Protein therapeutics for treatment of senescent cells

Abstract

Disclosed are methods of producing conditionally active proteins that target senescent cells and are conditionally active in the extracellular environment of senescent cells. The methods include methods of using an evolved protein library and testing using physiological concentrations of bodily fluid components. Also disclosed are conditionally active proteins, antibodies, and antibody fragments for killing or removing senescent cells, conjugates and pharmaceutical compositions using these conditionally active proteins, and methods of treating age-related diseases, conditions, or disorders using the pharmaceutical compositions. Conditionally active proteins can be further evolved, conjugated to other molecules, masked, and reduced in activity by linking cleavable moieties.

Description

[0001] Incorporate into the XML material sequence table via reference. The sequence list of the XML file “BIAT1023BWOSequenceListingXML” submitted with this application was created on February 29, 2024, and is 87,000 bytes in size, and is incorporated herein by reference in its entirety. Technical Field

[0002] This invention relates to the field of treating or eliminating senescent cells and / or treating diseases or disorders associated with senescent cells. In particular, this invention relates to conditionally active proteins that target senescent cells and methods for producing such conditionally active proteins. Background Technology

[0003] Senescent cells possess metabolic activity but are arrested in the G1 phase of the cell cycle; their lifespan is controlled by multiple dominant genes (Stanulis-Praeger, ...). Mech. Ageing Dev (Dimri et al., vol. 38, pp.1-48, 1987). Senescent cells differ from quiescent and terminally differentiated cells in several important ways, exhibiting characteristic morphological changes such as enlargement, flattening, and increased granularity (Dimri et al., vol. 38, pp.1-48, 1987). Proc. Nat. Acad. Sci. USA, vol. 92, pp. 9363-9367, 1995. Even when stimulated by mitogens, senescent cells do not divide (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.

[0004] The expression level of plasminogen activator inhibitor (PAI) is increased in senescent cells and they exhibit β-galactosidase activity staining at pH 6 (Sharpless et al., J. Clin. Invest Irreversible G1 arrest is mediated by the inactivation of a cell cycle-dependent kinase (CdK) complex phosphorylated by Rb. P21 accumulates in senescent cells and inhibits CdK4-CdK6. P16 also inhibits CdK4-CdK6 and accumulates in senescent cells in a manner proportional to β-galactosidase activity and cell volume (Stein et al., vol. 113, pp.160-168, 2004). Mol. Cell. Biol (., vol.19, pp.2109-2117, 1999). There is evidence that p21 is expressed at the onset of aging but is not necessary to maintain aging, while p16 expression helps maintain aging once it begins.

[0005] Because senescence is associated with the gradual shortening of telomeres during each cell division in some cases, senescence is triggered when certain chromosome telomeres reach a critical length (Mathon and Lloyd). Nat. Rev. Cancer , vol. 3,pp.203-213, 2001; Martins, UM Exp Cell Res (., vol. 256, pp.291-299, 2000). Senescence can be eliminated by the expression of telomerase, which elongates telomeres. For example, when fibroblasts are transfected to express telomerase, human fibroblasts replicate indefinitely. Most cancer cells express telomerase to maintain telomere length and replicate indefinitely. A minority of cancer cells that do not express telomerase possess a telomere elongation replacement mechanism (ALT).

[0006] Other causes of aging also exist. These other causes are generally referred to as stress-induced premature aging (SIPS). Oxidative stress can shorten telomeres, thereby inducing aging (von Zglinicki, Trends Biochem. Sci (Vol. 27, pp. 339-344, 2002). Hyperoxia has been shown to induce senescence. Gamma-ray irradiation of human fibroblasts in the early to mid-G1 phase induces senescence in a p53-dependent manner (Di Leonardo et al.). Genes Dev (Vol. 8, pp. 2540-2551, 1994). Ultraviolet radiation can also induce aging. Other agents that can induce aging include hydrogen peroxide (Krtolica et al., vol. 8, pp. 2540-2551, 1994). Proc. Nat. Acad. Sci. USA , vol. 98, pp.12072-12077,2001), sodium butyrate, 5-azacytidine, and transfection with the Ras oncogene (Tominaga, Mech. Ageing Dev (Roninson, vol. 123, pp. 927-936, 2002). Chemotherapy agents (including doxorubicin, cisplatin, and many other compounds) have been shown to induce senescence in cancer cells (Roninson, vol. 123, pp. 927-936, 2002). Cancer Res ., vol. 63, pp.2705-2715, 2003). 5-bromodeoxyuridine treatment leads to senescence in both normal and malignant cells (Michishita et al., vol. 63, pp.2705-2715, 2003). J. Biochem (., vol.126, pp.1052-1059, 1999). Generally speaking, agents that damage DNA can lead to aging.

[0007] Evidence suggests a relationship between senescence and aging. Cultured cells from older donors exhibit senescence after fewer growth cycles compared to 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 begin to age after fewer growth cycles compared to cells from long-lived species (Rohme, D., vol. 73, pp.3584-3588, 1976). Proc. Nat. Acad. Sci. USA, (vol. 78, pp. 5009-3320, 1981). Compared with cells from age-matched control groups, cultured cells from donors with hereditary progeria syndromes (e.g., Werner syndrome) showed senescence after fewer growth cycles.

[0008] Aging endows senescent cells with functional changes, and these senescent cells are associated with various age-related diseases and disorders (Chang et al., Proc. Nat. Acad. Sci. USA (Vol. 97, pp. 4291-4296, 2000). With age, senescent cells accumulate in an individual's tissues and organs and are found at age-related pathological sites. Given the causal relationship between senescent cells and certain aspects of age-related health decline, and the fact that senescent cells may contribute to certain diseases, and because necessary life-sustaining chemotherapy and radiation therapy can induce senescent cells, their presence can have detrimental effects on millions of patients worldwide. It is widely believed that selectively eliminating senescent cells can prevent and treat age-related diseases and disorders.

[0009] Senescent cells can also promote tumorigenesis. Senescent stromal cells express tumor-promoting factors, which exert paracrine effects on neighboring epithelial cells. These effects include promoting mitosis and inhibiting apoptosis (Chang et al., Proc. Nat. Acad. Sci. USA (Vol. 97, pp. 4291-4296, 2000). Senescent fibroblasts have been shown to stimulate precancerous and malignant epithelial cells, but not normal epithelial cells, to form tumors in mice. This occurs when as few as 10% of the fibroblasts are senescent (Krtolica et al., vol. 97, pp. 4291-4296, 2000). Proc. Nat. Acad. Sci. USA (2001, vol. 98, pp. 12072-12077). Tumor-promoting factors secreted by senescent cells are partially mediated by p21waf1 / cip1 / sdi1 (Roninson, vol. 98, pp. 12072-12077). Cancer Res(., vol. 63, pp.2705-2715, 2003). The threshold of senescent stromal cells appears to provide an environment that allows adjacent precancerous epithelial cells to survive, migrate, and divide (Campisi, vol. 63, pp.2705-2715, 2003). Nat. Rev. Cancer , vol. 3, pp. 339-349, 2003).

[0010] Therefore, therapies targeting senescent cells are promising treatment options for age-related diseases and disorders. US 2016 / 0038576 discloses an immunogenic composition for inducing a specific adaptive immune response 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 containing the polynucleotide for administration to an individual.

[0011] WO2015116740 discloses a method for administering a therapeutically effective amount of a small-molecule senolytic agent that selectively kills senescent cells without killing non-senescent cells, for the treatment of senescence-related diseases and disorders. Senescence-related diseases and disorders treatable by this method include cardiovascular diseases and disorders associated with or caused by atherosclerosis, such as atherosclerosis, idiopathic pulmonary fibrosis, chronic obstructive pulmonary disease, osteoarthritis, age-related ophthalmic diseases and disorders, and age-related skin diseases and disorders.

[0012] US 2015 / 0064137 discloses a polypeptide and a virus containing a polypeptide that can be used to selectively eliminate senescent cells. The polypeptide and the virus can induce apoptosis in senescent cells. The polypeptide is selected as a product of a pro-apoptotic gene. The virus contains a pro-apoptotic gene whose expression is regulated by a p16 promoter. The p16 promoter can be a typical p16 promoter or an atypical p16 promoter.

[0013] These therapies target one or more proteins in senescent cells to kill or remove them. However, these target proteins for senescent cells may also be present on other cell types, which could lead to undesirable side effects. Therefore, it is advantageous to develop a class of therapeutic proteins that preferably and / or specifically bind to targets on senescent cells while minimizing or eliminating binding to the same targets on other cell types. Summary of the Invention

[0014] In one embodiment, the present invention provides a method for preparing a conditionally active protein that binds to a target associated with senescent cells from a parent protein that binds to a target associated with senescent cells, the method comprising the following steps: (i) Using one or more evolution techniques to evolve the DNA encoding the parental protein to produce mutant DNA; (ii) Express the mutant DNA to obtain the mutant protein; (iii) Testing the mutant protein under extracellular conditions in the senescent cells, and then under normal physiological conditions; and (iv) Select conditionally active proteins from the mutant proteins, which exhibit at least one of the following properties: (a) The activity in tests under normal physiological conditions is decreased compared to the same activity of the parental protein in the same test, while the activity in tests under extracellular conditions in senescent cells is increased compared to the same activity of the conditionally active protein in tests under normal physiological conditions; and (b) The activity in the test under the normal physiological conditions is lower than that of the same parent protein in the same test, while the activity in the test under the extracellular conditions of the senescent cells is higher than that of the same parent protein in the test under the extracellular conditions of the senescent cells.

[0015] In some implementations, the parent protein may be selected from enzymes, antibodies, receptors, ligands, fragments of enzymes, fragments of antibodies, fragments of receptors, and fragments of ligands.

[0016] In each of the foregoing embodiments, the activity can be the binding activity to the target.

[0017] In each of the foregoing embodiments, the parent protein may be an enzyme, and the activity is the enzymatic activity using at least a portion of senescent cells as a substrate.

[0018] In each of the foregoing embodiments, the conditionally active protein may be a cyclic peptide. The length of the cyclic peptide may be from about 5 to about 500 amino acids, or from about 10 to about 50 amino acids.

[0019] In each of the foregoing embodiments, the target may be a surface molecule located on the outer surface of senescent cells. In each of the foregoing embodiments, the surface molecule may be a cell membrane protein of senescent cells. In each of the foregoing embodiments, the target may be selected from 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, LaminB1, LANCL1, LCMT2, LPHN1, MADCAM1, MAG, MA P3K14, MAPK, MEF2C, miR22, MMP3, MTHFD2, NAIP, NAPG, NCKAP1, Connecton 4, 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. Additionally, it should be recognized that the target can be any combination of the aforementioned.

[0020] In each of the foregoing embodiments, the ratio of the activity of the conditionally active protein in the test under extracellular conditions of the senescent cells to the activity of the conditionally active protein in the test 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 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.

[0021] In each embodiment, the extracellular conditions of senescent cells can be a pH 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.

[0022] In each of the foregoing embodiments, normal physiological conditions may be a pH range of about 7.2 to about 7.8, or a pH range of about 7.2 to about 7.6, or a pH range of about 7.4 to about 7.6.

[0023] In each of the aforementioned embodiments, the extracellular conditions of senescent cells may be that the concentration of deoxynucleotides is lower than the normal physiological concentration of the same type of deoxynucleotide.

[0024] In each of the aforementioned embodiments, the extracellular conditions of senescent cells may be that the oxygen concentration is lower than the normal physiological oxygen concentration.

[0025] In each of the aforementioned embodiments, the extracellular conditions of senescent cells may be that the NAD+ / NADH ratio is lower than the normal physiological NAD+ / NADH ratio.

[0026] In each of the foregoing embodiments, the extracellular conditions of senescent cells may be an increased concentration of at least one redox homeostatic metabolite selected from taurine, cysteine ​​sulfinic acid, cysteine-glutathione disulfide, γ-glutamyl alanine, γ-glutamyl methionine, pyridoxate, γ-glutamyl glutamine, and alanine, relative to the normal physiological concentration of the same redox homeostatic metabolite.

[0027] In each of the foregoing embodiments, the extracellular condition of senescent cells is an increased concentration of a nucleotide metabolite selected from at least one of 3-ureidopropionic acid, uric acid, 7-methylguanine, and hypoxanthine relative to the normal physiological concentration of the same nucleotide metabolite.

[0028] In each of the aforementioned embodiments, the extracellular conditions of senescent cells may be a decrease in the concentration of thymidine relative to the normal physiological concentration of thymidine.

[0029] In each of the foregoing embodiments, the extracellular condition of senescent cells is a reduced concentration of a dipeptide selected from at least one of glycyl isoleucine, glycylvaline, glycylleucine, isoleucylglycine, and valineglycine relative to the normal physiological concentration of the same dipeptide.

[0030] In each of the foregoing embodiments, the extracellular conditions of senescent cells are a concentration of at least one fatty acid selected from linoleic acid, di-homo-linoleic acid, and 10-heptadecenoic acid, which is reduced relative to the normal physiological concentration of said fatty acid.

[0031] In each of the foregoing embodiments, the extracellular conditions of senescent cells may be an increased concentration of a phospholipid metabolite selected from at least one of 2-hydroxypalmitic acid, 2-hydroxystearic acid, 3-hydroxydecanoic acid, 3-hydroxyoctanoic acid, and glycerophosphocholine relative to the normal physiological concentration of said phospholipid metabolite.

[0032] In each of the foregoing embodiments, the extracellular conditions of senescent cells may be an increased concentration of an amino acid metabolite selected from at least one of alanine, C-glycosyltryptophan, kynurenine, dimethylarginine, and orthithine, relative to the normal physiological concentration of said amino acid metabolite.

[0033] In each of the foregoing embodiments, the extracellular conditions of senescent cells may be a concentration of phenylpyruvate that is reduced relative to the normal physiological concentration of phenylpyruvate.

[0034] In each of the foregoing embodiments, the extracellular conditions of senescent cells may be an increased concentration of a metabolite selected from at least one of fumaric acid, malonic acid, eicosapentaenoic acid, and citric acid, relative to the normal physiological concentration of said metabolite.

[0035] In each of the aforementioned embodiments, the extracellular condition of senescent cells may be an increased ratio of glycerophosphocholine to phosphocholine relative to the normal physiological ratio.

[0036] In each of the foregoing embodiments, the extracellular condition of senescent cells can be an increase in the concentration of proteins secreted by the senescent cells compared to the normal physiological concentration of the proteins, and the proteins secreted by the senescent cells are selected from GM-CSF, GROa, GRC-α, GRC-β, GRC-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, bimodal protein, ENA-78, eosinophil activation chemokine-3, GCP-2, GITR, HGF, ICAM-1, IGFBP-1, IGFBP-2, IGFBP-3, IGFBP-4, IGFBP-5, IGFBP- 6. IL-13, IL-1β, MCP-4, MIF, MIP-3a, MMP-12, MMP-13, MMP-14, NAP2, Oncogene M, Osteoporosis Protectin, PIGF, RANTES, sgp130, TIMP-2, TRAIL-R3, Acrp30, Angiopoietin, AXL, bFGF, BLC, BTC, CTACK, EGF-R, Fas, FGF-7, G-CSF, GDNF, HCC-4, I-309, IFN-γ, IL-1R1, 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 At least one of 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 and BMP-6, and any combination thereof.

[0037] In each of the foregoing embodiments, the tests under the normal physiological conditions and the tests under the extracellular conditions of the senescent cells can be performed in a test solution containing at least one component selected from inorganic compounds, ions, and organic molecules. In this embodiment, the at least one component can have substantially the same concentration in the test solutions used for the tests under the normal physiological conditions and the tests used for the tests under the extracellular conditions of the senescent cells. In these embodiments, the at least one component can be an inorganic compound selected from boric acid, calcium chloride, calcium nitrate, diammonium phosphate, magnesium sulfate, monoammonium phosphate, monopotassium phosphate, potassium chloride, potassium sulfate, copper sulfate, ferric sulfate, manganese sulfate, zinc sulfate, magnesium sulfate, calcium nitrate, calcium chelate, copper chelate, iron chelate, iron chelate, manganese chelate, zinc chelate, ammonium molybdate, ammonium sulfate, calcium carbonate, magnesium phosphate, potassium bicarbonate, potassium nitrate, hydrochloric acid, carbon dioxide, sulfuric acid, phosphoric acid, carbonic acid, uric acid, hydrogen chloride, and urea. In these embodiments, the at least one component can be an ion selected from phosphate ions, sulfide ions, chloride ions, magnesium ions, sodium ions, potassium ions, ammonium ions, iron ions, zinc ions, and copper ions. In these embodiments, the at least one component may be selected from one or more of the following: uric acid with a concentration range of 2-7.0 mg / dL, calcium ions with a concentration range of 8.2-11.6 mg / dL, chloride ions with a concentration range of 355-381 mg / dL, iron ions with a concentration range of 0.028-0.210 mg / dL, potassium ions with a concentration range of 12.1-25.4 mg / dL, sodium ions with a concentration range of 300-330 mg / dL, and carbonic acid with 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, pyrrolidone, proline, selenocysteine, serine, and tyrosine. In these embodiments, the at least one component may be an organic acid selected from citric acid, α-ketoglutarate, succinic acid, malic acid, fumaric acid, acetoacetic acid, β-hydroxybutyric acid, lactic acid, pyruvic acid, α-keto 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, O-glycoside, N-glycoside, C-glycoside, and S-glycoside.In these embodiments, the at least one component may be selected from magnesium ions, sulfate ions, bisulfate ions, carbonate ions, bicarbonate ions, nitrate ions, nitrite ions, phosphate ions, hydrogen phosphate ions, dihydrogen phosphate ions, persulfate ions, monopersulfate ions, borate ions, and ammonium ions.

[0038] In each of the foregoing embodiments, the extracellular conditions of 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 tests are performed in a test solution containing at least one substance with a molecular weight of less than 900 a.m. and a pKa that differs from the first pH by at most 0.5, 1, 2, 3 or 4 pH units.

[0039] In each of the foregoing embodiments, the extracellular conditions of 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. The one or more tests may be performed in a test solution containing at least one substance with a molecular weight of less than 900 a.m., and the substance may have a pKa between the first pH and the second pH.

[0040] In each of the foregoing embodiments, the extracellular conditions of 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. The tests may be performed in a test solution containing at least one substance selected from histidine, histamine, hydrogenated adenosine diphosphate, hydrogenated adenosine triphosphate, citrate, bicarbonate, acetate, lactate, disulfide, hydrogen sulfide, ammonium, and dihydrogen phosphate.

[0041] In each of the foregoing embodiments, the selection step (iv) may include selecting a conditionally active protein exhibiting the following properties: (a) decreased activity in a test under the normal physiological conditions compared to the same activity of the parent protein in the same test, and increased activity in a test under extracellular conditions in the senescent cells compared to the same activity of the conditionally active protein in a test under the normal physiological conditions.

[0042] In each of the foregoing embodiments, the selection step (iv) may include selecting a conditionally active protein exhibiting the following properties: (b) its activity in a test under the normal physiological conditions is reduced compared to the same activity of the parent protein in the same test, while its activity in a test under extracellular conditions of senescent cells is increased compared to the same activity of the parent protein in a test under extracellular conditions of senescent cells.

[0043] In another embodiment, the present invention provides a conditionally active protein prepared by any of the foregoing 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 engineered to be part of a chimeric antigen receptor for T cells. The antibody may be a humanized antibody, a bispecific antibody, or a multispecific antibody.

[0044] In each of the foregoing embodiments, the conditionally active protein may be selected from receptors, regulatory proteins, soluble proteins, cytokines, fragments of receptors, fragments of regulatory proteins, fragments of soluble proteins, and fragments of cytokines.

[0045] In one embodiment, the present invention provides a isolated polypeptide that specifically binds to CD73, comprising: The heavy chain variable region includes three complementarity-determining regions (CDRs) with H1, H2, and H3 sequences, wherein: The H1 sequence is GFTFSSYAYS (SEQ ID NO: 52); The H2 sequence is AISGSGGRTYYADSVKG (SEQ ID NO: 53); and The H3 sequence is LGX1GRVDE (SEQ ID NO: 54); Where X1 is either Y or E, and The light chain variable region includes three complementarity-determining regions (CDRs) with L1, L2, and L3 sequences, wherein: The L1 sequence is SGSLSNIGRNPVN (SEQ ID NO: 47); The L2 sequence is X2DNLRLS (SEQ ID NO: 48); and The L3 sequence is ATWDDSHPGWT (SEQ ID NO: 51). Where X2 is L or D, and The condition is: X1 and X2 cannot be Y and L respectively at the same time, or The combination of the heavy chain variable region and the light chain variable region is not SEQ ID NO: 45 and 43.

[0046] In each of the foregoing embodiments, the H3 sequence may be selected from LGYGRVDE (SEQ ID NO: 55) and LGEGRVDE (SEQ ID NO: 56).

[0047] In each of the foregoing embodiments, the L3 sequence may be selected from LDNLRLS (SEQ ID NO: 49) and DDNLRLS (SEQ ID NO: 50).

[0048] In each of the foregoing embodiments, the isolated polypeptide may comprise a heavy chain variable region having a sequence selected from SEQ ID NO: 45 to 46.

[0049] In each of the foregoing embodiments, the isolated polypeptide may comprise a light chain variable region having a sequence selected from SEQ ID NO: 43 to 44.

[0050] In each of the foregoing embodiments, the isolated polypeptide may comprise a heavy chain variable region and a light chain variable region having any pair of sequences selected from SEQ ID NO: 45 and 44 and SEQ ID NO: 46 and 43.

[0051] In each of the foregoing embodiments, the isolated polypeptide may comprise a heavy chain variable region having at least 80%, 85%, 90%, 95%, 98%, or 99% identity with an amino acid sequence selected from the group consisting of amino acid sequences having SEQ ID NO: 45 to 46, and a light chain variable region having at least 80%, 85%, 90%, 95%, 98%, or 99% identity with an amino acid sequence selected from the group consisting of amino acid sequences having SEQ ID NO: 43 to 44; and wherein the isolated polypeptide specifically binds to human CD73. In each of these embodiments, the variation in the percentage of identity may be limited to regions of the polypeptide located outside the CDR.

[0052] In each of the foregoing embodiments, the isolated polypeptide may comprise a heavy chain variable region having at least 80%, 85%, 90%, 95%, 98%, or 99% identity with the amino acid sequence having SEQ ID NO: 45, and a light chain variable region having at least 80%, 85%, 90%, 95%, 98%, or 99% identity with the amino acid sequence having SEQ ID NO: 46; and wherein said isolated polypeptide specifically binds to human CD73. In each of these embodiments, the variation in the percentage of identity may be limited to regions of the polypeptide located outside the CDR.

[0053] In each of the foregoing embodiments, the isolated polypeptide may comprise a heavy chain variable region having at least 80%, 85%, 90%, 95%, 98%, or 99% identity with the amino acid sequence having SEQ ID NO: 45, and a light chain variable region having at least 80%, 85%, 90%, 95%, 98%, or 99% identity with the amino acid sequence having SEQ ID NO: 43; and wherein said isolated polypeptide specifically binds to human CD73. In each of these embodiments, the variation in the percentage of identity may be limited to regions of the polypeptide located outside the CDR.

[0054] In each of the foregoing embodiments, the isolated polypeptide may comprise a heavy chain variable region having at least 80%, 85%, 90%, 95%, 98%, or 99% identity with the amino acid sequence having SEQ ID NO: 44, and a light chain variable region having at least 80%, 85%, 90%, 95%, 98%, or 99% identity with the amino acid sequence having SEQ ID NO: 46; and wherein said isolated polypeptide specifically binds to human CD73. In each of these embodiments, the variation in the percentage of identity may be limited to regions of the polypeptide located outside the CDR.

[0055] In each of the foregoing embodiments, the isolated polypeptide may comprise a heavy chain variable region having at least 80%, 85%, 90%, 95%, 98%, or 99% identity with the amino acid sequence having SEQ ID NO: 44, and a light chain variable region having at least 80%, 85%, 90%, 95%, 98%, or 99% identity with the amino acid sequence having SEQ ID NO: 43; and wherein said isolated polypeptide specifically binds to human CD73. In each of these embodiments, the variation in the percentage of identity may be limited to regions of the polypeptide located outside the CDR.

[0056] In another embodiment, the present invention provides a conditionally active senescent cell clearance antibody or antibody fragment, which may comprise any of the aforementioned isolated polypeptides.

[0057] In each of the foregoing embodiments, the binding activity of conditionally active senescent cell clearance antibodies or antibody fragments to CD73 may be higher under extracellular conditions of senescent cells compared to normal physiological conditions. These conditions may be pH. Extracellular conditions for senescent cells may be a pH ranging from about 5.5 to about 7.0, while normal physiological conditions may be a pH ranging from about 7.2 to about 7.8.

[0058] In each of the foregoing embodiments, the ratio of the binding activity of the conditionally active senescent cell clearance antibody or antibody fragment to CD73 under extracellular conditions of senescent cells to its binding activity to CD73 under normal physiological conditions is at least about 1.5:1, at least about 2:1, at least about 3:1, at least about 4:1, at least about 5:1, at least about 6:1, at least about 7:1, at least about 8:1, at least about 9:1, at least about 10:1, at least about 20:1, at least about 30:1, at least about 50:1, at least about 70:1, or at least about 100:1.

[0059] In each of the foregoing embodiments, the conditionally active senescent cell clearance antibody or antibody fragment may be selected from multispecific antibodies and multispecific antibody fragments.

[0060] In each of the foregoing embodiments, the conditionally active senescent cell clearance antibody or antibody fragment may be selected from bispecific antibodies and bispecific antibody fragments.

[0061] In each of the foregoing embodiments, the conditionally active senescent cell clearance antibody or antibody fragment may be a single-chain antibody.

[0062] In another embodiment, the present invention provides a conjugate comprising any of the aforementioned conditionally active antibodies or antibody fragments conjugated to a masking moiety via a linker, wherein the masking moiety is identified by screening a diverse peptide library for peptides that bind to one or more variable regions of a conditionally active senescent cell clearance antibody. The masking moiety can reduce the activity of the conditionally active antibody binding to CD73 by at least 50%.

[0063] In another embodiment, the present invention provides a conjugate comprising any of the aforementioned conditionally active antibodies or antibody fragments conjugated to a drug via a linker, said drug being selected from cytotoxic drugs, cell growth inhibitors, and antiproliferative drugs. The linker may include a cleavage site capable of being cleaved by proteases in the extracellular environment of senescent cells.

[0064] In another embodiment, the present invention provides a conjugate comprising any of the aforementioned conditionally active antibodies or antibody fragments conjugated to an agent selected from the group consisting of: toxic agents, radioactive agents, or D retroinverso peptides. The amino acid sequence of the D retroinverso peptide may have at least 70% amino acid sequence identity with the reverse sequence of a fragment or full-length natural protein selected from FOXO4, AMPK, JNK, MST1, CK1, STAT3, p38, PRMT1, and ASK1. D-reverse peptide may contain one or more functional domains selected from PPRRRQRRKKRG (SEQ ID NO:10), GALFLGFLGA AGSTMGAWSQPKKKRKV (SEQ ID NO:11), KETWWETWWT EWSQPKKKRKV (SEQ ID NO:12), Ac-GLWRALWRLLRSLWRLLWRA-Cya (SEQ ID NO:13), LTLRKEPASE IAQSILEAYS QNGWANRRSG GKRP (SEQ ID NO:5), LTLRKEPASE IAQSILEAYS QNGWANRRSG GKRPPPRRRQ RRKKRG (SEQ ID NO:6), SEIAQSILEAYSQNGW (SEQ ID NO:7) and octargine.

