, the extracellular condition of the senescent cell may be a reduced concentration of at least one dipeptide selected from glycylisoleucine, glycylvaline, glycylleucine, isoleucylglycine, and valylglycine relative to normal physiological concentrations of the dipeptide.

[0029] In each of the foregoing embodiments, the extracellular condition of the senescent cell may be a reduced concentration of at least one fatty acid selected from linoleate, dihomolinoleate, and 10-heptadecanoate relative to normal physiological concentrations of the fatty acid.

[0030] In each of the foregoing embodiments, the extracellular conditions of the senescent cells may be an elevated concentration of at least one phospholipid metabolite selected from 2-hydroxypalmitate, 2-hydroxystearate, 3-hydroxydecanoate, 3-hydroxyoctanoate, and glycerophosphorylcholine relative to normal physiological concentrations of the phospholipid metabolite.

[0031] In each of the foregoing embodiments, the extracellular condition of the senescent cell may be an elevated concentration of at least one amino acid metabolite selected from alanine, C-glycosyltryptophan, kynurenine, dimethylarginine, and orthithine relative to a normal physiological concentration of the amino acid metabolite.

[0032] In each of the foregoing embodiments, the extracellular conditions of the senescent cell may be a reduced concentration of phenylpyruvate relative to the normal physiological concentration of said phenylpyruvate.

[0033] In each of the foregoing embodiments, the extracellular condition of the senescent cell may be an elevated concentration of at least one metabolite selected from fumarate, malonate, eicosapentaenoate, and citrate relative to a normal physiological concentration of the metabolite.

[0034] In each of the foregoing embodiments, the extracellular conditions of the senescent cells may be a high ratio of glycerophosphocholine to phosphocholine compared to the normal physiological ratio of glycerophosphocholine to phosphocholine.

[0035] In each of the foregoing embodiments, the extracellular condition of the senescent cells may be a high concentration of a protein secreted by the senescent cells relative to the normal physiological concentration of the protein, wherein the protein secreted by the senescent cells is selected from the group consisting of GM-CSF, GROα, GRC-α, β, γ, IGFBP-7, IL-1α, IL-6, IL-7, IL-8, MCP-1, MCP-2, MIP-1a, 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 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 above-mentioned embodiments, 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 have 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, and may be 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, 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, sulfur, chloride, magnesium, sodium, potassium, ammonium, iron, zinc, and copper ions. In these embodiments, the at least one component may be selected from one or more of uric acid in a concentration range of 2-7.0 mg / dL, calcium in a concentration range of 8.2-11.6 mg / dL, chloride in a concentration range of 355-381 mg / dL, iron in a concentration range of 0.028-0.210 mg / dL, potassium in a concentration range of 12.1-25.4 mg / dL, sodium in a concentration range of 300-330 mg / dL, and carbonate in a concentration range of 15-30 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, pyrrolysine, 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, nitrite ions, phosphate ions, hydrogen phosphate ions, dihydrogen phosphate ions, persulfate ions, monopersulfate ions, borate ions, and ammonium ions.

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

[0038] In each of the foregoing embodiments, 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 the one or more assays may be performed in an assay solution containing at least one molecular species having a molecular weight of less than 900 amu, the molecular species having a pKa between the first pH and the second pH.

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

[0040] In each of the foregoing embodiments, the selecting step (iv) may comprise: (a) selecting a conditionally active protein that exhibits a decreased activity in the assay under normal physiological conditions compared to the activity of a parent protein in the assay, and an increased activity in the assay under the 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 foregoing embodiments, the selecting step (iv) may include (b) selecting a conditionally active protein that exhibits a decreased activity in the assay under normal physiological conditions compared to the activity of the parent protein in the assay, and an increased activity in the assay under the extracellular conditions of the senescent cell compared to the activity of the parent protein in the assay under the extracellular conditions of the senescent cell.

[0042] In another embodiment, the present disclosure provides a conditionally active protein produced by any of the aforementioned methods. 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 engineering as part of a chimeric antigen receptor on T cells. The antibody may be a humanized antibody, a bispecific antibody, or a multispecific antibody.

[0043] In each of the foregoing embodiments, 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 foregoing embodiments, the conditionally active protein may be a conditionally active antibody, and the conditionally active antibody may be conjugated by a linker to a masking moiety that 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 foregoing embodiments, the linker may be covalently attached to the variable region of the conditionally active antibody.

[0046] In each of the foregoing embodiments, the masking moiety may specifically bind to a variable region of the conditionally active antibody.

[0047] In each of the foregoing embodiments, the linker may include a flexible region and a cleavage site.

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

[0049] In each of the foregoing embodiments, the conditionally active protein may be conjugated to a cytotoxic, cytostatic, or antiproliferative agent by a linker.

[0050] In each of the foregoing embodiments, the linker may comprise a cleavage site for at least one protease in the extracellular environment of the senescent cell, the at least one protease being selected from ADAM10, ADAM12, ADAM17, ADAMTS, ADAMTS5, BACE, caspases 1-14, 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 1-17, MT-SP1, neprilysin, NS3 / 4A, plasmin, PSA, PSMA, TRACE, TMPRSS 3, TMPRSS 4, and uPA.

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

[0052] In yet another embodiment, the present disclosure provides a method of treating aging or a senescent cell-associated disease or disorder, comprising administering any of the aforementioned conditionally active proteins or any of the aforementioned pharmaceutical compositions. In the aforementioned embodiments, the senescent cell-associated disease or disorder may be selected from cognitive diseases, cardiovascular diseases, metabolic diseases and disorders, motor function diseases and disorders, cerebrovascular diseases, emphysema, osteoarthritis, pulmonary diseases, inflammatory / autoimmune diseases and disorders, ophthalmological diseases or disorders, metastasis, chemotherapy or radiation therapy side effects, age-related diseases and disorders, and fibrotic diseases and disorders.

[0053] In yet another embodiment, the present disclosure provides a method for producing conditionally active molecules having a molecular weight of less than about 3000 amu from a parent organic compound, the method comprising: modifying the parent organic compound by introducing one or more partial charges or charged groups into 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 the activity in the assay under normal physiological conditions.

[0054] In yet another embodiment, the present disclosure provides a method for generating conditionally active molecules having a molecular weight of less than about 3000 amu from a parent organic compound, the method comprising: modifying the parent organic compound by removing one or more partial charges or charged groups from 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 the activity in the assay under normal physiological conditions.

[0055] In yet another embodiment, the present disclosure provides a method for producing conditionally active molecules having a molecular weight of less than about 3000 amu from a parent organic compound, the method comprising: modifying the parent organic compound by replacing 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 the activity in the assay under normal physiological conditions.

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

[0057] In each of the foregoing methods, the abnormal condition may be a value of an extracellular condition in a senescent cell, and the normal physiological condition is a different value of the same extracellular condition in a normal cell.

[0058] In each of the foregoing methods, the abnormal conditions may be a pH within 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 conditions are a pH within 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 foregoing methods, the conditionally active protein may be conjugated to an agent selected from a toxic agent, a radioactive agent, or a D retro-inverso peptide.

[0060] In each of the foregoing embodiments, the D retro-inverso peptide may comprise 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 drawings]

[0061] [Figure 1] 1 is a plot showing the selectivity of the conditionally active antibodies selected in Example 9 at pH 6.0 over pH 7.4. [Figure 2]1 is a diagram showing the formation of salt bridges in deoxyhemoglobin, where three amino acid residues form two salt bridges, stabilizing the T quaternary structure of deoxyhemoglobin and leading to a lower affinity for oxygen. [Figure 3] 1 is a diagram showing the structure of a chimeric antigen receptor (CAR). [Figure 4] 1 shows the binding activity of conditionally active antibodies to antigens assayed in different buffer solutions. [Figure 5] 1 shows the effect of varying the composition of Krebs buffer on the binding activity of a conditionally active antibody. [Figure 6] 1 shows that the binding activity of three different conditionally active antibodies was dependent on the presence and concentration of bicarbonate in H7.4, as described in Example 12. [Figure 7] The design principles of D-retro-inverso (DRI) peptides of natural or wild-type peptides are shown. [Figure 8] Signaling pathways regulating FOXO4 and other FOXO family members are shown, with "+p" indicating phosphorylation, "-p" indicating dephosphorylation, "+m" indicating methylation, arrows indicating activation, and lines with crossed bars indicating inhibition, all associated with target genes. [Figure 9A] Untreated MCF-7 cells are shown. [Figure 9B] 1 shows MCF-7 cells treated with 1 μM palbociclib isethionate. [Figure 9C] Separation of untreated and treated MCF-7 cells by fluorescence activated cell sorting (FACS) is shown. [Figure 9D] 1 shows the target expression profiles of untreated MCF-7 cells and MCF-7 cells treated with palbociclib isethionate. [Figure 10A] Untreated MDA-MB231 cells are shown. [Figure 10B] 1 shows MDA-MB231 cells treated with 1 μM palbociclib isethionate. [Figure 10C]1 shows separation of untreated and palbociclib isethionate-treated MDA-MB231 cells by FACS. [Figure 10D] 1 shows the target expression profiles of untreated MDA-MB231 cells and MDA-MB231 cells treated with palbociclib isethionate. [Figure 11A] Untreated MDA-MB468 cells are shown. [Figure 11B] 1 shows MDA-MB468 cells treated with 1 μM palbociclib isethionate. [Figure 11C] 1 shows that untreated MDA-MB468 cells and MDA-MB468 cells treated with palbociclib isethionate were not separated by FACS. [Figure 11D] Untreated and palbociclib isethionate-treated MDA-MB468 cells show similar target expression profiles. [Figure 12A] Untreated MDA-MB231 cells are shown. [Figure 12B] 1 shows MDA-MB231 cells treated with palbociclib isethionate. [Figure 13A] Untreated MDA-MB468 cells are shown. [Figure 13B] 1 shows MDA-MB468 cells treated with palbociclib isethionate. [Figure 14A] FACS cell sorting of untreated MDA-MB231 cells that were negative for B-gal staining is shown. [Figure 14B] 1 shows FACS cell sorting of MDA-MB231 cells treated with palbociclib isethionate that were negative for B-gal staining. [Figure 14C] FACS cell sorting of untreated MDA-MB231 cells that stained positive for B-gal is shown. [Figure 14D] 1 shows FACS cell sorting of MDA-MB231 cells treated with palbociclib isethionate that were positive for B-gal staining. [Figure 15A] FACS sorting of untreated MDA-MB231 cells is shown. [Figure 15B] 1 shows FACS sorting of MDA-MB231 cells treated with palbociclib isethionate. [Figure 16A] FACS cell sorting of untreated MDA-MB468 cells that were negative for B-gal staining is shown. [Figure 16B] 1 shows FACS sorting of MDA-MB468 cells treated with palbociclib isethionate that were negative for B-gal staining. [Figure 16C] FACS cell sorting of untreated MDA-MB468 cells that stained positive for B-gal is shown. [Figure 16D] 1 shows FACS cell sorting of MDA-MB468 cells treated with palbociclib isethionate that were positive for B-gal staining. [Figure 17A] FACS sorting of untreated MDA-MB468 cells is shown. [Figure 17B] 1 shows FACS sorting of MDA-MB468 cells treated with palbociclib isethionate. [Figure 18A] 1 shows CD73 expression levels in MDA-MB231 and MDA-MB468 cells before and after palbociclib isethionate treatment. [Figure 18B] 1 shows senescent cell killing by anti-CD73 conditional activity antibody. DETAILED DESCRIPTION OF THE INVENTION

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

[0063] The terms "about," "activity," "drug," "ambiguous base requirement," "amino acid," "amplification," "chimeric properties," "cognate," "comparison window," "conservative amino acid substitution," "corresponding to," "effective degradation," "defined sequence framework," "digestion," "directed ligation," "DNA shuffling," "drug" or "drug molecule," "effective amount," "electrolyte," "epitope," "enzyme," "evolution" or "evolving," "fragment," "derivative," "analog," "full range of single amino acid substitutions," "gene," "genetic instability," "heterologous," "homologous (or homeologous)," "industrial application," "identical" or "identity," "area of ​​identity," "isolated," "isolated nucleic acid," "ligand," "ligation," "linker" or "spacer," "microenvironment," "evolved molecular properties," "mutation," "naturally occurring," "normal physiological conditions" or "wild-type operating conditions," "nucleic acid molecule," "nucleic acid molecule," " "nucleic acid sequence encoding" or "DNA coding sequence for" or "nucleotide sequence encoding"," promoter sequence," "enzyme (protein)-encoding nucleic acid" or "DNA encoding enzyme (protein)" or "polynucleotide encoding enzyme (protein)," "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," "homologous," "operably linked," "operably linked to," "parent polynucleotide set," "patient" or "subject," "physiological condition," "population," "surrogate form," "pre-pro-form," "pseudo-random," "quasi-repeated unit," "random peptide library," "random peptide sequence," "receptor," "recombinant," "synthetic," "related polynucleotide," "reductive reassortment"The definitions of "reference sequence", "comparison window", "sequence identity", "percentage of sequence identity", "substantial identity", "reference sequence", "repetition index (RI)", "restriction site", "selective polynucleotide", "sequence identity", "similarity", "specifically binds", "specific hybridization", "specific polynucleotide", "stringent hybridization conditions", "substantially identical", "substantially pure enzyme", "substantially pure", "treating", "variable segment", "mutant", "wild-type", "wild-type protein" or "wild-type biological protein", "parent molecule" or "target protein", "working", "conditionally active antibody", "antibody-dependent cell-mediated cytotoxicity" or "ADCC", "cancer" and "cancerous", "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 and fragments of immunoglobulin molecules, such as Fab, Fab', (Fab')2, Fv, and SCA fragments, capable of binding to an epitope of an antigen. These antibody fragments, which retain some ability to selectively bind to the antigen (e.g., polypeptide antigen) of the derived antibody, can be produced using methods well known in the art (see, e.g., Harlow and Lane, supra) and 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. Antibodies can be used to isolate preparative quantities of antigens by immunoaffinity chromatography. Various other uses of such antibodies include diagnosing and / or staging disease (e.g., neoplasms), as well as for therapeutic applications to treat diseases such as neoplasms, autoimmune diseases, AIDS, cardiovascular diseases, infectious diseases, and the like. Chimeric, human-like, humanized or fully human antibodies are particularly useful for administration to human patients.

[0065] A Fab fragment consists of a monovalent antigen-binding fragment of an antibody molecule and can be produced by digestion of whole antibody molecules with the enzyme papain to yield a fragment consisting of an intact light chain and a portion of the heavy chain.

[0066] An Fab' fragment of an antibody molecule can be obtained by treating a whole antibody molecule with pepsin, followed by reduction, to yield a molecule consisting of an intact light chain and a portion of the heavy chain. Two Fab' fragments are obtained by treating an antibody molecule in this manner.

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

[0068] An Fv fragment is defined as a genetically engineered fragment containing the variable region of the light chain and the variable region of the heavy chain expressed as two chains.

[0069] Single-chain antibodies ("SCAs" or scFvs) are genetically engineered single-chain molecules that contain the variable region of a light chain and the variable region of a heavy chain connected by a suitable flexible polypeptide liner, and may contain additional amino acid sequences at the amino and / or carboxyl termini. For example, single-chain antibodies may contain a tether segment for linking to an encoding polynucleotide. Functional single-chain antibodies generally contain a sufficient portion of the variable region of the light chain and sufficient region of the variable region of the heavy chain so as to retain the properties of a full-length antibody for binding to a specific target molecule or epitope.

[0070] As used herein, the term "antigen" or "Ag" is defined as a molecule 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 any protein or peptide, can serve as an antigen. It will be readily apparent that antigens can be produced, synthesized, or derived from biological samples. Such biological samples may include, but are not limited to, tissue samples, tumor samples, cells, or biological fluids.

[0071] As used herein, the term "apoptosis" refers to a mechanism of cell death affecting single cells characterized by cell shrinkage, chromatin condensation, and cell fragmentation into membrane-bound bodies 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 inherent property of a compound to selectively induce apoptosis in (i) specific cell types and / or (ii) cells at a specific stage of development or differentiation upon internal or external stimuli. Skilled practitioners are aware of the existence of standard in vitro assays for measuring the apoptosis-inducing activity of compounds in cell culture, such as tests assessing the levels of cytoplasmic cytochrome C (a marker of apoptosis) and TUNEL (a marker of apoptosis). Using these standard assays, skilled practitioners can easily assess and compare the apoptosis-inducing activity of different compounds with respect to different cell types or cells at different developmental stages, for example, between senescent and non-senescent cells. Other standard apoptosis assays are the Annexin V assay and cleaved caspase-3 staining.