[0065] In another embodiment, the present invention provides an immune conjugate comprising any of the aforementioned conditionally active senescent cell clearance antibodies or antibody fragments.

[0066] In other embodiments, the present invention provides a pharmaceutical composition comprising any of the aforementioned polypeptides, antibodies or antibody fragments, conjugates, and immunoconjugates.

[0067] In another embodiment, the present invention provides a specific polypeptide that binds to B7H4, comprising: The heavy chain variable region includes three complementarity-determining regions (CDRs) with H1, H2, and H3 sequences, wherein: The H1 sequence is GYTFTDRTIH (SEQ ID NO: 64); The H2 sequence is SIYPRDGSTKYNEKFKD (SEQ ID NO: 65); and The H3 sequence is SVGYAX3DY (SEQ ID NO: 66); Where X3 is F or D, and The light chain variable region includes three complementarity-determining regions (CDRs) with L1, L2, and L3 sequences, wherein: The L1 sequence is RVSEGIDNYGFTFIH (SEQ ID NO: 61); The L2 sequence is RASNLQS (SEQ ID NO: 62); and The L3 sequence is QQSDKDPFT (SEQ ID NO: 63), and The conditions are: X3 cannot be F, or the combination of the heavy chain variable region and the light chain variable region is not SEQ ID NO:59 and 57.

[0068] In the above implementation scheme, the H3 sequence may be selected from SVGYAFDY (SEQ ID NO: 67) and SVGYADDY (SEQ ID NO: 68).

[0069] In each of the foregoing embodiments, the isolated polypeptide may comprise a heavy chain variable region having a sequence selected from SEQ ID NO: 57 to 58.

[0070] In each of the foregoing embodiments, the isolated polypeptide may comprise a light chain variable region having a sequence selected from SEQ ID NO: 59 to 60.

[0071] In each of the foregoing embodiments, the isolated polypeptide may comprise a heavy chain variable region having the sequence of SEQ ID NO: 58 and a light chain variable region having the sequence of SEQ ID NO: 60.

[0072] In each of the foregoing embodiments, the isolated polypeptide may comprise a heavy chain variable region having at least 80%, 85%, 90%, 95%, 98%, or 99% identity with an amino acid sequence selected from the group consisting of amino acid sequences having SEQ ID NO: 57 to 58, and a light chain variable region having at least 80%, 85%, 90%, 95%, 98%, or 99% identity with an amino acid sequence selected from the group consisting of amino acid sequences having SEQ ID NO: 59 to 60; and wherein the isolated polypeptide specifically binds to human B7H4. In each of these embodiments, the variation in the percentage of identity may be limited to regions of the polypeptide located outside the CDR.

[0073] In each of the foregoing embodiments, the isolated polypeptide may comprise a heavy chain variable region having at least 80%, 85%, 90%, 95%, 98%, or 99% identity with the amino acid sequence having SEQ ID NO: 58, and a light chain variable region having at least 80%, 85%, 90%, 95%, 98%, or 99% identity with the amino acid sequence having SEQ ID NO: 60; and wherein said isolated polypeptide specifically binds to human B7H4. In each of these embodiments, the variation in the percentage of identity may be limited to regions of the polypeptide located outside the CDR.

[0074] In another embodiment, the present invention provides a conditionally active senescent cell clearance antibody or antibody fragment, which may comprise any of the aforementioned isolated polypeptides that specifically bind to B7H4.

[0075] In each of the foregoing embodiments, the binding activity of conditionally active senescent cell clearance antibodies or antibody fragments to B7H4 may be higher under extracellular conditions of senescent cells compared to normal physiological conditions. These conditions may be pH. Extracellular conditions for senescent cells may be a pH ranging from about 5.5 to about 7.0, while normal physiological conditions may be a pH ranging from about 7.2 to about 7.8.

[0076] In each of the foregoing embodiments, the ratio of the binding activity of the conditionally active senescent cell clearance antibody or antibody fragment to B7H4 under extracellular conditions of senescent cells to its binding activity to B7H4 under normal physiological conditions is at least about 1.5:1, at least about 2:1, at least about 3:1, at least about 4:1, at least about 5:1, at least about 6:1, at least about 7:1, at least about 8:1, at least about 9:1, at least about 10:1, at least about 20:1, at least about 30:1, at least about 50:1, at least about 70:1, or at least about 100:1.

[0077] In each of the foregoing embodiments, the conditionally active senescent cell clearance antibody or antibody fragment that binds to B7H4 may be selected from multispecific antibodies and multispecific antibody fragments.

[0078] In each of the foregoing embodiments, the conditionally active senescent cell clearance antibody or antibody fragment that binds to B7H4 may be selected from bispecific antibodies and bispecific antibody fragments.

[0079] In each of the foregoing embodiments, the conditionally active senescent cell clearance antibody or antibody fragment that binds to B7H4 can be a single-chain antibody.

[0080] In another embodiment, the present invention provides a conjugate comprising any of the aforementioned conditionally active antibodies or antibody fragments conjugated to a masking portion via a linker, these antibodies or antibody fragments binding to B7H4, wherein the masking portion is identified by screening a diverse peptide library for peptides that bind to one or more variable regions of a conditionally active senescent cell clearance antibody. The masking portion can reduce the activity of the conditionally active antibody binding to B7H4 by at least 50%.

[0081] In another embodiment, the present invention provides a conjugate comprising any of the aforementioned conditionally active antibodies or antibody fragments that bind to B7H4, said antibody or antibody fragment being conjugated to a drug selected from cytotoxic drugs, cell growth inhibitors, and antiproliferative drugs. The linker may include a cleavage site capable of being cleaved by proteases in the extracellular environment of senescent cells.

[0082] In another embodiment, the present invention provides a conjugate comprising any of the aforementioned conditionally active antibodies or antibody fragments that bind to B7H4, said antibody or antibody fragment being conjugated to an agent selected from the group consisting of toxic agents, radioactive agents, or D-reverse peptides. The amino acid sequence of the D-reverse peptide may have at least 70% amino acid sequence identity with a fragment or full-length reverse sequence of a natural protein selected from FOXO4, AMPK, JNK, MST1, CK1, STAT3, p38, PRMT1, and ASK1. D-reverse peptide may contain one or more functional domains selected from PPRRRQRRKKRG (SEQ ID NO:10), GALFLGFLGA AGSTMGAWSQPKKKRKV (SEQ ID NO:11), KETWWETWWT EWSQPKKKRKV (SEQ ID NO:12), Ac-GLWRALWRLLRSLWRLLWRA-Cya (SEQ ID NO:13), LTLRKEPASE IAQSILEAYS QNGWANRRSG GKRP (SEQ ID NO:5), LTLRKEPASE IAQSILEAYS QNGWANRRSG GKRPPPRRRQ RRKKRG (SEQ ID NO:6), SEIAQSILEAYSQNGW (SEQ ID NO:7) and octargine.

[0083] In another embodiment, the present invention provides an immunoconjugate comprising any of the aforementioned conditionally active senescent cell clearance antibodies or antibody fragments that bind to B7H4.

[0084] In other embodiments, the present invention provides a pharmaceutical composition comprising any of the aforementioned polypeptides that bind to B7H4, an antibody or antibody fragment that binds to B7H4, a conjugate of an antibody or antibody fragment that binds to B7H4, and an immunoconjugate of an antibody or antibody fragment that binds to B7H4.

[0085] In each of the foregoing embodiments, a conditionally active senescent cell clearance antibody or antibody fragment can be conjugated to a masking portion via a linker. The masking portion can reduce 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 even 100%. The masking portion can specifically bind to the variable region of the conditionally active antibody. The linker can be covalently bonded to the variable region of the conditionally active antibody. The linker may include a flexible region and a cleavage site. The cleavage site can be cleaved by proteases in the extracellular environment of senescent cells.

[0086] In each of the foregoing embodiments, the conditionally active senescent cell clearance antibody can be conjugated to a cytotoxic drug, a cell growth inhibitor, or an antiproliferative drug via a linker, said linker possibly containing a cleavage site for at least one protease in the extracellular environment of senescent cells. The at least one protease is selected from ADAM10, ADAM12, ADAM17, ADAMTS, ADAMTS5, BACE, caspase 1-14, cathepsin A, cathepsin B, cathepsin D, cathepsin E, cathepsin K, cathepsin S, FAP, MT1-MMP, granzyme B, guanidinobenzoate, serine protease, human neutrophil elastase, lentinan, proteolytic enzyme 2, transmembrane peptidase, MMP1-17, MT-SP1, enkephalinase, NS3 / 4A, plasmin, PSA, PSMA, TRACE, TMPRSS 3, TMPRSS 4, and uPA.

[0087] In another embodiment, the present invention provides a method for treating aging or diseases or disorders related to senescent cells, comprising the step of administering to an individual any of the aforementioned conditionally active peptides, conditionally active senescent cell-clearing antibodies or antibody fragments, conjugates or immunoconjugates, or any of the aforementioned pharmaceutical compositions. In the foregoing embodiments, diseases or disorders related to senescent cells may be selected from cognitive disorders, cardiovascular diseases, metabolic diseases and disorders, motor function disorders and disorders, cerebrovascular diseases, emphysema, osteoarthritis, lung diseases, inflammatory / autoimmune diseases and disorders, ophthalmic diseases or conditions, metastasis, side effects of chemotherapy or radiotherapy, age-related diseases and disorders, and fibrotic diseases and disorders.

[0088] In another embodiment, the present invention provides a method for generating conditionally active molecules with a molecular weight of less than about 3000 a.m. from a parent organic compound. The method includes the steps of: modifying a parent organic compound by introducing one or more partially charged or charged groups to produce one or more modified organic compounds; and selecting a modified organic compound that exhibits higher activity in tests under abnormal conditions compared to the same activity in tests under normal physiological conditions.

[0089] In another embodiment, the present invention provides a method for generating conditionally active molecules with a molecular weight of less than about 3000 a.m. from a parent organic compound, comprising the steps of: modifying the parent organic compound by removing one or more partially charged or charged groups from the parent organic compound to generate one or more modified organic compounds; and selecting modified organic compounds that exhibit higher activity in tests under abnormal conditions compared with the same activity in tests under normal physiological conditions.

[0090] In another embodiment, the present invention provides a method for generating conditionally active molecules with a molecular weight of less than about 3000 a.m. from a parent organic compound, comprising the steps of: 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 generate one or more modified organic compounds; and selecting modified organic compounds that exhibit higher activity in tests under abnormal conditions compared with the same activity in tests under normal physiological conditions.

[0091] In each of the aforementioned methods, the molecular weight of the parent organic compound can range from about 100 a.mu to about 3000 a.mu, or about 100 a.mu to about 1500 a.mu, or about 150 a.mu to about 1250 a.mu, or about 300 a.mu to about 1100 a.mu, or about 400 a.mu to about 1000 a.mu.

[0092] In each of the aforementioned methods, the abnormal condition can be the value of the extracellular condition of senescent cells, and the normal physiological condition is a different value of the same extracellular condition of normal cells.

[0093] In each of the aforementioned methods, the abnormal conditions may be a pH range of about 5.0 to about 7.0, or a pH range of about 5.5 to about 7.0, or a pH range of about 6.0 to about 7.0, or a pH range of about 6.2 to about 6.8, and the normal physiological conditions are a pH range of about 7.0 to about 7.8, or a pH range of about 7.2 to about 7.8, or a pH range of about 7.2 to about 7.6.

[0094] In each of the aforementioned methods, the conditionally active peptide or conditionally active senescent cell clearance antibody may be conjugated with an agent selected from toxic agents, radioactive agents, or D-reverse peptides.

[0095] In each of the foregoing embodiments, the D-reverse peptide may comprise LTLRKEPASE IAQSILEAYS QNGWANRRSGGKRP (SEQ ID NO: 5), LTLRKEPASE IAQSILEAYS QNGWANRRSG GKRPPPRRRQ RRKKRG (SEQ ID NO: 6), or SEIAQSILEAYSQNGW (SEQ ID NO: 7).

[0096] In another embodiment, the present invention provides a method for treating a disease or disorder associated with senescent cells, the method comprising administering to an individual suffering from the disease or disorder any of the aforementioned conditionally active peptides, conditionally active senescent cell-clearing antibodies, their immunoconjugates, or pharmaceutical compositions comprising the aforementioned conditionally active peptides, conditionally active senescent cell-clearing antibodies, or their immunoconjugates, to kill or remove senescent cells associated with the disease or disorder. The disease or disorder may be selected from osteoarthritis, idiopathic pulmonary fibrosis, chronic obstructive pulmonary disease (COPD), and atherosclerosis. Attached Figure Description

[0097] Figure 1 A graph illustrating the selectivity of the conditionally active antibody selected in Example 9 at pH 6.0 versus pH 7.4.

[0098] Figure 2 The diagram illustrates the formation of salt bridges in deoxyhemoglobin, where three amino acid residues form two salt bridges that stabilize the T quaternary structure of deoxyhemoglobin, resulting in a lower affinity for oxygen.

[0099] Figure 3 A diagram illustrating the structure of a chimeric antigen receptor (CAR).

[0100] Figure 4 The binding activity of conditionally active antibodies to antigens, as measured in different buffer solutions, is shown.

[0101] Figure 5 The effect of changing the composition of Krebs buffer on the binding activity of conditionally active antibodies is shown.

[0102] Figure 6 The binding activity of three different conditionally active antibodies was shown to depend on the presence and concentration of bicarbonate at pH 7.4, as described in Example 12.

[0103] Figure 7 The design principles of D-reverse inversion (DRI) peptides from natural or wild-type peptides are shown.

[0104] Figure 8The signal transduction pathways regulating the FOXO family (including FOXO4) are shown. "+p" indicates phosphorylation, "-p" indicates dephosphorylation, "+m" indicates methylation, arrows indicate activation, and lines with a horizontal bar at the end indicate repression. Each of these is associated with a target gene.

[0105] Figure 9A Untreated MCF-7 cells are shown.

[0106] Figure 9B MCF-7 cells treated with 1 μM of Pabocilib Isethionate are shown.

[0107] Figure 9C The separation of untreated and treated MCF-7 cells by fluorescence activated cell sorting (FACS) is shown.

[0108] Figure 9D The target expression profiles of untreated MCF-7 cells and MCF-7 cells treated with Papositories hydroxyethyl sulfonate are shown.

[0109] Figure 10A Untreated MDA-MB231 cells are shown.

[0110] Figure 10B MDA-MB231 cells treated with 1 μM Paposibi hydroxyethyl sulfonate are shown.

[0111] Figure 10C The images show the separation of untreated MDA-MB231 cells and MDA-MB231 cells treated with Paposidipeptide hydroxyethyl sulfonate via FACS.

[0112] Figure 10D The target expression levels in untreated MDA-MB231 cells and MDA-MB231 cells treated with Paposidipeptide hydroxyethyl sulfonate are shown.

[0113] Figure 11A Untreated MDA-MB468 cells are shown.

[0114] Figure 11B MDA-MB468 cells treated with 1 μM Paposibi hydroxyethyl sulfonate are shown.

[0115] Figure 11C The results show that untreated MDA-MB468 cells and MDA-MB468 cells treated with Paposidipeptide hydroxyethyl sulfonate were not separated by FACS.

[0116] Figure 11DSimilar target expression levels were shown in untreated MDA-MB468 cells and MDA-MB468 cells treated with Paposidipeptide hydroxyethyl sulfonate.

[0117] Figure 12A Untreated MDA-MB231 cells are shown.

[0118] Figure 12B MDA-MB231 cells treated with Papositories hydroxyethyl sulfonate are shown.

[0119] Figure 13A Untreated MDA-MB468 cells are shown.

[0120] Figure 13B MDA-MB468 cells treated with Paposidipeptide hydroxyethyl sulfonate are shown.

[0121] Figure 14A FACS cell sorting of untreated, B-gal-negative MDA-MB231 cells is shown.

[0122] Figure 14B FACS cell sorting of B-gal-negative MDA-MB231 cells treated with Paposidipeptide hydroxyethyl sulfonate is shown.

[0123] Figure 14C FACS cell sorting of untreated B-gal-positive MDA-MB231 cells is shown.

[0124] Figure 14D The image shows FACS cell sorting of B-gal-positive MDA-MB231 cells treated with Paposidipeptide hydroxyethyl sulfonate.

[0125] Figure 15A The FACS sorting of untreated MDA-MB231 cells is shown.

[0126] Figure 15B FACS sorting of MDA-MB231 cells treated with Papositories hydroxyethyl sulfonate is shown.

[0127] Figure 16A FACS cell sorting of untreated MDA-MB468 cells that are negative for B-gal staining is shown.

[0128] Figure 16B The image shows FACS cell sorting of B-gal-negative MDA-MB468 cells treated with Paposidipeptide hydroxyethyl sulfonate.

[0129] Figure 16CFACS cell sorting of untreated B-gal-positive MDA-MB468 cells is shown.

[0130] Figure 16D The image shows FACS cell sorting of B-gal-positive MDA-MB468 cells treated with Paposidipeptide hydroxyethyl sulfonate.

[0131] Figure 17A The FACS sorting of untreated MDA-MB468 cells is shown.

[0132] Figure 17B FACS sorting of MDA-MB468 cells treated with Papositories hydroxyethyl sulfonate is shown.

[0133] Figure 18 The expression levels of CD73 in MDA-MB231 and MDA-MB468 cells before and after treatment with Paposibine hydroxyethyl sulfonate are shown.

[0134] Figure 19 Methods for inducing senescent cells and methods for using conditionally active antibodies to kill senescent cells are shown.

[0135] Figure 20 MCF-7 cells were treated for 7 days with different concentrations of pabosipi, doxorubicin, and paclitaxel. After 7 days, the presence of senescent cells in each treatment group was detected by quantitative flow cytometry by measuring SA-b-Gal levels. (CBA-232, Cell Biolabs). Clinical Cancer Research; 22 (8) April 15, 2016.

[0136] Figure 21 Untreated MCF-7 cells, T47D cells, and MDA-MB468 cells (left) are shown, as well as MCF-7 cells, T47D cells, and MDA-MB468 cells treated with 3 µM hydroxyethyl sulfonate for 6 days (right), and stained using a senescent β-galactosidase staining kit (Cell Signaling Technologies, catalog number 9860) to detect SA β-gal activity.

[0137] Figure 22A The image shows FACS cell sorting of MCF-7 cells treated with pabosipi hydroxyethyl sulfonate and untreated MCF-7 cells. These two groups of cells were subsequently treated with either PE-anti-human CD73 antibody or isotype PE-mouse antibody. Antibodies are used for treatment.

[0138] Figure 22B The expression levels of CD73 in MCF-7 cells treated with Pabosibi hydroxyethyl sulfonate and in untreated MCF-7 cells are shown.

[0139] Figure 22C The image shows FACS cell sorting of MCF-7 cells treated with pabosipi hydroxyethyl sulfonate and untreated MCF-7 cells. These two groups of cells were subsequently treated with either PE-anti-human B7H3 antibody or allotype PE-mouse antibody. Antibodies are used for treatment.

[0140] Figure 22D The expression levels of B7H3 in MCF-7 cells treated with Pabosibi hydroxyethyl sulfonate and untreated MCF-7 cells are shown.

[0141] Figure 22E The image shows FACS cell sorting of MCF-7 cells treated with pabosipi hydroxyethyl sulfonate and untreated MCF-7 cells. These two groups of cells were subsequently treated with either PE-anti-human B7H4 antibody or allotype PE-mouse antibody. Antibodies are used for treatment.

[0142] Figure 22F The expression levels of B7H4 in MCF-7 cells treated with Pabosibi hydroxyethyl sulfonate and untreated MCF-7 cells are shown.

[0143] Figure 22G The image shows FACS cell sorting of MCF-7 cells treated with pabosipi hydroxyethyl sulfonate and untreated MCF-7 cells. These two groups of cells were subsequently treated with either PE-anti-human CD54 antibody or isotype PE-mouse antibody. Antibodies are used for treatment.

[0144] Figure 22H The CD54 expression levels in MCF-7 cells treated with Pabosibi hydroxyethyl sulfonate and untreated MCF-7 cells are shown.

[0145] Figure 22I The image shows FACS cell sorting of MCF-7 cells treated with pabosipi hydroxyethyl sulfonate and untreated MCF-7 cells. These two groups of cells were subsequently treated with either PE-anti-human DPP4 antibody or isotype PE-mouse antibody. Antibodies are used for treatment.

[0146] Figure 22J The expression levels of DPP4 in MCF-7 cells treated with Pabosipi hydroxyethyl sulfonate and in untreated MCF-7 cells are shown.

[0147] Figure 23AThe image shows FACS cell sorting of T47D cells treated with pabosipi hydroxyethyl sulfonate and untreated T47D cells. These two groups of cells were subsequently treated with either PE-anti-human CD73 antibody or allotype PE-mouse antibody. Antibodies are used for treatment.

[0148] Figure 23B The expression levels of CD73 in T47D cells treated with paboseib hydroxyethyl sulfonate and untreated T47D cells are shown.

[0149] Figure 23C The image shows FACS cell sorting of T47D cells treated with pabosipi hydroxyethyl sulfonate and untreated T47D cells. These two groups of cells were subsequently treated with either PE-anti-human B7H3 antibody or allotype PE-mouse antibody. Antibodies are used for treatment.

[0150] Figure 23D The expression levels of B7H3 in T47D cells treated with Pabosibi hydroxyethyl sulfonate and untreated T47D cells are shown.

[0151] Figure 23E The image shows FACS cell sorting of T47D cells treated with pabosipi hydroxyethyl sulfonate and untreated T47D cells. These two groups of cells were subsequently treated with either PE-anti-human B7H4 antibody or allotype PE-mouse antibody. Antibodies are used for treatment.

[0152] Figure 23F The expression levels of B7H4 in T47D cells treated with Pabosibi hydroxyethyl sulfonate and untreated T47D cells are shown.

[0153] Figure 23G The image shows FACS cell sorting of T47D cells treated with pabosipi hydroxyethyl sulfonate and untreated T47D cells. These two groups of cells were subsequently treated with either PE-anti-human CD54 antibody or allotype PE-mouse antibody. Antibodies are used for treatment.

[0154] Figure 23H The expression levels of CD54 in T47D cells treated with paboseib hydroxyethyl sulfonate and untreated T47D cells are shown.

[0155] Figure 23I The image shows FACS cell sorting of T47D cells treated with pabosipi hydroxyethyl sulfonate and untreated T47D cells. These two groups of cells were subsequently treated with either PE-anti-human DPP4 antibody or allotype PE-mouse antibody. Antibodies are used for treatment.

[0156] Figure 23J The expression levels of DPP4 in T47D cells treated with paboseib hydroxyethyl sulfonate and in untreated T47D cells are shown.

[0157] Figure 24A The image shows FACS cell sorting of MBA-MD-468 cells treated with pabosipi hydroxyethyl sulfonate and untreated MBA-MD-468 cells. These two groups of cells were subsequently treated with either PE-anti-human CD73 antibody or allotype PE-mouse antibody. Antibodies are used for treatment.

[0158] Figure 24B The expression levels of CD73 in MBA-MD-468 cells treated with pabosebi hydroxyethyl sulfonate and untreated MBA-MD-468 cells are shown.

[0159] Figure 24C The image shows FACS cell sorting of MBA-MD-468 cells treated with pabosipi hydroxyethyl sulfonate and untreated MBA-MD-468 cells. These two groups of cells were subsequently treated with either PE-anti-human B7H3 antibody or allotype PE-mouse antibody. Antibodies are used for treatment.

[0160] Figure 24D The expression levels of B7H3 in MBA-MD-468 cells treated with pabosebi hydroxyethyl sulfonate and untreated MBA-MD-468 cells are shown.

[0161] Figure 24E The image shows FACS cell sorting of MBA-MD-468 cells treated with pabosipi hydroxyethyl sulfonate and untreated MBA-MD-468 cells. These two groups of cells were subsequently treated with either PE-anti-human B7H4 antibody or allotype PE-mouse antibody. Antibodies are used for treatment.

[0162] Figure 24F The expression levels of B7H4 in MBA-MD-468 cells treated with paboseib hydroxyethyl sulfonate and untreated MBA-MD-468 cells are shown.

[0163] Figure 24G The image shows FACS cell sorting of MBA-MD-468 cells treated with pabosipi hydroxyethyl sulfonate and untreated MBA-MD-468 cells. These two groups of cells were subsequently treated with either PE-anti-human CD54 antibody or allotype PE-mouse antibody. Antibodies are used for treatment.

[0164] Figure 24HThe expression levels of CD54 in MBA-MD-468 cells treated with pabosebi hydroxyethyl sulfonate and untreated MBA-MD-468 cells are shown.

[0165] Figure 24I The image shows FACS cell sorting of MBA-MD-468 cells treated with pabosipi hydroxyethyl sulfonate and untreated MBA-MD-468 cells. These two groups of cells were subsequently treated with either PE-anti-human DPP4 antibody or allotype PE-mouse antibody. Antibodies are used for treatment.

[0166] Figure 24J The expression levels of DPP4 in MBA-MD-468 cells treated with pabosebi hydroxyethyl sulfonate and untreated MBA-MD-468 cells are shown.

[0167] Figure 25A Antibody-dependent cell-mediated cytotoxicity (ADCC) activity of cells was demonstrated by detecting CD73-ADCC in untreated MCF-7 cells at pH 6.0.

[0168] Figure 25B ADCC activity in cells was demonstrated by detecting CD73-ADCC in untreated MCF-7 cells at pH 7.4.

[0169] Figure 25C ADCC activity in cells was demonstrated by detecting CD73-ADCC in MCF-7 cells treated with Pabosipi at pH 6.0.

[0170] Figure 25D ADCC activity in cells was demonstrated by detecting CD73-ADCC in MCF-7 cells treated with Pabosipi at pH 7.4.

[0171] Figure 26A Antibody-dependent cell-mediated cytotoxicity (ADCC) activity of cells was demonstrated by detecting B7H4-ADCC in untreated T47D cells at pH 6.0.

[0172] Figure 26B ADCC activity in cells was demonstrated by detecting B7H4-ADCC in untreated T47D cells at pH 7.4.

[0173] Figure 26C ADCC activity in cells was demonstrated by detecting B7H4-ADCC in T47D cells treated with Pabocepi at pH 6.0.

[0174] Figure 26DADCC activity in cells was demonstrated by detecting B7H4-ADCC in T47D cells treated with Pabocepi at pH 7.4.

[0175] Figure 27A shows the CD73 variable light chain sequence of BAP-147-VL-WT (SEQ ID NO: 43), in which LCDR1 (SGSLSNIGRNPVN (SEQ ID NO: 47)), LCDR2 (LDNLRLS (SEQ ID NO: 49)) and LCD3 (ATWDDSHPGWT (SEQ ID NO: 51)) are underlined; and the CD73 variable light chain sequence of BAP-147-VL-L051D (SEQ ID NO: 44), in which (SGSLSNIGRNPVN (SEQ ID NO: 47)), LCDR2 (DDNLRLS (SEQ ID NO: 50)) and LCDR3 (ATWDDSHPGWT (SEQ ID NO: 51)) are underlined.

[0176] Figure 27B shows the CD73 variable heavy chain sequence of BAP-147-VH-WT (SEQ ID NO: 45), in which HCDR1 (GFTFSSYAYS (SEQ ID NO: 52)), HCDR2 (AISGSGGRTYYADSVKG (SEQ ID NO: 53)) and HCDR3 (LGYGRVDE (SEQ ID NO: 55)) are underlined; and the CD73 variable heavy chain sequence of BAP-147-VH-Y101E (SEQ ID NO: 46), in which HCDR1 (GFTFSSYAYS (SEQ ID NO: 52)), HCDR2 (AISGSGGRTYYADSVKG (SEQ ID NO: 53)) and HCDR3 (LGEGRVDE (SEQ ID NO: 56)) are underlined.

[0177] Figure 28A shows the B7H4 variable light chain sequence of BA-091-00-2E2 (SEQ ID NO: 57), in which LCDR1 (RVSEGIDNYGFTFIH (SEQ ID NO: 61)), LCDR2 (RASNLQS (SEQ ID NO: 62)), and LCDR3 (QQSDKDPFT (SEQ ID NO: 63)) are underlined; and the B7H4 variable light chain sequence of BA-091-02-04-04 (SEQ ID NO: 58), in which LCDR1 (RVSEGIDNYGFTFIH (SEQ ID NO: 61)), LCDR2 (RASNLQS (SEQ ID NO: 62)), and LCDR3 QQSDKDPFT (SEQ ID NO: 63)) are underlined.

[0178] Figure 28B shows the B7H4 variable heavy chain sequence of BA-091-00-2E2 (SEQ ID NO: 59), in which HCDR1 (GYTFTDRTIH (SEQ ID NO: 64)), HCDR2 (SIYPRDGSTKYNEKFKD (SEQ ID NO: 65)), and HCDR3 (SVGYAFDY (SEQ ID NO: 67)) are underlined; and the B7H4 variable heavy chain sequence of BA-091-02-04-04 (SEQ ID NO: 60), in which HCDR1 (GYTFTDRTIH (SEQ ID NO: 64)), HCDR2 (SIYPRDGSTKYNEKFKD (SEQ ID NO: 65)), and HCDR3 (SVGYADDY (SEQ ID NO: 68)) are underlined.

[0179] definition To facilitate understanding of the embodiments provided herein, some frequently used methods and / or terms will be defined.

[0180] The following terms are defined identically to those in WO 2016 / 138071: “approximately”, “activity”, “pharmaceutical”, “ambiguous base requirement”, “amino acid”, “amplification”, “chimeric property”, “cognate”, “comparison window”, “conserved amino acid substitution”, “corresponding to”, “degradation-effective”, “defined sequence frame”, “digestion”, “directed ligation”, “DNA shuffling”, “drug” or “drug molecule”, “effective amount”, “electrolyte”, “antigenic epitope”, “enzyme”, “evolution or evolving”, “fragment”, “derivative”, “analyte”, “full range substitution of a single amino acid”, “gene”, “genetically unstable”, “heterologous”, “homologous” or “partially homologous”, “industrial application”, “identical” or “identity”, “identity region”, “isolated”, “isolated nucleic acid”, “ligand”, “linker” or “spacer”, “microenvironment”, “molecular property to be evolved”, “mutation”, “naturally occurring”, “normal physiological conditions” or “wild-type” “Working conditions”, “nucleic acid molecules”, “nucleic acid sequence used to encode…” or “DNA coding sequence of…” or “nucleotide sequence encoding…”, “promoter sequence”, “nucleic acid encoding enzyme (protein)” or “DNA encoding enzyme (protein)” or “polynucleotide encoding enzyme (protein)”, “specific type of nucleic acid molecule”, “assembling working nucleic acid samples into a nucleic acid library”, “nucleic acid library”, “nucleic acid construct” or “nucleotide construct” or “DNA construct”, “construct”, “oligonucleotide” or “oligonucleotide”, “homologous”, “operable ligation”, “operable ligation to”, “parental polynucleotide combination”, “patient” or “individual”, “physiological condition”, “population”, “pro-form”, “pre-pro-form”, “pseudo-random”, “quasi-repeated unit”, “random peptide library”, “random peptide sequence”, “receptor”, “recombination”, “synthesis”, “related polynucleotide”, “reductive”“reassortment”, “reference sequence”, “comparison window”, “sequence identity”, “sequence identity percentage”, “substantial identity”, “reference sequence”, “repetition index (RI)”, “restriction site”, “optional polynucleotide”, “sequence identity”, “similarity”, “specific binding”, “specific hybridization”, “specific polynucleotide”, “strict hybridization conditions”, “substantially identical”, “substantially pure enzyme”, “substantially pure”, “therapeutic”, “variable segment”, “variant”, “wild type”, “wild type protein” or “wild type biological protein”, “parent molecule” or “target protein”, “work”, “conditional active antibody”, “antibody-dependent cell-mediated cytotoxicity” or “ADCC”, “cancer” and “cancer”, “multispecific antibody”, “full-length antibody”, “library”, “recombinant antibody” and “person” or “individual”.

[0181] As used herein, the term "antibody" refers to both complete immunoglobulin molecules capable of binding to antigenic epitopes and fragments of immunoglobulin molecules, such as Fab, Fab', (Fab')2, Fv, and SCA fragments. These antibody fragments retain some ability to selectively bind to the antigen (e.g., a polypeptide antigen) from which the antibody is derived ("antigen-binding antibody fragments"), and these antibody fragments can be prepared using methods well known in the art (see, for example, Harlow and Lane, ibid.), and these antibodies are further described below. Antibodies that can be used to implement the claimed invention may be IgG1, IgG2, IgG3, IgG4, IgM, IgA1, IgA2, sIgA, IgD, or IgE. Antibodies can be used to prepare quantities of antigen by immunoaffinity chromatography. Various other uses of such antibodies include diagnosing and / or staging diseases (e.g., tumorigenesis) and for therapeutic applications to treat diseases such as tumorigenesis, autoimmune diseases, AIDS, cardiovascular diseases, infections, etc. Administration of chimeric, human-like, humanized, or fully human antibodies to human patients is particularly useful.

[0182] Fab fragments consist of monovalent antigen-binding fragments of antibody molecules and can be generated by digesting intact antibody molecules with papain to produce fragments consisting of intact light chains and partial heavy chains.

[0183] The Fab' fragment of an antibody molecule can be obtained by the following steps: treating the intact antibody molecule with pepsin, followed by reduction to produce a molecule consisting of a complete light chain and a portion of the heavy chain. Treating each antibody molecule in this manner yields two Fab' fragments.

[0184] The (Fab')2 fragment of an antibody can be obtained by treating the intact antibody molecule with pepsinase without subsequent reduction. The (Fab')2 fragment is a dimer of two Fab' fragments linked together by two disulfide bonds.

[0185] Fv fragments are defined as genetically engineered fragments containing both light and heavy chain variable regions, represented as two strands.

[0186] Single-chain antibodies (“SCA” or scFv) are genetically engineered single-chain molecules containing variable regions on both the light and heavy chains, linked by suitable flexible polypeptide linkers, and may include additional amino acid sequences at the amino- and / or carboxyl-termini. For example, single-chain antibodies may include tether segments for linking polynucleotides. Functional single-chain antibodies typically contain a sufficient portion of the light chain variable region and a sufficient region of the heavy chain variable region to retain the properties for binding full-length antibodies to specific target molecules or epitopes.

[0187] As used herein, the term "antigen" or "Ag" is defined as a molecule capable of triggering an immune response. This immune response may involve antibody production, or activation of specific immune-active cells, or both. Those skilled in the art will understand that any macromolecule (including virtually all proteins or peptides) can be used as an antigen. It will be apparent that antigens can be generated, synthesized, or derived from biological samples. Such biological samples may include, but are not limited to, tissue samples, tumor samples, cells, or biological fluids.

[0188] As used herein, the term "apoptosis" refers to the cell death mechanism affecting individual cells, characterized by cell contraction, chromatin condensation, and cell fragmentation into membrane-bound bodies, which are eliminated through phagocytosis. The term "apoptosis" is often used synonymously with the term "programmed cell death."

[0189] As used herein, the term "apoptotic activity" refers to the intrinsic property of a compound to selectively induce apoptosis in cells of (i) a specific cell type and / or (ii) a specific developmental or differentiation stage, due to internal or external stimuli. Those skilled in the art are aware of the existence of standardized in vitro assays for determining the apoptotic-inducing activity of compounds in cell cultures, such as tests assessing cytochrome C (apoptotic marker) and TUNEL (apoptotic marker) levels in the cytoplasm. Using these standardized assays, those skilled in the art can readily assess and compare the apoptotic-inducing activity of different compounds in different cell types or cells at different developmental stages (e.g., senescent versus non-senescent cells). Other standardized apoptotic assays include annexin V assay and caspase-3 staining.

[0190] The use of the terms “biosimilar” or “follow-up biologic” is consistent with the working definition issued by the U.S. Food and Drug Administration (FDA), which defines a biosimilar as “very similar” to a reference product (despite minor clinical differences in inactive components). In practice, there are no clinically meaningful differences between the reference product and the biosimilar product in terms of safety, purity, and potency (Public Health Service (PHS) § 262). A biosimilar can also be a biosimilar that meets one or more of the guidelines adopted by the European Medicines Agency’s Committee on Medicinal Products for Human Use (CHMP) on 30 May 2012 and published by the European Union as “Guidelines for Biosimilars Containing Monoclonal Antibodies – Non-clinical and Clinical Issues” (document cited as EMA / CHMP / BMWP / 403543 / 2010). For example, an “antibody biosimilar” refers to a follow-up version of an innovator antibody (reference antibody) typically manufactured by a different company. Differences between antibody biosimilars and reference antibodies can include post-translational modifications, such as by attaching other biochemical groups like phosphate esters, various lipids, and carbohydrates to the antibody; by post-translational proteolytic cleavage; by altering the chemical properties of amino acids (e.g., formylation); or by many other mechanisms. Other post-translational modifications may result from manufacturing process operations—for example, glycosylation can occur when the product is exposed to reducing sugars. In some cases, storage conditions may allow certain degradation pathways (such as oxidation, deamidation, or aggregation) to occur. Because all these product-related variants can be included in antibody biosimilars...

[0191] The terms “cancer” and “cancerous” refer to or describe a physiological condition in mammals typically characterized by unregulated cell growth / proliferation. A “tumor” contains one or more cancer cells. Examples of cancer include, but are not limited to, carcinoma, lymphoma, blastoma, sarcoma, and leukemia or lymphoma. More specific examples of such cancers include squamous cell carcinoma (e.g., epithelial squamous cell carcinoma), lung cancer (including small cell lung cancer, non-small cell lung cancer (“NSCLC”), lung adenocarcinoma, and lung squamous cell carcinoma), peritoneal cancer, hepatocellular carcinoma, gastric cancer or stomach cancer (including gastrointestinal cancer), pancreatic cancer, glioblastoma, cervical cancer, ovarian cancer, liver cancer, bladder cancer, hepatoma, breast cancer, colon cancer, rectal cancer, colorectal cancer, endometrial cancer or uterine cancer, salivary gland cancer, kidney cancer or renal cancer, prostate cancer, vulvar cancer, thyroid cancer, liver cancer, anal cancer, penile cancer, and head and neck cancer.

[0192] The term "conditionally active protein" refers to a variant or mutant of a parent protein that exhibits higher or lower activity under one or more abnormal conditions compared to a control or its equivalent under normal physiological conditions. This conditionally active protein also exhibits activity in selected areas of the body and / or increased or decreased activity under abnormal or permissible physiological conditions. Normal physiological conditions are those considered within the normal range at a particular site in an individual (e.g., at the site of administration or in a tissue or organ at the site of action). Abnormal conditions are those that deviate from the normal acceptable range at that site. In one aspect, a conditionally active protein is practically inactive under normal physiological conditions but active under abnormal or permissible conditions. For example, in one aspect, an evolved conditionally active protein is practically inactive at body temperature but active at lower or higher temperatures. In another aspect, a conditionally active protein may be reversibly or irreversibly inactivated under normal physiological or control conditions. In another aspect, a conditionally active protein is a therapeutic protein. In another aspect, a conditionally active protein is used as a drug or therapeutic agent. On the other hand, conditionally active proteins are more or less active in highly oxygenated blood (e.g., after flowing through the lungs) or in the lower pH environment found in the kidneys. Conditionally active proteins can be conditionally active biological proteins.

[0193] As used herein, the term "cyclic peptide" refers to a polypeptide chain in which its amino and carboxyl terms (i.e., between the α-carboxyl group of one residue and the α-amino group of another residue) are linked together by peptide bonds to form a cyclic chain. For the purposes of this application, cyclic peptides may also include bonds other than peptide bonds, such as non-α-amide bonds, and thioether bonds between Trp residues and Cys residues. The length of a cyclic peptide may be 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. Furthermore, cyclic peptides may contain amino acids other than naturally occurring amino acids, such as β-alanine, phenylglycine, and homoarginine.

[0194] As used in this article, the abbreviation "DRI" refers to the D retro inversoisoform of an L-peptide, in which the amino acid sequence is reversed compared to a fragment or full-length segment of the native or wild-type protein, and at least a portion of the amino acid residues in the DRI peptide are D amino acid residues, rather than L amino acid residues found in the native or wild-type protein. Figure 7 D-reverse peptides can be prepared by identifying a fragment or full-length amino acid sequence of a natural protein, reversing that sequence, and synthesizing the D-reverse peptide using known methods to provide a peptide having a sequence opposite to the fragment or full-length amino acid sequence of the natural protein and containing a sufficient number of D amino acids to provide the desired function.

[0195] The terms “disease or symptom where the removal of senescent cells is beneficial,” “disease or symptom associated with the presence of senescent cells,” and “disorder where the removal of senescent cells is beneficial” are used interchangeably to refer to any disease or symptom in mammals (e.g., humans) in which the removal or elimination or reduction of the vitality of senescent cells is beneficial to the individual suffering from said disease or symptom. This term includes situations where senescent cells are one or the sole cause of the disease or contribute to its progression. This term also relates to situations where senescent cells may in the future become a cause of the disease or symptom in said individual. For example, treatment of a disease or symptom in which the removal of senescent cells is beneficial involves preventing, halting, or alleviating the disease or symptom by removing senescent cells. For example, chemotherapy agents and radiation therapy are known to induce cellular senescence. These senescent cells are removed to prevent the onset of a disease or symptom associated with cellular senescence. This term also includes diseases or symptom in which the removal of senescent cells alleviates or reduces the symptoms of the disease or symptom.

[0196] Removing senescent cells is beneficial if it can cure or prevent diseases or conditions, or if it can alleviate or reduce the symptoms of diseases or conditions. The removal of senescent cells can be achieved by inducing apoptosis in them. For example, the removal of senescent cells is beneficial for diseases or conditions selected from atherosclerosis, chronic inflammatory diseases (such as arthritis or arthropathy), cancer, osteoarthritis, diabetes, diabetic ulcers, kyphosis, sclerosis, liver failure, cirrhosis, Hutchinson-Gilford progeria syndrome (HGPS), laminopathies, osteoporosis, dementia, cardiovascular disease, obesity, metabolic syndrome, acute myocardial infarction, emphysema, insulin sensitivity, Southern European spotted 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, urinary incontinence, hearing loss (such as deafness), vision loss (such as blindness), sleep disorders, pain (joint pain or leg pain), imbalance, fear, depression, difficulty breathing, weight loss, hair loss, muscle loss, bone density loss, weakness, and / or declining health. The removal of senescent cells is beneficial for diseases or conditions that are associated with or related to inflammation (especially chronic inflammation) in mammals (e.g., humans) or individuals, wherein the inflammation is caused or mediated by senescent cells. In some embodiments, the senescent cells causing or mediating the inflammation are at least partially co-located in the same organ or tissue affected by the disease or condition, more preferably the same tissue.

[0197] As used herein, the term "disease or condition associated with the presence of senescent cells" refers to any disease or condition in a mammal (e.g., a human) or individual in which the presence of senescent cells or cellular senescence is associated with the disease or condition in that individual. In this context, "associated with" can specifically mean that senescent cells or cellular senescence are (i) at least partly a cause of the disease or condition, or (ii) at least partly a cause of the symptoms. In some implementations, the diseases or conditions associated with the presence of senescent cells are selected from atherosclerosis, chronic inflammatory diseases (such as arthritis or arthropathy), cancer, osteoarthritis, diabetes, diabetic ulcers, kyphosis, sclerosis, liver failure, cirrhosis, Hutchinson-Gilford progeria syndrome (HGPS), laminopathies, osteoporosis, dementia, cardiovascular disease, obesity, metabolic syndrome, acute myocardial infarction, emphysema, insulin sensitivity, Southern European spotted 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, urinary incontinence, hearing loss (such as deafness), vision loss (such as blindness), sleep disorders, pain (joint pain or leg pain), imbalance, fear, depression, difficulty breathing, weight loss, hair loss, muscle loss, bone density loss, frailty, and / or declining health. The specific diseases or conditions for which the removal of senescent cells is beneficial are those related to or associated with inflammation (typically chronic inflammation) in individuals, such as mammals (e.g., humans), where the inflammation is caused or mediated by senescent cells. In some embodiments, the senescent cells causing or mediating the inflammation are at least partially co-located in the same organ or tissue as the organ or tissue affected by the disease or condition, for example, in the same tissue.

[0198] As used herein, the term "extracellular condition of senescent cells" refers to the conditions in the extracellular environment that directly surrounds one or more senescent cells, which differ from the same conditions surrounding non-senescent cells. The extracellular environment of senescent cells may include, for example, any extracellular matrix or fluid adjacent to the senescent cells.

[0199] As used herein, the terms “FOXO4 peptide” and “FOXO4 protein” refer to proteins translated from transcripts of the forkhead box protein O4 (FOXO4) gene. FOXO4 has two variants (SEQ ID NO: 1 and SEQ ID NO: 2). The term “FOXO4DRI peptide” refers to a D-trans peptide having the reverse amino acid sequence of at least one segment of the FOXO4 protein and containing some (e.g., all) of the D amino acid residues.

[0200] The term "full-length antibody" refers to an antibody that contains an antigen-binding variable region (V). H or V L Antibodies consist of a light chain constant domain (CL) and heavy chain constant domains CH1, CH2, and CH3. The constant domain can be a natural sequence constant domain (e.g., the human natural sequence constant domain) or a variant of its amino acid sequence. Full-length antibodies can be classified into different “classes” based on the amino acid sequence of their heavy chain constant domain. There are five main classes of full-length antibodies: IgA, IgD, IgE, IgG, and IgM, some of which can be further divided into “subclasses” (isotypes), such as IgG1, IgG2, IgG3, IgG4, IgA, and IgA2. The heavy chain constant domains corresponding to different antibody classes are called α, δ, ε, γ, and μ, respectively.

[0201] "Individual" or "person" refers to mammals. Mammals include, but are not limited to, domesticated animals (such as cows, sheep, cats, dogs, and horses), primates (such as humans and non-human primates such as monkeys), rabbits, and rodents (such as mice and rats).

[0202] As used herein, the term "library" refers to a collection of proteins in a single pool. Libraries can be generated using DNA recombination techniques. For example, a collection of cDNA or any other protein-coding DNA can be inserted into an expression vector to generate a protein library. Alternatively, a collection of cDNA or protein-coding DNA can be inserted into a phage genome to generate a phage display library of wild-type proteins. Collections of cDNA can be generated from selected cell populations or tissue samples, for example, using the methods disclosed by Sambrook et al. (Molecular Cloning, Cold Spring Harbor Laboratory Press, 1989). Collections of cDNA generated from selected cell types are also commercially available from suppliers such as Stratagene®. The wild-type protein libraries used herein are not collections of biological samples.

[0203] As used herein, the term "ligand" refers to a molecule that is recognized by a specific receptor and binds specifically 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., opioids, steroids, etc.), lectins, sugars, polynucleotides, nucleic acids, oligosaccharides, proteins, and monoclonal antibodies. Typically, a ligand comprises two structural parts: a first part that participates in the binding of the ligand to its receptor and a second part that does not participate in such binding.

[0204] As used herein, the term "multispecific antibody" refers to an antibody that has binding specificity to at least two different epitopes. An exemplary multispecific antibody may bind to both BBB-R and a brain antigen. Multispecific antibodies can be prepared as full-length antibodies or antibody fragments (e.g., an F(ab')2 bispecific antibody). Engineered antibodies having two, three, or more (e.g., four) functional antigen-binding sites are also considered (see, for example, US 2002 / 0004587A1).

[0205] 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 any D-amino acid, amino acids with side chains not found in nature, and peptide mimics. Examples of peptide mimics include, but are not limited to, b-peptides, g-peptides, and d-peptides; oligomers having a backbone that can adopt a helical or sheet-like conformation, such as compounds having a backbone utilizing bipyridine segments, compounds having a backbone utilizing solubilizer interactions, compounds having a backbone utilizing side-chain interactions, compounds having a backbone utilizing hydrogen bonding interactions, and compounds having a backbone utilizing metal coordination. Non-naturally occurring amino acids also include residues having side chains designed to enhance the presentation of antimicrobial peptides in biological fluids to resist nonspecific protein adsorption, and / or polymerizable side chains that can utilize non-natural amino acid residues within the peptide as monomer units to synthesize polymer brushes.

[0206] As used herein, the term "parental protein" refers to a polypeptide or protein that can be evolved using the methods of the present invention to produce a conditionally active polypeptide or protein. A parental protein can be a wild-type protein or a non-naturally occurring protein. For example, therapeutic polypeptides or proteins, or mutant or variant polypeptides or proteins, can be used as parental polypeptides or proteins. A parental protein can also be a fragment of another naturally occurring protein, a wild-type protein, a therapeutic protein, or a mutant protein. Examples of parental 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.

[0207] As used herein, the term "peptide" refers to a polymer in which the monomers are amino acids linked together by peptide bonds or disulfide bonds. A peptide can be a full-length naturally occurring amino acid chain or a fragment thereof, a mutant, or a variant, such as a selected region of the amino acid chain of interest in binding interactions. A peptide can also be a synthetic amino acid chain, or a combination of a naturally occurring amino acid chain or a fragment thereof with a synthetic amino acid chain. A fragment refers to an amino acid sequence that is part of a full-length protein, typically of about 8 amino acids to about 500 amino acids in length, preferably about 8 amino acids to about 300 amino acids, more preferably about 8 amino acids to about 200 amino acids, and even more preferably about 10 amino acids to about 50 or 100 amino acids. Furthermore, amino acids other than naturally occurring amino acids (e.g., β-alanine, phenylglycine, and homoarginine) can be included in the peptide. Non-genetically encoded amino acids that are commonly encountered can also be included in the peptide. The amino acid can be a D-optical isomer or an L-optical isomer. D-isomers are preferred for specific situations further described below. In addition, other peptide mimics are also useful, for example, in linker sequences of peptides (see Spatola, 1983, in Chemistry and Biochemistry of Amino Acids. Peptides and Proteins, Weinstein, ed., Marcel Dekker, New York, p. 267). Generally, the term "protein" is not intended to express any significant difference from the term "peptide," except that it includes structures comprising two or more polypeptide chains held together by covalent or non-covalent bonds.

[0208] As used herein, the term "protein" refers to a polymer in which the monomers are amino acids linked together by peptide bonds or disulfide bonds. A protein can be a full-length naturally occurring amino acid chain or a fragment thereof, a mutant, or a variant, such as a selected region of an amino acid chain of interest in an interaction. A protein can be a cyclic peptide, wherein the amino acid polymer forms a cyclic structure using all or part of the polymer. A protein can also be a synthetic amino acid chain, an amino acid chain containing non-natural amino acids, or a combination of a naturally occurring amino acid chain or a fragment thereof with a synthetic amino acid chain. A fragment refers to an amino acid sequence that is part of a full-length protein, typically of about 8 amino acids to about 500 amino acids in length, preferably about 8 amino acids to about 300 amino acids, more preferably about 8 amino acids to about 200 amino acids, and even more preferably about 10 amino acids to about 50 or 100 amino acids. Furthermore, amino acids other than naturally occurring amino acids (e.g., β-alanine, phenylglycine, and homoarginine) can be included in the polypeptide. Non-genetically encoded amino acids that are commonly encountered can also be included in the polypeptide. Amino acids can be D-optical isomers or L-optical isomers. D-isomers are preferred for specific cases as further described below. Additionally, other peptide mimics are also useful, for example, for linker sequences of peptides (see Spatola, 1983, in Chemistry and Biochemistry of Amino Acids. Peptides and Proteins, Weinstein, ed., Marcel Dekker, New York, p. 267). Generally, the term "protein" is not intended to express any significant difference from the term "peptide," except that it includes structures comprising two or more polypeptide chains held together by covalent or non-covalent bonds.

[0209] As used herein, the term "receptor" refers to a molecule that has an affinity for a given ligand. Receptors can be naturally occurring or synthetic molecules. Receptors can be used in their unchanged state or as an aggregate with other substances. Receptors can be directly or covalently or non-covalently linked to a binding member via a specific binding substance. Examples of receptors include, but are not limited to, antibodies, including monoclonal antibodies and antisera that react with specific antigenic determinants (e.g., viruses, cells, or other substances), cell membrane receptors, complex carbohydrates and glycoproteins, enzyme and hormone receptors. The binding of a ligand to its receptor refers to the combination of the ligand and its receptor molecules through specific molecular recognition to form a complex, which can be detected by a variety of ligand-receptor binding assays known to those skilled in the art.

[0210] As used in this article, the term "senescence" or "cellular senescence" refers to the transition from an actively dividing cell to a metabolically active, non-dividing cell. The term "senescence" also refers to the state a cell enters after multiple rounds of division, in which future cell divisions will not occur even if the cell remains metabolically active.

[0211] As used herein, the term "senescent cell" refers to a metabolically active cell that has permanently exited the cell cycle (see, for example, Campisi). Cell (Vol. 120, pp. 513-522, 2005). Senescent cells do not replicate and possess one or more of the following additional characteristics attributed to senescent cells: cell cycle arrest in the G1 phase; enlarged, flattened morphology; increased granularity; staining at pH 6 for β-galactosidase activity; senescence-associated heterochromatic foci; and characteristic gene expression partially regulated by p16 and p21. Examples of senescent cells include senescent preadipocytes, senescent endothelial cells, senescent fibroblasts, senescent neurons, senescent epithelial cells, senescent mesenchymal cells, senescent smooth muscle cells, senescent macrophages, and senescent chondrocytes.

[0212] As used herein, the term "senolytic agent" refers to an agent that selectively (preferably or to a greater extent) destroys, kills, removes, or promotes 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. Senescent agent may be a small compound or biomolecule, such as a protein or polynucleotide. In one embodiment, the senolytic agent is a senescent cell-clearing antibody or a fragment thereof. The amount and duration of use of the senolytic agent are sufficient to selectively kill mature senescent cells, but insufficient to kill (destroy, cause death) a clinically or biologically significant number of non-senescent cells. In some embodiments, the senolytic agent described herein alters at least one signaling pathway in a manner that induces (initiates, stimulates, triggers, activates, promotes) and causes (i.e., induces, causes) senescent cell death. For example, by antagonizing proteins within cell survival and / or inflammatory pathways in senescent cells, the senolytic agent may, for example, alter one or both of cell survival signaling pathways (e.g., the Akt pathway) or inflammatory pathways.