[0073] The terms "biosimilar" and "follow-on product" are used in a manner consistent with the working definition published by the U.S. Food and Drug Administration (FDA), which defines a biosimilar as a product that is "highly similar" to a reference product (albeit with minor differences in clinically inactive ingredients). In practice, there may be no clinically meaningful differences between the reference and biosimilar products with respect to safety, purity, and potency (Public Health Service (PHS) Act §262). Biosimilars 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. Differences between a biosimilar antibody and a reference antibody may include post-translational modifications, such as those resulting from the attachment of other biochemical groups to the antibody, such as phosphates, various lipids, and carbohydrates; post-translational proteolytic cleavage; changes in the chemical nature of amino acids (e.g., formylation); or many other mechanisms. Other post-translational modifications may be the result of manipulation of the manufacturing process; for example, glycation may occur upon exposure of the product to reducing sugars. In some instances, storage conditions may allow certain degradation pathways, such as oxidation, deamidation, or aggregation, to occur. All of these product-related variants may therefore be included in the biosimilar antibody.

[0074] The terms "cancer" and "cancerous" refer to or describe the physiological condition in mammals that is typically characterized by unregulated cell growth / proliferation. A "tumor" contains one or more cancerous cells. Examples of cancer include, but are not limited to, carcinoma, lymphoma, blastoma, sarcoma, and leukemia or lymphoid malignancies. More 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 cancer such as adenocarcinoma of the lung and squamous cell carcinoma of the lung, cancer of the peritoneum, 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 its activity under control or normal physiological conditions. The conditionally active protein also exhibits activity in selected regions of the body and / or exhibits increased or decreased activity under abnormal or acceptable physiological conditions. Normal physiological conditions are conditions considered within the normal range at a given location in a subject, such as the tissue or organ at the site of administration or action in a subject. Abnormal conditions are conditions that deviate from the normally acceptable range for that condition at that location. In one embodiment, the conditionally active protein is effectively inactive under normal physiological conditions but active under abnormal or acceptable conditions. For example, in one embodiment, an evolved conditionally active protein is effectively 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 after passing through the lungs or in the lower pH environment found in the kidney. The conditionally active protein can 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 termini are themselves linked to one another by peptide bonds (i.e., between the carboxyl of one residue and the alpha amine of another) to form a cyclic chain. For purposes of this application, cyclic peptides may also contain linkages other than peptide bonds, such as non-alpha amide linkages and thioether linkages between Trp and Cys residues. The length of a cyclic peptide may range from about 5 to about 500 amino acids, or from about 8 to about 300 amino acids, or from about 8 to about 200 amino acids, or from about 10 to about 100 amino acids, or from about 10 to about 50 amino acids. Additionally, amino acids other than naturally occurring amino acids, such as β-alanine, phenylglycine, and homoarginine, may be included within the cyclic peptide.

[0077] As used herein, the abbreviation "DRI" refers to a D retro-inverso isoform of an L-peptide whose amino acid sequence is in reverse orientation relative to a fragment or full-length of a native or wild-type protein, and at least some of the amino acid residues in the DRI peptide are D amino acid residues in place of L amino acid residues in the native or wild-type protein (Figure 7). D retro-inverso peptides can be made by identifying the amino acid sequence of a fragment or full-length of a native protein, inverting the sequence, and synthesizing the D retro-inverso peptide using known methods to provide a peptide having the reverse orientation of the amino acid sequence of the fragment or full-length of the native protein and containing a sufficient number of D amino acids to provide the desired function in the D retro-inverso peptide.

[0078] The terms "disease or condition in which the removal of senescent cells would be beneficial," "disease or condition associated with the presence of senescent cells," and "disorder in which the removal of senescent cells would be beneficial" are used interchangeably and refer to any disease or condition in a mammalian subject, e.g., a human subject, in which the removal, clearance, or reduced viability of senescent cells would be beneficial to the subject suffering from the disease or condition. The term encompasses situations in which senescent cells are a cause or sole cause of the disease or contribute to the progression of the disease. The term also relates to situations in which senescent cells may become the cause of the disease or condition in the subject in the future. For example, a disease or condition treatment in which the removal of senescent cells would be beneficial is a disease or condition that is prevented, can be prevented, or is ameliorated by removing senescent cells. For example, chemotherapeutic agents and radiation therapy are known to induce cellular senescence. Removing these senescent cells to prevent the onset of a disease or condition associated with cellular senescence is advantageous. The term also encompasses diseases or conditions in which the removal of senescent cells alleviates or reduces the symptoms of the disease or condition.

[0079] Removal of senescent cells is beneficial, particularly if the disease or condition can be cured, prevented, or the symptoms of the disease or condition can be reduced or alleviated. Removal of senescent cells may be achieved by inducing apoptosis in senescent cells. For example, diseases or conditions that would benefit from the removal 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), laminoplasty, osteoporosis, dementia, (cardio)vascular diseases, obesity, metabolic syndrome, acute myocardial infarction, emphysema, insulin sensitivity, Boutonneux 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 deafness, vision loss such as blindness, sleep disorders, pain such as joint pain or leg pain, imbalance, fear, depression, difficulty breathing, weight loss, hair loss, muscle loss, bone mineral density loss, frailty and / or poor health. A disease or condition that would benefit from the removal of senescent cells is a disease or condition associated with or leading to inflammation, particularly chronic inflammation in a mammal, e.g., a human subject, 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, and more preferably in the same tissue, as the organ or tissue affected by the disease or condition.

[0080] As used herein, the term "disease or condition associated with the presence of senescent cells" refers to any disease or condition in a mammalian, e.g., human, subject, in which the presence of senescent cells or cellular senescence in the mammalian, e.g., human, subject leads to said disease or condition in said subject. In this context, "leading to" can refer, inter alia, to senescent cells or cellular senescence (i) as at least a partial cause of the disease or condition, or (ii) as at least a partial cause of the symptoms. In some embodiments, the disease or condition associated with the presence of senescent cells is selected from the group formed by atherosclerosis, chronic inflammatory diseases such as arthritis or arthropathy, cancer, osteoarthritis, diabetes, diabetic ulcers, kyphosis, sclerosis, liver failure, cirrhosis, Hutchinson-Gilford Progeria Syndrome (HGPS), laminoplasty, osteoporosis, dementia, (cardio)vascular disease, obesity, metabolic syndrome, acute myocardial infarction, emphysema, insulin sensitivity, Boutonneux 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 deafness, vision loss such as blindness, sleep disorders, pain such as joint pain or leg pain, imbalance, fear, depression, difficulty breathing, weight loss, hair loss, muscle loss, bone mineral density loss, frailty and / or poor health. A particular disease or condition in which removal of senescent cells would be beneficial is a disease or condition associated with or leading to inflammation, typically chronic inflammation in a mammalian subject, e.g., a human, where the inflammation is induced or mediated by senescent cells. In some embodiments, the inflammation-induced or mediated senescent cells are at least partially present in the same organ, e.g., the same 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 conditions in the extracellular environment immediately surrounding one or more senescent cells that differ from the same conditions surrounding non-senescent cells. The extracellular environment of senescent cells can 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 a protein translated from the transcription product of the forkhead box 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 retro-inverso peptide having the reverse amino acid sequence of at least a fragment of the FOXO4 protein and containing some, e.g., all, D amino acid residues.

[0083] The term "full antibody" refers to an antibody that contains an antigen-binding variable region (V H or V L ), a light chain constant domain (CL), and heavy chain constant domains CH1, CH2, and CH3. The constant domains may be native sequence constant domains (e.g., human native sequence constant domains) or amino acid sequence variants thereof. Depending on the amino acid sequence of the constant domain of their heavy chain, full-length antibodies can be assigned to different "classes." There are five major 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 that correspond to the different classes of antibodies are called alpha, delta, epsilon, gamma, and mu, respectively.

[0084] An "individual" or "subject" is a mammal, including, but not limited to, domestic animals (e.g., cows, sheep, cats, dogs, and horses), primates (e.g., humans and non-human primates, such as monkeys), rabbits, and rodents (e.g., mice and rats).

[0085] The term "library" as used herein refers to a collection of proteins in a single pool. Libraries may be generated using recombinant DNA technology. For example, a collection of cDNAs or any other protein-encoding DNA may be inserted into an expression vector to generate a protein library. Similarly, a collection of cDNAs or protein-encoding DNA may be inserted into a phage genome to generate a bacteriophage display library of wild-type proteins. A collection of cDNAs 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). Collections of cDNAs from selected cell types are also commercially available from vendors such as Stratagene®. A library of wild-type proteins as used herein is not a collection of biological samples.

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

[0087] As used herein, the term "multispecific antibody" refers to an antibody having binding specificities for at least two different epitopes. An exemplary multispecific antibody 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). Engineered antibodies with two, three, or more (e.g., four) functional antigen-binding sites are also contemplated (see, e.g., US2002 / 0004587 A1).

[0088] As used herein, the term "non-naturally occurring amino acid" refers to any amino acid not found in nature. Non-naturally occurring 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, b-peptides, g-peptides, and d-peptides; oligomers with backbones that can adopt helical or sheet conformations, such as compounds with backbones that utilize bipyridine segments, compounds with backbones that utilize solvophobic interactions, compounds with backbones that utilize side chain interactions, compounds with backbones that utilize hydrogen bonding interactions, and compounds with backbones that utilize metal coordination. Non-naturally occurring amino acids also include residues with side chains that resist nonspecific protein adsorption, which can be designed to increase the presentation of antimicrobial peptides in biological fluids, and / or with polymerizable side chains that allow the synthesis of polymer brushes using non-natural amino acid residues in peptides as monomer units.

[0089] As used herein, the term "parent protein" refers to a polypeptide or protein that can be evolved to generate a conditionally active polypeptide or protein using the methods of the present invention. The parent protein may be a wild-type protein or a non-naturally occurring protein. For example, a therapeutic polypeptide or protein, or a mutant or variant polypeptide or protein may be used as a parent polypeptide or protein. A parent protein may also be a fragment of another naturally occurring 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 fragments thereof, hormones and fragments thereof, ligands and fragments thereof, receptors and fragments thereof, regulatory proteins and fragments thereof, and growth factors and fragments thereof.

[0090] As used herein, the term "polypeptide" refers to a polymer in which the monomers are amino acids and are linked together through peptide or disulfide bonds. A polypeptide may be a full-length naturally occurring amino acid chain or a fragment, mutant, or variant thereof, such as a selected region of the amino acid chain in a binding interaction. A polypeptide may also be a synthetic amino acid chain, or a combination of natural amino acids or fragments thereof with a synthetic amino acid chain. A fragment refers to an amino acid sequence that is a portion of a full-length protein and is typically 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 length. In addition, amino acids other than naturally occurring amino acids, such as β-alanine, phenylglycine, and homoarginine, may be included in a polypeptide. Commonly encountered amino acids that are not genetically encoded may also be included in a polypeptide. The amino acids may be D- or L-optical isomers. D-isomers are preferred for use in specific situations, as described further below. In addition, other peptidomimetics are also useful, for example, in linker sequences of polypeptides (see Spatola, 1983, Chemistry and Biochemistry of Amino Acids. Peptides and Proteins, Weinstein, ed., Marcel Dekker, New York, p. 267). In general, the term "protein" is not intended to include any significant difference from the term "polypeptide" other than including structures comprising 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 the monomers are amino acids and are linked together through peptide or disulfide bonds. A protein may be an extended naturally occurring amino acid chain or a fragment, mutant, or variant thereof, such as a selected region of the amino acid chain in a binding interaction. A protein may also be a cyclic peptide, having an amino acid polymer that utilizes all or part of the polymer to form a ring structure. A protein may also be a synthetic amino acid chain, an amino acid chain containing unnatural amino acids, or a combination of a natural amino acid chain or a fragment thereof with a synthetic amino acid chain. A fragment refers to an amino acid sequence that is a portion of a full-length protein and 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 length. In addition, amino acids other than naturally occurring amino acids, such as β-alanine, phenylglycine, and homoarginine, may be included in a polypeptide. Commonly encountered amino acids that are not genetically encoded may also be included in a polypeptide. The amino acids may be D- or L-optical isomers. D-isomers are preferred for use in specific situations, as described further below. In addition, other peptidomimetics are also useful, for example, in linker sequences of polypeptides (see Spatola, 1983, Chemistry and Biochemistry of Amino Acids. Peptides and Proteins, Weinstein, ed., Marcel Dekker, New York, p. 267). In general, the term "protein" is not intended to include any significant difference from the term "polypeptide" other than including structures comprising two or more polypeptide chains held together by covalent or non-covalent 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 unmodified or as aggregates with other molecular species. Receptors can be covalently or noncovalently attached to the binding membrane directly or via a specific binding substance. 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. Binding of a ligand to a receptor indicates the combination of the ligand and receptor molecule through specific molecular recognition to form a complex that can be detected by various ligand-receptor binding assays known to those skilled in the art.

[0093] As used herein, the term "senescence" or "cellular senescence" refers to the progression from an actively dividing cell to a metabolically active, non-dividing cell. The term "senescence" refers to the state that cells enter after multiple divisions, in which future cell divisions are prevented from occurring, even though the cell remains metabolically active.

[0094] As used herein, the term "senescent cells" refers to metabolically active but permanently withdrawn cells from the cell cycle (see, for example, Campisi, Cell, vol. 120, pp.513-522, 2005). Senescent cells do not replicate and have one or more of the following additional characteristics attributed to senescent cells: cell cycle arrest in G1 phase; enlarged and flat morphology; increased granularity; staining for β-galactosidase activity at pH 6; senescence-associated heterochromatin formation; and characteristic gene expression partially regulated by p16 and p21. Examples of senescent cells include senescent preadipocytes, senescent endothelial cells, senescent fibroblasts, senescent nerve cells, senescent epithelial cells, senescent mesenchymal cells, senescent smooth muscle cells, senescent macrophages, and senescent chondrocytes.

[0095] As used herein, the term "senolytic agent" refers to an agent that selectively (preferentially or to a greater extent) destroys, kills, removes, or facilitates the selective destruction of senescent cells. In other words, the senolytic agent destroys or kills senescent cells in a biologically, clinically, and / or statistically significant manner compared to its ability to destroy or kill non-senescent cells. The senolytic agent may be a small compound or a biological molecule such as a protein or polynucleotide. The senolytic agent is used in an amount and for a time sufficient to selectively kill defined senescent cells but insufficient to kill (cause destruction or death of) a clinically or biologically significant number of non-senescent cells. In certain embodiments, the senolytic agents described herein alter at least one signaling pathway in a manner that induces (initiates, stimulates, triggers, activates, promotes) and results in (i.e., induces, leads to) the death of senescent cells. The senolytic agent may, for example, modify either or both cell survival signaling pathways (e.g., the Akt pathway) or inflammatory pathways, e.g., by antagonizing proteins in viable cells and / or inflammatory pathways 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, or more preferably less than 500 a.mu, or 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 "senescent cell-associated target" refers to a molecule, e.g., a protein, that is located on the surface of a senescent cell (e.g., a cell membrane protein), or that is present in a senescent cell, or that is secreted by a senescent cell into the extracellular environment of the senescent cell.

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

[0099] As used herein, the term "therapeutically effective amount" refers to any amount that results in, but is not limited to, a cure, prevention, or amelioration of a disease, disorder, or side effect, or slows the rate of progression of a disease or disorder, compared to a corresponding subject not receiving such amount. The term also includes within its scope an amount effective to enhance normal physiological function, and an amount effective to induce physiological function in a patient that enhances or supports the therapeutic effect of a second pharmaceutical agent.

[0100] As used herein, the terms "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 dosage and treatment regimen provides a senolytic agent in an amount sufficient to provide therapeutic and / or prophylactic benefit. Therapeutic benefits to a subject to whom a senolytic agent described herein is administered include improved clinical outcomes, for example, where the agent prevents, delays, or arrests (reduces) undesirable physiological changes associated with a disease, or prevents, delays, or arrests (reduces) the progression or severity of such a disease.

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

[0102] Detailed Description It should be noted that as used in this specification and the appended claims, the singular forms "a," "an," and "the" include plural referents unless clearly indicated otherwise. Furthermore, the terms "a" (or "an"), "one or more," and "at least one" may be used interchangeably herein. The terms "including," "comprising," "having," and "composed of" may also be used interchangeably.

[0103] Unless otherwise indicated, all numbers expressing quantities of raw materials, properties, e.g., molecular weights, percentages, ratios, reaction conditions, and the like, used in the specification and claims, whether the term "about" is present or absent, should be understood to be modified in all instances by the term "about." Accordingly, unless indicated to the contrary, the numerical parameters set forth in the specification and claims are approximations that may vary depending upon the desired properties sought to be obtained by the present disclosure. At the very least, and not as an attempt to limit the application of the doctrine of equivalents to the claims, each numerical parameter should be construed at least in light of the number of reported significant digits and by applying ordinary rounding techniques. Notwithstanding that the numerical ranges and parameters setting forth the broad scope of the present disclosure are approximations, the numerical values ​​set forth in the specific examples are reported as precisely as possible. Any numerical value, however, inherently contains certain errors necessarily resulting from the standard deviation found in their respective testing measurements.

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

[0105] Likewise, it should be understood that each amount / value or amount / value range for each component, compound, substituent, or parameter disclosed herein is to be interpreted as disclosed in combination with each amount / value or amount / value range disclosed for any other component(s), compound(s), substituent(s), or parameter(s) disclosed herein, and that any combination of amount / values ​​or amount / value ranges for two or more components, compounds, substituent(s), or parameters disclosed herein are thus disclosed in combination with each other for 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 significant digits. Thus, a range of 1 to 4 should be interpreted as representing a disclosure of values ​​of 1, 2, 3, and 4. It will also be understood that each lower limit of each range disclosed herein should be interpreted as a disclosure in combination with each upper limit of each range and each specific value within each range disclosed herein for the same component, compound, substitution, or parameter. Thus, the present 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 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 an ingredient, compound, substituent, or parameter disclosed in the specification or examples should be construed as disclosing either the lower or upper limit of a range, and thus can be combined with any other lower or upper limit of a range or specific amount / value of the same ingredient, compound, substituent, or parameter elsewhere in this application to form a range for that ingredient, compound, substituent, or parameter.

[0108] The present invention provides a method for generating a conditionally active protein having activity towards senescent cells from a parent protein that binds to said target associated with senescent cells, the method comprising: (i) evolving DNA encoding the parent protein using one or more evolutionary techniques to generate mutant DNA; (ii) expressing the mutant DNA to obtain a mutant protein; (iii) subjecting the mutant protein to assay under extracellular conditions of the senescent cells and under normal physiological conditions; (iv) (a) a decrease in 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 the extracellular conditions of the senescent cell, compared to the activity of the parent 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 the normal physiological conditions, and an increase in the activity of the parent protein in the assay under the extracellular conditions of the senescent cells compared to the activity of the parent protein in the assay under the extracellular conditions of the senescent cells; selecting the conditionally active protein from the mutant proteins that exhibit at least one of: Includes.

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

[0110] In the case of an antibody, ligand, or receptor, the parent protein may bind to a target associated with senescent cells, and the activity may be binding activity to the target. In the case of an enzyme, the parent protein may utilize at least a portion of senescent cells, as the substrate and the activity is an 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] The target associated with senescent cells is typically a protein in senescent cells. In some instances, the target is a protein on the cell membrane of senescent cells. In some embodiments, the target is selected from DEP-1, NTAL, EBP50, STX4, VAMP3, ARMCX-3, LANCL1, B2MG, PLD3, and VPS26A. These proteins are recognized as biomarkers of 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 Fas protein or 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 TNF-related apoptosis-inducing ligand (TRAIL), see Guicciardi et al., "Life and death by death receptors," FASEB J. vol. 23, pp. 1625-1637, 2009. Examples of DR include DR4 and DR5.

[0114] In some embodiments, the senescent cell-associated target 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 have BH1-BH4 domains (BCL-2 (i.e., BCL-2 protein members of the BCL-2 anti-apoptotic protein family), BCL-xL, BCL-w, A1, 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, e.g., 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 associated with 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 senescent cell-associated target is selected from misfolded forms of proteins selected from prion protein (PrP), CD38, Notch-1, CD44, CD59, Fas ligand, TNF receptor, and EGF receptor, as described in US 2016 / 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 is 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 use of an assay under conditions, such as a pH below 7.0, e.g., from 5.0 to below 7.0, or from 5.5 to below 7.0, or from 6.0 to below 7.0, or from 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 with a pKa between 6 and 7.5, preferably between 6 and 7, and more preferably between 6.2 and 6.8, for example. Examples of such small molecules are described herein.

[0119] In some embodiments, the parent protein is an antibody. In some embodiments, the parent protein has one or more suitable characteristics based on which it 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 at 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 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 in one or more extracellular conditions of senescent cells.

[0121] In other embodiments, the parent antibody is selected based on internalization efficiency, which 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 the extracellular conditions of senescent cells. In such embodiments, the parent antibody is selected based on having the most similar binding activity and / or the most similar combination of one or more characteristics under both normal physiological conditions and the extracellular conditions of senescent cells. For example, if the normal physiological conditions and the extracellular conditions of senescent cells are pH 7.4 and 6.4, respectively, an antibody with the most similar binding activity at pH 7.4 and 6.4 may be selected as the parent antibody over an antibody with a 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 of only 8 amino acids.

[0124] After a parent protein has been selected, the DNA encoding the parent protein may be evolved using suitable evolutionary techniques to generate mutant DNA, which may then be expressed to generate mutant proteins for screening to identify conditionally active proteins. Suitable techniques for evolving the DNA encoding the parent protein, expressing the mutant DNA to generate mutant proteins, and screening the mutant proteins are described in WO2016 / 138071.

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

[0126] The selected conditionally active protein may also be produced using a polypeptide-expressing cellular production host or organism. To make the production process more efficient, the DNA encoding the conditionally active protein may be subjected to codon optimization for the cellular production host or organism.Codon optimization in the mouse system is described by 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. 7250-7253, 2001; codon optimization in the yeast system is described by 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 codon optimization in Escherichia coli is described by 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. 2000; how codon usage affects protein secretion in E. coli has been described in previous studies, 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 production host may be a mammalian cell production host selected from the group consisting of CHO, HEK293, IM9, DS-I, THP-I, Hep G2, COS, NIH 3T3, C33a, A549, A375, SK-MEL-28, DU 145, PC-3, HCT 116, Mia PACA-2, ACHN, Jurkat, MML-1, Ovcar 3, HT 1080, Panc-1, U266, 769P, BT-474, Caco-2, HCC 1954, MDA-MB-468, LnCAP, NRK-49F, and SP2 / 0 cell lines; and mouse splenocytes and rabbit PBMCs. The mammalian cell production host may be, for example, a CHO or HEK293 cell line. In one specific embodiment, the mammalian cell production host is a CHO-S cell line. In another embodiment, the mammalian cell production host is a HEK293 cell line.

[0128] In some embodiments, the cell production host is a yeast cell, such as an S. cerevisiae yeast cell or a Pichia yeast cell. In some embodiments, the cell production host is a prokaryotic cell such as Escherichia coli (Owens, RJ and Young, RJ, J. Immunol. Meth., vol. 168, p. 149, 1994; Johnson S and Bird RE, Methods Enzymol., vol. 203, p. 88, 1991). The conditionally active protein can also be produced in plant cells or plants (Firek et al., Plant Mol. Biol., vol. 23, p. 861, 1993).

[0129] The conditionally active proteins may be modified through natural processes or using chemical modification techniques as described in WO2016 / 138071. The conditionally active proteins may also be synthesized using solid phase chemical peptide synthesis methods, as also described in WO2016 / 138071.

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

[0131] The conditions are the same but have different values ​​in an assay under the extracellular conditions of senescent cells compared to an assay under normal physiological conditions; for example, the condition may be pH, where the pH value under normal physiological conditions may be 7.2 to 7.8 or 7.2 to 7.6, and the pH value under the extracellular conditions of senescent cells may be 6.0 to 7.0 or 6.2 to 6.8.

[0132] The activity may be any activity relevant to the treatment of any senescent cell, such as, for example, the binding activity of a conditionally active antibody to the target or specific epitope, 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 on at least a portion of the senescent cell 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 surrounding the senescent cells that are the result of the special characteristic(s) of the senescent cells, e.g., compared to those of normal cells. One group of special characteristics of senescent cells that is useful in the present invention is the metabolic activity of senescent cells. For example, senescent cells may exhibit one or more of the following special characteristics: (1) the growth arrest of senescent cells is essentially permanent and cannot be reversed by known physiological stimuli; (2) senescent cells increase in size, sometimes enlarging to more than twice the size of their non-senescent counterparts; (3) senescent cells express senescence-associated β-galactosidase (SAP-gal), which in part reflects an increase in lysosomal mass; (4) many senescent cells express p16INK4a, 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 termed DNA segments, where chromatin changes reinforce senescence (DNA-SCARS, such as dysfunctional telomeres or telomere dysfunction-induced foci (TIFs)), contain activated DDR proteins, and are distinguishable from transient damage foci; (7) Senescent cell nuclei lose structural proteins such as laminin B1 or chromatin-associated 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 environment of senescent cells is low pH, induced by increased glycolytic metabolism 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 sugar to form two pyruvates and two ATPs, where pyruvate may be converted to lactate and excreted, thereby 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, and the glycolytic metabolism of cancer cells lowers the pH in the tumor microenvironment. Thus, the extracellular conditions of senescent cells can be an 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 the senescent cells may have a lower concentration of deoxynucleotides compared to the normal physiological concentration of deoxynucleotides in a 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 may lead to a lower concentration of deoxynucleotides in the extracellular environment of the senescent cells compared to the extracellular concentration of deoxynucleotides in the extracellular environment of normal cells. Thus, the extracellular conditions of the senescent cells may be selected to have a lower concentration of deoxynucleotides compared to the normal physiological concentration of deoxynucleotides in the extracellular environment of normal cells, where the corresponding normal physiological condition is the concentration of deoxynucleotides in the extracellular environment of normal cells.

[0136] In some embodiments, the extracellular conditions of the senescent cells may be at a lower concentration of oxygen compared to the physiological concentration of oxygen 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, which may result in a lower concentration of oxygen in the extracellular environment of senescent cells compared to the extracellular environment of normal cells. Thus, the extracellular conditions of the senescent cells may be selected to be at a lower concentration of oxygen compared to the normal physiological concentration of oxygen in the extracellular environment of normal cells, where the corresponding normal physiological condition is the concentration of oxygen in the extracellular environment of normal cells.

[0137] In some embodiments, the extracellular conditions of the senescent cells may have a lower NAD+ / NADH ratio than the NAD+ / NADH ratio 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). Thus, 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, where the corresponding normal physiological condition is 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 an elevated concentration of a redox homeostasis metabolite selected from hypotaurine, cysteine ​​sulfinic acid, cysteine-glutathione disulfide, gamma-glutamylalanine, gamma-glutamylmethionine, pyridoxate, gamma-glutamylglutamine, and alanine compared to the normal concentration of the same redox homeostasis metabolite in the extracellular environment of a normal growing, confluent, or quiescent cell (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 result in a higher concentration of a redox homeostatic metabolite compared to the normal physiological concentration of the same in the extracellular environment of a normal cell, which may be selected from a growing cell, a confluent cell, or a quiescent cell, the corresponding normal physiological condition being the concentration of the redox homeostatic metabolite in the extracellular environment of the normal cell.

[0139] In some embodiments, the extracellular conditions of the senescent cells may be selected to result in a higher concentration of a nucleotide metabolite selected from 3-ureidopropionate, urate, 7-methylguanine, and hypoxanthine compared to the concentration of the nucleotide metabolite in the extracellular environment of a normal growing cell, a confluent cell, or a quiescent cell (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 result in a higher concentration of the nucleotide metabolite compared to the normal physiological concentration of the nucleotide metabolite in the extracellular environment of a normal cell, which may be selected from a growing cell, a confluent cell, or a quiescent cell, where the corresponding normal physiological condition is the concentration of the nucleotide metabolite in the extracellular environment of the normal cell.

[0140] In some embodiments, the extracellular conditions of the senescent cells may have a lower concentration of thymidine compared to the concentration of thymidine 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 have a lower concentration of thymidine compared to the normal physiological concentration of thymidine in the extracellular environment of normal cells, which may be selected from growing cells, confluent cells, or quiescent cells, where the corresponding normal physiological condition is the concentration of thymidine in the extracellular environment of normal cells.

[0141] In some embodiments, the extracellular conditions of the senescent cells may be selected from glycylisoleucine, glycylvaline, glycylleucine, isoleucylglycine, and valylglycine at a reduced concentration of a dipeptide relative to the concentration of the dipeptide in the extracellular environment of a normal growing cell, a confluent cell, or a quiescent cell (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 result in a reduced concentration of the dipeptide relative to the normal physiological concentration of the dipeptide in the extracellular environment of a normal cell, which may be selected from a growing cell, a confluent cell, or a quiescent cell, and the corresponding normal physiological condition is the concentration of the dipeptide in the extracellular environment of the normal cell.

[0142] In some embodiments, the extracellular conditions of the senescent cells may be selected to result in a reduced 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 a normal growing cell, a confluent cell, or a quiescent cell (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 result in a reduced concentration of a fatty acid selected from linoleate, dihomolinoleate, and 10-heptadecenoate compared to the normal physiological concentration of the fatty acid in the extracellular environment of a normal cell, which may be selected from a growing cell, a confluent cell, or a quiescent cell, where the corresponding normal physiological condition is the concentration of the fatty acid in the extracellular environment of the normal cell.

[0143] In some embodiments, the extracellular conditions of the senescent cells may be selected to result in a higher concentration of a phospholipid metabolite selected from 2-hydroxypalmitate, 2-hydroxystearate, 3-hydroxydecanoate, 3-hydroxyoctanoate, and glycerophosphorylcholine compared to the concentration of the phospholipid metabolite in the extracellular environment of a normal growing cell, a confluent cell, or a quiescent cell (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 result in a higher concentration of the phospholipid metabolite compared to the normal physiological concentration of the phospholipid metabolite in the extracellular environment of a normal cell, which may be selected from a growing cell, a confluent cell, or a quiescent cell, and the corresponding normal physiological condition is the concentration of the phospholipid metabolite in the extracellular environment of the normal cell.

[0144] In some embodiments, the extracellular conditions of the senescent cells may be selected to result in a higher concentration of an amino acid metabolite selected from alanine, C-glycosyltryptophan, kynurenine, dimethylarginine, and ortithine compared to the concentration of the same amino acid metabolite in the extracellular environment of a normal growing cell, a confluent cell, or a quiescent cell (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 result in a higher concentration of the same amino acid metabolite compared to the normal physiological concentration of the same amino acid metabolite in the extracellular environment of a normal cell, which may be selected from a growing cell, a confluent cell, or a quiescent cell, where the corresponding normal physiological condition is the concentration of the amino acid metabolite in the extracellular environment of the normal cell.

[0145] In some embodiments, the extracellular conditions of the senescent cells may be a reduced concentration of phenylpyruvate relative to the concentration of phenylpyruvate 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 a reduced concentration of phenylpyruvate relative to the normal physiological concentration of phenylpyruvate in the extracellular environment of normal cells, the corresponding normal physiological condition being the concentration of phenylpyruvate in the extracellular environment of normal cells.

[0146] In some embodiments, the extracellular conditions of the senescent cells may be selected to result in a higher concentration of a metabolite selected from fumarate, malonate, eicosapentaenoate, and citrate compared to the concentration of the metabolite in the extracellular environment of a normal growing cell, a confluent cell, or a quiescent cell (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 result in a higher concentration of a metabolite selected from fumarate, malonate, eicosapentaenoate, and citrate compared to the normal physiological concentration of the metabolite in the extracellular environment of a normal cell, the corresponding normal physiological condition being the concentration of the metabolite in the extracellular environment of a normal cell.

[0147] In some embodiments, the extracellular conditions of senescent cells may be selected to have a higher ratio of glycerophosphocholine to phosphocholine compared to the ratio of glycerophosphocholine to phosphocholine 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 have a higher ratio of glycerophosphocholine to phosphocholine compared to the extracellular environment of normal, non-quiescent cells, the corresponding normal physiological condition being the ratio of glycerophosphocholine to phosphocholine in the extracellular environment of normal, non-quiescent cells.

[0148] Senescent cells secrete a variety of different proteins, collectively referred to as the senescence cell-associated secretory phenotype (SASP), including GM-CSF, GROα, GRC-α, β, and γ, IGFBP-7, IL-1α, IL-6, IL-7, IL-8, MCP-1, MCP-2, MIP-1a, 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-1β, 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-1Rl, IL-11, IL-15, IL-2R-α, IL-6R, I-TAC, leptin, LIF, MMP-2, MSP-α, 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 -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.Additional proteins secreted by senescent cells include IGF-2 and IGF-2R, IGFBP-3, IGFBP-7, TGF-β, WNT2, CXCR2-binding chemokine, 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 condition of the senescent cell is either an elevated concentration of one or more of the secreted proteins, or the presence of one or more of the proteins, relative to the concentrations of these secreted proteins in the extracellular environment of a normal cell and normal physiological conditions or normal physiological concentrations in the absence of the secreted protein(s) in the extracellular environment of a normal cell.

[0149] The conditionally active proteins of the invention may be used as senolytic agents to kill or remove senescent cells from a subject. Interaction of the conditionally active proteins with senescent cells may inhibit or kill the senescent cells through 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 (i.e., initiate, trigger, stimulate, or in some manner remove or inhibit the suppression of cell death pathways) cell death pathways, such as apoptotic pathways, in senescent cells, leading to the death of the senescent cells.

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

[0151] The src kinase signaling pathway is involved in the regulation of cell proliferation, differentiation, apoptosis, cell adhesion and stress response (see, for example, 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, for example, Byeon et al., Mediators of Inflammation, vol. 2012, article ID 512926, 2012) and acute inflammatory responses (see, for example, Okutani et al., Am. J. Physiol. Lung Cell Mol. Physiol., vol. 291, pp. L129-L141, 2006). Thus, conditionally active proteins that alter the src kinase signaling pathway may alter both signaling and inflammatory pathways.