[0213] As used herein, the term "small molecule" refers to a molecule or ion with a molecular weight of less than 900 a.mu, or more preferably less than 500 a.mu, or more preferably less than 200 a.mu, or even more preferably less than 100 a.mu. In the testing and environment of this invention, small molecules can generally exist as a mixture of molecules and deprotonated ions of molecules, depending primarily on the pH of the testing or environment.

[0214] As used herein, the term "targets associated with senescent cells" refers to molecules, such as proteins, located on the surface of senescent cells (e.g., cell membrane proteins) or present in or secreted by senescent cells into the extracellular environment of senescent cells.

[0215] As used herein, the term "therapeutic protein" refers to any protein and / or polypeptide that, for example, is intended to be administered to a mammal to elicit a biological or medical response in a tissue, system, animal, or human, as sought by researchers or clinicians. Therapeutic proteins can elicit more than one biological or medical response. Examples of therapeutic proteins include antibodies, enzymes, hormones, cytokines, regulatory proteins, and fragments thereof.

[0216] As used herein, the term "therapeuticly effective amount" means any amount that, compared to a corresponding individual who has not received such an amount, results in, but is not limited to, the cure, prevention, or improvement of a disease, disorder, or side effect, or a reduction in the rate of progression of a disease or disorder. The term also includes amounts that effectively enhance normal physiological function within their scope, as well as amounts that effectively induce physiological function in a patient that enhances or contributes to the therapeutic effect of a second agent.

[0217] As used herein, the term “treatment” refers to the medical management of an individual’s (i.e., a patient’s) disease, disorder, or condition (see, for example, Stedman’s Medical Dictionary). Generally, appropriate dosages and treatment regimens provide sufficient amounts of senescent cell scavengers to achieve therapeutic and / or preventative benefits. The therapeutic benefits to an individual from the administration of the senescent cell scavengers described herein include, for example, improved clinical outcomes where the aim is to prevent or slow or delay (mitigate) adverse physiological changes associated with the disease, or to prevent or slow or delay (mitigate) the spread or severity of such disease.

[0218] As used in this article, the term "tumor microenvironment" refers to the microenvironment within a solid tumor and the microenvironment surrounding the solid tumor, which supports the growth and metastasis of tumor cells. The tumor microenvironment includes surrounding blood vessels, immune cells, fibroblasts, other cells, soluble factors, signaling molecules, extracellular matrix, and mechanotransmitters that promote tumorigenic transformation, support tumor growth and invasion, protect the tumor from host immune attack, promote therapeutic resistance, and provide niches for dormant metastases to grow. Tumors and their surrounding microenvironment are closely related and constantly interact. Tumors can influence their microenvironment by releasing extracellular signals, promoting tumor angiogenesis, and inducing peripheral immune tolerance, while immune cells within the microenvironment can influence the growth and evolution of cancer cells. See Swarts et al. “Tumor Microenvironment Complexity: Emerging Roles in Cancer Therapy,” Cancer Res , vol., 72, pp. 2473-2480, 2012; Weber et al., “The tumor microenvironment,” Surgical Oncology , vol. 21, pp. 172-177, 2012; Blagosklonny, “Antiangiogenictherapy and tumor progression,” Cancer Cell , vol. 5, pp. 13-17, 2004; Siemann, “Tumor microenvironment,” Wiley, 2010; and Bagley, “The tumormicroenvironment,” Springer, 2010. Detailed Implementation

[0219] It should be noted that, as used herein and in the appended claims, the singular forms “a” and “the” include plural references unless the context clearly specifies otherwise. Furthermore, the terms “a”, “one or more”, and “at least one” are used interchangeably herein. The terms “comprising,” “including,” “having,” and “consisting of” are also used interchangeably.

[0220] Unless otherwise stated, all figures used in the specification and claims to indicate the amount and properties of components (such as molecular weight, percentage, ratio, reaction conditions, etc.) should be understood to be modified by the term "about" in all cases, regardless of whether the term "about" is present. Therefore, unless otherwise stated, the numerical parameters set forth in the specification and claims are approximate values ​​that may vary according to the desired properties sought to be obtained according to the invention. At least, without attempting to limit the application of the equivalence principle to the scope of the claims, each numerical parameter should be interpreted at least according to the number of significant figures reported and by applying conventional rounding techniques. Although the numerical ranges and parameters describing the broad scope of the invention are approximate, in specific embodiments we report the listed values ​​as precisely as possible. However, any numerical value necessarily inherently contains some error caused by the standard deviation found in the respective test measurements of these values.

[0221] It should be understood that each component, compound, substituent or parameter disclosed herein should be interpreted as disclosed for use alone or in combination with one or more of each other component, compound, substituent or parameter disclosed herein.

[0222] It should also be understood that each amount / value or range of each component, compound, substituent, or parameter disclosed herein is to be interpreted as being disclosed in combination with each amount / value or range of any other component, compound, substituent, or parameter disclosed herein, and it is understood that for the purposes of this specification, any combination of amounts / values ​​or ranges of two or more components, compounds, substituents, or parameters disclosed herein is also disclosed in combination with each other.

[0223] It should also be understood that each range disclosed herein should be interpreted as a disclosure of each specific value within the disclosed range having the same number of significant digits. Therefore, the range 1-4 should be interpreted as an explicit disclosure of the values ​​1, 2, 3, and 4. It should also be understood that each lower limit of each range disclosed herein should be interpreted as a combination of each upper limit of each range disclosed herein and each specific value of the same component, compound, substituent, or parameter within each range. Therefore, the invention is interpreted as a disclosure of all ranges obtained by combining each lower limit of each range with each upper limit of each range or each specific value within each range, or by combining each upper limit of each range with each specific value within each range.

[0224] Furthermore, the specific volume / value of a component, compound, substituent, or parameter disclosed in the specification or examples should be interpreted as a disclosure of the lower or upper limit of a range, and thus can be combined with any other lower or upper limit or specific volume / value of the range of the same component, compound, substituent, or parameter disclosed elsewhere in this application to form a range for that component, compound, substituent, or parameter.

[0225] This invention provides a method for preparing a conditionally active protein that is active against senescent cells from a parent protein that binds to a target associated with senescent cells. The method includes the following steps: (i) Using one or more evolution techniques to evolve the DNA encoding the parental protein to produce mutant DNA; (ii) Express the mutant DNA to obtain the mutant protein; (iii) Testing the mutant protein under extracellular conditions in the senescent cells, and then under normal physiological conditions; and (iv) Select from the mutant proteins a conditionally active protein that exhibits at least one of the following properties: (a) The activity in tests under normal physiological conditions is decreased compared to the same activity of the parental protein in the same test, while the activity in tests under extracellular conditions in senescent cells is increased compared to the same activity of the conditionally active protein in tests under normal physiological conditions; and (b) The activity in the test under the normal physiological conditions is lower than that of the same parent protein in the same test, while the activity in the test under the extracellular conditions of the senescent cells is higher than that of the same parent protein in the test under the extracellular conditions of the senescent cells.

[0226] Parental proteins can be antibodies, ligands, receptors, enzymes, or fragments of any of the aforementioned substances. Examples of ligands include cytokines and their fragments, hormones and their fragments, regulatory proteins and their fragments, and growth factors and their fragments.

[0227] In the case of antibodies, ligands, or receptors, the parent protein binds to a target associated with senescent cells, and the activity can be the binding activity to the target. For enzymes, the parent protein can use at least a portion of senescent cells as its substrate, and the activity is the enzyme activity using at least a portion of senescent cells as its substrate.

[0228] In some implementations, the parent protein may be a therapeutic protein or a biosimilar.

[0229] Targets associated with senescent cells are typically proteins of senescent cells. In some instances, targets are proteins on the cell membrane of senescent cells. In some embodiments, targets are selected from DEP-1, NTAL, EBP50, STX4, VAMP3, ARMCX-3, LANCL1, B2MG, PLD3, and VPS26A. As described in WO 2015 / 181526, these proteins are considered biomarkers of senescent cells. In some implementations, the targets are selected from ITGAV, RAC1, ARHGAP1, RAPGEF1, CRKL, NCKAP1, CDC42, CAPNS2, EBP, FGF1, ISG20, KITLG, LPHN1, MAG, MEF2C, OSBPL3, PFN1, POU5F1, PPP1CB, p16INK4a, 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.

[0230] In some implementations, the target is the Fas protein or death receptor (DR). Fas is sometimes also referred to as tumor necrosis factor receptor superfamily member 6A (TNFSF6). This is a membrane receptor that is readily available from outside senescent cells. DR is a TNF-associated apoptosis-inducing ligand (TRAIL), see Guicciardi et al., “Life and death by death receptors,” FASEB J . vol. 23, pp. 1625-1637, 2009. Examples of DR include DR4 and DR5.

[0231] In some implementations, the targets associated with senescent cells are selected from proteins of the MDM2, AKT (AKT1, AKT2, and AKT3), NOTCH3, DcR2 (TNFRSF10D), and BCL-2 anti-apoptotic protein families. Proteins in this family possess 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 only the BH3 domain (BIK, BAD, BID, BIM, BMF, HRK, NOXA, and PUMA) (see, for example, Cory et al., Nature Reviews Cancer, vol. 2, pp. 647-56,2002; Cory et al., Cancer Cell , vol. 8, pp. 5-6, 2005; Adams et al, Oncogene (vol. 26, pp. 1324-1337, 2007). Further targets related to senescent cells applicable to this invention are described in Althubiti et al., Cell Death and Disease , vol. 5, p. el528, 2014.

[0232] In some embodiments, such as those described in US 2016 / 0115237, the targets associated with senescent cells are selected from misfolded proteins, including prion proteins (PrP), CD38, Notch-1, CD44, CD59, Fas ligand, TNF receptor, and EGF receptor. The target may also be p16INK4a, or a protein selected from Tables 1-3 of US 2016 / 0038576.

[0233] In some embodiments, once a target associated with senescent cells is selected, a parent protein that binds to the target can be selected. This parent protein is an enzyme that binds to the selected target and uses at least a portion of the senescent cell as a substrate, or an antibody, ligand, or receptor that binds to the target. Some examples of suitable parent proteins for use in this invention are described in the “Target Wild-type Proteins” section of WO 2016 / 138071.

[0234] As described in WO 2016 / 138071, the parent protein may be selected from a library. In some embodiments, the parent protein is selected from the library, for example, by using a test under conditions below 7.0 (e.g., in the pH range of 5.0 to below 7.0, or 5.5 to below 7.0, or 6.0 to below 7.0, or 6.2 to 6.8).

[0235] In some other embodiments, a screening solution is used to select parental proteins from a library, the screening solution being free of small molecules with a pKa of 6 to 7.5, preferably 6 to 7, more preferably 6.2 to 6.8. Examples of such small molecules are described in this application.

[0236] In some embodiments, the parent protein is an antibody. In some embodiments, the parent antibody has one or more advantageous characteristics, which are used as the parent antibody based on these advantageous characteristics. For example, in some embodiments, the parent antibody may be selected based on good binding activity under one or more extracellular conditions in senescent cells (e.g., in a pH range of 5.0 to less than 7.0).

[0237] In some implementations, parental antibodies are selected based on their binding activity to a specific epitope. Selection based on binding activity to a specific epitope can be combined with one or more other selection criteria, such as selection based on good binding activity under one or more extracellular conditions in senescent cells.

[0238] In other implementations, parental antibodies are selected based on internalization efficiency. Selection based on internalization efficiency can 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 in senescent cells.

[0239] In other embodiments, the parent antibody may exhibit similar binding activity and / or characteristics under both normal physiological and extracellular conditions of senescent cells. In such embodiments, the parent antibody is selected based on the most similar binding activity and / or the most similar combination of one or more characteristics under both normal physiological and extracellular conditions of senescent cells. For example, if the normal physiological and extracellular conditions of senescent cells are pH 7.4 and pH 6.4, respectively, then the antibody exhibiting the most similar binding activity at pH 7.4 and pH 6.4 may be selected as the parent antibody, while antibodies exhibiting less similar binding activity at pH 7.4 and pH 6.4 may not be selected.

[0240] In some embodiments, the parent protein can be a fragment of a naturally occurring protein. For example, the parent protein can 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 can be a peptide or cyclic peptide of as few as eight amino acid units.

[0241] After selecting a parental protein, the DNA encoding the parental protein is evolved using appropriate evolution techniques to generate mutant DNA. This mutant DNA can then be expressed to produce mutant proteins for screening to identify conditionally active proteins. Appropriate techniques for evolving the DNA encoding the parental protein, expressing the mutant DNA to generate mutant proteins, and screening for mutant proteins are described in WO 2016 / 138071.

[0242] As described in WO 2016 / 138071, once selected, conditionally active proteins can be synthesized optionally in the form of "mimetic" or "peptidomimetic".

[0243] Peptide expression cells can also be used to produce selected conditionally active proteins from host or organism production. To make the production process more efficient, the DNA encoding the conditionally active protein can be codon-optimized for the host or organism. Codon optimization has been previously described, for example in Narum et al., “Codon optimization of genefragments encoding Plasmodium falciparum merzoite proteins enhances DNA vaccine protein expression and immunogenicity in mice,” Infect. Immun. In , vol.69, pp. 7250-3, 2001, codon optimization in the mouse system is described; in Outchkourov et al., “Optimization of the expression of Equistatin in Pichia pastoris, protein expression and purification,” Protein Expr. Purif. In , vol. 24, pp. 18-24, 2002, it describes codon optimization in yeast systems; in Feng et al., “High level expression and mutagenesis of recombinant human phosphatidylcholine transfer protein using asynthetic gene: evidence for a C-terminal membrane binding domain” Biochemistry In , vol. 39, pp. 15399-409, 2000, codon optimization in E. coli is described; in Humphreys et al., “High-level periplasmic expression in Escherichia coliusing a eukaryotic signal peptide: importance of codon usage at the 5' end of the coding sequence”, Protein Expr. Purif In ., vol. 20, pp. 252-64, 2000, it describes how codon usage affects protein secretion in E. coli.

[0244] The cell production host can be a mammalian cell production host selected from one of the following cell lines: CHO, HEK293, IM9, DS-I, THP-I, Hep G2, COS, NIH 3T3, C33a, A549, A375, SK-MEL-28, DU145, 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; as well as mouse spleen cells and rabbit PBMCs. A mammalian cell production host, for example, is selected from the CHO or HEK293 cell lines. In one specific aspect, the mammalian cell production host is the CHO-S cell line. In another implementation, the mammalian cell production host is the HEK293 cell line.

[0245] In some implementations, the cell production host is a yeast cell, such as *Saccharomyces cerevisiae*. S. cerevisiae The cells are either Pichia pastoris cells or Pichia cells. In some embodiments, the cell production host is a prokaryotic cell, such as Escherichia coli. E. coli (Owens, RJ and Young, RJ, J. Immunol. Meth ., vol. 168, p.149,1994; Johnson S and Bird RE, Methods Enzymol ., vol. 203, p.88, 1991). Conditionally active proteins can also be produced in plant cells or plants (Firek et al., ). Plant Mol. Biol (vol. 23, p.861, 1993).

[0246] As described in WO 2016 / 138071, conditionally active proteins can be modified by natural processes or using chemical modification techniques. Also as described in WO 2016 / 138071, conditionally active proteins can be synthesized using solid-phase chemical peptide synthesis methods.

[0247] Conditionally active proteins can be selected using tests under extracellular conditions of senescent cells and / or under normal physiological conditions. The selected conditionally active proteins exhibit at least one of the following properties: (a) The activity in tests under normal physiological conditions is decreased compared to the same activity of the parental protein in the same test, while the activity in tests under extracellular conditions in senescent cells is increased compared to the same activity of the conditionally active protein in tests under normal physiological conditions; and (b) The activity in the test under the normal physiological conditions is lower than that of the same parent protein in the same test, while the activity in the test under the extracellular conditions of the senescent cells is higher than that of the same parent protein in the test under the extracellular conditions of the senescent cells.

[0248] The conditions are the same, but the conditions have different values ​​in the extracellular conditions of senescent cells compared to the tests under normal physiological conditions. For example, the condition can be pH. The pH value under normal physiological conditions can be 7.2-7.8 or 7.2-7.6, while the pH value under the extracellular conditions of senescent cells can be 6.0-7.0 or 6.2-6.8.

[0249] The activity can be any activity related to the treatment of any senescent cells, such as the binding activity of a conditionally active antibody to a target or specific epitope, the internalization efficiency of a protein, or for an enzyme, the activity can be, for example, the enzymatic activity of a conditionally active enzyme on at least a portion of the senescent cells as a substrate.

[0250] The extracellular conditions of senescent cells are selected from one or more differences arising in the extracellular environment immediately adjacent to the senescent cells, resulting from comparisons of specific characteristics of senescent cells with, for example, those of normal cells. A set of specific characteristics of senescent cells that can be used in this invention is the metabolic activity of senescent cells. For example, senescent cells may exhibit one or more of the following specific characteristics: (1) growth arrest in senescent cells is essentially permanent and cannot be reversed by known physiological stimuli; (2) the size of senescent cells increases, sometimes more than twice the size of non-senescent cells; (3) senescent cells express senescence-associated β-galactosidase (SAP-gal), which partially reflects an increase in lysosomal mass; (4) many senescent cells express p16INK4a, which is not typically expressed by quiescent or terminally differentiated cells; (5) some senescent cells with persistent DNA damage response (DDR) signaling exhibit persistent nuclear foci. (6) DNA fragments with enhanced senescence-related chromatin alterations (DNA-SCARS, such as dysfunctional telomeres or telomere dysfunction-induced lesions (TIFs)), containing activated DDR proteins and distinguishable from transient damage lesions; (7) senescent cells express and can secrete senescence-related molecules, which can be observed in some cases in the presence of persistent DDR signaling; (8) the nucleus of senescent cells loses structural proteins (e.g., lamin B1) or chromatin-related proteins (e.g., histones and HMGB1). See, 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.

[0251] In some implementations, the extracellular condition of senescent cells is caused by a low pH resulting from 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 (Willey and Campisi, “From Ancient Pathways to Aging Cells—Connecting Metabolism and Cellular Senescence,” vol. 14, pp. 1854-71, 2015). Glycolysis involves the breakdown of glucose to form two pyruvates and two ATP molecules, of which pyruvates can be converted to lactate and excreted, thereby lowering the pH of 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 analogous to the tumor microenvironment, where glycolytic metabolism in cancer cells lowers the pH of the tumor microenvironment. Therefore, the extracellular conditions of senescent cells can be an acidic pH 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, which ranges from about 7.2 to about 7.8, preferably about 7.2 to about 7.6, or more preferably about 7.4 to about 7.6.

[0252] In some implementations, the extracellular condition of senescent cells can be a low concentration of deoxyribonucleotides compared to the normal physiological concentrations found 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). Compared to the extracellular concentration of deoxynucleotides in the extracellular environment of normal cells, some senescent cells may lose the ability to synthesize deoxynucleotides, resulting in a lower concentration of deoxynucleotides in the extracellular environment of senescent cells. Therefore, relative to the normal physiological concentration of the same deoxynucleotide in the extracellular environment of normal cells, the extracellular conditions of senescent cells can be selected as a lower concentration of deoxynucleotides, and the corresponding normal physiological conditions are the concentration of the same deoxynucleotide in the extracellular environment of normal cells.

[0253] In some implementations, the extracellular conditions of senescent cells can be characterized by lower oxygen concentrations compared to the physiological oxygen concentrations 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). Compared with non-senescent cells, senescent cells have increased oxygen consumption, which may result in a lower concentration of oxygen in the extracellular environment of senescent cells compared to that of normal cells. Therefore, the extracellular conditions of senescent cells can be selected as an oxygen concentration lower than the normal physiological concentration of oxygen in the extracellular environment of normal cells, and the corresponding normal physiological condition is the oxygen concentration in the extracellular environment of normal cells.

[0254] In some implementations, the extracellular condition of senescent cells can be a lower NAD+ / NADH ratio than that of normal cells in their extracellular environment (Wiley and Campisi, “From Ancient Pathways to Aging Cells—Connecting Metabolism and Cellular Senescence”). Cell Metab(., vol. 23, pp. 1013-21, 2016). Therefore, the extracellular conditions of senescent cells can be selected as a lower NAD+ / NADH ratio than the normal physiological ratio in the extracellular environment of normal cells, and the corresponding normal physiological conditions are the normal NAD+ / NADH ratio in the extracellular environment of normal cells.

[0255] In some embodiments, the extracellular conditions of senescent cells may be characterized by an increased concentration of the same redox homeostatic metabolite in the extracellular environment compared to the normal concentration of the same redox homeostatic metabolite in the extracellular environment of normally growing fused or quiescent cells. This redox homeostatic metabolite is selected from taurine, cysteine ​​sulfinic acid, cysteine-glutathione disulfide, γ-glutamyl alanine, γ-glutamyl methionine, pyridoxate, γ-glutamyl glutamine, and alanine (James et al., “Senescent human fibroblasts show increased glycolysis and redox homeostasis with extracellular metabolomes that overlap with those of irreparable DNA damage, aging, and disease,”). J Proteome Res (vol. 14, pp. 1854-71, 2015). Therefore, the extracellular conditions of senescent cells can be selected as an increase in the concentration of redox homeostatic metabolites (relative to the normal physiological concentration of the same redox homeostatic metabolite in the extracellular environment of normal cells, which can be selected from fused or quiescent cells in growth), and the corresponding normal physiological condition is the concentration of redox homeostatic metabolites in the extracellular environment of normal cells.

[0256] In some embodiments, the extracellular condition of senescent cells can be an increased concentration of nucleotide metabolites (compared to the concentration of the same nucleotide metabolite in the extracellular environment of normal proliferating cells, fused cells, or quiescent cells), said nucleotide metabolites being selected from 3-ureidopropionic acid, uric acid, 7-methylguanine, and hypoxanthine (James et al., “Senescent human fibroblasts show increased glycolysis and redox homeostasis with extracellular metabolomes that overlap with those of irreparable DNA damage, aging, and disease,” J Proteome Res (., vol. 14, pp. 1854-71, 2015). Therefore, the extracellular condition of senescent cells can be selected as an increase in the concentration of nucleotide metabolites (relative to the normal physiological concentration of the same nucleotide metabolites in the extracellular environment of normal cells, which can be selected from proliferating cells, fused cells, or quiescent cells), and the corresponding normal physiological condition is the concentration of nucleotide metabolites in the extracellular environment of normal cells.

[0257] In some implementations, the extracellular condition of senescent cells can be a decrease in thymidine concentration compared to the extracellular environment of normal proliferating cells, fused 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). Therefore, the extracellular conditions of senescent cells can be selected as a decrease in thymidine concentration relative to the normal physiological concentration of thymidine in the extracellular environment of normal cells, which can be selected from proliferating cells, fused cells, or quiescent cells, and the corresponding normal physiological conditions are the concentration of thymidine in the extracellular environment of normal cells.

[0258] In some embodiments, the extracellular condition of senescent cells can be a reduced concentration of dipeptides (compared to the concentration of the same dipeptide in the extracellular environment of normal proliferating cells, fused cells, or quiescent cells), said dipeptides being selected from glycyl isoleucine, glycylvaline, glycylleucine, isoleucylglycine, and valineglycine (James et al., “Senescent human fibroblasts show increased glycolysis and redox homeostasis with extracellular metabolomes that overlap with those of irreparable DNA damage, aging, and disease,” J Proteome Res (., vol. 14, pp. 1854-71, 2015). Therefore, the extracellular conditions of senescent cells can be selected as a decrease in dipeptide concentration (relative to the normal physiological concentration of the same dipeptide in the extracellular environment of normal cells, which can be selected from proliferating cells, fused cells, or quiescent cells), and the corresponding normal physiological condition is the concentration of the same dipeptide in the extracellular environment of normal cells.

[0259] In some embodiments, the extracellular condition of senescent cells can be a reduced concentration of fatty acids (compared to the concentration of the same fatty acids in the extracellular environment of normal proliferating cells, fused cells, or quiescent cells), said fatty acids being selected from linoleic acid, dihomolinoleic acid, and 10-heptadecenoic acid (James et al., “Senescent human fibroblasts show increased glycolysis and redox homeostasis with extracellular metabolites that overlap with those of irreparable DNA damage, aging, and disease,” J Proteome Res (., vol. 14, pp. 1854-71, 2015). Therefore, the extracellular conditions of senescent cells can be selected as a decrease in the concentration of fatty acids selected from linoleic acid, dihomolinoleic acid, and 10-heptadecenoic acid (relative to the normal physiological concentration of the same fatty acids in the extracellular environment of normal cells, which can be selected from proliferating cells, fused cells, or quiescent cells), and the corresponding normal physiological conditions are the concentration of the same fatty acids in the extracellular environment of normal cells.

[0260] In some embodiments, the extracellular condition of senescent cells can be an increased concentration of phospholipid metabolites (compared to the concentration of the same phospholipid metabolite in the extracellular environment of normal proliferating cells, fused cells, or quiescent cells), said phospholipid metabolites being selected from 2-hydroxypalmitic acid, 2-hydroxystearic acid, 3-hydroxydecanoic acid, 3-hydroxyoctanoic acid, and glycerophosphocholine (James et al., “Senescent human fibroblasts show increased glycolysis and redox homeostasis with extracellular metabolomes that overlap with those ofirreparable DNA damage, aging, and disease,” J Proteome Res (., vol. 14, pp.1854-71, 2015). Therefore, the extracellular conditions of senescent cells can be selected as an increase in the concentration of phospholipid metabolites (relative to the normal physiological concentration of the same phospholipid metabolites in the extracellular environment of normal cells, which can be selected from proliferating cells, fused cells, or quiescent cells), and the corresponding normal physiological condition is the concentration of the same phospholipid metabolites in the extracellular environment of normal cells.

[0261] In some embodiments, the extracellular condition of senescent cells can be an increased concentration of amino acid metabolites (compared to the concentration of the same amino acid metabolites in the extracellular environment of normal proliferating cells, fused cells, or quiescent cells), said amino acid metabolites being selected from alanine, C-glycosyltryptophan, kynurenine, dimethylarginine, and orthithine (James et al., “Senescent human fibroblasts show increased glycolysis and redox homeostasis with extracellular metabolomes that overlap with those of irreparable DNA damage, aging, and disease,” J Proteome Res (vol. 14, pp. 1854-71, 2015). Therefore, the extracellular conditions of senescent cells can be selected as an increase in the concentration of amino acid metabolites (relative to the normal physiological concentration of the same amino acid metabolites in the extracellular environment of normal cells, which can be selected from proliferating cells, fused cells, or quiescent cells), and the corresponding normal physiological condition is the concentration of the same amino acid metabolites in the extracellular environment of normal cells.

[0262] In some implementations, the extracellular condition of senescent cells can be a decrease in phenylpyruvate concentration compared to the extracellular environment of normal proliferating cells, fused 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). Therefore, the extracellular conditions of senescent cells can be selected as a decrease in the concentration of phenylpyruvate relative to the normal physiological concentration of phenylpyruvate in the extracellular environment of normal cells, and the corresponding normal physiological conditions are the concentration of phenylpyruvate in the extracellular environment of normal cells.