[0152] Modifying a cell signaling pathway and / or inflammatory pathway may affect the function of one or more downstream proteins or the interaction of one or more downstream proteins with other components of each cell signaling or inflammatory pathway. For example, a conditionally active protein that modifies the src kinase signaling pathway or the PBK / Akt pathway may modify the function of one or more downstream proteins in each pathway or affect the interaction of one or more downstream proteins with other components of each pathway (see, e.g., Example 1; Figures 2B-2D). Exemplary proteins that are upregulated in senescent cells include P38 / MAPK, ERK1 / 2, and PBK (complex). In certain embodiments, the PBK / Akt pathway, a cell signaling pathway, is activated during senescence, 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 under extracellular conditions of senescent cells and assays under normal physiological conditions comprise components selected from, for example, citrate buffers such as sodium citrate, phosphate buffers, bicarbonate buffers such as Krebs buffer, phosphate-buffered saline (PBS) buffer, Hank's buffer, Tris buffer, HEPES buffer, etc. Other buffers known to those skilled in the art that are suitable for the assay may also 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 body fluids of mammals or animals, including humans, as described in detail in WO2016 / 138071.

[0155] The conditionally active protein may interact with one or more of inorganic compounds, ions, and organic molecules. Such interactions between the conditionally active protein and the component, 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 lower pH, such as 5.5 to 7.2, 6.0 to 7.0, or 6.2 to 6.8, and normal physiological conditions are normal physiological pH, such as 7.2 to 7.8. The assay solution for the extracellular pH may contain a component with a pKa between the lower pH of the extracellular conditions and normal physiological pH. The pKa may be, 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, the component has a molecular weight of less than 900 a.m. and may be selected from, for example, histidine, histamine, adenosine hydrogen diphosphate, adenosine hydrogen 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 percentage) of charged amino acid residues compared to the amino acid residues of the parent protein from which the conditionally active protein is derived. There are three positively charged amino acid residues: lysine, arginine, and histidine, and two negatively charged amino acid residues: aspartic acid and glutamic acid. These charged amino acid residues are over-represented in certain conditionally active proteins compared to the parent protein from which the conditionally active protein is derived. As a result, due to the increased number of charged amino acid residues in the conditionally active protein, the conditionally active protein is 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 have different activities in the presence of different molecular species in the assay solution. A molecular species with at least two ionization states: an uncharged or low-charged state at one value of a condition, such as pH, and a charged or higher-charged state at a different value 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 the charged amino acid residues present in the conditionally active protein. This mechanism can 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 can be one factor used to determine the molecular species suitable for affecting the activity of the conditionally active protein. In some embodiments, the conditionally active protein may have more positively charged amino acid residues, such as lysine, arginine, and histidine, than the parent protein. Thus, the conditionally active protein can 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 desired, and / or to have a desired level of interaction with specific molecular species present under normal physiological conditions where low activity is desired.

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

[0161] In some embodiments, the interaction between the charged species and the conditionally active protein may result in the formation of salt bridges between different moieties on the protein, particularly charged or polarized moieties. The formation of salt bridges is known to stabilize polypeptide structures (Donald, et al., "Salt Bridges: Geometrically Specific, Designable Interactions," Proteins, 79(3): 898-915, 2011; Hendsch, et al., "Do salt bridges stabilize proteins? A continuum electrostatic analysis," Protein Science, 3:211-226, 1994). The salt bridges can stabilize or fix protein structures that undergo constant, minute structural changes, commonly referred to as "breathing" (Parak, "Proteins in action: the physics of structural fluctuations and conformational changes," Curr Opin Struct Biol., 13(5):552-557, 2003). Because structural variations allow the conditionally active protein to efficiently recognize and bind to its partner, the "breathing" of protein structure is important for protein function and its binding with its partner (Karplus, et al., "Molecular dynamics and protein functions," PNAS, vol. 102, pp. 6679-6685, 2015). Possibly, the salt bridge may directly block the partner's access to the binding site, so the formation of a salt bridge may make the binding site on the conditionally active protein, particularly the binding pocket, less accessible to the partner. Even salt bridges remote from the binding site may have allosteric effects that alter the conformation of the binding site and inhibit binding. Therefore, after a salt bridge stabilizes (fixes) the structure of the conditionally active protein, the protein may become less active upon binding to its partner, leading to reduced activity.

[0162] Hemoglobin is a well-known example of how proteins and their structures are stabilized by salt bridges. Structural and chemical studies have revealed that at least two combinations of chemical groups, namely the amino terminus and side chains of histidines β146 and α122, which have pKa values ​​near pH 7, are responsible for salt bridges. In deoxyhemoglobin, the terminal carboxylic acid group of β146 forms a salt bridge with a lysine residue in the α subunit of another αβ dimer. This interaction anchors the side chain of histidine β146 in a position where it can participate in a salt bridge with the negatively charged aspartic acid 94 in the same chain, provided that the imidazole group of the histidine residue is protonated (Figure 2). At high pH, ​​the side chain of histidine β146 is not protonated, and a salt bridge does not form. However, as the pH decreases, the side chain of histidine β146 becomes protonated, and a salt bridge forms between histidine β146 and aspartic acid β94, which stabilizes the quaternary structure of deoxyhemoglobin, making it more prone to release oxygen (at lower pH) during active tissue metabolism. Hemoglobin exhibits pH-dependent binding activity for oxygen; at low pH, the binding activity to oxygen decreases due to the formation of the salt bridge, whereas at high pH, ​​the binding activity to oxygen increases due to the absence of the salt bridge.

[0163] Similarly, small molecules such as bicarbonate may reduce the binding activity of a conditionally active protein to its partner by forming salt bridges in the conditionally active protein. For example, at a pH below pKa 6.4, bicarbonate is protonated and therefore uncharged. Uncharged bicarbonate cannot form salt bridges and thus has little effect on the binding between the conditionally active protein and its partner. Therefore, the conditionally active protein has high binding activity with its partner at low pH. On the other hand, at a high pH greater than the pKa of bicarbonate, bicarbonate ionizes by releasing a proton and thus becomes negatively charged. Negatively charged bicarbonate will form salt bridges between positively charged or polarized moieties on the conditionally active protein, stabilizing the structure of the conditionally active protein. This will block or reduce the binding between the conditionally active protein and 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] In the absence of molecular species such as bicarbonate in the assay solution, the conditionally active protein may lose its conditional activity. This may be due to the absence of salt bridges on the conditionally active protein that stabilize (fix) the protein's structure. Thus, the partner will have similar accessibility to the binding site on the conditionally active protein at any pH, resulting in similar activity at the first and second pHs.

[0165] Although salt bridges (ionic bonds) are the strongest and most common way for molecular species to affect the activity of the conditionally active protein, it should be understood that other interactions between such molecular species and the conditionally active protein may also contribute to stabilizing (fixing) the structure of the conditionally active protein. Other interactions include hydrogen bonds, hydrophobic interactions, and van der Waals interactions.

[0166] In some embodiments, the conditionally active protein is evolved by comparing the conditionally active protein with a parent protein to determine whether the conditionally active protein has a higher proportion of negatively or positively charged amino acid residues, in order to select an appropriate compound or ion as the molecular species. Compounds with an appropriate charge at normal physiological pH can then be selected to affect 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 appropriate compound will typically be negatively charged at normal physiological pH to interact with the conditionally active protein. On the other hand, if the conditionally active protein has a higher proportion of negatively charged amino acid residues than the parent protein, the appropriate small molecule will typically be positively charged at normal physiological pH to interact with the conditionally active protein.

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

[0168] Disulfide has a pKa of 7.05. Therefore, at higher pH, 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 activity. On the other hand, at lower pH, 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 disulfide, 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 the present application, such as "CRC Handbook of Chemistry and Physics," 96th Edition, CRC Press, 2015; "Chemical Properties Handbook," McGraw-Hill Education, 1998.

[0170] For example, the molecular species have a low molecular weight and / or a relatively small conformation to ensure maximum access to the small pocket on the conditionally active protein by minimizing steric hindrance. To this end, small molecules typically have a molecular weight of less than 900 a.mu, or more preferably less than 500 a.mu, more preferably less than 200 a.mu, and more preferably less than 100 a.mu. For example, hydrogen sulfide, disulfide, and bicarbonate all have low molecular weights and small structures, providing access to the pocket on the conditionally active protein.

[0171] The concentration of the molecular species in the assay solution is, for example, at or near the physiological concentration of the molecular species in a subject. For example, the physiological concentration of bicarbonate (in human serum) is in the range of 15 to 30 mM. Thus, the concentration of bicarbonate in the assay solution may be 10 to 40 mM, or 15 to 30 mM, or 20 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 the 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 are present, such as a combination of bicarbonate and histidine, and 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 components of the assay solution or 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 pH dependence. Thus, 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 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 increased activity at the lower pH 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 the mutant proteins generated by the evolutionary steps, hi another embodiment, an assay solution containing an 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 at substantially the same concentrations to both the assay solutions for normal physiological conditions and for the extracellular conditions of senescent cells, e.g., both bicarbonate and histidine are added to both assay solutions.

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

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

[0181] In some embodiments, the assay solution may be designed to select conditionally active biological proteins 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. An assay solution for selecting such conditionally active proteins 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. An assay solution for the 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, the assay solutions for both normal physiological conditions and the extracellular conditions of senescent cells may also contain ions to support binding between the mutant protein and the binding partner, thereby increasing the number of hits for conditionally active proteins.

[0182] In some embodiments, certain serum components may be minimized or excluded from the assay solution for this 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 mutant binding antibodies that simply bind to components present in serum rather than conditionally active under a variety of different conditions, may thereby give false positives. Therefore, careful selection of assay components to minimize or exclude components that could potentially bind to mutant proteins in the assay can reduce the number of false positive mutant proteins that may inadvertently be identified as positive for conditional activity due to binding to components in the assay other than the desired binding partner. For example, in some embodiments in which 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 instances to achieve similar goals that will be well understood by those skilled in the art.

[0183] In some embodiments, the evolution step may generate mutant proteins that may simultaneously possess other desirable properties in addition to the conditional activity characteristics discussed above. Suitable other desirable properties that may be evolved may include binding affinity, expression, humanization, etc. Thus, the present invention may be used to generate conditionally active proteins that also possess improvements in at least one or more of these other desirable properties.

[0184] In some embodiments, the conditionally active protein may be further mutated, e.g., in a second evolutionary step, using one of the mutagenesis techniques disclosed herein to improve another property of the conditionally active protein, such as binding affinity, expression, humanization, etc. After the second evolutionary step, the mutant proteins may be screened for both the conditional activity and the improved property.

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

[0186] The selected first conditionally active protein may then be subjected to one or more additional evolution, expression and selection steps to produce a similar product. (a) 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, and an increase in the activity of the conditionally active protein in the assay under the extracellular conditions of the senescent cell, 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 the normal physiological conditions, and an increase in the activity of the parent protein in the assay under the extracellular conditions of the senescent cells compared to the activity of the parent protein in the assay under the extracellular conditions of the senescent cells; At least one second conditionally active protein may be selected that exhibits at least one of the following. The second activity may be similar to the first activity, in which case it is desirable for the second conditionally active protein to have 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 that have a ratio of activity under the extracellular conditions of senescent cells to activity under normal physiological conditions of greater than 1.0 (e.g., high selectivity between the two conditions). The ratio or selectivity of activity under extracellular conditions in senescent cells relative 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 in 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 comprising an antibody or antibody fragment (collectively referred to as "antibody") conjugated to a masking moiety (MM) through a linker (L). The probody is more active in the extracellular environment of senescent cells compared to that 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 for the target senescent cells. The masking moiety is cleaved from the antibody by proteases present in the extracellular environment of senescent cells, thereby exposing the antibody to the free binding activity for the target senescent cells. Therefore, the probody has higher binding activity for the target senescent cells in the extracellular environment of senescent cells than for the same target in the extracellular environment of normal cells.

[0190] Antibody fragments that can be included in a probody include antibodies (V L , V H ), variable or hypervariable regions of the light and / or heavy chains of the antibody (I), 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 moiety functions to reduce the binding activity of the antibody in the probody to the target senescent cells compared to the binding activity of the antibody without the masking moiety (e.g., after the masking moiety is cleaved from the probody). The binding activity of the antibody to the target senescent cells can be reduced by the masking moiety 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 can be for 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 the 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 the linker form a single chain conjugated to the side chain of an amino acid in one or more variable regions. In yet another example, when the probody comprises only an antibody fragment (such as only the variable regions), the masking moiety is conjugated to the C-terminus of one or more variable regions. In some embodiments, the probody has an N- to C-terminal structure of MM-L-Ab. In another embodiment, the probody has an N- to C-terminal structure of Ab-L-MM.

[0193] In some embodiments, the masking moiety may be identified by screening a diverse peptide library for peptides that bind to one or more variable regions of an antibody (Desnoyers et al., "Tumor-specific activation of an EGFR-targeting probody enhances therapeutic index," Sci Transl Med., vol. 5, pp. 207ra144, 2013). A peptide that, when conjugated to an antibody via a linker, can specifically bind to the antibody and block the antibody's binding to target senescent cells is selected as the masking moiety. The screening may be performed 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, random peptide libraries (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 more than 40 amino acids) may be used in screening methods to identify suitable masking moieties. For example, masking moieties with specific binding affinity to an antibody can be identified through a screening procedure that includes providing a library of peptide scaffolds, each peptide scaffold consisting of a transmembrane protein and a candidate substance. The library is then contacted with an antibody to identify one or more suitable masking moieties with detectable binding activity to the antibody. Screening may include another round of magnetic-activated sorting or fluorescence-activated cell sorting.

[0195] Thus, the present invention contemplates that the masking moiety may be specific to the antibody in the probody. One masking moiety that works well for a particular antibody may be less optimal for another antibody. Thus, screening diverse peptide libraries using the antibodies in the probody for the best masking moiety for 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 certain level of similarity to the 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 changing one or more amino acid residues to slightly reduce its binding activity to the antibody. In other embodiments, 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 with 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 disrupt 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 moiety cannot specifically bind to the antibody, but rather merely interferes with the binding of the antibody to the target senescent cell through one or more non-specific interactions, such as steric hindrance. For example, the masking moiety may be positioned within the probody such that the structure of the probody allows the masking moiety to mask the antibody through charge-based interactions, thereby holding the masking moiety in place and preventing access of the antibody to the binding site.

[0199] The linker of the probody is located between the masking moiety and the antibody. The linker contains a cleavage site (CS), where proteases present in the extracellular environment of senescent cells cleave the linker, releasing the masking moiety from the probody. The antibody is then exposed and available to bind to the target senescent cell. The linker may further contain one or more flexible regions (FR) flanking one or both sides of the cleavage moiety. For example, the linker may have the structure -FR-CS-FR-, -FR-CS-, -CS-FR-, -FR-FR-CS-FR-, -FR-CS-FR-FR-, -FR-FR-CS-FR-FR-, -FR-FR-CS-FR-FR-.

[0200] The flexible region provides conformational flexibility for the masking moiety, allowing it to reach the antibody's binding site and prevent binding. The flexible region consists essentially of small amino acids such as glycine, serine, and alanine, with small side chains to provide maximum flexibility. Glycine and glycine-serine polymers are relatively unstructured and can therefore act as neutral tethers between components. Glycine approaches the phi-psi space significantly more than alanine and is much less constrained than residues with longer side chains (see Scheraga, Rev. Computational Chem., pp. 11173-11142, 1992).

[0201] Suitable flexible regions may have different lengths, such as from 1 to 20 amino acids, from 2 to 15 amino acids, from 3 to 12 amino acids, from 4 to 10 amino acids, from 5 to 9 amino acids, from 6 to 8 amino acids, or from 7 to 8 amino acids, and may be 1, 2, 3, 4, 5, 6, or 7 amino acids in length.

[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), GSGSG (SEQ ID NO: 21), GSGGG (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. The cleavage site is generally included as part of the linker. However, in some instances, the cleavage site may be part of a masking moiety, such that all or part of the cleavage site facilitates masking of the antibody when the probody is in an inhibited, uncleaved, or masked state.

[0204] The cleavage site may be selected based on the proteases present in the extracellular environment of senescent cells. Senescent cells are known to secrete proteases, such as matrix metalloproteinases (MMPs), into the extracellular environment. Examples of MMP family members include stromelysin-1 and -2 (MMP-3 and -10, respectively) and collagenase-1 (MMP-1). Other MMPs include MMP1, MMP2, MMP7, MMP8, MMP9, MMP13, and MMP14. Natural substrates of these proteases are also known, which may aid in the design of cleavage sites used in probodies. 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. Members of the serine protease family include urokinase-type and tissue-type plasminogen activators (uPA and 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 for a matrix metalloprotease, and thus cleavable by an MMP to release the making moiety. In another embodiment, the cleavage site is a substrate for serine uPA or PSA. In some embodiments, the probody may contain more than one cleavage site, each of which may be a substrate for a different protease. Exemplary cleavage sites that can be substrates for proteases include ADAM10, ADAM12, ADAM17, ADAMTS, ADAMTS5, BACE, caspases 1-14, 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 1-17, MT-SP1, neprilysin, NS3 / 4A, plasmin, PSA, PSMA, TRACE, TMPRSS 3, TMPRSS 4, and uPA. Some exemplary cleavage sites are PLGLWA (SEQ ID NO: 33), which can be cleaved by MMPs, and GPQGIAGQ (SEQ ID NO: 34), which can be cleaved by collagenases. Other examples of cleavage 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 itself has conditional activity. In particular, the antibody itself may have higher binding activity to the target in the extracellular environment of senescent cells than in normal physiological conditions. Such a probody provides a two-fold amplification upon reaching the extracellular environment of senescent cells by (1) cleaving the masking moiety to release the antibody binding site from the masking moiety, and (2) the antibody has higher binding activity to the target in the extracellular environment of senescent cells than in normal physiological conditions.

[0207] In one embodiment, the conditionally active protein is an antibody intended to be conjugated with another agent. The conditionally active antibody may have a high ratio of activity under extracellular conditions in senescent cells to activity under normal physiological conditions, such as 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. This can be particularly important when the conjugated agent is, for example, toxic or radioactive, so that the conjugated agent can be concentrated at the disease or treatment site, if desired.

[0208] In some embodiments, the conjugated agent is a D retro-inverso peptide ("DRI peptide"). The DRI peptides have an inverted D amino acid sequence, which allows them to maintain a similar amino acid side chain topology to the native protein from which they are derived. Additionally, the DRI peptides are more resistant to proteolysis and therefore tend to have a significantly longer half-life than the native protein from which they are derived. Furthermore, the DRI peptides have a structure similar to that of the native protein from which they are derived. Finally, the DRI peptides have bioavailability comparable to that of the native protein from which they are derived. Therefore, the DRI peptides can be functional substitutes for and compete with the native protein from which they are derived. Therefore, DRI peptides are considered promising pharmaceutical agents.

[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 embryonic lethal, and foxo3- / - mice exhibit reproductive defects, whereas foxo4- / - mice do not harbor a significantly defective 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 conditional somatic foxo3- / - mice exhibit significantly shortened lifespans, whereas conditional somatic foxo1- / - and foxo4- / - mice do not (Paik, JH et al., Cell, vol. 128, pp. 309-323 (2007)). Somatic triple 1, 3, 4- / - mice show increased lymphomas, indicating functional redundancy of each FOXO protein (ibid.). Notably, however, single somatic foxo4- / - mice do not show any shortened lifespan or altered tumor-free survival. Unlike its counterparts FOXO1 and FOXO3, FOXO4 mRNA and protein expression are significantly increased in response to senescence-induced levels of DNA damage.

[0210] Senescence induced by ionizing radiation (X-ray)-induced DNA damage is characterized by the formation of persistent nuclear foci called DNA-SCARS (or DNA Segments with Chromatin Alterations Reinforcing Senescence), which are required 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 indicates that FOXO4 is a pivotal factor in the molecular decision between cellular senescence and apoptosis in response to genotoxic stress.

[0211] The mechanism by which FOXO4 favors senescence and restricts apoptosis involves its physical association with the p53 tumor suppressor protein. p53 is 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 its interacting 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 favors apoptosis over cell cycle arrest (Bulavin, DV et al., EMBO J., vol. 18, pp. 6845-6854 (1999)). However, Ser46 is also phosphorylated in response to multiple 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-damaging conditions, p53 Ser46 phosphorylation 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 suppressing the apoptotic function of p53 signaling, favoring senescence. Inhibition of FOXO4, particularly its interaction with p53, would switch senescent cells to apoptosis.

[0212] The human FOXO4 protein has two variants (SEQ ID NOs: 1 and 2). In some embodiments, any fragment of the FOXO4 protein may be used as a basis for designing a FOXO4 DRI peptide. In one embodiment, the FOXO4 fragment comprises at least a portion of a functional domain of the FOXO4 protein, such as the DNA-binding domain (SEQ ID NO: 3) or the p53-interacting domain (SEQ ID NO: 4).

[0213] Any FOXO4 DRI peptide that can inhibit the function of FOXO4 and / or interfere with its interaction with p53 can be used as a conjugate for the 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 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 substituted 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 ability to repress p53 and inducing apoptosis in senescent cells.

[0214] FOXO4 is itself regulated by other proteins. Referring to Figure 8, FOXO family members, including FOXO4, are activated by other proteins through phosphorylation or methylation, including AMPK, JNK, MST1, CK1, STAT3, and p38 through phosphorylation, and 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 serve as the basis for designing DRI peptides useful in 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] Take the JNK protein as an example. JNK is a c-Jun N-terminal kinase that can phosphorylate and activate FOXO4. Human JNK has the amino acid sequence of SEQ ID NO:8. DRI peptides based on JNK protein can allosterically and selectively modulate JNK by blocking access to substrates 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] In addition, activators of AMPK, JNK, MST1, CK1, STAT3, p38 and PRMT1 can also be used as natural proteins for designing the DRI peptide. For example, ASK1 is an apoptosis signal-regulating kinase 1 that activates JNK. Human ASK1 has GenBank accession number No. NP_005914. ASK1 protein can also be a natural protein for designing DRI peptides. Such DRI peptides can inhibit ASK1, thereby suppressing the activity of JNK and leading to the inhibition of FOXO4.

[0217] In some embodiments, the natural 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 natural 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 different molecular species, which means that DRI peptides designed based on orthologs can function in different 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 of senescent cells in humans.

[0218] In one embodiment, a fragment of a natural protein is used to design the DRI peptide. In another embodiment, the full length of a natural protein is used to design the DRI peptide. 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 of the natural 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 the exact reverse 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 to the reverse sequence of the fragment or full-length native protein.

[0220] The DRI peptide may be a small peptide, for example to allow 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, 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, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 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 peptides consist of all D amino acid residues, although some functional DRI peptides may contain a combination of L and D amino acid residues. In some embodiments, the DRI peptides have at most 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% amino acid residues that are L amino acid residues.

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

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

[0224] A functional domain within the DRI peptide may not itself have any apoptosis-inducing activity, but may instead function to enhance the apoptosis-inducing activity of another portion of the DRI peptide. The functional domain may comprise 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 peptides according to the invention have apoptosis-inducing activity in senescent cells if they kill, purge, remove, inactivate, or reduce the viability of senescent cells. In some embodiments, the DRI peptides can kill, purge, remove, inactivate, or reduce the viability of at least 5, 10, 15, 20, 25, 30, 40, 50, 60, 70, 80, 90, or 95% of the cells in a senescent cell culture.

[0226] In some embodiments, the DRI peptides selectively exhibit apoptosis-inducing activity in senescent cells, i.e., have little or no apoptosis-inducing activity in non-senescent cells, and may induce apoptosis in senescent cells relative to apoptosis in non-senescent cells by 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 assess whether the DRI peptides of the present invention exhibit apoptosis-inducing activity in senescent cells using standard in vitro tests. For example, cell cultures of senescent cells can be obtained by subjecting the cell cultures to ionizing radiation or chemotherapeutic agents and then mixing them with non-senescent cells. Other methods for providing senescent cells include (i) serial passage until repeated senescence occurs (= telomere shortening), (ii) the use of oxidative stressors such as H2O2 and rotenone, (iii) chromatin remodelers such as sodium dibutyrate, or (iv) the expression of hyperactivated oncogenes such as RASG12V or BRAFV600E. The presence of senescent cells can be determined by testing for SA-B-GAL.

[0228] The second step involves administering a peptide according to the present invention to a cell culture and measuring one or more markers of apoptosis, such as (i) cytoplasmic cytochrome c staining or (ii) TUNEL staining. Cytochrome c data can be quantified by counting the number of cells in which cytochrome c has been released from mitochondria into the cytosol (cells can be indicated using DAPI) or (at later stages) the number of cells in which it has completely disappeared. Because 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) are unable to actually die. An advantage of this assay is that it allows for the acquisition of cumulative senescence counts over several days (e.g., 5 days). For TUNEL staining, the percentage of nuclei staining positively with TUNEL (DAPI positive) is counted. This can be easily performed by naked eye, or a software tool called Cellprofiler (freeware) can be utilized.

[0229] In some embodiments, the conditionally active protein is covalently linked to a peptide linker, and the peptide linker also includes 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 linked peptide linker, the drug is not in an active form. The peptide linker can be cleaved by a protease in the extracellular environment of senescent cells, thus releasing the covalently linked drug from the active form of the conditionally active protein.

[0230] The peptide linker between the drug and the conditionally active protein may contain the same cleavage site used in the probodies described herein (e.g., the cleavage sites of SEQ ID NOs:33-37). The same proteases in the extracellular environment of senescent cells that can release the antibodies in the probodies will cleave the peptide linker, liberating 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 legumain. Such peptide linkers include a cleavage site for legumain. Some exemplary cleavage 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); 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); and NN (SEQ ID NO:67).

[0232] The drug covalently attached to the peptide linker in the prodrug may be a cytotoxic, cytostatic or antiproliferative drug. Alkaloids: docetaxel, etoposide, irinotecan, paclitaxel, teniposide, tepotecan, vinblastine, vincristine, vindesine, Alkylating agents: busulfan, improsulfan, piposulfan, benzodepa, carboquone, meturedepa, uredepa, altretamine, triethylenemelamine, triethylenephosphoramide, triethylenethiophosphoramide, chlorambucil, chloranaphazine, cyclophosphamide, estramustine, ifosfamide, mechlorethamine, mechlorethamine oxide hydrochloride, melphalan, novemebichin, perfosfamide, phenesterine, prednimustine, trofosfamide, uracil mustard, carmustine, chlorozotocin, fotemustine, lomustine, nimustine, semustine, ranimustine, dacarbazine, manomustine, mitobronitol, mitolactol, pipobroman, temozolomide Antibiotics and their analogues: aclacinomycin, actinomycin, anthramycin, azaserine, bleomycin, cactinomycin, carubicin, carzinophilin, chromomycin, dactinomycin, daunorubicin, 6-diazo-5-oxo-L-norleucine, doxorubicin, epirubicin, idarubicin, menogaril, mitomycin, mycophenolic acid, nogalamycin, olivomycin, peplomycin, pirarubicin, plicamycin, porfiromycin, puromycin, streptonigrin, streptozocin, tubercidin, zinostatin, zorubicin, Antimetabolites: denopterin, edatrexate, methotrexate, piritrexim, pteropterin, tomudex, trimetrexate, cladridine, fludarabine, 6-mercaptopurine, pentostatin, thiamiprine, thioguanine, ancitabine, azacitidine, 6-azauridine, carmoftir, cytarabine, doxifluridine, emitefur, floxuridine, fluorouracil, gemcitabine, tegafur, Platinum complexes: caroplatin, cisplatin, miboplatin, oxaliplatin, Others: aceglatone, amsacrine, bisantrene, dephosfamide, demecolcine, diaziquone, eflornithine, elliptinium acetate, etoglucide, etopside, fenretinide, gallium nitrate, hydroxyurea, lonidamine, miltefosine, mitoguazone, mitoxantrone, mopidamol, nitracrine, pentostatin, phenamet, podophyllic acid, 2-ethylhydrazide, procarbazine, razoxane, sobuzoxane, spirogermanium, teniposide, tenuazonic acid, triaziquone, 2,2',2''-trichlorotriethylamine, urethane This is embodied by:

[0233] The drug covalently attached to the peptide linker in the prodrug may be a chemotherapeutic agent. Chemotherapeutic agents can inhibit senescent cells in different ways. Chemotherapeutic agents can damage DNA templates by alkylation, cross-linking, or double-strand breaks in DNA. Other chemotherapeutic agents can 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 various drug groups, including but not limited to alkylating agents, antimetabolites, antitumor antibiotics, vinca alkaloids, epipodophyllotoxins, nitrosoureas, hormones and antihormones, and toxins. Some examples are as follows: Examples of alkylating agents include cyclophosphamide, chlorambucil, busulfan, melphalan, thiotepa, ifosfamide, and nitrogen mustard. Examples of antimetabolites include methotrexate, 5-fluorouracil, cytosine arabinoside, 6-thioguanine, and 6-mercaptopurine. Examples of antitumor antibiotics include doxorubicin, daunorubicin, idrubicin, nimitoxantron, dactinomycin, bleomycin, mitomycin, and plicamycin. Examples of vinca alkaloids and epipodophyllotoxins include vincristine, vinblastine, vindestin, etoposide, and teniposide. Examples of nitrosoureas include carmustine, lomustine, semustine, and streptozocin. Examples of hormones and antihormones include adrenocorticorticoids, estrogens, antiestrogens, progestins, aromatase inhibitors, androgens, and antiandrogens. Random synthetic drugs include dacarbazine, hexamethylmelamine, hydroxyurea, mitotane, procarbazide, cisplatin, and carboplatin.

[0234] In another aspect, the present invention provides conditionally active molecules or conditionally active pharmaceutical agents (CAMs) that are more active under abnormal physiological conditions than under normal physiological conditions. The conditionally active molecules are organic compounds and / or salts thereof derived from a parent organic compound having a molecular weight of less than about 3000 amu. The parent organic compound can be a therapeutically active compound having a molecular weight within the range of about 100 amu to about 1500 amu, or about 150 amu to about 1250 amu, or about 300 amu to about 1100 amu, or about 400 amu to about 1000 amu.

[0235] The parent organic compound may be selected from the group of drugs consisting of anti-cancer agents, anti-bacterial agents, immunomodulatory agents, anti-obesity agents, anti-diabetic agents, anti-fungal agents, anti-viral agents, contraceptives, analgesics, anti-inflammatory agents (e.g., steroidal or non-steroidal anti-inflammatory drugs (NSAIDs)), anti-emetics, vasorelaxants, 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, anidulafungin, or voriconazole; antiviral agents such as acyclovir or ganciclovir; thiothixene or midazoline. anti-inflammatory agents such as hydrocortisone, methylprednisolone, indomethacin, ketoprofen or parecoxib; immunomodulators such as methotrexate; antiemetics such as aprepitant, dolasetron, fosaprepitant, granisetron, ondansetron, metoclopramide, hycosine or promethazine; cardiovascular agents such as atenolol, dobutamine or epoprostenol; anesthetics such as methohexital; and pharmaceutically acceptable salts or combinations thereof.

[0236] In some embodiments, the present invention provides a method for generating conditionally active molecules from parent organic compounds, comprising the steps of 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 compounds a conditionally active molecule that exhibits higher activity under abnormal conditions compared to under normal physiological conditions.

[0237] The modification of a parent organic compound may be achieved by replacing one or more uncharged and / or partially charged groups on the parent organic compound with one or more partially charged or charged groups, or by adding one or more partially charged or charged groups. The addition of one or more partially charged or charged groups to a parent organic compound may be achieved by replacing one or more atoms, such as hydrogen atoms, or neutral groups on the parent organic compound with one or more partially charged or charged groups. The partially charged or charged groups may be positively or negatively charged. Examples of suitable charged groups include -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 produced and modified organic compounds are subjected to assays under normal physiological conditions and abnormal conditions. In some embodiments, the abnormal conditions are values ​​of the extracellular conditions of senescent cells, such as a pH 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 values ​​different from the conditions in the extracellular environment of normal cells, such as a pH 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. (a) 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, and an increase in the activity of the conditionally active protein in the assay under abnormal conditions, 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 the normal physiological 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 the abnormal conditions; The assay solutions used in the assays under abnormal conditions and under normal physiological conditions may contain small molecules and / or molecular species as discussed above.

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

[0242] In certain embodiments, the conditionally active molecule has a ratio of activity under abnormal conditions to activity under normal physiological conditions of greater than 1.0 (eg, greater selectivity between the two conditions). The ratio of activity 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 engineered as described in WO2016 / 138071. The conditionally active protein may be engineered through antibody conjugation, engineered to generate multispecific antibodies, engineered to generate bispecific conditionally active antibodies against immune effector cell surface antigens, engineered to generate masked conditionally active proteins, and / or engineered antibody Fc regions, each of which is described in WO2016 / 138071. The conditionally active protein may be used to engineer conditionally active viral particles as described in WO2015 / 175375.

[0244] T cells are used by mammalian immune systems to fight against substances or cells bearing foreign antigens. CAR-T technology utilizes genetic engineering to reprogram natural circulating T cells by inserting a chimeric antigen receptor (CAR) into T cells to generate highly specific CAR-T cells, where the CAR specifically binds to antigens on the surface of the target tissue, thereby directing the engineered CAR-T cells to the target tissue. Thus, the CAR-T cells can specifically target tumor cells, making them much more effective than natural circulating T cells. The CAR-T cells may also be engineered to target senescent cells.