[0263] In some embodiments, the extracellular condition of senescent cells can be an increased concentration of metabolites (compared to the concentration of the same metabolite in the extracellular environment of normal proliferating cells, fused cells, or quiescent cells), said metabolites being selected from fumaric acid, malonic acid, eicosapentaenoic acid, and citric acid (James et al., “Senescent human fibroblasts show increased glycolysis and redox homeostasis with extracellular metabolites that overlap with those of irreparable DNA damage, aging, and disease,” J Proteome Res (., vol. 14, pp. 1854-71, 2015). Therefore, the extracellular conditions of senescent cells can be selected as an increase in the concentration of metabolites selected from fumaric acid, malonic acid, eicosapentaenoic acid, and citric acid (relative to the normal physiological concentration of the same metabolites in the extracellular environment of normal cells), and the corresponding normal physiological condition is the concentration of the same metabolites in the extracellular environment of normal cells.

[0264] In some implementations, the extracellular condition of senescent cells can be an increased glycerophosphocholine to phosphocholine ratio (compared to the glycerophosphocholine to phosphocholine ratio in the extracellular environment of normal non-stationary cells) (Gey and Seeger, “Metabolic changes during cellular senescence investigated by proton NMR-spectroscopy”). Mech Ageing Dev. (vol. 134, pp. 130-8, 2013). Therefore, the extracellular conditions of senescent cells can be selected as an increased glycerophosphocholine to phosphocholine ratio (relative to the same glycerophosphocholine to phosphocholine ratio in the extracellular environment of normal non-stationary cells), and the corresponding normal physiological condition is the glycerophosphocholine to phosphocholine ratio in the extracellular environment of normal non-stationary cells.

[0265] Senescent cells secrete a variety of different proteins, collectively known as the senescence-associated secretory phenotype (SASP). These secreted proteins include, for example, GM-CSF, GROa, GRC-α, GRC-β, GRC-γ, IGFBP-7, IL-1α, IL-6, IL-7, IL-8, MCP-1, MCP-2, MIP-1a, MMP-1, MMP-10, MMP-3, bimodalin, ENA-78, eosinophil activation chemokine-3, GCP-2, GITR, HGF, ICAM-1, IGFBP-2, IGFBP-4, IGFBP-5, IGFBP-6, and IL-13. IL-Iβ, MCP-4, MIF, MIP-3a, MMP-12, MMP-13, MMP-14, NAP2, Oncogene M, Osteoporosis Protectant, PIGF, RANTES, sgp130, TIMP-2, TRAIL-R3, Acrp30, Angiopoietin, Axl, bFGF, BLC, BTC, CTACK, EGF-R, Fas, FGF-7, G-CSF, GDNF, HCC-4, I-309, IFN-γ, IGFBP-1, IGFBP-3, IL-1 R1, IL-11, IL-15, IL-2R-a, IL-6R, I-TAC, leptin, LIF, MMP-2, MSP-a, PAI-1, PAI-2, PDGF-BB, SCF, SDF-1, sTNF RI, sTNF RⅡ, thrombopoietin, TIMP-1, tPA, uPA, uPAR, VEGF, MCP-3, IGF-1, TGF-β3, MIP-1-δ, 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.Other proteins secreted by senescent cells include IGF-2, IGF-2R, IGFBP-3, IGFBP-7, TGF-β, WNT2, CXCR2 binding chemokines, WNT16B, SFRP2, SPINK1, ENPP5, EREG, ANGPTL4, CSGALNACT, CCL26, AREG, ANGPT1, CCK, THBD, CXCL14, NOV, GAL, NPPC, FAM150B, CST1, MUCL1, NPTX2, TMEM155, EDN1, PSG9, ADAMTS3, CD24, PPBP, CXCL3, CST2, PSG8, PCOLCE2, PSG7, TNFSF15, C17orf67, CALCA, FGF18, BMP-2, MATN3, TFP1, SERPINI 1, TNFRSF25, and IL-23A. In some implementations, the extracellular condition of senescent cells is the presence or increased concentration of one or more of these secretory proteins (compared to the concentration of the same protein in the extracellular environment of normal cells), and the normal physiological condition is the absence of the same secretory protein or the normal physiological concentration of the same secretory protein in the extracellular environment of normal cells.

[0266] The conditionally active protein of this invention can be used as a senescent cell scavenger to kill or remove senescent cells from an individual. The interaction between the conditionally active protein and senescent cells can inhibit or even kill senescent cells by suppressing cell survival signaling pathways and / or inflammatory pathways activated during cellular senescence. Inhibition of cell survival signaling pathways and / or inflammatory pathways can induce (i.e., initiate, trigger, stimulate, or somehow eliminate or prevent inhibition) cell death pathways in senescent cells, such as apoptosis, which will lead to the death of senescent cells.

[0267] 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-κB pathway.

[0268] 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 response (see, for example, Okutani et al, 2012) Am. J. Physiol. Lung Cell Mol. Physiol (., vol. 291, pp. L129-L141, 2006). Therefore, altering the conditionally active protein in the src kinase signaling pathway can change both the signaling and inflammatory pathways.

[0269] Altering cellular signaling and / or inflammatory pathways can affect the function of one or more downstream proteins, or can affect the interaction of one or more downstream proteins with other components of the corresponding cellular signaling or inflammatory pathway. For example, altering the conditionally active protein in the src kinase signaling pathway or the PBK / Akt pathway can alter the function of one or more downstream proteins in the corresponding pathway, or can affect the interaction of one or more downstream proteins with another component of the corresponding pathway (see, for example, Example 1). Figure 2 Exemplary proteins upregulated in senescent cells include P38 / MAPK, ERK1 / 2, and PBK (complex). In some embodiments, the PBK / Akt pathway, as a cell signaling pathway, is activated during senescence, and the conditionally active proteins described herein inhibit the PBK / Akt pathway to enhance or induce apoptosis in senescent cells.

[0270] Test solutions for tests of senescent cells under extracellular conditions and under normal physiological conditions may include, for example, components selected from citrate buffer (e.g., sodium citrate), phosphate buffer, bicarbonate buffer (e.g., Krebs buffer), phosphate-buffered saline (PBS) buffer, Hank's buffer, Tris buffer, HEPES buffer, etc. Other buffers suitable for testing and known to those skilled in the art may be used.

[0271] The test solution of the present invention may contain at least one component selected from inorganic compounds, ions, and organic molecules, such as components commonly found in the bodily fluids of mammals (e.g., humans or animals). These inorganic compounds, ions, and organic molecules are described in detail in WO 2016 / 138071.

[0272] Conditionally active proteins can interact with one or more inorganic compounds, ions, and organic molecules. These interactions between conditionally active proteins and components selectable from inorganic compounds, ions, and organic molecules include hydrogen bonding, hydrophobic interactions, and van der Waals interactions.

[0273] In some embodiments, the extracellular conditions for senescent cells are a lower pH ranging from 5.5 to 7.2, or from 6.0 to 7.0, or from 6.2 to 6.8, while normal physiological conditions are normal physiological pH, for example, a pH in the range of 7.2 to 7.8. The test solution for the pH of the extracellular conditions may include a component having a pKa between the lower pH of the extracellular conditions and the normal physiological pH. This pKa differs from the lower pH of the extracellular conditions by, for example, up to 0.5, 1, 1.5, 2, 2.5, or 3 units. In some embodiments, the component has a molecular weight of less than 900 a.m. and may be selected, for example, from histidine, histamine, adenosine diphosphate, adenosine triphosphate, citrate, bicarbonate, acetate, lactate, bisulfide, hydrogen sulfide, ammonium, dihydrogen phosphate, and any combination thereof.

[0274] It has been observed that some conditionally active proteins contain an increased number (or proportion) of charged amino acid residues compared to their parent proteins. 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 overexpressed in some conditionally active proteins compared to their parent proteins. As a result, conditionally active proteins are more likely to interact with charged substances in the test solution due to the increased number of charged amino acid residues in the conditionally active proteins. This, in turn, affects the activity of the conditionally active proteins.

[0275] It was also observed that certain conditionally active proteins typically exhibit different activities when different substances are present in the test solution. Substances having at least two ionization states (an uncharged or less-charged state at a given pH value, and a charged or more-charged state at different values ​​of the same condition) can alter the activity of conditionally active proteins. The charged or more-charged state of a substance can increase the interaction between the substance and the charged amino acid residues present in the conditionally active protein. This mechanism can be used to enhance the selective and / or pH-dependent activity of conditionally active proteins.

[0276] The nature of the charge on a conditionally active protein can be a factor in determining suitable substances that influence its activity. In some embodiments, the conditionally active protein may have more positively charged amino acid residues, such as lysine, arginine, and histidine, compared to the parent protein. In some embodiments, the conditionally active protein may have more negatively charged amino acid residues, such as aspartic acid and glutamic acid, compared to the parent protein. Therefore, conditionally active proteins can be selected to interact at desired levels with specific substances present in the extracellular environment of senescent cells (where activity is required), or to interact at desired levels with specific substances present under normal physiological conditions (where reduced activity is required).

[0277] The position of charged amino acid residues on a conditionally active protein can also affect its activity. For example, the proximity of charged amino acid residues to the binding site of a conditionally active protein can influence its activity.

[0278] In some implementations, the interaction between a charged substance and a conditionally active protein may lead to the formation of salt bridges between different parts of the protein, particularly charged or polarized portions. The formation of salt bridges is known to stabilize peptide structures (Donald, et al., “Salt Bridges: Geometrically Specific, Designable Interactions,”). Proteins, 79(3): 898–915, 2011; Hendsch, et al., “Do saltbridges stabilize proteins? A continuum electrostatic analysis,” Protein Science (Parak, 3:211-226, 1994). Salt bridges can stabilize or immobilize protein structures that typically undergo constant, minute structural changes known as "breathing" (Parak, "Proteins in action: the physics of structural fluctuations and conformational changes"). Curr Opin Struct Biol ., 13(5):552-557, 2003). The “breathing” of protein structure is important for protein function and its binding to its partner, because structural fluctuations allow conditionally active proteins to efficiently recognize and bind to their partner (Karplus, et al., “Molecular dynamics and protein functions,”PNAS (See, vol. 102, pp. 6679-6685, 2015). By forming salt bridges, binding sites on conditionally active proteins, especially binding pockets, may become less accessible to their mates, possibly because the salt bridge may directly prevent the mate from entering the binding site. Even if the salt bridge is far from the binding site, an allosteric effect may alter the conformation of the binding site and inhibit binding. Therefore, after the structure of a conditionally active protein is stabilized (fixed) by a salt bridge, the binding of the protein to its mate may become less active, leading to reduced activity.

[0279] A known example of how proteins and their structures are stabilized by salt bridges is hemoglobin. Structural and chemical studies have shown that at least two sets of chemical groups are involved in the formation of salt bridges: the amino terminus and side chain of histidine β146 and α122, with pKa values ​​close to pH 7. In deoxyhemoglobin, the terminal carboxylate group of β146 forms a salt bridge with a lysine residue in the α subunit of another αβ dimer. This interaction locks the side chain of histidine β146 at a position where it can participate in a salt bridge with a 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 unprotonated and does not form a salt bridge. However, as pH decreases, the side chain of histidine β146 becomes protonated, and a salt bridge forms between histidine β146 and aspartic acid β94. This stabilizes the quaternary structure of deoxyhemoglobin, leading to a greater tendency for oxygen to be released in metabolically active tissues (with lower pH). Hemoglobin exhibits pH-dependent oxygen-binding activity; at low pH, oxygen-binding activity decreases due to the formation of the salt bridge. On the other hand, at high pH, ​​oxygen-binding activity increases due to the absence of a salt bridge.

[0280] Similarly, small molecules, such as bicarbonates, can reduce the binding activity of conditionally active proteins to their mating bodies by forming salt bridges within the protein. For example, at pH values ​​below their pKa (6.4), bicarbonates are protonated and thus uncharged. Uncharged bicarbonates cannot form salt bridges and therefore have little effect on the binding of conditionally active proteins to their mating bodies. Therefore, conditionally active proteins exhibit high binding activity to their mating bodies at low pH values. On the other hand, at high pH values ​​above the pKa of bicarbonates, they ionize by losing protons, becoming negatively charged. Negatively charged bicarbonates will form salt bridges between positively charged or polarized portions of the conditionally active protein, stabilizing its structure. This will block or reduce the binding of the conditionally active protein to its mating body. Therefore, conditionally active proteins exhibit low activity at high pH values. Thus, conditionally active proteins exhibit pH-dependent activity in the presence of bicarbonates, with higher binding activity at low pH values ​​than at high pH values.

[0281] When substances such as bicarbonate are absent in the test solution, conditionally active proteins may lose their conditional activity. This may be due to the lack of salt bridges on the conditionally active protein to stabilize (fix) its structure. Therefore, the partner protein has a similar pathway to enter the binding site on the conditionally active protein at any pH, producing similar activity at the first and second pH.

[0282] It should be understood that although salt bridges (ionic bonds) are the strongest and most common way in which substances influence the activity of conditionally active proteins, other interactions between these substances and conditionally active proteins may also contribute to stabilizing (fixing) the structure of conditionally active proteins. These other interactions include hydrogen bonds, hydrophobic interactions, and van der Waals interactions.

[0283] In some embodiments, to select suitable compounds or ions as substances, the conditionally active protein is compared with a parent protein from which it evolved to determine whether the conditionally active protein has a higher proportion of negatively charged or positively charged amino acid residues. A compound with a suitable charge at normal physiological pH can then be selected to influence the activity of the conditionally active protein. For example, when the conditionally active protein has a higher proportion of positively charged amino acid residues than the parent protein, a suitable compound should generally be negatively charged at normal physiological pH to interact with the conditionally active protein. On the other hand, when the conditionally active protein has a higher proportion of negatively charged amino acid residues than the parent protein, a suitable small molecule should generally be positively charged at normal physiological pH to interact with the conditionally active protein.

[0284] Therefore, suitable substances can be inorganic or organic molecules that transition from an uncharged or less charged state at the lower pH of extracellular conditions in senescent cells to a charged or more charged state at normal physiological pH. This substance should typically have a pKa between the lower pH and normal physiological pH. For example, bicarbonate has a pKa of 6.4. Therefore, at higher pH levels, such as pH 7.4, negatively charged bicarbonate will bind to charged amino acid residues in conditionally active proteins and reduce their activity. On the other hand, at lower pH levels, such as pH 6.0–6.2, less charged bicarbonate will not bind to conditionally active proteins in the same amount, thus resulting in higher activity of the conditionally active proteins.

[0285] The pKa of disulfides is 7.05. Therefore, at higher pH levels, such as pH 7.4, the more negatively charged disulfides will bind to the positively charged amino acid residues in conditionally active proteins and reduce their activity. On the other hand, at lower pH levels, such as pH 6.0–6.8, the less charged hydrogen sulfide / disulfide will not bind to conditionally active proteins at the same level, thus allowing for higher activity of the conditionally active proteins.

[0286] Some of the substances are selected from disulfides, hydrogen sulfide, histidine, histamine, citrate, bicarbonate, acetate, and lactate. Each of these small molecules has a pKa between 6.2 and 7.0. Other suitable small molecules can be found in textbooks using the principles of this application, such as the CRC Handbook of Chemistry and Physics, 96th Edition, by CRC press, 2015; Chemical Properties Handbook, McGraw-Hill Education, 1998.

[0287] For example, substances have low molecular weight and / or relatively small conformations to ensure maximum access to small pockets on conditionally active proteins by minimizing steric hindrance. Therefore, the molecular weight of small molecules is typically less than 900 Å·mu, or more preferably less than 500 Å·mu, or more preferably less than 200 Å·mu, or even more preferably less than 100 Å·mu. For example, hydrogen sulfide, disulfides, and bicarbonates all have low molecular weight and small structures to access pockets on conditionally active proteins.

[0288] The concentration of a substance in the test solution is, for example, at or near the physiological concentration in an individual. For example, the physiological concentration of bicarbonate (in human serum) is in the range of 15 to 30 mM. Therefore, the concentration of bicarbonate in the test solution can be 10 mM to 40 mM, or 15 mM to 30 mM, or 20 mM to 25 mM, or approximately 20 mM. The physiological concentration of disulfide is also relatively low. The concentration of disulfide in the test solution can be 3 to 500 nM, or 5 to 200 nM, or 10 to 100 nM, or 10 to 50 nM.

[0289] The substance can be present at substantially the same concentration (e.g., about 20 μM for bicarbonate) in test solutions for extracellular conditions of senescent cells and test solutions for normal physiological conditions.

[0290] In some implementations, the conditionally active protein is pH-dependent when two or more different small molecules (e.g., a combination of bicarbonate and histidine) are present. Therefore, these two or more small molecules are present in the test solution.

[0291] The substances in the test solution can be formed in situ from the components of the test solution or directly contained within the test solution. For example, CO2 from the air can dissolve in the test solution to provide bicarbonate as a substance in the test solution. As another example, sodium dihydrogen phosphate can be added to the test solution to provide dihydrogen phosphate as a substance in the test solution.

[0292] When this substance is absent, conditionally active proteins may lose their pH dependence. Therefore, in the absence of this substance, conditionally active proteins can exhibit similar activity between the lower pH of extracellular conditions in senescent cells and the normal physiological pH in the absence of this substance. The same result can be achieved based on any extracellular conditions of senescent cells that differ from normal physiological conditions.

[0293] In some implementations, in the presence of accessory proteins, the activity of conditionally active proteins in the lower pH conditions of senescent cells' extracellular environment exhibits increased activity compared to the same activity at normal physiological pH. Accessory proteins can be proteins present in blood or human serum. A suitable protein may be albumin, particularly mammalian albumin, such as bovine albumin or human albumin.

[0294] In one respect, accessory proteins (such as albumin) are present in test solutions used to screen and select conditionally active proteins from mutant proteins generated in the evolutionary process. In another respect, test solutions containing accessory proteins (such as albumin) are also used to test the activity of selected conditionally active proteins under the same or different conditions.

[0295] In some embodiments, two or more of the inorganic compounds, ions, and organic molecules discussed herein are added at substantially the same concentration to two test solutions for both normal physiological conditions and extracellular conditions of senescent cells. For example, both bicarbonate and histidine are added to both test solutions.

[0296] In one implementation, human serum can be added to two test solutions used for normal physiological conditions and extracellular conditions of senescent cells at substantially the same concentration. Because human serum contains a large number of inorganic compounds, ions, and organic molecules (including proteins), the test solutions will contain a wide variety of components selected from inorganic compounds, ions, and organic molecules, present at substantially the same concentration in both test solutions.

[0297] In some other embodiments, at least one of two or more components is added at different concentrations to a test solution for extracellular conditions of normal physiological conditions and senescent cells. For example, both bicarbonate and histidine are added to the test solution. The bicarbonate concentration may differ between the test solutions, while the histidine concentration may be the same in both test solutions.

[0298] In some embodiments, test solutions can be designed to select conditionally active biological proteins that exhibit activity dependent on two or more conditions. In one exemplary embodiment, the conditionally active protein may have pH- and bicarbonate-dependent activity. The test solution used to select such a conditionally active protein may be a test solution with a pH of 7.2-7.6 and a bicarbonate concentration of 25-30 mM for normal physiological conditions. The test solution for extracellular conditions in senescent cells may have a pH of 6.4-6.8 and a bicarbonate concentration of 10-20 mM. Optionally, the test solutions for normal physiological conditions and extracellular conditions in senescent cells may also contain ions to promote binding between the mutant protein and its binding partner, thereby increasing the number of hits for the conditionally active protein.

[0299] In some embodiments, certain components of serum can be intentionally minimized or omitted from the test solution. For example, when screening for antibodies, serum components that bind to or adsorb antibodies can be minimized or omitted from the test solution. Such bound antibodies can produce false positives, including bound mutant antibodies that lack conditional activity but bind only to components present in serum under various conditions. Therefore, careful selection of test components to minimize or omit components that may bind to the mutant protein in the test can reduce the number of false positive mutant proteins that might be inadvertently identified as conditionally active positives due to binding to components in the test other than the desired mate. For example, in some embodiments screening for mutant proteins that tend to bind to components in human serum, bovine serum albumin can be used in the test solution to reduce or eliminate the possibility of false positives caused by the binding of mutant proteins to human serum components. Other similar substitutions can be made in specific cases to achieve the same objective, as is well known to those skilled in the art.

[0300] In some embodiments, the evolution step can produce a mutant protein that, in addition to the conditional activity characteristics discussed above, may also possess other desired properties. Suitable other desired properties that can be evolved may include binding affinity, expression, humanization, etc. Therefore, the present invention can be used to produce conditionally active proteins that also possess at least one or more improvements of these other desired properties.

[0301] In some implementations, one of the mutagenesis techniques disclosed herein can be used to further mutate the conditionally active protein in, for example, a second evolution step, to improve another property of the conditionally active protein, such as binding affinity, expression, humanization, etc. Following the second evolution step, the conditional activity and improved properties of the mutant protein can be screened.

[0302] In some embodiments, after evolving the parent protein to produce a mutant protein, a first conditionally active protein is selected that exhibits at least one of the following properties: (a) The activity in tests under normal physiological conditions is decreased compared to the same activity of the parental protein in the same test, while the activity in tests under extracellular conditions in senescent cells is increased compared to the same activity of the conditionally active protein in tests under normal physiological conditions; and (b) The activity in the test under the normal physiological conditions is lower than that of the same parent protein in the same test, while the activity in the test under the extracellular conditions of the senescent cells is higher than that of the same parent protein in the test under the extracellular conditions of the senescent cells.

[0303] The selected first conditionally active protein can then undergo one or more additional evolution, expression, and selection steps to select at least one second conditionally active protein that also exhibits at least one of the following properties: (a) The activity in tests under normal physiological conditions is decreased compared to the same activity of the parental protein in the same test, while the activity in tests under extracellular conditions in senescent cells is increased compared to the same activity of the conditionally active protein in tests under normal physiological conditions; and (b) The activity in tests under normal physiological conditions is reduced compared to the same activity of the parent protein in the same test, while the activity in tests under extracellular conditions in senescent cells is increased compared to the same activity of the parent protein in tests under extracellular conditions in senescent cells. The second activity may be the same as the first activity, in which case a larger ratio between the activity of the second conditionally active protein under extracellular conditions and its activity under normal physiological conditions is desired compared to the first conditionally active protein. In some embodiments, the second activity may be an activity different from the first activity, in which case an activity such as internalization efficiency or binding to a specific epitope may be the second activity.

[0304] In some embodiments, the present invention aims to produce conditionally active proteins whose activity under extracellular conditions in senescent cells is greater than 1.0 compared to their activity under normal physiological conditions (e.g., high selectivity between the two conditions). The ratio of activity or selectivity under extracellular conditions in senescent cells to activity under normal physiological conditions may be at least about 1.3:1, or at least about 2:1, or at least about 3:1, or at least about 4:1, or at least about 5:1, or at least about 6:1, or at least about 7:1, or at least about 8:1, or at least about 9:1, or at least about 10:1, or at least about 11:1, or at least about 12:1, or at least about 13:1, or at least about 14:1, or at least about 15:1, or at least about 16:1, or at least about 17:1, or at least about 18:1, or at least about 19:1, or at least about 20:1, or at least about 30:1, or at least about 40:1, or at least about 50:1, or at least about 60:1, or at least about 70:1, or at least about 80:1, or at least about 90:1, or at least about 100:1.

[0305] In one embodiment, the conditionally active protein is an antibody whose activity under extracellular conditions in senescent cells is 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.

[0306] In some embodiments, the conditionally active protein is a precursor comprising an antibody or antibody fragment (collectively, “antibody”) conjugated to a masking moiety (MM) via a linker (L). This precursor is more active in the extracellular environment of senescent cells compared to the extracellular environment of normal cells. Specifically, in the extracellular environment of normal cells, the masking moiety of the precursor masks the activity of the antibody, resulting in lower binding activity of the antibody against the target senescent cells. The masking moiety is cleaved from the antibody by a protease present in the extracellular environment of senescent cells. Therefore, the antibody is unmasked and freely binds to the target senescent cells. Thus, the binding activity of this precursor against the target senescent cells in the extracellular environment of senescent cells is increased compared to its binding activity against the same target in the extracellular environment of normal cells.

[0307] Antibody fragments that may be included in the precursor may include variable or hypervariable regions (V) of the antibody's light and / or heavy chains. L V H ), 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), BHH or BNAR type single-domain heavy chain immunoglobulins and single-domain light chain immunoglobulins.

[0308] Compared to the binding activity of the same antibody without the masking portion (e.g., after the masking portion has been cleaved from the precursor), the masking portion reduces the binding activity of the antibody in the precursor to target senescent cells. The binding activity of the antibody to target senescent cells can be reduced by the masking portion by at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or even 100%. The reduction in binding activity can persist for, for example, at least 2, 4, 6, 8, 12, 28, 24, 30, 36, 48, 60, 72, 84, or 96 hours.

[0309] In one embodiment, the masking portion (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 portion may be conjugated to the N-terminus of one or more variable regions. The masking portion and the linker form a single chain conjugated to the N-terminus of one or more variable regions. In another example, the masking portion may be conjugated to the side chains of amino acids in one or more variable regions, in which case the masking portion and the linker form a single chain conjugated to the side chains of amino acids in one or more variable regions. In yet another example, when the precursor contains only a fragment of the antibody (e.g., only the variable region), the masking portion is conjugated to the C-terminus of one or more variable regions. In some embodiments, the precursor has an MM-L-Ab structure from the N-terminus to the C-terminus. In other embodiments, the precursor has an Ab-L-MM structure from the N-terminus to the C-terminus.

[0310] In some implementations, the masking moiety can be identified by screening a diverse peptide library to find peptides that bind to one or more variable regions of the antibody (Desnoyers et al., “Tumor-specific activation of an EGFR-targeting probody enhances therapeutic index”). Sci Transl Med (Rice et al., vol. 5, 207ra144, 2013). Peptides that can specifically bind to antibodies and block antibody binding to target senescent cells when conjugated to antibodies via linkers are selected as masking moieties. Screening can be performed using known techniques, including but not limited to panning, fluorescence-activated cell sorting, and magnetic separation 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).

[0311] In some implementations, a random peptide library (e.g., a peptide 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) can be used in a screening method to identify suitable masking moieties. For example, masking moieties with specific binding affinity to antibodies can be identified by a screening procedure that includes providing a peptide backbone library (where each backbone consists of a transmembrane protein and a candidate). 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 one or more rounds of magnetically activated sorting or fluorescently activated cell sorting.

[0312] Therefore, this invention proposes that the masking portion can be specific to the antibody in the precursor. A masking portion effective for a particular antibody may not be optimal for another. Therefore, for some embodiments of this invention, it may be important to screen a diverse peptide library using the antibody in the precursor to find the optimal masking portion for the antibody.

[0313] In some embodiments, masking moieties are screened from different synthetic peptide libraries. This type of masking moieties can have a certain level of similarity to target senescent cells (the natural binding partner of the antibody). In some embodiments, the masking moieties can be modeled to mimic the natural binding partner of the antibody. For example, the natural binding partner can be modified by altering one or more amino acid residues to slightly reduce its binding activity to the antibody. In other embodiments, the sequence identity of the masking moieties to the natural binding partner of the antibody is no more than 5%, 7%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, or 80%.

[0314] The structural properties of the masking portion depend on several factors, such as the minimum amino acid sequence required for the interfering antibody to bind to target senescent cells, the size of the antibody (full-length antibody or fragment), and the length of the linker. In some embodiments, the masking portion is covalently coupled to the antibody. In one example, the antibody is coupled to the masking portion via a cysteine-cysteine ​​disulfide bond between the linker and the antibody. In another example, the antibody is coupled to the masking portion via a peptide bond between the linker and the antibody.

[0315] In some implementations, the masking portion may not specifically bind to the antibody, but rather interfere with the binding of the antibody to the target senescent cells only through one or more nonspecific interactions (e.g., steric hindrance). For example, the masking portion may be located in a precursor such that the structure of the precursor allows the masking portion to mask the antibody through charge-based interactions, thereby keeping the masking portion in place to interfere with access to the binding site on the antibody.

[0316] The precursor's linker is located between the masking portion and the antibody. The linker contains a cleavage site (CS), where a protease present in the extracellular environment of senescent cells cleaves the linker to release the masking portion from the precursor. The antibody is then exposed and can be used to bind to target senescent cells. The linker may also contain one or more flexible regions (FRs) located on one or both sides of the cleavage site. For example, the linker may have the following structures: -FR-CS-FR-, -FR-CS-, -CS-FR-, -FR-FR-CS-, -CS-FR-FR-, -FR-FR-CS-FR-, -FR-CS-FR-FR-, -FR-FR-CS-FR-FR-.

[0317] The flexible region provides conformational flexibility to the masking portion, allowing it to reach and interfere with antibody binding sites. The flexible region is primarily composed of small amino acids (e.g., glycine, serine, and alanine) with small side chains to provide maximum flexibility. Glycine and glycine-serine polymers are relatively unstructured and can therefore be used as neutral linkers between components. Glycine occupies... It has much more space than alanine and is less restricted compared to residues with longer side chains (see Scheraga, Rev. Computational Chem. (pp. 11173-11142, 1992).

[0318] Suitable flexible regions can have different lengths, such as 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, 6 to 8 amino acids, or 7 to 8 amino acids, and the length can be 1, 2, 3, 4, 5, 6 or 7 amino acids.

[0319] Exemplary flexible regions include glycine polymers (G)n, glycine-serine polymers (including, for example, (GS)n, (GGS)n, (GSGGS)n (SEQ ID NO: 14), (GSGGS)n (SEQ ID NO: 15), and (GGGS)n (SEQ ID NO: 16), 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: 17), GGSGG (SEQ ID NO: 18), GGSSG (SEQ ID NO: 19), GSGGG (SEQ ID NO: 20), GGGSG (SEQ ID NO: 21) and GSSSG (SEQ ID NO: 22), GSSGGSGGSGGSG (SEQ ID NO: 23), GSSGGSGGSGG (SEQ ID NO: 23) ID NO: 24), GSSGGSGGSGGS (SEQ ID NO: 25), GSSGGSGGSGGSGGGS (SEQ ID NO: 26), GSSGGSGGSG (SEQ ID NO: 27) or GSSGGSGGSGS (SEQ ID NO: 28), GSSGT (SEQ ID NO: 29) or GSSG (SEQ ID NO: 30).

[0320] Cleavage sites are substrates for proteases in the extracellular environment of senescent cells. Cleavage sites are typically included as part of the adaptor. However, in some cases, cleavage sites can be part of a masking portion, such that all or part of the cleavage site helps to mask antibodies when the antibody is in an inhibited, uncleaved, or masked state.

[0321] Cleavage sites can be selected based on proteases present in the extracellular environment of senescent cells. Senescent cells are known to secrete proteases, such as matrix metalloproteinases (MMPs), into their extracellular environment. Examples of MMP family members include lysosometin-1 and lysosometin-2 (MMP-3 and MMP-10, respectively) and collagenase-1 (MMP-1). Other MMPs include MMP1, MMP2, MMP7, MMP8, MMP9, MMP13, and MMP14. The natural substrates of these proteases are also known, which can help design cleavage sites used in precursors. For example, these MMPs can cleave MCP-1, MCP-2, and MCP-4, as well as IL-8. Several other CXCL / CCL family members can also be cleaved by MMP-9, MMP-2, or MMP-7. Serine proteases are also present in the extracellular environment of senescent cells. Members of serine proteases include urokinase or tissue-type plasminogen activator (uPA or tPA, respectively). See Coppe et al., “The Senescence-Associated Secretory Phenotype: The Dark Side of Tumor Suppression,” Annu Rev Pathol ., vol. 5, pp. 99-118, 2010.

[0322] In one exemplary embodiment, the cleavage site is a substrate of a matrix metalloproteinase (MMP) and can therefore be cleaved by MMP to release the masking portion. In another embodiment, the cleavage site is a substrate of serine uPA or PSA. In some embodiments, the precursor may contain more than one cleavage site, and each cleavage site may be a substrate of a different protease. Exemplary cleavage sites that may be substrates of proteases include: ADAM10, ADAM12, ADAM17, ADAMTS, ADAMTS5, BACE, caspase 1-14, cathepsin A, cathepsin B, cathepsin D, cathepsin E, cathepsin K, cathepsin S, FAP, MT1-MMP, granzyme B, guanidinobenzoic acid enzyme, serine protease (Hepsin), human neutrophil elastase, lentinan, proteolytic enzyme 2, meprin, MMP1-17, MT-SP1, enkephalin, NS3 / 4A, plasmin, PSA, PSMA, TRACE, TMPRSS 3, TMPRSS 4, and uPA. Some exemplary cleavage sites are PLGLWA (SEQ ID NO:31), which can be cleaved by MMP, and GPQGIAGQ (SEQ ID NO:32), which can be cleaved by collagenase. Other examples of cleavage sites include YGLLGIAGPPGP (SEQ ID NO:33), SPGRVVRG (SEQ ID NO:34), and VRG.

[0323] In some implementations, the antibody in the precursor is itself conditionally active. In particular, the antibody itself may exhibit higher binding activity to its target under conditions in the extracellular environment of senescent cells compared to its homologous binding activity to the same target under normal physiological conditions. Once the precursor reaches the extracellular environment of senescent cells, it provides a dual enhancement by: (1) cleaving the masked portion to release the antibody binding site from the masked portion, and (2) having an antibody with increased binding activity to the target under conditions in the extracellular environment of senescent cells compared to its binding activity under normal physiological conditions.

[0324] In one embodiment, the conditionally active protein is an antibody designed to conjugate with another pharmaceutical agent. The conditionally active antibody exhibits a high ratio of activity under extracellular conditions in senescent cells to activity under normal physiological conditions, wherein the ratio is at least about 10:1, or at least about 11:1, or at least about 12:1, or at least about 13:1, or at least about 14:1, or at least about 15:1, or at least about 16:1, or at least about 17:1, or at least about 18:1, or at least about 19:1, or at least about 20:1, 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 may be particularly important when the conjugate is, for example, toxic or radioactive, as such a conjugate ideally concentrates at the affected or treated site.

[0325] In some embodiments, the conjugating agent is a D-reverse peptide (“DRI peptide”). Due to the D amino acid in the reverse sequence, the DRI peptide can maintain the side-chain topology of the amino acids (similar to the side-chain topology of the native protein from which it is derived). Furthermore, the DRI peptide is more resistant to proteolytic degradation and therefore tends to have a much longer half-life than the native protein from which it is derived. In addition, the DRI peptide has a structure similar to that of the native protein from which it is derived. Finally, the DRI peptide has comparable bioavailability to the native protein from which it is derived. Therefore, the DRI peptide can be a functional substitute for and can compete with the native protein from which it is derived. Thus, the DRI peptide is considered a promising pharmaceutical agent.

[0326] FOXO4 is a molecular pivot that determines whether damaged cells undergo senescence or apoptosis. The FOXO protein family, including FOXO1, FOXO3, and FOXO4, 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, PLet al., Cancer Res , vol. 70, pp. 8526-8536 (2010); Essers, MA et al., EMBOJ ., vol. 23, pp. 4802-4812 (2004)). Constitutive foxo1- / - mice are embryo-lethal and foxo3- / - mice exhibit reproductive defects, but foxo4- / - mice do not show obvious defective phenotypes (Hosaka, T. et al., ., vol. 23, pp. 4802-4812 (2004)). Proc. Natl. Acad. Sci. U.S.A, vol. 101, pp. 2975-2980 (2004); Castrillon, DH etal., Science , vol. 301, pp. 215-218 (2003)). The lifespan of some conditional somatic foxo3- / - mice is slightly shortened, while this is not the case for conditional somatic foxo1- / - and foxo4- / - mice (Paik, JH et al., vol. 301, pp. 215-218 (2003)). Cell (vol. 128, pp. 309-323 (2007)). Somatic cell triploid foxo1,3,4- / - mice showed increased lymphoma size, thus indicating that the FOXO protein is functionally redundant in this respect (ibid.). However, it is noteworthy that single-cell foxo4- / - mice did not show any shortened lifespan or any change in tumor-free survival. Furthermore, unlike its counterparts FOXO1 and FOXO3, FOXO4 mRNA and protein expression increased significantly with the level of senescence-induced DNA damage.

[0327] Senescence caused by X-ray ionizing radiation (XRAY)-induced DNA damage is characterized by the formation of persistent nuclear foci called DNA-SCARS (or DNA segments with chromatin alterations that enhance senescence), which are required for growth arrest (Rodier, F. et al.). J Cell Sci (2011) , vol. 124, pp. 68-81). Under these DNA damage conditions, the use of stable short hairpin-based RNA interference (shRNA) led to the loss of FOXO4 expression, inducing apoptosis rather than senescence. This suggests that FOXO4 is a key molecular determinant of senescence or apoptosis in response to genotoxic stress.

[0328] The mechanism by which FOXO4 inhibits apoptosis, a process favoring senescence, involves its physical binding to the p53 tumor suppressor protein. p53 is well known to regulate cell fate following DNA damage (Rodier, F. et al., Nucleic Acids Res, vol.35, pp. 7475-7484 (2007)), is a major component of DNA-SCARS (Rodier, F. et al., Nat. Cell Biol ., vol. 11, pp. 973-979 (2009)). p53 can induce senescence and apoptosis, depending on its post-translational modifications and its interacting mates (Vousden, KH et al., vol. 11, pp. 973-979 (2009)). Nat. Rev. Mol. Cell Biol., vol. 8, pp. 275-283 (2007)). When phosphorylated on Ser46, p53 strongly promotes apoptosis rather than cell cycle arrest (Bulavin, DV et al., vol. 8, pp. 275-283 (2007)). EMBO J ., vol. 18, pp. 6845-6854 (1999)). However, Ser46 is phosphorylated in response to several senescence-inducing stimuli, including activated oncogenes (Feng, L. et al., Cell Cycle , vol. 5, pp. 2812-2819 (2006); Bischof, O. et al., EMBO J ., vol.21, pp. 3358-3369 (2002)). Under conditions of DNA damage, Ser46 phosphorylation of p53 is increased and interferes with HIPK2 kinase (which is responsible for Ser46 phosphorylation) (Dauth, I. et al., vol.21, pp. 3358-3369 (2002)). Cancer Res (2007) , vol. 67, pp. 2274-2279, impaired apoptotic responses induced by FOXO4 depletion. Therefore, FOXO4 inhibits apoptosis in senescent cells by suppressing the apoptotic function of p53 signaling, which is conducive to senescence. Inhibition of FOXO4, particularly its interaction with p53, will induce apoptosis in senescent cells.

[0329] The human FOXO4 protein has two variants (SEQ ID NO:1 and 2). In some embodiments, any fragment of the FOXO4 protein can be used as a basis for designing the FOXO4 DRI peptide. In one embodiment, the FOXO4 fragment contains at least a portion of the functional domains of the FOXO4 protein, such as its DNA-binding domain (SEQ ID NO:3) or p53-interacting domain (SEQ ID NO:4).

[0330] 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 conditionally active antibodies. In particular, three FOXO4 DRI peptides are preferred for effectively interfering with the interaction between FOXO4 and p53: LTLKEPASE IAQSILEAYS QNGWANRRSG GKRP (SEQ ID NO:5), LTLKEPASE IAQSILEAYS QNGWANRRSG GKRPPPRRRQ RRKKRG (SEQ ID NO:6), and SEIAQSILEAYS QNGW (SEQ ID NO:7). 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 can be substituted with L amino acid residues without significantly reducing their ability to induce apoptosis in senescent cells. These FOXO4 DRI peptides interfere with the interaction between FOXO4 and p53, thereby hindering the function of FOXO4 in inhibiting p53, which leads to apoptosis in senescent cells.

[0331] FOXO4 itself is regulated by other proteins. (Reference) Figure 8 As shown, members of the FOXO family, including FOXO4, are activated by other proteins through phosphorylation or methylation: phosphorylation by AMPK, JNK, MST1, CK1, STAT3, and p38, and methylation by PRMT1. 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. Any protein that activates FOXO4 can be the basis (i.e., a natural or wild-type protein) for designing DRI peptides that can be used in this invention. In some embodiments, the natural protein is selected from the group consisting of AMPK, JNK, MST1, CK1, STAT3, p38, and PRMT1.

[0332] Take 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 SEQ ID NO:8. DRI peptides based on JNK protein can allosterically and selectively regulate JNK by using a competitive mechanism to block access to its substrate (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)). An exemplary JNK DRI peptide is DQSRPVQPFLQLTTPRKP (SEQ ID NO:9).

[0333] In addition, activators of AMPK, JNK, MST1, CK1, STAT3, p38, and PRMT1 can also be used as native proteins for designing DRI peptides. For example, ASK1 is apoptosis signal-regulated kinase 1, which activates JNK. The GenBank accession number for human ASK1 is NP_005914. The ASK1 protein can be a native protein for designing DRI peptides. Such DRI peptides can inhibit ASK1, thereby inhibiting JNK activity, which will lead to the inhibition of FOXO4.

[0334] In some embodiments, the natural proteins used to design 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 used to design the DRI peptides of the present invention are mammalian proteins, such as primate or mouse proteins FOXO4, AMPK, JNK, MST1, CK1, STAT3, p38, PRMT1, and ASK1. It is generally understood that orthologous proteins can also function in another species, meaning that DRI peptides designed based on direct homology can function in another species. For example, a DRI peptide designed based on mouse FOXO4 may function on human FOXO4 and can therefore be used as a conjugate of the present invention for inducing apoptosis in senescent human cells.

[0335] In one embodiment, a fragment of the natural protein is used to design the DRI peptide. In another embodiment, the full length of the natural protein is used to design the DRI peptide. In these embodiments, the amino acid sequence of the DRI peptide is exactly the opposite of the amino acid sequence of a fragment or the full length of the natural proteins FOXO4, AMPK, JNK, MST1, CK1, STAT3, p38, PRMT1, and ASK1.

[0336] In some embodiments, the amino acid sequence of the DRI peptide is not exactly the reverse of a fragment or full-length amino acid sequence of the natural proteins FOXO4, AMPK, JNK, MST1, CK1, STAT3, p38, PRMT1, and ASK1. In such embodiments, the amino acid sequence of the DRI peptide may have at least 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the reverse sequence of the natural protein fragment or full-length.

[0337] DRI peptides can be, for example, small peptides used to facilitate their entry into senescent cells. In some embodiments, the DRI peptide contains the following components: 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 or more amino acid residues.

[0338] Although in some embodiments, DRI peptides consist entirely of D amino acid residues, some functional DRI peptides may contain a combination of L-amino acid residues and D amino acid residues. In some embodiments, up to 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 35%, 40%, 45%, 50%, 55%, or 60% of the amino acid residues in the DRI peptide are L-amino acid residues.

[0339] In some embodiments, the DRI peptide may also include one or more functional domains that are not part of the natural protein on which the DRI peptide is used as a basis for designing the DRI peptide. In one embodiment, the DRI peptide includes the sequence “PPRRRQRRKKRG” (SEQ ID NO:10), which promotes the entry of the DRI peptide into senescent cells to induce apoptosis. Those skilled in the art will understand that this functional domain can be replaced by any other protein domain that promotes the entry of the DRI peptide into senescent cells.

[0340] Other functional domains that may be included in the DRI peptide include cell-permeable peptides (“CPP”), such as the primary amphiphilic peptide MPG (GALFLGFLGA AGSTMGAWSQ PKKKRKV, SEQ ID NO:11), Pep-1 (KETWWETWWTEWSQPKKKRKV, SEQ ID NO:12), the secondary amphiphilic peptide CATY (Ac-GLWRALWRLLRSLWRLLWRA-Cya, SEQ ID NO:13), or octargine (R(8)).

[0341] The functional domains in the DRI peptide do not possess any apoptosis-inducing activity themselves, but can be used to enhance the apoptosis-inducing activity of another part of the DRI peptide. The functional domains contain at least 1, 2, 3, 4, 5, 6, 7, or 10 D amino acid residues, more preferably all amino acid residues in the functional domains are D amino acid residues.

[0342] If the DRI peptide can kill, eliminate, remove, inactivate, or reduce the viability of senescent cells, then the DRI peptide according to the present invention has apoptosis-inducing activity in senescent cells. In some embodiments, the DRI peptide can kill, eliminate, remove, inactivate, or reduce the viability of at least 5%, 10%, 15%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 95% of cells in a culture of senescent cells.

[0343] In some embodiments, the DRI peptide selectively exhibits apoptosis-inducing activity in senescent cells, and therefore has very low or no apoptosis-inducing activity in non-senescent cells. The ratio of the apoptosis-inducing activity of the DRI peptide in senescent cells to that in non-senescent cells is 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.

[0344] Using common sense, those skilled in the art can assess whether the DRI peptide of the present invention exhibits apoptosis-inducing activity in senescent cells using standard in vitro assays. For example, cell cultures of senescent cells can be obtained by treating cell cultures with ionizing radiation or chemotherapeutic agents and then mixing them with non-senescent cells. Other methods of providing senescent cells include (i) continuous passage until replicative senescence (= telomere shortening) occurs, (ii) the use of oxidative stressors such as H2O2 and rotenone, (iii) chromatin remodeling agents such as sodium dibutyrate, or (iv) expression of overactivated oncogenes such as RASG12V or BRAFV600E. The presence of senescent cells can be determined by detecting SA-B-GAL.

[0345] The second step involves administering the peptides of this invention to the cell culture and measuring one or more apoptosis markers, such as (i) staining the cytoplasm with cytochrome C or (ii) TUNEL staining. Cytochrome C data can be quantified by counting the number of cells (where cytochrome C has been released from mitochondria into the cytosol) (DAPI can be used to indicate cells) or (in later stages) by counting the number of cells that have completely disappeared. This test can be performed in the presence of a caspase inhibitor, so that cells about to undergo apoptosis (indicated by the release of cytochrome C into the cytosol) do not actually die, since cell death requires caspase. The benefit of this test is that it provides a cumulative count of senescence over several days (e.g., 5 days). In TUNEL staining, the percentage of TUNEL-positive cell nuclei (DAPI-positive) is counted. This can be easily done visually, but can also be done using a software tool called CellProfiler (free software).

[0346] In some embodiments, the conditionally active protein comprises a prodrug covalently bonded to a peptide linker, which in turn is conjugated to the conditionally active protein. The prodrug is a drug conjugated to the peptide linker. Due to the presence of the covalently bonded peptide linker, the drug is not in its active form. The peptide linker can be cleaved by proteases in the extracellular environment of senescent cells, thereby releasing the covalently bonded drug from the conditionally active protein in its active form.

[0347] The peptide linker between the drug and the conditionally active protein may contain the same cleavage site as used in the prodrugs described in this application (e.g., the cleavage site and VRG of SEQ ID NO: 31-34). The same protease capable of releasing the antibody from the prodrug in the extracellular environment of senescent cells will also cleave the peptide linker to release the prodrug from the conditionally active protein in the extracellular environment of senescent cells in its active form.

[0348] In some embodiments, the peptide linker can be cleaved by an enzyme (skin podase). This peptide linker contains a cleavage site for the skin podase. Some exemplary cleavage sites are: PTN, PNN, PAN, PPN, TTN, TNN, TAN, TPN, NTN, NNN, NAN, NPN, ATN, ANN, AAN, APN, TTNL (SEQ ID NO: 35), TTNA (SEQ ID NO: 36), PTNL (SEQ ID NO: 37), PTNA (SEQ ID NO: 38), PNNL (SEQ ID NO: 39), PNNA (SEQ ID NO: 40), TNNL (SEQ ID NO: 41), TNNA (SEQ ID NO: 42), NK, NL, NA, NE, ND, and NN. Drugs covalently bonded to the peptide linker in a prodrug can be cytotoxic, cell-inhibiting, or antiproliferative drugs. Examples of such drugs are as follows: Alkaloids: Docetaxel, Etoposide, Irinotecan, Paclitaxel, Teniposide, Topotecan, Vincristine, Vincristine, Vincristine.

[0349] Alkylating agents: Busulfan, Indomethacin, Piperazine, Benzotipane, Carboquinone, Metoprolol, Uretoprolol, Hexamethylmelamine, Tratamide, Triethylenephosphoramide, Triethylenethiophosphoramide, Chloranaphazine, Cyclophosphamide, Estramide, Ifosfamide, Nitrogen Mustard, Methoxymethacin Hydrochloride, Melphalan, Novembichin, Perfosfamide Phenesterine, Prednisolone, Trafenisol, Uracil Mustard, Carmustine, Chlorouracil, Formosine, Lomustine, Nimustine, Semustine, Ramustine, Dacarbazine, Mannomustine, Dibromomannitol, Dibromoeusine, Piperazine, Temozolomide.

[0350] Antibiotics and analogues: Akanamycin, Actinomycins, Atrazoserine, Bleomycin, Cactinomycin C, Carrubicin, Carzinophilin, Chromomycin, Dactinomycin D, Daunorubicin, 6-Diazo-5-oxo-L-leucine, Doxorubicin, Epirubicin, Idarubicin, Menoril, Mitomycin, Mycophenolic acid, Nogalamycine, Oligomycin, Pelosimycin, Pirarubicin, Prucidiacin, Methylmitromycin, Puromycin, Streptomycin, Streptozotocin, Tuberculin, Netostatin, Zolarubicin.

[0351] Antimetabolites: Folic acid, edaraxal, methotrexate, pirarubicin, pteroxetine, raltitrexed, trimethoprim, cladridine, fludarabine, 6-mercaptopurine, pentostatine, thiamiprine, thioguanine, ancitabine, azacitidine, 6-azouridine, carmoflurane, vidarabine, deoxyfluorouridine, ethirimol, fluorouridine, fluorouracil, gemcitabine, tegafur; Platinum complexes: carboplatin, cisplatin, miplatin, oxaliplatin; Other medications: aceglucan lactone, acridine, bisantrene, defosfamide, colchicine, acetoquinone, elenisolone, elenisolone, etoposide, etoposide, fenretinide, gallium nitrate, hydroxyurea, chlorpheniramine, mitefoxine, mitoguazone, mitoxantrone, mopidazole, nitracorine, pentostatin, phennamet, podophillinic acid 2-Ethyl-Hydrazide, procarbazine, Razoxane, Sobuzoxane, Spirogermanium, Teniposide, Tenuazonicacid, Triaziquone, 2,2',2''-trichlorotriethylamine, and ethyl carbamate (Urethan).

[0352] Drugs covalently bonded to peptide linkers in prodrugs can also be chemotherapeutic agents. Chemotherapy agents may inhibit senescent cells in different ways. Chemotherapy agents can disrupt DNA templates through alkylation, cross-linking, or double-strand cleavage of DNA. Other chemotherapeutic agents can block RNA synthesis through insertion. Some chemotherapeutic agents are spindle toxins, antimetabolites that inhibit enzyme activity, or hormones and antihormones. Chemotherapy agents can be selected from a wide variety of agents, including but not limited to alkylating agents, antimetabolites, antitumor antibiotics, vinca alkaloids, epipodophyllotoxin, nitrosoureas, hormones and antihormones, and toxins. Some examples are as follows: Examples of alkylating agents include cyclophosphamide, chlorambucil, busulfan, mefarnall, thiotepa, ifosphamide, and nitrogen mustard.

[0353] Examples of antimetabolites include methotrexate, 5-fluorouracil, cytosine arabinoside, 6-thioguanine, and 6-mercaptopurine.

[0354] Examples of antitumor antibiotics include doxorubicin, daunorubicin, doxorubicin, nimosinolone, actinomycin, bleomycin, mitomycin, and pricamycin.

[0355] Examples of vinca alkaloids and epipodophyllotoxins include vincristine, vinca alkaloid, vinca alkaloid, etoposide, and teniposide.

[0356] Examples of nitrosoureas include carmustin, lomustin, semustin, and streptozotocin.

[0357] Examples of hormones and anti-hormones include adrenocortical hormones, estrogens, anti-estrogens, progestins, aromatase inhibitors, androgens, and anti-androgens.

[0358] Examples of random synthetic agents include dacarbazine, hexamethylmelamine, hydroxyurea, mitotane, procarbazine, cisplatin, and carboplatin.

[0359] On the other hand, the present invention provides conditionally active molecules or conditionally active pharmaceutical ingredients (CAMs) that are more active under abnormal conditions than under normal physiological conditions. Conditionally active molecules are organic compounds and / or salts thereof derived from a parent organic compound with a molecular weight of less than about 3000 a.m. The parent organic compound may be a therapeutically active compound with a molecular weight of about 100 a.m. to about 1500 a.m., or about 150 a.m. to about 1250 a.m., or about 300 a.m. to about 1100 a.m., or about 400 a.m. to about 1000 a.m.

[0360] The parent organic compound can be selected from anticancer agents, antibacterial agents, immunomodulators, anti-obesity drugs, antidiabetic drugs, antifungal agents, antiviral agents, contraceptives, analgesics, anti-inflammatory drugs (such as steroids or nonsteroidal anti-inflammatory drugs (NSAIDs)), antiemetics, vasodilators, vasoconstrictors, and cardiovascular drugs. Specifically, the parent compound may include, but is not limited to, anticancer agents such as azacitidine, bendamustine, bortezomib, cisplatin, carboplatin, cyclophosphamide, carmustine, daunorubicin, doxorubicin, etoposide, fludarabine, gemcitabine, melphalan, mitomycin, oxaliplatin, pemetrexed, pentostatin, streptozotocin, thiotepa, topotecan, or vincristine; cytoprotective agents such as amifostine; antibacterial agents such as tigecycline, doxycycline, chloramphenicol, azithromycin, or cefazolin; antifungal agents such as caspofungin, micafungin, anisofungin, or voriconazole; antiviral agents such as acyclovir or ganciclovir; and antipsychotic drugs such as thiothiophene. Or midazolam; anti-ulcer agents, such as esomeprazole, lansoprazole, or pantoprazole; analgesics, such as metamizole, hydromorphone, or remifentanil; anti-inflammatory drugs, such as hydrocortisone, methylprednisolone, indomethacin, ketoprofen, or parecoxib; immunomodulators, such as methotrexate; antiemetics, such as aprepitant, dolasetron, fosapirant, granisetron, ondansetron, metoclopramide, hycosine, or promethazine; cardiovascular drugs, such as atenolol, dobutamine, or eprostol; anesthetics, such as mesobital; and pharmaceutically acceptable salts thereof, or combinations thereof.