[0245] The CARs of the present invention comprise at least one antigen-specific targeting region (ASTR), an extracellular spacer domain (ESD), a transmembrane domain (TM), one or more costimulatory domains (CSDs), and an intracellular signaling domain (ISD). See FIG. 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 explore the antigen-binding properties of antibodies to reprogram CAR-T cell specificity and reactivity toward targets selected in a non-MHC-restricted manner. Non-MHC-restricted antigen recognition confers the CAR-T cell 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 dual specificity and specifically binds to two different antigens or epitopes. The conditionally active protein of the present invention can be engineered into 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 the side effects caused by T cell attack on normal tissues. This allows a higher dose of T cells to be used to increase therapeutic efficacy, improving the subject's tolerance to treatment.

[0246] ASTRs may include conditionally active proteins such as antibodies, particularly single-chain antibodies, or fragments thereof, that specifically bind to antigens on senescent cells. Some examples of proteins suitable for ASTRs include linked cytokines (which direct recognition of cells bearing cytokine receptors), affibodies, ligand-binding domains from naturally occurring receptors, and soluble protein / peptide ligands for receptors on senescent cells.

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

[0248] In yet another embodiment, the ASTR comprises a diabody. In a diabody, the scFv is created with a linker peptide that is too short to allow the two variable regions to fold 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 also be used in ASTRs.

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

[0250] In some embodiments, the extracellular spacer domain and transmembrane domain may be resistant to ubiquitylation, enhancing CAR-T cell signaling and thus 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 external to the CAR-T cell and thus exposed to different conditions, potentially generating conditional ubiquitylation resistance.

[0251] The conditionally active proteins of the invention may be comprised in a pharmaceutical composition, device, kit or article of manufacture 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-associated diseases and disorders, such as age-related diseases and disorders, in a subject in need thereof. Examples of senescent cell-associated diseases, disorders, or conditions that can be treated by administering the conditionally active proteins or pharmaceutical compositions described herein include cognitive diseases (e.g., mild cognitive impairment (MCI), Alzheimer's disease and other dementias; Huntington's disease); cardiovascular diseases (e.g., atherosclerosis, 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, cardiac fibrosis); 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; pulmonary diseases (e.g., idiopathic pulmonary fibrosis, chronic obstructive pulmonary disease (COPD), pulmonary fibrosis, and the like). inflammatory / autoimmune diseases and disorders (e.g., osteoarthritis, eczema, psoriasis, osteoporosis, mucositis, transplant-related diseases and disorders); ophthalmologic diseases or disorders (e.g., age-related macular degeneration, cataracts, glaucoma, vision loss, presbyopia); diabetic ulcers; metastasis; chemotherapy side effects, radiation therapy side effects; age-related diseases and disorders (e.g., kyphosis, renal failure or dysfunction, frailty, hair loss, hearing loss, muscle wasting, skin diseases, sarcopenia, and herniated discs) and other age-related diseases induced by aging (e.g., diseases / disorders resulting from radiation, chemical exposure, smoking, eating a high-fat / high-sugar diet, and environmental factors); wound healing: skin nevi; and fibrotic diseases and disorders (e.g., cystic fibrosis, renal fibrosis, liver fibrosis, pulmonary fibrosis, oral submucous fibrosis, cardiac fibrosis, and pancreatic fibrosis).

[0253] In more specific embodiments, methods are provided for treating a senescent cell-associated disease or disorder by administering the conditionally active protein or pharmaceutical composition to kill or remove senescent cells (i.e., confirmed senescent cells) associated with the disease or disorder in a subject having the disease or disorder. In certain exemplary embodiments, the present invention is used to treat osteoarthritis; idiopathic pulmonary fibrosis; chronic obstructive pulmonary disease (COPD); or atherosclerosis.

[0254] Subjects (i.e., patients, individuals (human or non-human animals)) who may benefit from the use of the methods described herein, including administering the conditionally active protein or pharmaceutical composition, also include subjects who may have cancer. Subjects treated by these methods may be considered in partial or complete remission (also referred to as cancer remission). As discussed in detail herein, the conditionally active protein or pharmaceutical composition for use in the methods for selectively killing or removing senescent cells is not intended to be used as a cancer treatment, i.e., in a manner that kills or destroys cancer cells in a statistically significant manner. Therefore, the methods disclosed herein do not encompass the use of the conditionally active protein or pharmaceutical composition in a manner that would be considered a primary therapy for the treatment of cancer. Even if the conditionally active protein, alone or with other chemotherapeutic or radiotherapeutic agents, is not used in a manner sufficient to be considered a primary 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 (ie, the subject has not been diagnosed as having cancer by one of ordinary skill in the medical arts).

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

[0256] In certain embodiments, methods are provided for treating senescent cell-associated cardiovascular diseases associated with or induced by arteriosclerosis (i.e., hardening of arteries). The cardiovascular diseases may be any one or more of atherosclerosis (e.g., coronary artery disease (CAD) and carotid artery disease); angina pectoris, congestive heart failure, and peripheral vascular disease (e.g., peripheral arterial disease (PAD)). Methods for treating cardiovascular diseases associated with or induced by arteriosclerosis may reduce the likelihood of high blood pressure / hypertension, angina pectoris, stroke, and heart attack (i.e., coronary thrombosis, myocardial infarction (MI)). In certain embodiments, methods are provided for stabilizing atherosclerotic plaque(s) in a subject's blood vessel (e.g., artery), thereby reducing the likelihood of or delaying the occurrence of a thrombotic event such as stroke or MI. In certain embodiments, these methods involving administration of a conditionally active protein reduce the lipid content (i.e., induce a reduction in lipid content) and / or increase the thickness of the fibrous cap (i.e., induce an increase in the thickness of the fibrous cap, promote or facilitate thickening) of atherosclerotic plaques in a subject's blood vessel (e.g., artery).

[0257] In one embodiment, a method is provided for inhibiting the formation of atherosclerotic plaques (or reducing, eliminating, inducing a decrease in the formation of atherosclerotic plaques) 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 plaque. A reduction in the amount of plaque in a blood vessel (e.g., an artery) may be determined, for example, by a reduction in the surface area of ​​the plaque or a reduction in the extent or degree (e.g., percent) of embolization of the blood vessel (e.g., an artery), and can be measured by angiography or other visualization methods used in the cardiovascular artery field. Also provided herein is a method for increasing the stability (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 a subject.

[0258] The effectiveness of the conditionally active protein or pharmaceutical composition for treating or preventing (i.e., reducing or lowering the likelihood of onset or occurrence of) cardiovascular disease (e.g., atherosclerosis) can be readily determined by those skilled in the medical and clinical arts. One or any combination of diagnostic methods, including physical examination, assessment and monitoring of clinical symptoms, and performance of analytical tests and methods described herein and practiced in the art (e.g., angiography, electrocardiography, stress tests, non-stress tests), can be used to monitor the health status of a 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 suffering from or at risk of cardiovascular disease who have received the treatment with those of patients who have not received such treatment or who have received a placebo.

[0259] Inflammatory and autoimmune diseases and disorders In certain embodiments, the senescent cell-associated disease or disorder is an inflammatory disease or disorder, such as, but not limited to, osteoarthritis, which can be treated or prevented (i.e., the likelihood of occurrence is reduced) according to the methods described herein, including 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 disc, and the lung diseases COPD and idiopathic pulmonary fibrosis.

[0260] Unexpectedly, by selectively killing senescent cells, the conditionally active proteins or pharmaceutical compositions reduce the likelihood of occurrence, reduce or inhibit loss or erosion of the proteoglycan layer in joints, reduce inflammation within affected joints, and promote (i.e., stimulate, enhance, induce) collagen (e.g., type 2 collagen) production. Removal of senescent cells can induce a reduction in the amount (i.e., level) of inflammatory cytokines, such as IL-6, produced within the joint, reducing inflammation. Provided herein are methods for treating osteoarthritis by selectively killing or removing senescent cells, possibly present in the subject's osteoarthritic joints, and / or inducing collagen (such as type 2 collagen) production in the subject's joints in need thereof, by administering at least one conditionally active protein to the subject. The conditionally active protein may also be used to reduce (inhibit, reduce) the production of metalloproteinase 13 (MMP-13), which degrades collagen in joints, and restore the proteoglycan layer, or inhibit the loss and / or degradation of the proteoglycan layer. Treatment with the conditionally active protein or pharmaceutical composition can thereby prevent or reduce the likelihood of bone erosion, inhibit or reduce the erosion, or slow the erosion. As described in detail herein, in certain embodiments, the conditionally active protein or pharmaceutical composition is directly administered to osteoarthritic joints (e.g., by intraarticular, topical, transdermal, intradermal, or subcutaneous delivery). Treatment with the conditionally active protein or pharmaceutical composition can restore or improve joint strength, or inhibit the deterioration of strength. In addition, a method comprising administering the conditionally active protein or pharmaceutical composition can reduce joint pain, and is therefore useful for pain management in osteoarthritic joints.

[0261] The effectiveness of one or more conditionally active proteins for treating or preventing osteoarthritis in a subject and for monitoring a subject receiving one or more senolytic drugs can be readily determined by those skilled in the medical and clinical arts. One or any combination of diagnostic methods, including physical examination (e.g., measuring the flexibility, swelling, or redness of the affected joint), assessing and monitoring clinical symptoms (e.g., pain, stiffness, mobility), and performing analytical tests and methods described herein and practiced in the art (e.g., measuring inflammatory cytokine or chemokine levels; X-ray imaging to measure cartilage loss indicated by narrowing of the space between bones in the joint; and magnetic resonance imaging (MRI), which provides detailed images of bones and soft tissues, including cartilage), may be used to monitor the subject's health. The effectiveness of treatment with one or more senolytic drugs can be analyzed by comparing the symptoms of patients suffering from or at risk of an inflammatory disease or disorder, such as osteoarthritis, who have received the treatment with those of patients who have not received such treatment or who have received a placebo treatment.

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

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

[0264] In yet another embodiment, inflammatory / autoimmune disorders that can be treated or prevented (i.e., the 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 the diseases are performed according to methods and diagnostic tests routinely practiced in the art, including blood tests, colonoscopy, flexible sigmoidoscopy, barium enema, CT scan, MRI, endoscopy, and small bowel enema.

[0265] In other embodiments, the methods described herein can be useful for treating subjects with herniated discs. These herniated disc subjects show increased prevalence of cellular senescence in the blood and blood vessel walls (see, for example, 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 aging degenerated disc tissue, suggesting a role for senescent cells (see, for example, Chang-Qing et al. Ageing Res. Rev., vol. 6, pp. 247-61, 2007). Animal models may be used to characterize the effectiveness of senolytic agents in treating herniated discs; disc degeneration is induced in mice by compression and pelvic strength assessment (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., the likelihood of occurrence is reduced) by using the conditionally active protein or pharmaceutical composition include eczema, psoriasis, osteoporosis, and pulmonary diseases (e.g., chronic obstructive pulmonary disease (COPD), idiopathic pulmonary fibrosis (IPF), asthma), inflammatory bowel disease, and mucositis (such as radiation-induced oral mucositis in some instances). Specific fibrotic or fibrotic conditions of organs, such as renal fibrosis, liver fibrosis, pancreatic fibrosis, cardiac fibrosis, skin wound healing, and oral submucosal fibrosis, may also be treated using the conditionally active protein or pharmaceutical composition.

[0267] In certain embodiments, the senescent cell-associated disorder is an inflammatory skin disorder, such as, but not limited to, psoriasis and eczema, which can be treated or prevented (i.e., the likelihood of occurrence is reduced) by the methods described herein, including administering the conditionally active protein or pharmaceutical composition. The effectiveness of the conditionally active protein or pharmaceutical composition for treating psoriasis and eczema, as well as monitoring subjects receiving such treatment, can be readily measured by 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 performing analytical tests and methods described herein and practiced in the art (e.g., measuring levels of pro-inflammatory cytokines).

[0268] Pulmonary Diseases and Disorders In one embodiment, a method is provided for treating or preventing (i.e., reducing the likelihood of occurrence of) a senescent cell-associated disease or disorder by administering the conditionally active protein or pharmaceutical composition to a subject with a pulmonary disease or disorder, thereby killing or eliminating senescent cells (i.e., confirmed senescent cells) associated with the disease or disorder. Senescence-associated pulmonary 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 observation that the incidence of the disease increases with age and that lung tissue from IPF patients contains abundant SA-P-Gal-positive cells and high levels of the senescence marker p21 (see, e.g., Minagawa et al., Am. J. Physiol. Lung Cell. Mol. Physiol., vol. 300, pp. L391-L401, 2011). Short telomeres are a common risk factor for both IPF and cellular senescence (see, e.g., Alder et al., Proc. Natl. Acad. Sci. USA, vol. 105, pp. 13051-56, 2008). Without wishing to be bound by theory, reports suggest that cellular senescence contributes to IPF by promoting the differentiation of fibroblasts into myofibroblasts and the epithelial-to-mesenchymal transition, resulting in extensive remodeling of the extracellular matrix in the alveolar and interstitial spaces (see, e.g., Minagawa et al., supra).

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

[0270] The methods described herein for treating or preventing (i.e., reducing the likelihood of occurrence of) aging-related lung diseases or disorders may be used to treat aging subjects and 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. Administration of a senolytic drug to an aging subject (including asymptomatic middle-aged adults) may kill and remove senescent cells from the respiratory tract, slowing or preventing the decline in lung function. The effects of treatment with the conditionally active protein or pharmaceutical composition may be analyzed using techniques known in the art, such as comparing the symptoms of treated patients with or at risk of a lung disease to those of patients who have not received such treatment or who have received a placebo. In addition, methods and techniques for assessing lung mechanical function, such as measuring lung volume, elastance, and airway hyperresponsiveness, may be performed. To measure and monitor pulmonary function throughout treatment, numerous measurements may be obtained: expiratory reserve volume (ERV), forced vital capacity (FVC), FEV to standard vital capacity (FEV) (e.g., FEV in 1 second, FEV1), FEV1 / FEV ratio, forced expiratory flow 25%-75%, and any one of maximal ventilation (MVV), peak expiratory flow (PEF), and stable vital capacity (SVC). Overall lung volumes include total lung capacity (TLC), vital capacity (VC), residual volume (RV), and functional residual capacity (FRC). Gas exchange across the alveolar-capillary membrane may be measured using the diffusing capacity for carbon monoxide (DLCO). Peripheral capillary oxygen saturation (SpO2) may also be measured.

[0271] Neurological Diseases and Disorders Senescent cell-associated diseases or disorders that can be treated by administering the conditionally active protein or pharmaceutical composition include neurological diseases or disorders.Such senescent cell-associated 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 aging are glaucoma, vision loss, presbyopia, and cataracts.

[0272] Aging of dopamine-producing neurons is thought to contribute to the cell death observed in PD through the generation of reactive oxygen species (see, e.g., 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 treating and preventing Parkinson's disease.

[0273] Methods for detecting, monitoring, or quantifying neurodegenerative deficits and / or locomotor deficits associated with Parkinson's disease are known in the art, such as histological tests, biochemical tests, and behavioral assessments (see, e.g., US2012 / 0005765). Symptoms of Parkinson's disease are known in the art and include, but are not limited to, difficulty initiating or ending voluntary movements, convulsions, rigid movements, muscle atrophy, tremors, changes in heart rate but normal reflexes, bradykinesia, and postural instability.

[0274] The effectiveness of the conditional proteins or pharmaceutical compositions described herein in subjects receiving one or more senolytic drugs can be readily determined by those skilled in the medical and clinical fields. One or any combination of diagnostic methods, including physical examination, assessment and monitoring of clinical symptoms, and performing the analytical tests and methods described herein, can be used to monitor the subject's health status. The effect of administering the conditionally active proteins or pharmaceutical compositions can be analyzed using techniques known in the art, such as comparing the symptoms of treated patients suffering from or at risk of Alzheimer's disease with those of patients not receiving such treatment or treated with a placebo.

[0275] Mild cognitive impairment (MCI) MCI is a brain function syndrome involving the onset and development of cognitive impairment beyond that expected based on an individual's age and education, but 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 deficits associated with MCI are known in the art (see, e.g., US 2012 / 0071468), such as morphological analysis of astrocytes, acetylcholine release, silver staining to assess neurodegeneration, and PiB PET imaging to detect beta-amyloid deposits. Methods for detecting, monitoring, quantifying, or assessing behavioral abnormalities associated with MCI are also known in the art (see, e.g., US 2012 / 0071468). Methods for detecting, monitoring, quantifying, or assessing behavioral abnormalities associated with MCI are also known in the art (see, e.g., US 2012 / 0071468), such as the eight-arm radial maze paradigm, the non-matching-to-sample task, the allocentric place determination task in a water maze, the Morris maze test, the visuospatial task, and the delayed-response spatial memory task, and the olfactory novelty test (ibid.).