[0361] In some embodiments, the present invention provides a method for generating conditionally active molecules from a parent organic compound. The method includes the steps of modifying a parent organic compound by introducing one or more charged groups to generate a modified organic compound; testing the modified organic compound under normal physiological conditions and under abnormal conditions; and selecting conditionally active molecules from the modified organic compounds that exhibit higher activity under abnormal conditions compared to those under normal physiological conditions.

[0362] Modification of a parent organic compound can 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 to the parent organic compound. Adding one or more partially charged or charged groups to the parent organic compound can be achieved by replacing one or more atoms (e.g., hydrogen atoms or neutral groups on the parent organic compound) with one or more partially charged or charged groups. Partially charged or charged groups can be positively or negatively charged. Examples of suitable charged groups include, but are not limited to, -COO. - -SO3- -PO4 - -PO3 - -PO2 - -BO3 - -NH2 + -NH3 + And other charged groups. Examples of suitable partially charged groups include polar groups or polar side chains.

[0363] In other embodiments, the parent organic compound can be modified by removing one or more partially charged or charged groups from the parent organic compound.

[0364] The resulting modified organic compounds are tested under normal physiological conditions and under abnormal conditions. In some embodiments, abnormal conditions are values ​​of extracellular conditions of senescent cells, such as a pH range of about 5.0 to less than 7.0, or about 5.5 to less than 7.0, or about 6.0 to less than 7.0, or about 6.2 to about 6.8. Normal physiological conditions are different values ​​of conditions in the extracellular environment of normal cells, such as a pH 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.

[0365] The activity of the modified organic compound was measured in two tests. The conditionally active molecule can be selected from modified organic compounds having at least one of the following properties: (a) The activity in the test under the normal physiological conditions is decreased compared to the same activity of the parental protein in the same test, while the activity in the test under the abnormal conditions is increased compared to the same activity of the conditionally active protein in the test under the normal physiological conditions; and (b) The activity in the test under the normal physiological conditions is reduced compared to the same activity of the parent protein in the same test, while the activity in the test under the abnormal conditions is increased compared to the same activity of the parent protein in the test under the abnormal conditions. The test solutions used for the tests under abnormal conditions and the tests under normal physiological conditions may also contain the aforementioned small molecules and / or substances.

[0366] The activity measured in both abnormal and normal physiological conditions can be the binding activity of a molecule to its target.

[0367] In some embodiments, the ratio of the activity of the conditionally active molecule under abnormal conditions to its activity under normal physiological conditions is greater than 1.0 (e.g., greater selectivity between the two conditions). The activity ratio can 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.

[0368] Conditionally active proteins can be further engineered as described in WO 2016 / 138071. Conditionally active proteins can be engineered via antibody conjugation to produce multispecific antibodies, to produce bispecific conditionally active antibodies against immune effector cell surface antigens, to produce masked conditionally active proteins, and / or to engineer the Fc region of antibodies, each of which can be as described in WO 2016 / 138071. Conditionally active proteins can also be used to engineer conditionally active viral particles, as described in WO 2015 / 175375.

[0369] The mammalian immune system uses T cells to fight substances or cells with foreign antigens. CAR-T technology uses genetic engineering methods to reprogram naturally circulating T cells by inserting a chimeric antigen receptor (CAR) into them, producing highly specific CAR-T cells. The CAR specifically binds to antigens on the surface of the target tissue, guiding the engineered CAR-T cells to the target tissue. Therefore, CAR-T cells can specifically target tumor cells, making them more effective than naturally circulating T cells. CAR-T cells can also be engineered to target senescent cells.

[0370] The CAR of this invention includes at least one antigen-specific targeting region (ASTR), an extracellular spacer domain (ESD), a transmembrane domain (TM), one or more co-stimulatory domains (CSD), and an intracellular signal transduction domain (ISD). See [link to relevant documentation]. Figure 3 and Jensen et al., “Design and implementation of adoptive therapy with chimeric antigen receptor-modified T cells,”Immunol Rev ., vol. 257, pp. 127–144, 2014. After the ASTR specifically binds to the target antigen, the ISD activates intracellular signaling in CAR-T cells. For example, the ISD can utilize the antigen-binding properties of the antibody to specifically and reliably redirect CAR-T cells to a selected target in a non-MHC-restricted manner. Non-MHC-restricted antigen recognition endows CAR-T cells with the ability to recognize senescent cells and initiate antigen processing. In one embodiment, ESD and / or CSD are optional. In another embodiment, the ASTR is bispecific, allowing it to bind specifically to two different antigens or epitopes. The conditionally active protein of the present invention can be engineered as an ASTR or a portion thereof to make the CAR more active in the extracellular environment of senescent cells. Such CARs can preferentially deliver T cells to senescent cells, thereby significantly reducing side effects caused by T cell attack on normal tissues. This allows for the use of higher doses of T cells to increase therapeutic efficacy and improve individual tolerance to treatment.

[0371] ASTRs can contain conditionally active proteins, such as antibodies, especially single-chain antibodies, or antibody fragments that specifically bind to antigens on senescent cells. Some examples of proteins suitable for ASTRs include linked cytokines (which enable recognition of cells with cytokine receptors), affinities, ligand-binding domains from naturally occurring receptors, and soluble protein / peptide ligands of receptors on senescent cells.

[0372] In some embodiments, the CAR of the present invention comprises at least two ASTRs that target at least two different antigens or two epitopes on the same antigen. In one embodiment, the CAR comprises three or more ASTRs that target at least three or more different antigens or epitopes. When multiple ASTRs are present in the CAR, the ASTRs can be arranged in tandem and separated by adaptor peptides. Figure 3 ).

[0373] In another embodiment, the ASTR comprises a diabody. In the diabody, an scFv is created with a linker peptide that is too short for the two variable regions to fold together, thereby driving the scFv to dimerize. Shorter linkers (one or two amino acids) result in the formation of trimers, known as triabove-strand antibodies. Quadruple-strand antibodies can also be used in ASTRs.

[0374] Target antigens include surface proteins found on senescent cells, such as those discussed above.

[0375] In some implementations, the extracellular septal domain and transmembrane domain can be ubiquitylation-resistant, which can enhance CAR-T cell signaling and thus enhance their activity (Kuniiet la., “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 located outside the CAR-T cell, and therefore, exposure to different conditions may conditionally induce ubiquitination resistance.

[0376] As described in detail in WO 2016 / 138071, the conditionally active proteins of the present invention may be included in pharmaceutical compositions, medical devices, kits or articles for human pharmaceutical or diagnostic purposes.

[0377] The conditionally active proteins and pharmaceutical compositions of the present invention can be used to treat senescent cell-related diseases and disorders in individuals in need, including age-related diseases and disorders. Examples of senescent cell-related conditions, disorders, or diseases that can be treated by administration of the conditionally active proteins or pharmaceutical compositions described herein include cognitive disorders (e.g., mild cognitive impairment (MCI), Alzheimer's disease and other dementias; Huntington's disease); cardiovascular diseases (e.g., atherosclerosis, diastolic dysfunction, aortic aneurysm, angina pectoris, arrhythmia, cardiomyopathy, congestive heart failure, coronary artery disease, myocardial infarction, endocarditis, hypertension, carotid artery disease, peripheral vascular disease, cardiac stress response, myocardial 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; lung diseases (e.g., idiopathic pulmonary fibrosis, chronic obstructive pulmonary disease (COPD), emphysema, obstructive pulmonary disease). Bronchiolitis, asthma; pulmonary insufficiency; inflammatory / autoimmune diseases and disorders (e.g., osteoarthritis, eczema, psoriasis, osteoporosis, mucositis, transplant-related diseases and disorders); ophthalmic diseases or conditions (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 fatigue, skin condition, sarcopenia, and herniated discs) and other age-related diseases caused by aging (e.g., radiation, chemical exposure, smoking, high-fat / high-sugar diets, and diseases / disorders caused by environmental factors); wound healing; skin nevi and fibrotic diseases and disorders (e.g., cystic fibrosis, renal fibrosis, liver fibrosis, pulmonary fibrosis, oral submucosal fibrosis, cardiac fibrosis, and pancreatic fibrosis).

[0378] In one embodiment, the present invention provides a isolated polypeptide that specifically binds to CD73, comprising: The heavy chain variable region includes three complementarity-determining regions (CDRs) with H1, H2, and H3 sequences, wherein: The H1 sequence is GFTFSSYAYS (SEQ ID NO: 52); The H2 sequence is AISGSGGRTYYADSVKG (SEQ ID NO: 53); and The H3 sequence is LGX1GRVDE (SEQ ID NO: 54); Where X1 is Y or E, and The light chain variable region includes three complementarity-determining regions (CDRs) with L1, L2, and L3 sequences, wherein: The L1 sequence is SGSLSNIGRNPVN (SEQ ID NO: 47); The L2 sequence is X2DNLRLS (SEQ ID NO: 48); and The L3 sequence is ATWDDSHPGWT (SEQ ID NO: 51). Where X2 is L or D, and The conditions are: X1 and X2 cannot be Y and L at the same time, or the combination of the above heavy chain variable region and the light chain variable region is not SEQ ID NO: 45 and 43.

[0379] In each of the foregoing embodiments, the H3 sequence may be selected from LGYGRVDE (SEQ ID NO: 55) and LGEGRVDE (SEQ ID NO: 56).

[0380] In each of the foregoing embodiments, the L3 sequence may be selected from LDNLRLS (SEQ ID NO: 49) and DDNLRLS (SEQ ID NO: 50).

[0381] In each of the foregoing embodiments, the isolated polypeptide may comprise a heavy chain variable region having a sequence selected from SEQ ID NO: 45 to 46.

[0382] In each of the foregoing embodiments, the isolated polypeptide may comprise a light chain variable region having a sequence selected from SEQ ID NO: 43 to 44.

[0383] In each of the foregoing embodiments, the isolated polypeptide may comprise a heavy chain variable region and a light chain variable region having any pair of sequences selected from SEQ ID NO: 45 and 44 and SEQ ID NO: 46 and 43.

[0384] In each of the foregoing embodiments, the isolated polypeptide may comprise a heavy chain variable region having at least 80%, 85%, 90%, 95%, 98%, or 99% identity with an amino acid sequence selected from the group consisting of amino acid sequences having SEQ ID NO: 45 to 46, and a light chain variable region having at least 80%, 85%, 90%, 95%, 98%, or 99% identity with an amino acid sequence selected from the group consisting of amino acid sequences having SEQ ID NO: 43 to 44; and wherein the isolated polypeptide specifically binds to human CD73. In each of these embodiments, the variation in the percentage of identity may be limited to regions of the polypeptide located outside the CDR.

[0385] In each of the foregoing embodiments, the isolated polypeptide may comprise a heavy chain variable region having at least 80%, 85%, 90%, 95%, 98%, or 99% identity with the amino acid sequence having SEQ ID NO: 45, and a light chain variable region having at least 80%, 85%, 90%, 95%, 98%, or 99% identity with the amino acid sequence having SEQ ID NO: 46; and wherein said isolated polypeptide specifically binds to human CD73. In each of these embodiments, the variation in the percentage of identity may be limited to regions of the polypeptide located outside the CDR.

[0386] In each of the foregoing embodiments, the isolated polypeptide may comprise a heavy chain variable region having at least 80%, 85%, 90%, 95%, 98%, or 99% identity with the amino acid sequence having SEQ ID NO: 45, and a light chain variable region having at least 80%, 85%, 90%, 95%, 98%, or 99% identity with the amino acid sequence having SEQ ID NO: 43; and wherein said isolated polypeptide specifically binds to human CD73. In each of these embodiments, the variation in the percentage of identity may be limited to regions of the polypeptide located outside the CDR.

[0387] In each of the foregoing embodiments, the isolated polypeptide may comprise a heavy chain variable region having at least 80%, 85%, 90%, 95%, 98%, or 99% identity with the amino acid sequence having SEQ ID NO: 44, and a light chain variable region having at least 80%, 85%, 90%, 95%, 98%, or 99% identity with the amino acid sequence having SEQ ID NO: 46; and wherein said isolated polypeptide specifically binds to human CD73. In each of these embodiments, the variation in the percentage of identity may be limited to regions of the polypeptide located outside the CDR.

[0388] In each of the foregoing embodiments, the isolated polypeptide may comprise a heavy chain variable region having at least 80%, 85%, 90%, 95%, 98%, or 99% identity with the amino acid sequence having SEQ ID NO: 44, and a light chain variable region having at least 80%, 85%, 90%, 95%, 98%, or 99% identity with the amino acid sequence having SEQ ID NO: 43; and wherein said isolated polypeptide specifically binds to human CD73. In each of these embodiments, the variation in the percentage of identity may be limited to regions of the polypeptide located outside the CDR.

[0389] In another embodiment, the present invention provides a conditionally active senescent cell clearance antibody or antibody fragment, which may comprise any of the aforementioned isolated polypeptides.

[0390] In each of the foregoing embodiments, the binding activity of conditionally active senescent cell clearance antibodies or antibody fragments to CD73 may be higher under extracellular conditions of senescent cells compared to normal physiological conditions. These conditions may be pH. Extracellular conditions for senescent cells may be a pH range of approximately 5.5 to 7.0, while normal physiological conditions may be a pH range of approximately 7.2 to 7.8.

[0391] In each of the foregoing embodiments, the ratio of the binding activity of the conditionally active senescent cell clearance antibody or antibody fragment to CD73 under extracellular conditions of senescent cells to its binding activity to CD73 under normal physiological conditions is at least about 1.5:1, at least about 2:1, at least about 3:1, at least about 4:1, at least about 5:1, at least about 6:1, at least about 7:1, at least about 8:1, at least about 9:1, at least about 10:1, at least about 20:1, at least about 30:1, at least about 50:1, at least about 70:1, or at least about 100:1.

[0392] In each of the foregoing embodiments, the conditionally active senescent cell clearance antibody or antibody fragment may be selected from multispecific antibodies and multispecific antibody fragments.

[0393] In each of the foregoing embodiments, the conditionally active senescent cell clearance antibody or antibody fragment may be selected from bispecific antibodies and bispecific antibody fragments.

[0394] In each of the foregoing embodiments, the conditionally active senescent cell clearance antibody or antibody fragment may be a single-chain antibody.

[0395] In another embodiment, the present invention provides a conjugate comprising any of the aforementioned conditionally active antibodies or antibody fragments conjugated to a masking moiety via a linker, wherein the masking moiety is identified by screening a diverse peptide library for peptides that bind to one or more variable regions of a conditionally active senescent cell clearance antibody. The masking moiety can reduce the activity of the conditionally active antibody binding to CD73 by at least 50%.

[0396] In another embodiment, the present invention provides a conjugate comprising any of the aforementioned conditionally active antibodies or antibody fragments conjugated to a drug via a linker, said drug being selected from cytotoxic drugs, cell growth inhibitors, and antiproliferative drugs. The linker may include a cleavage site capable of being cleaved by proteases in the extracellular environment of senescent cells.

[0397] In another embodiment, the present invention provides a conjugate comprising any of the aforementioned conditionally active antibodies or antibody fragments conjugated to an agent selected from the group consisting of: toxic agents, radioactive agents, or D-reverse peptides. The amino acid sequence of the D-reverse peptide may have at least 70% amino acid sequence identity with the reverse sequence of a fragment or full-length of a natural protein selected from FOXO4, AMPK, JNK, MST1, CK1, STAT3, p38, PRMT1, and ASK1. D-reverse peptide may contain one or more functional domains selected from PPRRRQRRKKRG (SEQ ID NO:10), GALFLGFLGA AGSTMGAWSQ PKKKRKV (SEQ ID NO:11), KETWWETWWT EWSQPKKKRKV (SEQ ID NO:12), Ac-GLWRALWRLLRSLWRLLWRA-Cya (SEQ ID NO:13), LTLRKEPASE IAQSILEAYS QNGWANRRSG GKRP (SEQ ID NO:5), LTLRKEPASE IAQSILEAYSQNGWANRRSG GKRPPPRRRQ RRKKRG (SEQ ID NO:6), SEIAQSILEAYSQNGW (SEQ ID NO:7) and octargine.

[0398] In another embodiment, the present invention provides an immune conjugate comprising any of the aforementioned conditionally active senescent cell clearance antibodies or antibody fragments.

[0399] In other embodiments, the present invention provides a pharmaceutical composition comprising any of the aforementioned polypeptides, antibodies or antibody fragments, conjugates, and immunoconjugates.

[0400] In another embodiment, the present invention provides a specific polypeptide that binds to B7H4, comprising: The heavy chain variable region includes three complementarity-determining regions (CDRs) with H1, H2, and H3 sequences, wherein: The H1 sequence is GYTFTDRTIH (SEQ ID NO: 64); The H2 sequence is SIYPRDGSTKYNEKFKD (SEQ ID NO: 65); and The H3 sequence is SVGYAX3DY (SEQ ID NO: 66); Where X3 is F or D, and The light chain variable region includes three complementarity-determining regions (CDRs) with L1, L2, and L3 sequences, wherein: The L1 sequence is RVSEGIDNYGFTFIH (SEQ ID NO: 61); The L2 sequence is RASNLQS (SEQ ID NO: 62); and The L3 sequence is QQSDKDPFT (SEQ ID NO: 63), and The conditions are: X3 cannot be F, or the combination of the heavy chain variable region and the light chain variable region is not SEQ ID NO:59 and 57.

[0401] In the above implementation scheme, the H3 sequence may be selected from SVGYAFDY (SEQ ID NO: 67) and SVGYADDY (SEQ ID NO: 68).

[0402] In each of the foregoing embodiments, the isolated polypeptide may comprise a heavy chain variable region having a sequence selected from SEQ ID NO: 57 to 58.

[0403] In each of the foregoing embodiments, the isolated polypeptide may comprise a light chain variable region having a sequence selected from SEQ ID NO: 59 to 60.

[0404] In each of the foregoing embodiments, the isolated polypeptide may comprise a heavy chain variable region having the sequence of SEQ ID NO: 58 and a light chain variable region having the sequence of SEQ ID NO: 60.

[0405] In each of the foregoing embodiments, the isolated polypeptide may comprise a heavy chain variable region having at least 80%, 85%, 90%, 95%, 98%, or 99% identity with an amino acid sequence selected from the group consisting of amino acid sequences having SEQ ID NO: 57 to 58, and a light chain variable region having at least 80%, 85%, 90%, 95%, 98%, or 99% identity with an amino acid sequence selected from the group consisting of amino acid sequences having SEQ ID NO: 59 to 60; and wherein the isolated polypeptide specifically binds to human B7H4. In each of these embodiments, the variation in the percentage of identity may be limited to regions of the polypeptide located outside the CDR.

[0406] In each of the foregoing embodiments, the isolated polypeptide may comprise a heavy chain variable region having at least 80%, 85%, 90%, 95%, 98%, or 99% identity with the amino acid sequence having SEQ ID NO: 58, and a light chain variable region having at least 80%, 85%, 90%, 95%, 98%, or 99% identity with the amino acid sequence having SEQ ID NO: 60; and wherein said isolated polypeptide specifically binds to human B7H4. In each of these embodiments, the variation in the percentage of identity may be limited to regions of the polypeptide located outside the CDR.

[0407] In another embodiment, the present invention provides a conditionally active senescent cell clearance antibody or antibody fragment, which may comprise any of the aforementioned isolated polypeptides that specifically bind to B7H4.

[0408] In each of the foregoing embodiments, the binding activity of conditionally active senescent cell clearance antibodies or antibody fragments to B7H4 may be higher under extracellular conditions of senescent cells compared to normal physiological conditions. These conditions may be pH. Extracellular conditions for senescent cells may be a pH ranging from about 5.5 to about 7.0, while normal physiological conditions may be a pH ranging from about 7.2 to about 7.8.

[0409] In each of the foregoing embodiments, the ratio of the binding activity of the conditionally active senescent cell clearance antibody or antibody fragment to B7H4 under extracellular conditions of senescent cells to its binding activity to B7H4 under normal physiological conditions is at least about 1.5:1, at least about 2:1, at least about 3:1, at least about 4:1, at least about 5:1, at least about 6:1, at least about 7:1, at least about 8:1, at least about 9:1, at least about 10:1, at least about 20:1, at least about 30:1, at least about 50:1, at least about 70:1, or at least about 100:1.

[0410] In each of the foregoing embodiments, the conditionally active senescent cell clearance antibody or antibody fragment that binds to B7H4 may be selected from multispecific antibodies and multispecific antibody fragments.

[0411] In each of the foregoing embodiments, the conditionally active senescent cell clearance antibody or antibody fragment that binds to B7H4 may be selected from bispecific antibodies and bispecific antibody fragments.

[0412] In each of the foregoing embodiments, the conditionally active senescent cell clearance antibody or antibody fragment that binds to B7H4 can be a single-chain antibody.

[0413] In another embodiment, the present invention provides a conjugate comprising any of the aforementioned conditionally active antibodies or antibody fragments conjugated to a masking portion via a linker, these antibodies or antibody fragments binding to B7H4, wherein the masking portion is identified by screening a diverse peptide library for peptides that bind to one or more variable regions of a conditionally active senescent cell clearance antibody. The masking portion can reduce the activity of the conditionally active antibody binding to B7H4 by at least 50%.

[0414] In another embodiment, the present invention provides a conjugate comprising any of the aforementioned conditionally active antibodies or antibody fragments that bind to B7H4, said antibody or antibody fragment being conjugated to a drug selected from cytotoxic drugs, cell growth inhibitors, and antiproliferative drugs. The linker may include a cleavage site capable of being cleaved by proteases in the extracellular environment of senescent cells.

[0415] In another embodiment, the present invention provides a conjugate comprising any of the aforementioned conditionally active antibodies or antibody fragments that bind to B7H4, said antibody or antibody fragment being conjugated to an agent selected from the group consisting of toxic agents, radioactive agents, or D-reverse peptides. The amino acid sequence of the D-reverse peptide may have at least 70% amino acid sequence identity with a fragment or full-length reverse sequence of a natural protein selected from FOXO4, AMPK, JNK, MST1, CK1, STAT3, p38, PRMT1, and ASK1. D-reverse peptide may contain one or more functional domains selected from PPRRRQRRKKRG (SEQ ID NO:10), GALFLGFLGA AGSTMGAWSQPKKKRKV (SEQ ID NO:11), KETWWETWWT EWSQPKKKRKV (SEQ ID NO:12), Ac-GLWRALWRLLRSLWRLLWRA-Cya (SEQ ID NO:13), LTLRKEPASE IAQSILEAYS QNGWANRRSG GKRP (SEQ ID NO:5), LTLRKEPASE IAQSILEAYS QNGWANRRSG GKRPPPRRRQ RRKKRG (SEQ ID NO:6), SEIAQSILEAYSQNGW (SEQ ID NO:7) and octargine.

[0416] In another embodiment, the present invention provides an immunoconjugate comprising any of the aforementioned conditionally active senescent cell clearance antibodies or antibody fragments that bind to B7H4.

[0417] In other embodiments, the present invention provides a pharmaceutical composition comprising any of the aforementioned polypeptides that bind to B7H4, an antibody or antibody fragment that binds to B7H4, a conjugate of an antibody or antibody fragment that binds to B7H4, and an immunoconjugate of an antibody or antibody fragment that binds to B7H4.

[0418] In more specific embodiments, methods are provided for treating diseases or disorders associated with senescent cells, which kill or remove senescent cells (i.e., mature senescent cells) associated with the disease or disorder by administering a conditionally active protein or pharmaceutical composition to an individual suffering from the disease or disorder. In some exemplary embodiments, the invention is used to treat osteoarthritis, idiopathic pulmonary fibrosis, chronic obstructive pulmonary disease (COPD), or atherosclerosis.

[0419] Individuals who may benefit from the methods described herein, including the administration of conditionally active proteins or pharmaceutical compositions (i.e., patients, individuals (human or non-human animals)) include those who may also have cancer. Individuals treated by these methods may be considered to have partial or complete remission (also known as cancer remission). As discussed in detail herein, the conditionally active proteins or pharmaceutical compositions in methods for selectively killing or removing senescent cells are not intended for the treatment of cancer, i.e., in a manner that statistically significantly kills or destroys cancer cells. Therefore, the methods disclosed herein do not include the use of conditionally active proteins or pharmaceutical compositions in a manner considered a primary therapy for cancer. Although conditionally active proteins used alone or with other chemotherapeutic agents or radiotherapeutic agents are not used in a manner sufficient to be considered a primary cancer therapy, the conditionally active proteins or pharmaceutical compositions described herein may be used in a manner intended to inhibit metastasis (e.g., for a short course of treatment). In some embodiments, the individual receiving the conditionally active protein or pharmaceutical composition does not have cancer (i.e., the individual has not been diagnosed with cancer by a person skilled in the medical field).

[0420] Cardiovascular diseases and disorders Diseases or conditions related to senescent cells that can be treated with conditionally active proteins or pharmaceutical compositions can be cardiovascular diseases. Cardiovascular diseases can be any one or more of the following: angina pectoris, arrhythmia, atherosclerosis, cardiomyopathy, congestive heart failure, coronary artery disease, carotid artery disease, endocarditis, heart attack (coronary thrombosis, myocardial infarction), hypertension / high blood pressure, aortic aneurysm, cerebral aneurysm, cardiac fibrosis, diastolic dysfunction, hypercholesterolemia / hyperlipidemia, mitral valve prolapse, peripheral vascular disease (e.g., peripheral artery disease), cardiac stress resistance, and stroke.

[0421] In some embodiments, methods are provided for treating cardiovascular diseases associated with or caused by atherosclerosis (i.e., hardening of the arteries). Cardiovascular diseases can be any one or more of atherosclerosis (e.g., coronary artery disease (CAD) and carotid artery disease), angina, congestive heart failure, and peripheral vascular diseases (e.g., peripheral artery disease (PAD)). Methods for treating cardiovascular diseases associated with or caused by atherosclerosis can reduce the likelihood of developing hypertension / high blood pressure, angina, stroke, and heart attack (i.e., coronary thrombosis, myocardial infarction (MI)). In some embodiments, methods are provided for stabilizing atherosclerotic plaques in an individual's blood vessels (e.g., arteries), thereby reducing the likelihood of thrombotic events or delaying the occurrence of thrombotic events (e.g., stroke or myocardial infarction (MI)). In some embodiments, these methods, including the administration of conditionally active proteins, can reduce (i.e., decrease) the lipid content of atherosclerotic plaques in an individual's blood vessels (e.g., arteries) and / or increase the fibrous cap thickness (i.e., increase, enhance, or promote the thickening of the fibrous cap).

[0422] In one embodiment, a method is provided for inhibiting (or reducing, shrinking, or diminishing) the formation of atherosclerotic plaques by administering a conditionally active protein or pharmaceutical composition. In other embodiments, methods are provided for reducing (shrinking, shrinking) the amount (i.e., level) of plaque. A reduction in the amount of plaque in a blood vessel (e.g., an artery) can be determined, for example, by a reduction in plaque surface area, or by a reduction in the extent or degree (e.g., percentage) of obstruction in the blood vessel (e.g., an artery), which can be determined by angiography or other visualization methods used in cardiovascular techniques. This document also provides a method for increasing (or improving, promoting, or enhancing) the stability of atherosclerotic plaques present in one or more blood vessels (e.g., one or more arteries) of an individual, the method comprising administering a conditionally active protein or pharmaceutical composition to the individual.