[0276] Motor neuron dysfunction (MND) MNDs are a group of progressive neurological disorders that destroy motor neurons, the 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, SMA2, SMA3, also known as Kugelberg-Welander disease, and Kennedy disease), post-polio syndrome, and hereditary spastic paraplegia. Administration of the conditionally active protein can reduce or inhibit MNDs by killing or removing senescent cells. Methods for detecting, monitoring, or quantifying locomotor deficits and / or other deficiencies associated with Parkinson's disease, such as MNDs, are known in the art (see, e.g., US20120005765). Methods for detecting, monitoring, quantifying, or assessing motor and histopathological deficits associated with MND, including histopathological, biochemical, and electrophysiological testing and motor activity analysis, are known in the art (see, e.g., 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] Ophthalmological Diseases and Disorders In certain embodiments, the senescent cell-associated disease or disorder is an ocular disease, disorder, or condition, such as presbyopia, macular degeneration, or cataract. In other specific embodiments, the senescent cell-associated disease or disorder is glaucoma. Macular degeneration is a neurodegenerative disease that causes 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, e.g., 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). The reduction of microRNAs contributes to senescent cell profiles; DICER1 ablation induces premature aging. Diagnosis and monitoring of subjects with macular degeneration can be achieved by those skilled in the art of ophthalmology by following art-accepted procedures for regular eye examinations and subject reporting of symptoms.

[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 due, at least in part, to changes in the tissue composition.

[0279] Research suggests that type IV collagen influences cellular function by inferring the placement of the basement membrane beneath the epithelial layer, and data support a role for type IV collagen in tissue stabilization. Posterior capsule opacification (PCO) occurs as a complication in approximately 20–40% of patients within several years of cataract surgery (see, e.g., Awasthi et al., Arch Ophthalmol., vol. 127, pp. 555–62, 2009). PCO results from the proliferation and activity of remaining 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). Adhesion of type IV collagen, fibronectin, and laminin to intraocular lenses can inhibit cell migration and reduce the risk of PCO (see, e.g., Raj et al., Int. J. Biomed. Sci., vol. 3, pp. 237-50, 2007).

[0280] Without wishing to be bound by any particular theory, selective killing or removal of the conditionally active proteins described herein may slow or interfere (delay, inhibit, or prevent) the breakdown of type IV collagen network. Removal of senescent cells and the resulting inflammatory effects of SASP may reduce or inhibit epithelial cell migration, delaying (suppressing) the onset of presbyopia or slowing or slowing the progression of disease severity (such as slowing the progression from mild to moderate or from moderate to severe). The conditionally active proteins and pharmaceutical compositions described herein may also be useful in reducing the likelihood of PCO after cataract surgery.

[0281] BubR1 hypomorphic 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 examination 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 a subject at risk of developing presbyopia, cataracts, or macular degeneration. Treatment with the conditionally active protein may begin when the human subject is at least 40 years old to delay or inhibit the onset or development of cataracts, presbyopia, and macular degeneration. Because nearly all humans will develop presbyopia, in certain embodiments, a senolytic drug may be administered to a human subject after the subject reaches 40 years old in the manner described herein to delay or inhibit the onset or development of presbyopia.

[0283] In certain embodiments, the aging-related disease or disorder is glaucoma.Glaucoma is a broad term used to describe a group of diseases that often do not have other major symptoms and cause visual field loss.When the cellular network that is required for fluid outflow is subjected to SA-P-Gal staining, a four-fold increase in aging has been observed in glaucoma patients (see, for example, Liton et al., Exp. Gerontol., vol. 40, pp. 745-748, 2005).

[0284] To monitor the effect of treatment on inhibiting the progression of glaucoma, standard automated perimetry (visual field testing) is the most widely used technique. In addition, several algorithms for progression detection have been developed (see, for example, Wesselink et al., Arch Ophthalmol., vol. 127, pp. 270-274, 2009, and references therein). Additional methods include gonioscopy (examining the trabecular meshwork and angle where fluid drains from the eye); imaging techniques (e.g., scanning laser tomography (e.g., HRT3), laser polarimetry (e.g., GDX), optical coherence tomography); ophthalmoscopy; and pachymetry, which measures the thickness of the central cornea.

[0285] metabolic diseases or disorders Senescence-related diseases or disorders that can be treated by administering the conditionally active protein or pharmaceutical composition include metabolic diseases or disorders. Such senescence-related diseases and disorders include diabetes, metabolic syndrome, diabetic ulcers, and obesity. The conditionally active protein described herein may be used to treat type 2 diabetes, particularly type 2 diabetes 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 from obese mice showed induction of senescence markers SA-P-Gal, p53, and p21 (see, e.g., Minamino et al., Nat. Med., vol. 15, pp. 1082-1087, 2009). Concurrent 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., supra). Because pro-inflammatory SASP components have also been suggested to contribute to type 2 diabetes (see, e.g., Tchkonia et al., supra), the induction of senescent cells in obesity has potential clinical implications. Similar patterns of upregulation of senescence markers and SASP components are associated with diabetes in both mice and humans (see, e.g., Minamino et al., supra). Thus, the methods described herein, which involve administering a senolytic agent, may be useful for treating or preventing obesity and metabolic syndrome, in addition to type 2 diabetes. Without wishing to be bound by theory, contacting senescent preadipocytes with a senolytic agent, thereby killing the senescent preadipocytes, may provide clinical and health benefits to individuals with any one of diabetes, obesity, or metabolic syndrome.

[0287] A disease or disorder associated with diabetes and aging is diabetic ulcers (i.e., diabetic wounds). Ulcers are breakdowns in the skin that can expand to involve subcutaneous tissue or even muscle or bone. These lesions occur particularly on the lower extremities. Patients with diabetic venous ulcers show an 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 pro-inflammatory cytokine phenotype of senescent cells plays a role in the pathogenesis.

[0288] The effectiveness of the conditionally active protein can be readily determined by those skilled in the medical and clinical arts. One or any combination of diagnostic methods, including physical examination, assessment and monitoring of clinical symptoms, and performing analytical tests and methods such as those described herein, can be used to monitor the health status of a subject. Subjects receiving one or more senolytic drugs described herein for the treatment or prevention of diabetes can be monitored, for example, by assaying glucose and insulin resistance, energy expenditure, body composition, adipose tissue, skeletal muscle, and liver inflammation, and / or lipid toxicity (muscle and liver lipids by in vivo imaging, and 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] The subject 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 cardiovascular disease, hepatic steatosis, hyperlipidemia, diabetes and insulin resistance.The subject with metabolic syndrome may exhibit a group of metabolic disorders or metabolic abnormalities, for example, may include one or more of hypertension, type 2 diabetes, hyperlipidemia, dyslipidemia (for example, hypertriglyceridemia, hypercholesterolemia), insulin resistance, hepatic steatosis (steatohepatitis), hypertension, atherosclerosis and other metabolic disorders.

[0290] skin diseases or disorders Senescent cell-associated diseases or disorders treatable by administering the conditionally active proteins or pharmaceutical compositions described herein include skin diseases or disorders. Such senescent cell-associated diseases and disorders include psoriasis and eczema, which are also inflammatory diseases and are discussed in more detail above. Other skin diseases and disorders associated with aging include fine lines (age wrinkles); psychogenic pruritus (associated with diabetes and aging); dysesthesia (a side effect of chemotherapy associated with diabetes and multiple sclerosis); psoriasis (as mentioned) and other papular-scaling disorders, such as erythroderma, lichen planus, and lichenoid dermatoses; atopic dermatitis (related to eczema forms and inflammation); and eczematous rash (commonly observed in elderly patients and associated with the side effects of certain medications). Other skin diseases and disorders related to aging include eosinophilic dermatosis (associated with certain types of blood cancer); reactive neutrophilic dermatosis (associated with underlying conditions such as inflammatory bowel syndrome); pemphigus (an autoimmune disease in which autoantibodies form against desmoglein); pemphigoid and other immunobullous dermatoses (autoimmune blistering of the skin); age-related fibrohistiocytic 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 manifestation of lupus erythematosus. Late-onset lupus may be associated with impaired (i.e., decline) function of T cells and B cells and cytokines associated with aging (immunosenescence).

[0291] metastasis In certain embodiments, the conditionally active protein or pharmaceutical composition can 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 can benefit from the administration of the conditionally active protein or pharmaceutical composition to inhibit metastasis. Such a conditionally active protein or pharmaceutical composition can 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 by PET scan, MRI, CAT scan, biopsy, etc. The effect of a therapeutic agent on tumor growth can also be evaluated by examining tumor cell differentiation.

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

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

[0294] In certain embodiments, when chemotherapy or radiation therapy is administered in treatment cycles of at least one day on-therapy (i.e., chemotherapy or radiation therapy) followed by at least one week off-therapy, the conditionally active protein or pharmaceutical composition is administered on one or more days during the time interval between on-therapy, beginning on or after day two of the time interval between on-therapy and ending on or before the last day of the time interval between on-therapy. In more specific embodiments, when chemotherapy or radiation therapy is administered in treatment cycles of at least one day on-therapy (i.e., chemotherapy or radiation therapy) followed by at least one week off-therapy, the conditionally active protein or pharmaceutical composition is administered on day one, which is day six of the time interval between on-therapy. In other specific embodiments, when chemotherapy or radiation therapy is administered in treatment cycles of at least one day on-therapy (i.e., chemotherapy or radiation therapy) followed by at least two weeks off-therapy, the conditionally active protein or pharmaceutical composition is administered beginning on day six of the time interval between on-therapy and ending at least one day or at least two days before the first day of the subsequent chemotherapy or radiation therapy treatment course.

[0295] In another embodiment for treating metastasis, the conditionally active protein or pharmaceutical composition may be administered after a chemotherapy or radiation therapy treatment regimen has been completed, hi certain embodiments, the conditionally active protein or pharmaceutical composition is administered one or more days within a 14-day treatment window (i.e., a senolytic treatment course) after chemotherapy or radiation therapy has been completed.

[0296] The method described herein is also useful for inhibiting, preventing or slowing the progression of metastatic cancer of any one of the tumor types described in the medical technology field.Cancer (tumor) types include: adrenocortical carcinoma, childhood adrenocortical carcinoma, AIDS-related cancer, anal cancer, appendix cancer, basal cell carcinoma, childhood basal cell carcinoma, bladder cancer, childhood bladder cancer, bone cancer, brain tumor, childhood astrocytoma, childhood brain stem glioma, childhood central nervous system atypical teratoid / rhabdomyosarcoma-like tumor, childhood central nervous system embryonal tumor, childhood central nervous system germ cell tumor, childhood craniopharyngioma brain tumor, childhood ependymoma brain tumor, breast cancer, Childhood bronchial tumors, carcinoid tumors, childhood carcinoid tumors, gastrointestinal carcinoid, cancer of unknown primary site, childhood cancer of unknown primary site, 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 trophoblastic tumor, glioma, head and neck cancer, childhood head and neck cancer, hepatocellular (liver) cancer, hypopharyngeal cancer, kidney cancer, renal cell kidney cancer, 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, liver cancer (primary), childhood liver cancer (primary), 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 (eye) melanoma, Merkel cell carcinoma, malignant mesothelioma, childhood malignant mesothelioma, occult primary metastatic squamous cell carcinoma of the neck, midline tract carcinoma related to the NUT genecarcinoma), oral cancer, childhood multiple endocrine neoplasia, mycosis fungoides, myelodysplastic syndrome, myelodysplastic neoplasm, myeloproliferative 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 (insulin cell tumor), childhood papillomatosis, paraganglioma, paranasal sinus cancer, parathyroid cancer, penile cancer, pharyngeal cancer, pheochromocytoma, pituitary tumor, plasma cell tumor, 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, Sezary 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, endometrial cancer of the uterus, vaginal cancer, vulvar cancer, Waldenstrom's macroglobulinemia.

[0297] Side effects of chemotherapy and radiation therapy In another embodiment, the senescent cell-associated disorder or disease is a side effect of chemotherapy or radiation therapy. Examples of chemotherapeutic agents that induce senescence in non-cancerous cells include anthracyclines (doxorubicin, daunorubicin, etc.); taxol (e.g., paclitaxel); gemcitabine; pomalidomide; and lenalidomide. One or more senolytic agents administered as described herein can be used to treat and / or prevent (i.e., reduce the likelihood of) chemotherapy or radiation therapy side effects. Removal or destruction of senescent cells can ameliorate acute toxicities, such as those involving energy imbalance in chemotherapy or radiation therapy. Acute toxic side effects include, but are not limited to, gastrointestinal toxicity (e.g., nausea, vomiting, constipation, loss of appetite, diarrhea), peripheral neuropathy, fatigue, lethargy, low physical activity, hematotoxicity (e.g., anemia), hepatotoxicity, alopecia (hair loss), pain, infection, mucositis, fluid retention, skin toxicity (e.g., rash, dermatitis, hyperpigmentation, hives, photosensitivity, nail changes), oral problems (e.g., oral mucositis), gum or throat problems, or any toxic side effects caused by chemotherapy or radiotherapy.For example, the toxic side effects caused by radiotherapy or chemotherapy (see, for example, the National Cancer Institute website) can be improved by the methods described herein. Thus, in certain embodiments, provided herein are methods for ameliorating (reducing, inhibiting, or preventing (i.e., reducing the likelihood of) the occurrence of) the acute toxicity of chemotherapy or radiation therapy, or both, or reducing the severity of its toxic side effects (i.e., adverse side effects) in a subject receiving treatment, comprising administering to the subject an agent that selectively kills, removes, or destroys, or facilitates the selective destruction of, senescent cells.

[0298] Administration of the conditionally active protein or pharmaceutical composition to treat, reduce the likelihood of occurrence, or reduce the severity of chemotherapy or radiotherapy side effects can be achieved by the same course of treatment as described above for the treatment / prevention of metastasis. As described for treating or preventing metastasis (i.e., reducing the likelihood of occurrence), the conditionally active protein or pharmaceutical composition is administered during a time interval between chemotherapy or radiotherapy breaks or after the chemotherapy or radiotherapy treatment regimen has been completed.

[0299] In more specific embodiments, the acute toxicity is an acute toxicity involving energy imbalance, which may include one or more of weight loss, endocrinological change(s) (e.g., hormone imbalance, changes in hormone signaling), and body composition change(s). In certain embodiments, the acute toxicity involving energy imbalance is associated with a decrease or reduction in the subject's ability to be physically active, as evidenced by lower or eliminated energy expenditure than observed in subjects who did not receive the medical therapy. As a non-limiting example, such acute toxic effects involving energy imbalance include reduced physical activity. In other specific embodiments, the energy imbalance includes fatigue or lethargy.

[0300] In one embodiment, the chemotherapy side effect 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 who is being treated with an anthracycline (e.g., doxorubicin, daunorubicin, etc.) can be treated with one or more senolytic drugs described herein that reduce, ameliorate, or diminish the cardiotoxicity of the anthracycline. As is well understood in the medical arts, the cardiotoxicity associated with anthracyclines limits the maximum lifetime dose a subject can receive, even if the cancer responds to the drug. Administration of one or more of the conditionally active proteins can reduce cardiotoxicity, allowing additional amounts of anthracycline to be administered to the subject, resulting in an improved prognosis associated with the cancer disease. In one embodiment, the cardiotoxicity results from the administration of an anthracycline, such as doxorubicin. Doxorubicin is an anthracycline topoisomerase inhibitor approved for treating patients with ovarian cancer after failure of platinum-based therapy; Kaposi's sarcoma after failure of or intolerance to primary systemic chemotherapy; or multiple myeloma in combination with bortezomib in patients who have not previously received bortezomib or who have received at least one prior therapy. Doxorubicin is administered to patients at a total lifetime dose of 550 mg / m 2 If exceeded, it can 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 can be used in the methods provided herein to ameliorate chronic or long-term side effects. Chronic toxic side effects typically result from repeated exposure to or administration of chemotherapy or radiation therapy over a long period of time. Certain toxic effects appear long after treatment (also known as delayed toxic effects) and result from damage to organs or systems caused by the treatment. Organ dysfunction (e.g., neurological, pulmonary, cardiovascular, and endocrine dysfunction) has been observed in patients treated for cancer in childhood (see, e.g., Hudson et al., JAMA, vol. 309, pp. 2371-81, 2013). Without wishing to be bound by any particular theory, destroying senescent cells, particularly certain normal cells induced into senescence by chemotherapy or radiation therapy, can reduce the likelihood of chronic side effects, reduce or eliminate the severity of chronic side effects, or delay the onset of chronic side effects. Chronic and / or late toxic side effects that occur in subjects receiving chemotherapy or radiation therapy include, but are not limited to, cardiomyopathy, congestive heart disease, inflammation, premature menopause, osteoporosis, infertility, impaired cognitive function, peripheral neuropathy, secondary cancers, 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 to kill or remove senescent cells in a subject with cancer, sensitivity to chemotherapy or radiotherapy can be increased in a clinically or statistically significant manner compared to when the conditionally active protein or pharmaceutical composition is not administered. In other words, the development of resistance to chemotherapy or radiotherapy can be inhibited when the conditionally active protein or pharmaceutical composition is administered to a subject treated with chemotherapy or radiotherapy.

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

[0304] Age-related diseases or conditions include, for example, renal dysfunction, kyphosis, herniated disc, weakness, hair loss, hearing loss, visual loss (blindness or visual impairment), muscle fatigue, skin disorders, skin nevi, diabetes, metabolic syndrome, and sarcopenia. Visual loss refers to the lack of vision that a 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, but are not limited to, treating one or more of the following skin conditions: wrinkles, such as superficial fine lines; hyperpigmentation; scars; keloids; dermatitis; psoriasis; eczema (such as seborrheic eczema); rosacea; vitiligo; ichthyosis vulgaris; dermatomyositis; and actinic keratosis.

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

[0306] The effectiveness of the conditionally active proteins or pharmaceutical compositions described herein for treating senescent cell-associated diseases or disorders can be readily determined by those skilled in the medical and clinical arts. One or any combination of diagnostic methods appropriate for a particular disease or disorder, including physical examination, patient self-assessment, evaluation and monitoring of clinical symptoms, laboratory tests, physical fitness tests, and analytical tests and procedures including exploratory surgery, known to those skilled in the art, may be used, for example, to monitor the subject's health status and the effectiveness of the senolytic agent. The effectiveness of the treatment methods described herein may be analyzed using techniques known in the art, such as comparing the symptoms of patients suffering from or at risk for a particular disease or disorder who 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 can include beneficial or desired clinical results, including, but not limited to, relief, reduction, or alleviation of symptoms resulting from or associated with the disease being treated; a reduction in the occurrence of symptoms; improved quality of life; a longer disease-free state (i.e., a reduced likelihood or tendency for a subject to exhibit symptoms based on a diagnosis of the disease); a reduction in the extent of the disease; a stable (i.e., non-progressing) state of the disease; a delay or slowing of disease progression; an improvement or alleviation of the disease state; and remission (whether partial or complete), whether detectable or undetectable; and / or overall survival. The efficacy of the conditionally active protein or pharmaceutical composition can also mean an increase in survival compared to the expected survival if the subject had not received the conditionally active protein or pharmaceutical composition.

[0308] A subject, patient, or individual in need of treatment with the conditionally active proteins or pharmaceutical compositions described herein may be a human who has developed symptoms of a senescent cell-associated disease or disorder or who is at risk of developing a senescent cell-associated disease or disorder, or may be a non-human primate or other animal (i.e., for 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, domestic animals (e.g., pigs, miniature pigs), horses, dogs, cats, cows, elephants, bears, and other domestic, livestock, and zoo animals.

[0309] Example Examples 1-9 for generating 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 antibodies (ratio of activity in the assay at pH 6.0 to activity in the assay at pH 7.4) was affected by the buffer used in the assay. Conditionally active antibodies evolved from wild-type mAb 048-02 showed significantly higher selectivity in Krebs buffer than in phosphate buffer (Figure 4).

[0313] Example 11: Conditional Activity Antibodies and Bicarbonate Selectivity

[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 components in Krebs buffer that most significantly contributed to the higher selectivity observed in Example 10. The selectivity of one conditionally active antibody was retested in buffers derived from Krebs buffers in which various components were subtracted, one component at a time (Figure 5, left group of bars). When complete Krebs buffer was used, the selectivity of the conditionally active antibody was high, with an activity ratio at pH 6.0 / 7.4 of approximately 8. When components A through F were individually subtracted from the Krebs buffer, the selectivity of the conditionally active antibody was not lost, but the conditionally active antibody became less selective when components C and D were individually 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 is at least partially responsible for the high selectivity of the conditionally active antibody in Krebs buffer.

[0315] The selectivity of the same conditionally active antibody was then 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 was 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) was measured at pH 7.4 in buffers containing different bicarbonate concentrations ranging from 0 to physiological bicarbonate concentrations (approximately 20 mM) (Figure 6). The binding activity of all three conditionally active antibodies at pH 7.4 was observed to decrease in a dose-dependent manner as the bicarbonate concentration increased from 0 to physiological concentrations (Figure 6). Meanwhile, the binding activity of the wild-type antibody was unaffected by bicarbonate. This study suggested that the selectivity of the conditionally active antibodies in the presence of bicarbonate may be due, at least in part, to the loss of binding activity of the conditionally active antibodies at pH 7.4 due to their interaction with bicarbonate.

[0318] Example 13: Induction of senescent cells

[0319] Cell seeding: In a 6-well plate, 1.0 x 10 cells were seeded for blank and treatment in 2 mL of medium per well. 5 Cells MDA-MB468 (P10), MDA-MB231 (Px), and 2.0 × 10 5 MCF-7 (Px) cells were seeded and cultured overnight. MCF-7 is an ERa+ cell line. Palbociclib has antiproliferative activity in this cell line, arresting cell proliferation and inducing senescent cells. MDA-MB231 is an ERa- cell line. Palbociclib has antiproliferative activity in this cell line, arresting 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 arrest growth or induce senescence.

[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 HO to generate a 87.15 mM solution of palbociclib as a stock solution. 2.3 μL of the stock solution was mixed with 198 μL of HO to generate a 1 mM palbociclib solution.

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

[0322] Detection of senescent cells by FAC (B-gal and antibody co-staining): 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 Zombie NIR live / dead dye was applied. 1. Wash the cells twice with PBS and detach the cells with Detachin™ cell detachment solution. 2. Stop the reaction of Detachin™ with DMEM and count the cells. 3. 2 mM CFDG in PBS (final 33 µM), antibody (5 µL, 1 x 10 6 Cells were stained with Zombie NIR dye (1:1000) for 1 hour on ice. 4. Wash the cells twice with PBS and fix them with 4% PFA for 10 minutes at room temperature. 5. Wash with PBS and collect FACs in 100 μL PBS. 6. Apply FITC-PE-APC / Cy7. 7. Co-stain the cells with the following antibodies to detect the expression of the corresponding antigens: a) PE anti-human CD54 clone HCD54, 200 μg / mL, isotype: Ms IgG1. Biolegend, Cat. No. 322707, Lot No. B232865, 5 μl / 10 6 cell b) PE anti-human CD73 clone AD2, isotype: Ms IgG1. Biolegend, Cat. No. 344004, Lot No. B216193, 5 μl / 10 6 cell c) PE anti-human CD261 (DR4, TRAIL-R1) clone DJR1, 200 μg / mL, isotype: MS IgG1. Biolegend, Cat. No. 307205, Lot No. B189821, 5 μl / 10 6 cell d) PE anti-human CD95 (Fas) clone DX2, 100 μg / mL, isotype: Ms IgG1. Biolegend, Cat. No. 305607, Lot No. B203942, 5 μl / 10 6 cell e) PE anti-human CD39 clone A1, 50 μg / mL, isotype: Ms IgG1. Biolegend, Catalog No. 328208, Lot No. B199643, 5 μl / 10 6 cell f) PE anti-human Nectin4, isotype: Ms IgG1. R&D systems, Catalog No. FAB2659P, Lot No. AAAAO0217031, 5 μl / 10 6 cell g) PE-isotype mouse anti-IgG1, k: clone MOPC-21, 0.2 mg / mL. Biolegend, Cat. No. 400112, Lot No. B220359, 5 μl / 10 6 cell.

[0323] Induced senescent cells were detected by FACS. Stained cells were washed with PBS and fixed with 4% paraformaldehyde (PFA) for 10 minutes at room temperature before FACS analysis. SA-B-gal (Senescence Associated B-Gal) staining using the CBA-230 kit from Cell Biolabs 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 the 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 (Figures 9A-9B). MCF-7 cells formed clusters with an extracellular environment suitable for senescent cells (Figure 9B). FACS analysis clearly demonstrated that palbociclib-treated cells (senescent cells) were different from untreated cells (non-senescent cells) (Figure 9C). The target profile of palbociclib-treated cells (senescent cells) was found to be different from that of untreated cells (non-senescent cells) (Figure 9D). Specifically, targets 1, 2, and 6 were more highly expressed in senescent cells, with target 2 showing the greatest increase in expression level.

[0326] Similarly, MDA-MB231 cells were also induced to become senescent cells in response to palbociclib treatment (Figures 10A-10B). MCF-MB231 cells also formed clusters with an extracellular environment (Figure 10B). FACS analysis clearly demonstrated that palbociclib-treated cells (senescent cells) were distinct from untreated cells (non-senescent cells) (Figure 10C). The target profile of palbociclib-treated cells (senescent cells) was found to be distinct 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 thus this treatment did not induce them to become senescent (Figures 11A-11B). FACS and target profile analysis did not reveal any 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 plated in a 6-well plate at 1 × 10 5 Cells were seeded at 1000 x g / well and cultured overnight. The cultured cells were then separated into two batches: one batch was treated with 1 µM palbociclib isethionate for 7 days, while the other batch was left untreated. Both batches were harvested by detaching the cells from the wells.

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

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

[0332] Untreated cells were mostly B-gal staining-negative, but they were separated by FACS sorting for CD73 activity (Figure 14A). B-gal-positive cells were present in significantly lower numbers, but they were also separated by FACS sorting for CD73 activity (Figure 14C). In contrast, palbociclib-treated cells had approximately equal numbers of B-gal-negative and B-gal-positive cells (Figures 14B and 14D). Similarly, treated cells, regardless of whether they were B-gal-negative or B-gal-positive, were separated by FACS sorting for CD73 activity (Figures 14B and 14D).

[0333] The results of FACS sorting for MDA-MB231 cells are summarized in Figures 15A-15B. Figure 15A shows that the number of senescent cells was significantly lower and the cells had significantly lower levels of CD73 activity compared to treated cells, which contained a higher 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. The 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. Untreated and treated cells appeared morphologically similar under a microscope.

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

[0337] Although untreated cells were mostly negative for B-gal staining, they were separated by FACS sorting for CD73 activity (Figure 16A). The separation was not as clear as for the MDA-MB231 cells in Example 14. Although there were significantly fewer B-gal-positive cells, they were also separated by FACS sorting for CD73 activity (Figure 16C). Similarly, treated cells were mostly negative for B-gal (Figures 16B and 16D). Similarly, treated cells, whether B-gal-negative or B-gal-positive, were separated by FACS sorting for CD73 activity, although the separation was not as clear as for the MDA-MB231 cells in Example 14 (Figures 14B and 14D).

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

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

[0340] The CD73 expression levels of MDA-MB231 cells and MDA-MB468 cells after palbociclib treatment were measured (Figure 18A). Palbociclib treatment significantly increased the CD73 expression level in MDA-MB231 cells (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 the CD73 expression level 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 Advanced Targeting Systems ZAP assay kit.

[0343] Because palbociclib treatment was observed to induce senescent MDA-MB231 cells with increased CD73 expression, a cell killing assay (ZAP assay) was performed on MDA-MB231 cells. Briefly, cells were cultured at 4 × 10 in a 96-well plate. 3 Cells were seeded at 1000 x g / well 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 not treated with palbociclib isethionate.

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

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

[0346] B12 appeared to have a smaller effect on palbociclib-treated cells compared to untreated cells, indicating that B12 has a smaller and less significant cell-killing potential on senescent cells. Interestingly, saporin also induced a similarly smaller reduction in the number of senescent cells compared to B12. See Figure 18B.

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

[0348] All documents mentioned in this specification are incorporated herein by reference, either in their entirety or to the extent that they provide a specifically relied upon disclosure. The applicant(s) have no intention of offering any disclosed embodiments to the public, and insofar as any disclosed improvements or modifications may not be literally encompassed within the scope of the claims, they are considered part thereof under the doctrine of equivalents.

[0349] However, although numerous features and advantages of the present invention have been set forth in the foregoing description, together with details of the structure and function of the invention, it is to be understood that the disclosure is illustrative only, and that changes may be made in details, particularly as to the shape, size, and arrangement of parts, within the principles of the invention to all extents indicated in the broad sense of the terms expressed in the appended claims.

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 subjected to the assay of step (iii). (a) a decrease in the binding activity of the parent antibody or antibody fragment to the target in the assay under normal physiological conditions 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 conditionally active senolytic antibody or antibody fragment to the target in the assay under the extracellular conditions of the senescent cells 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, and an increase in the binding activity of the parent 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 parent antibody or antibody fragment to the target in the assay under the extracellular conditions of the senescent cell; selecting said conditionally active senolytic antibodies or antibody fragments that exhibit at least one of the following: wherein the target is selected from at least one of CD54, CD73, and nectin 4; 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 carried out in an assay solution having a bicarbonate concentration of at least 10 mM.

2. 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 1.3:1, or 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.

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

8.

4. The method of any one of claims 1 to 3, wherein the second pH is in the range of 7.2 to 7.6, or 7.4 to 7.

6.

5. 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.

6. 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.

7. The method of any one of claims 1 to 6, further comprising the step of conjugating the conditionally active antibody to a masking moiety by a linker.

8. 8. The method of claim 7, 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%.

9. 9. The method of claim 7 or 8, wherein the linker is covalently attached to the variable region of the conditionally active antibody.

10. The method of any one of claims 7 to 9, wherein the masking moiety specifically binds to the variable region of the conditionally active antibody.

11. 11. The method of claim 10, 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.

12. The method of claim 10 or 11, wherein the linker comprises a flexible region and a cleavage site.

13. 13. The method of claim 12, wherein the flexible region consists essentially of at least one amino acid selected from glycine, alanine, and serine.

14. 14. The method of claim 12 or 13, 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.

15. The method of any one of claims 12 to 14, wherein the cleavage site can be cleaved by a protease in the extracellular environment of the senescent cell.

16. The method of claim 15, 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.

17. 5. The method of any one of claims 1 to 4, further comprising the step of conjugating the conditionally active senolytic antibody or antibody fragment to a cytotoxic, cytostatic, or antiproliferative agent via a linker.

18. 18. The method of claim 17, wherein the linker comprises a cleavage site that can be cleaved by a protease in the extracellular environment of the senescent cell.

19. The method of claim 18, 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.

20. 18. The method of claim 17, wherein the antiproliferative agent is a chemotherapeutic agent.

21. 5. The method of any one of claims 1 to 4, 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.

22. 22. The method of claim 21, 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.

23. 23. The method of claim 22, wherein the native protein is selected from at least one of FOXO4, AMPK, JNK, MST1, CK1, STAT3, p38, PRMT1, and ASK1.

24. 24. The method of any one of claims 21-23, 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.

25. 25. The method of any one of claims 21 to 24, 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.

26. 25. The method of any one of claims 21 to 24, wherein the D retro-inverso peptide has 100% D amino acid residues.

27. 27. The method of any one of claims 21 to 26, 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.

28. 28. The method of any one of claims 22 to 27, 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).

29. 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) a decrease in the binding activity of the parent antibody or antibody fragment to the target in the assay under normal physiological conditions 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 conditionally active senolytic antibody or antibody fragment to the target in the assay under the extracellular conditions of the senescent cells 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, and an increase in the binding activity of the parent 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 parent antibody or antibody fragment to the target in the assay under the extracellular conditions of the senescent cell; selecting said conditionally active senolytic antibodies or antibody fragments from said modified antibodies or antibody fragments that exhibit at least one of: wherein the target is selected from at least one of CD54, CD73, and nectin 4; 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 carried out in an assay solution having a bicarbonate concentration of at least 10 mM.

30. 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 removing one or more partial charges or charged groups from 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) a decrease in the binding activity of the parent antibody or antibody fragment to the target in the assay under normal physiological conditions 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 conditionally active senolytic antibody or antibody fragment to the target in the assay under the extracellular conditions of the senescent cells 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, and an increase in the binding activity of the parent 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 parent antibody or antibody fragment to the target in the assay under the extracellular conditions of the senescent cell; selecting said conditionally active senolytic antibodies or antibody fragments from said modified antibodies or antibody fragments that exhibit at least one of: wherein the target is selected from at least one of CD54, CD73, and nectin 4; 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 carried out in an assay solution having a bicarbonate concentration of at least 10 mM.

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 replacing one or more groups of the parent antibody or antibody fragment with one or more partially charged or charged groups 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) a decrease in the binding activity of the parent antibody or antibody fragment to the target in the assay under normal physiological conditions 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 conditionally active senolytic antibody or antibody fragment to the target in the assay under the extracellular conditions of the senescent cells 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, and an increase in the binding activity of the parent 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 parent antibody or antibody fragment to the target in the assay under the extracellular conditions of the senescent cell; selecting said conditionally active senolytic antibodies or antibody fragments from said modified antibodies or antibody fragments that exhibit at least one of: wherein the target is selected from at least one of CD54, CD73, and nectin 4; 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 carried out in an assay solution having a bicarbonate concentration of at least 10 mM.

32. 32. The method of any one of claims 29 to 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 any one of claims 29 to 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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