[0423] The effectiveness (i.e., reduction or decrease in the likelihood of the development or occurrence of cardiovascular disease) of conditionally active proteins or pharmaceutical compositions used to treat or prevent cardiovascular disease (e.g., atherosclerosis) can be readily determined by those skilled in the art of medicine and clinical practice. An individual's health status can be monitored using diagnostic methods including one or any combination of the following: physical examination, assessment and monitoring of clinical symptoms, and performance of analytical tests and methods described herein and implemented in this technical field (e.g., angiography, electrocardiography, stress testing, non-stress testing). The therapeutic effects of conditionally active proteins or pharmaceutical compositions can be analyzed using techniques known in the art, for example, by comparing the symptoms of patients receiving treatment who have cardiovascular disease or are at risk of developing cardiovascular disease with those of patients who did not receive such treatment or who received a placebo.

[0424] Inflammatory and autoimmune diseases and diseases In some implementations, senescent cell-related diseases or disorders are inflammatory diseases or disorders. For example, as a non-limiting example, osteoarthritis (i.e., reducing the likelihood of its occurrence) can be treated or prevented according to the methods described herein, including the administration of conditionally active proteins or pharmaceutical compositions. Other inflammatory or autoimmune diseases or disorders include osteoporosis, psoriasis, stomatitis, rheumatoid arthritis, inflammatory bowel disease, eczema, kyphosis, herniated discs, lung diseases, COPD, and idiopathic pulmonary fibrosis.

[0425] Surprisingly, by selectively killing senescent cells, conditionally active proteins or pharmaceutical compositions can reduce the likelihood of bone erosion: reduce or inhibit the loss or erosion of the proteoglycan layer in the joint, reduce inflammation in the affected joint, and promote (i.e., stimulate, enhance, induce) collagen production (e.g., type 2 collagen). Removal of senescent cells can lead to a decrease in the amount (i.e., level) of inflammatory cytokines (e.g., IL-6) produced in the joint, and reduce inflammation. This article provides a method for treating osteoarthritis comprising: selectively killing or removing senescent cells that may be located in the osteoarthritis-affected joints of an individual by administering at least one conditionally active protein to the individual, and / or inducing the production of collagen (e.g., type 2 collagen) in the joints of the individual in need. Conditionally active proteins can also be used to reduce (inhibit, decrease) the production of metalloproteinase 13 (MMP-13, which degrades collagen in the joint) and to restore the proteoglycan layer or inhibit the loss and / or degradation of the proteoglycan layer. Therefore, treatment with conditionally active proteins or pharmaceutical compositions can prevent or reduce the likelihood of bone erosion, inhibit or reduce bone erosion, or delay bone erosion. As described in detail herein, in some embodiments, conditionally active proteins or pharmaceutical compositions are applied directly to the joints of osteoarthritis (e.g., via intra-articular, external, transdermal, intradermal, or subcutaneous delivery). Treatment with conditionally active proteins or pharmaceutical compositions can also restore, improve, or inhibit the deterioration of joint strength. Furthermore, methods including the application of conditionally active proteins or pharmaceutical compositions can reduce joint pain and are therefore used for pain control in osteoarthritis-affected joints.

[0426] The effectiveness of one or more conditionally active proteins for the treatment or prevention of osteoarthritis in individuals and the monitoring of individuals receiving one or more senescent cell scavengers can be readily determined by a person skilled in the medical and clinical fields. An individual's health status can be monitored using diagnostic methods including one or any combination of the following: physical examination (e.g., determining tenderness, swelling, or redness of affected joints), assessment and monitoring of clinical symptoms (e.g., pain, stiffness, mobility), and execution of analytical tests and methods described herein and implemented in the art (e.g., determining levels of inflammatory cytokines or chemokines; X-ray imaging to determine cartilage loss, such as narrowing of the space between bones in a joint; magnetic resonance imaging (MRI), providing detailed images of bone and soft tissue (including cartilage)). The therapeutic effect of one or more senescent cell scavengers can be analyzed by comparing the symptoms of patients who have received treatment for an inflammatory disease or disorder (e.g., osteoarthritis) or are at risk for such disease with those who have not received such treatment or have received a placebo.

[0427] In some implementations, conditionally active proteins or pharmaceutical compositions may be used to treat and / or prevent (i.e. reduce or decrease the likelihood of occurrence) rheumatoid arthritis (RA).

[0428] Chronic inflammation can 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 senescent cell scavengers to treat kyphosis can be determined in preclinical animal models used in this field. For example, kyphosis develops in TTD mice (see, for example, de Boer et al.). Science (, vol. 296, pp. 1276-1279, 2002); other mice that can be used include BubRl H / H Mice, which are known to also develop kyphosis (see, for example, Baker et al.). Nature ( , vol. 479, pp. 232-36, 2011). Kyphosis formation was visually measured over time. The level of senescent cells reduced by treatment with senescent cell scavengers could be determined by detecting the presence of one or more senescent cell-related markers (e.g., by SA-P-Gal staining).

[0429] In other embodiments, inflammatory / autoimmune conditions, including irritable bowel syndrome (IBS) and inflammatory bowel diseases such as ulcerative colitis and Crohn's disease, can be treated or prevented (i.e., have a reduced likelihood of occurrence) using the conditionally active proteins or pharmaceutical compositions described herein. Diagnosis and monitoring of the disease are performed according to methods and diagnostic tests conventionally practiced in the art, including blood tests, colonoscopy, flexible sigmoidoscopy, barium enema, CT scans, MRI, endoscopy, and small bowel imaging.

[0430] In other embodiments, the methods described herein can be used to treat individuals with herniated discs. Individuals with herniated discs exhibit elevated levels of cellular senescence in their blood and blood vessel walls (see, for example, Roberts et al.). Eur. Spine J (15 Suppl 3: S312-316, 2006). Increased levels of pro-inflammatory molecules and matrix metalloproteinases have also been found in aging and degenerated intervertebral disc tissue, indicating the role of senescent cells (see, for example, Chang-Qing et al.). Ageing Res. Rev Animal models can be used to characterize the effectiveness of senescent cell scavengers in treating intervertebral disc herniation; and to induce intervertebral disc degeneration in mice through compression and increased disc strength (see, for example, Lotz et al., vol. 6, pp. 247-61, 2007). Spine , vol. 23, pp. 2493-506, 1998).

[0431] Other inflammatory or autoimmune diseases that can be treated or prevented (i.e., reduced in likelihood of occurrence) by using conditionally active proteins or pharmaceutical compositions include eczema, psoriasis, osteoporosis, and lung diseases (e.g., chronic obstructive pulmonary disease (COPD), idiopathic pulmonary fibrosis (IPF), asthma), inflammatory bowel disease, and mucositis (including oral mucositis, in some cases induced by radiation). Certain fibrotic or degenerative conditions of organs, such as renal fibrosis, liver fibrosis, pancreatic fibrosis, cardiac fibrosis, skin wound healing disorders, and oral submucosal fibrosis, can be treated using conditionally active proteins or pharmaceutical compositions.

[0432] In some embodiments, the senescent cell-related disorder is an inflammatory condition of the skin, such as psoriasis and eczema, which, as a non-limiting example, can be treated or prevented (i.e., have a reduced likelihood of occurrence) by the methods described herein, including the administration of conditionally active proteins or pharmaceutical compositions. The effectiveness of conditionally active proteins or pharmaceutical compositions for treating psoriasis and eczema, and the monitoring of individuals receiving such treatment, can be readily determined by a person skilled in the medical or clinical field. Diagnostic methods include one or any combination of the following: physical examination (e.g., skin appearance), assessment and / or monitoring of clinical symptoms (e.g., itching, swelling, and pain), and the execution of analytical tests and methods described herein and practiced in this art (i.e., determining the levels of pro-inflammatory cytokines).

[0433] Lung diseases and disorders In one embodiment, a method is provided for treating or preventing (i.e., reducing the likelihood of occurrence) a disease or disorder (lung disease and disorder) associated with senescent cells, wherein the method kills or removes senescent cells (i.e., mature senescent cells) associated with the disease or disorder in an individual suffering from the disease or disorder by administering a conditionally active protein or pharmaceutical composition. Age-related lung diseases and disorders include, for example, idiopathic pulmonary fibrosis (IPF), chronic obstructive pulmonary disease (COPD), asthma, cystic fibrosis, bronchiectasis, and emphysema. The incidence of IPF has been observed to increase with age, and the lung tissue of IPF patients is rich in SA-P-Gal positive cells and contains elevated levels of the senescence marker p21, suggesting that IPF is associated with cellular senescence (see, for example, Mingawa et al.). Am. J. Physiol. Lung Cell. Mol. Physiol (., vol. 300, pp. L391-L401, 2011). Short telomeres are a common risk factor for IPF and cellular senescence (see, for example, Alder et al, ). Proc. Natl. Acad. Sci. USA( , vol. 105, pp.13051-56, 2008). Not wanting to be bound by theory, reports indicate that the role of cellular senescence in IPF is that the SASP components of senescent cells (e.g., IL-6, IL-8, and IL-1β) promote fibroblast differentiation into myofibroblasts and epithelial-mesenchymal transition, leading to extensive remodeling of the extracellular matrix in the alveoli and intercellular spaces (see, for example, Mingawa et al., ibid.).

[0434] Other lung diseases or disorders that can be treated with the use of conditionally active proteins or pharmaceutical compositions include, for example, emphysema, asthma, bronchiectasis, and cystic fibrosis (see, for example, Fischer et al.). Am J Physiol Lung Cell Mol Physiol ., vol. 304, pp. L394-400, 2013).

[0435] The methods described herein for treating or preventing (i.e., reducing the likelihood of occurrence) age-related lung diseases or conditions can also be used to treat individuals who are aging and have lost (or degenerated) lung function (i.e., whose lung function is reduced or impaired compared to younger individuals) and / or whose lung tissue is degenerating. By administering a senescent cell scavenger to aging individuals (including asymptomatic middle-aged adults), the decline in lung function can be slowed or inhibited by killing and removing senescent cells from the airways. The therapeutic effects of conditionally active proteins or pharmaceutical compositions can be analyzed using techniques known in the art, such as comparing the symptoms of patients who have received treatment for lung disease or are at risk of developing such disease with those who have not received such treatment or have received a placebo. Furthermore, methods and techniques for assessing the mechanical function of the lungs can be performed, such as techniques for measuring lung volume, elasticity, and airway hypersensitivity. To determine and monitor lung function throughout treatment, any of a variety of measurements can be obtained: expiratory reserve volume (ERV), forced vital capacity (FVC), forced expiratory volume (FEV) (e.g., FEV1 in one second), FEV1 / FEV ratio, forced expiratory flow (25% to 75%), maximum spontaneous ventilation (MVV), peak expiratory flow (PEF), and slow vital capacity (SVC). Total lung volume includes total lung capacity (TLC), vital capacity (VC), residual volume (RV), and functional residual capacity (FRC). Gas exchange at the alveolar-capillary membrane can be measured using carbon monoxide diffusion capacity (DLCO). Peripheral capillary oxygen saturation (SpO2) can also be measured.

[0436] Neurological diseases and disorders Senescence-related cell diseases or conditions that can be treated with the application of conditionally active proteins or pharmaceutical compositions include neurological diseases or conditions. These senescence-related diseases and disorders include Parkinson's disease, Alzheimer's disease (and other dementias), motor neuron dysfunction (MND), mild cognitive impairment (MCI), Huntington's disease, and eye diseases and disorders such as age-related macular degeneration. Other age-related eye conditions include glaucoma, vision loss, presbyopia, and cataracts.

[0437] The aging of dopamine-producing neurons is thought to be the cause of cell death observed in PD by producing reactive oxygen species (see, for example, Cohen et al.). J. Neural Transm. Suppl . 19:89-103(1983)). Therefore, the conditionally active proteins and pharmaceutical compositions described herein can be used for the treatment and prevention of Parkinson's disease.

[0438] Methods for detecting, monitoring, or quantifying neurodegenerative and / or motor deficits associated with Parkinson's disease are known in the art, such as histological studies, biochemical studies, and behavioral assessments (see, for example, US 2012 / 0005765). Symptoms of Parkinson's disease are known in the art and include, but are not limited to, difficulty initiating or completing voluntary movements, cramps, rigidity, muscle atrophy, tremor and changes in heart rate (but normal reflexes), bradykinesia, and postural instability.

[0439] The effectiveness of the conditionally active protein or pharmaceutical composition described herein in individuals receiving one or more senescent cell scavengers can be readily determined by those skilled in the art of medicine and clinical practice. The health status of subjects can be monitored using diagnostic methods including one or any combination of the following: physical examination, assessment and monitoring of clinical symptoms, and performance of the analytical tests and methods described herein. The effects of administration of the conditionally active protein or pharmaceutical composition can be analyzed using techniques known in the art, for example, by comparing the symptoms of treated patients with Alzheimer's disease or at risk of developing Alzheimer's disease with those of patients who did not receive such treatment or received a placebo.

[0440] Mild cognitive impairment (MCI) MCI is a neurological syndrome involving the occurrence and evolution of cognitive deficits beyond what is expected based on an individual's age and education level, but not sufficient to interfere with the individual's daily activities. Administration of conditionally active proteins can 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 and include astrocyte morphology analysis, acetylcholine release, silver staining for assessing neurodegeneration, and PiB PET imaging for detecting β-amyloid deposits (see, for example, US 2012 / 0071468). Methods for detecting, monitoring, quantifying, or assessing behavioral deficits associated with MCI are also known in the art and include the eight-armed radial maze paradigm, the non-matching-to-sample task, the allocentric place determination task in a water maze, the Morris maze test, visuospatial tasks and delayed-response spatial memory tasks, and the olfactory novelty test (see ibid.).

[0441] Motor neuron dysfunction (MND) MND is a group of progressive neurological disorders that destroy motor neurons, the cells that control basic voluntary muscle activities such as speaking, walking, breathing, and swallowing. Examples of MND 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 diseases, 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 conditionally active proteins can reduce or inhibit MND by killing or removing senescent cells. Methods for detecting, monitoring, or quantifying motor deficits and / or other deficits associated with Parkinson's disease (e.g., MND) are known in the art (see, for example, US20120005765). Methods for detecting, monitoring, quantifying, or assessing motor and histopathological defects associated with MND are known in the art, including histopathological, biochemical, and electrophysiological studies and motor activity analysis (see, for example, Rich et al., J Neurophysiol , vol. 88, pp. 3293-3304, 2002; Appel et al, Proc. Natl. Acad. Sci. USA , vol. 88, pp. 647-51, 1991).

[0442] Eye diseases and disorders In some implementations, the disease or impairment associated with senescent cells is an eye disease, impairment, or condition, such as presbyopia, macular degeneration, or cataracts. In other implementations, the disease or condition associated with senescent cells is glaucoma. Macular degeneration is a neurodegenerative disease that results in the loss of photoreceptor cells in the central part of the retina (called the macula). While the exact cause of age-related macular degeneration is unclear, the number of senescent retinal pigment epithelial (RPE) cells increases with age. Age, along with certain genetic and environmental factors, are risk factors for developing ARMD (see, for example, Lyengar et al.). Am. J. Hum. Genet ., vol. 74, pp. 20-39, 2004; Kenealy et al, Mol. Vis ., vol. 10, pp. 57-61, 2004; Gorin et al, Mol. Vis (., vol. 5, p. 29, 1999). The reduction of microRNAs contributes to the senescent cell profile, while DICER1 ablation induces premature aging. Diagnosis and monitoring of individuals with macular degeneration can be performed by ophthalmologists based on established routine eye examination procedures and individual symptom reports.

[0443] Age-related changes in the mechanical properties of the anterior and posterior lens capsules indicate that the mechanical strength of the posterior lens capsule decreases significantly with age (see, for example, 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 lamellar structure of the cyst can also change and can be caused at least in part by changes in the composition of the tissue.

[0444] Studies have shown that collagen IV affects cellular function, inferred from its location on the basement membrane beneath the epithelial layer, and data support the role of collagen IV in tissue stability. Posterior capsule opacification (PCO) develops as a complication in approximately 20–40% of patients in the years following cataract surgery (see, e.g., Awasthi et al.). Arch OphthalmolPosterior capsule opacification is caused by the proliferation and activity of lens epithelial cells remaining along the posterior capsule, and its response is similar to wound healing. Growth factors (e.g., 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 in this paper, these factors and cytokines produced by senescent cells contribute to SASP. Conversely, in vitro studies have shown that collagen IV promotes lens epithelial cell adhesion (see, for example, Olivero et al., vol. 127, pp. 555-62, 2009). Invest. Ophthalmol. Vis. Sci (., vol. 34, pp. 2825-34, 1993). Collagen IV, fibronectin, and laminin adhere to the intraocular lens, inhibiting cell migration and reducing the risk of PCO (see, for example, Rajet al., 1993). Int. J. Biomed. Sci. , vol. 3, pp. 237-50, 2007).

[0445] Without being bound by any particular theory, the selective killing or removal of senescent cells by the conditionally active proteins described herein can slow down or hinder (delay, inhibit, or prevent) the disorganization of the type IV collagen network. Removing senescent cells and thus eliminating the inflammatory effects of SASP can reduce or inhibit epithelial cell migration and may also delay (inhibit) the onset of presbyopia or reduce or slow the progressive severity of the condition (e.g., slowing the progression from mild to moderate or moderate to severe). The conditionally active proteins and pharmaceutical compositions described herein can also be used to reduce the likelihood of PCO after cataract surgery.

[0446] BubR1 hypomorphic mice developed bilateral posterior subcapsular cataracts early in life, suggesting that aging may play a role (see, for example, Baker et al.). Nat. Cell Biol (., vol. 10, pp.825-36, 2008). The presence and severity of cataracts can be monitored through eye examinations using methods routinely performed by ophthalmologists.

[0447] In some implementations, at least one conditionally active protein that selectively kills senescent cells can be administered to individuals at risk of developing presbyopia, cataracts, or macular degeneration. Treatment with the conditionally active protein can be initiated when the human individual is at least 40 years old to delay or inhibit the onset or progression of cataracts, presbyopia, and macular degeneration. Because presbyopia occurs in almost everyone, in some implementations, after an individual reaches age 40, a senescent cell scavenger can be administered to the human individual in the manner described herein to delay or inhibit the onset or progression of presbyopia.

[0448] In some implementations, the age-related disease or disorder is glaucoma. Glaucoma is a broad term used to describe a group of diseases that cause visual field loss, often without any other major symptoms. A fourfold increase in aging was observed in glaucoma patients when the cellular network required for fluid outflow was stained with SA-P-Gal (see, for example, Liton et al.). Exp. Gerontol ., vol. 40, pp. 745-748, 2005).

[0449] To monitor the effectiveness of treatment in inhibiting glaucoma progression, standard automated visual field testing (VAT) is the most widely used technique. In addition, several algorithms for progression detection have been developed (see, for example, Wesslink et al., Wesslink et al.). Arch Ophthalmol (See references in ., vol. 127, pp. 270-274, 2009). Other methods include gonioscopy (to examine the trabecular meshwork and the angle at which fluid flows out of the eye); imaging techniques such as scanning laser tomography (e.g., HRT3), laser polarimetry (e.g., GDX), and ocular coherence tomography; fundus examination; and pachymetry to determine central corneal thickness.

[0450] Metabolic diseases or disorders Senescence-related diseases or disorders, including metabolic diseases or disorders, can be treated by the application of conditionally active proteins or pharmaceutical compositions. These include diabetes, metabolic syndrome, diabetic ulcers, and obesity. The conditionally active proteins described herein can be used to treat type 2 diabetes, particularly type 2 diabetes associated with age, diet, and obesity.

[0451] Senescent cells are thought to be involved in metabolic diseases (such as obesity and type 2 diabetes) as a response to damage or metabolic dysfunction (see, for example, Tchkonia et al.). Aging Cell , vol. 9, pp. 667-684, 2010). Adipose tissue from obese mice showed induction of aging markers SA-P-Gal, p53, and p21 (see, for example, Minamino et al, , vol. 9, pp. 667-684, 2010). Nat. Med(See, vol. 15, pp. 1082-1087, 2009). A concomitant upregulation of pro-inflammatory cytokines (e.g., tumor necrosis factor-α and Ccl2 / MCP1) has been observed in the same adipose tissue (see, e.g., Minamino et al, ibid.). The induction of senescent cells in obesity may have clinical significance, as pro-inflammatory SASP components may also contribute to type 2 diabetes (see, e.g., Tchkonia et al, ibid.). Similar patterns of upregulation of senescence markers and SASP components are associated with diabetes in mice and humans (see, e.g., Minamino et al, ibid.). Therefore, the methods described herein, including the administration of senescent cell scavengers, could be used to treat or prevent type 2 diabetes, as well as obesity and metabolic syndrome. Without wishing to be bound by theory, contacting senescent preadipocytes with senescent cell scavengers to kill senescent preadipocytes could provide clinical and health benefits to individuals with any of diabetes, obesity, or metabolic syndrome.

[0452] A condition or symptom associated with diabetes and aging is diabetic ulcer (i.e., diabetic wound). Ulcers are breaks in the skin that can extend into the subcutaneous tissue and even into the muscle or bone. These lesions are particularly common in the lower extremities. Patients with diabetic venous ulcers exhibit elevated cellular senescence at the site of chronic wounds (see, for example, Stanley et al.). J. Vas. Surg Chronic inflammation has also been observed at sites of chronic wounds, such as diabetic ulcers (see, for example, Goren et al., vol. 33, pp. 1206-1211, 2001). Am. J. Pathol (vol. 168, pp. 65-77), indicating that the pro-inflammatory cytokine phenotype of senescent cells plays a certain role in pathology.

[0453] The effectiveness of conditionally active proteins can be readily determined by those skilled in the art of medicine and clinical practice. An individual's health status can be monitored using diagnostic methods including one or any combination of those described herein: physical examination, assessment and monitoring of clinical symptoms, and execution of the analytical tests and methods described herein. Individuals receiving one or more senescent cell scavengers described herein for the treatment or prevention of diabetes can be monitored, for example, by measuring glucose and insulin tolerance, energy expenditure, body composition, adipose tissue, skeletal muscle and liver inflammation, and / or lipotoxicity (in vivo imaging of lipids in muscle and liver, accumulation of lipids in muscle, liver, bone marrow and pancreatic β-cells, and histological inflammation). Other characteristics or phenotypes of type 2 diabetes are known and can be determined as described herein using other methods and techniques known and routinely practiced in the art.

[0454] Individuals with type 2 diabetes or at risk of developing type 2 diabetes may have metabolic syndrome. Metabolic syndrome in humans is typically associated with obesity and is characterized by one or more of cardiovascular disease, hepatic steatosis, hyperlipidemia, diabetes, and insulin resistance. Individuals with metabolic syndrome may exhibit a range of metabolic disorders or abnormalities, which may include, for example, hypertension, type 2 diabetes, hyperlipidemia, dyslipidemia (e.g., hypertriglyceridemia, hypercholesterolemia), insulin resistance, hepatic steatosis (steatohepatitis), hypertension, atherosclerosis, and one or more other metabolic disorders.

[0455] Skin diseases or disorders Diseases or disorders related to senescent cells that can be treated with the application of the conditionally active proteins or pharmaceutical compositions described herein include skin diseases or disorders. These diseases and disorders related to senescent cells include psoriasis and eczema, which are also inflammatory diseases and have been discussed in more detail above. Other age-related skin diseases or conditions include wrinkles (wrinkles caused by aging); pruritus (associated with diabetes and aging); sensory dullness (a chemotherapy side effect associated with diabetes and multiple sclerosis); psoriasis (as described above) and other papulosquamous diseases, such as erythroderma, lichen planus, and lichenoid dermatitis; atopic dermatitis (a form of eczema associated with inflammation); and eczematous eruptions (commonly observed in older patients and associated with side effects of certain medications). Other age-related skin conditions or disorders include eosinophilic dermatitis (associated with certain types of blood cancers); reactive neutrophilic dermatitis (associated with underlying conditions such as inflammatory bowel syndrome); pemphigus (an autoimmune disease in which autoantibodies are formed against desmosome core glycoprotein); bullous pemphigoid and other immune skin diseases (autoimmune bullous skin); age-related fibrosis of the skin; and cutaneous lymphoma, which is more common in older populations. Another skin condition that can be treated according to the methods described in this article includes cutaneous lupus, a symptom of lupus erythematosus. Late-onset lupus may be associated with a decline (i.e., decrease) in the function of age-related T cells and B cells and cytokines (immunosenescence).

[0456] transfer In one specific implementation, a conditionally active protein or pharmaceutical composition can be used to treat or prevent metastasis from one organ or tissue in the body to another (i.e., the spread and propagation of cancer cells or tumor cells). Individuals with cancer may benefit from administration of a conditionally active protein or pharmaceutical composition to inhibit metastasis. Such a conditionally active protein or pharmaceutical composition can inhibit tumor proliferation. Metastasis of cancer occurs when cancer cells (i.e., tumor cells) spread beyond their anatomical site of origin and initially colonize other areas within an individual's body. Tumor proliferation can be determined by tumor size, which can be measured in various ways familiar to those skilled in the art, such as by PET scans, MRI, CAT scans, and biopsies. The effect of a therapeutic agent on tumor proliferation can also be assessed by examining the differentiation of tumor cells.

[0457] As used herein and in the art, the terms cancer or tumor are clinical descriptive terms that encompass diseases typically characterized by cells exhibiting abnormal cell proliferation. The term cancer is generally used to describe malignant tumors or disease states caused by tumors. Alternatively, abnormal growth may be referred to as neoplasm in the art. The term tumor, such as in tissue-related contexts, generally refers to any abnormal tissue growth characterized at least in part by excessive and abnormal cell proliferation. Tumors can be metastatic and capable of spreading beyond their anatomically originating site and initially colonizing other areas within an individual's body. Cancer can include solid tumors or can include "liquid" tumors (e.g., leukemia and other blood cancers).

[0458] Cellular senescence is induced by cancer therapies such as radiation and certain chemotherapy drugs. The presence of senescent cells increases the secretion of inflammatory molecules (see the description of senescent cells in this article), promotes tumor progression (which may include promoting tumor growth and increasing tumor size), promotes metastasis, and alters differentiation. When senescent cells are destroyed, tumor progression is significantly suppressed, resulting in small tumor size and little or no metastatic growth (see, for example, WO2013 / 090645). Therefore, conditionally active proteins or pharmaceutical compositions can be administered after chemotherapy or radiation therapy to kill or remove these senescent cells. As discussed herein and understood in the art, the establishment of senescence, for example through the presence of a senescent cell-associated secretory phenotype (SASP), occurs within a few days. Therefore, once senescence has been established, the administration of senescent cell scavengers is initiated to kill senescent cells, thereby reducing the likelihood of metastasis or the extent of metastasis.

[0459] In one particular embodiment, when chemotherapy or radiotherapy is administered during a treatment cycle consisting of at least one day of treatment (i.e., chemotherapy or radiotherapy) followed by at least one week of off-therapy, the conditionally active protein or pharmaceutical composition is administered on one or more days during the off-therapy interval, beginning on or after the second day of the off-therapy interval and ending on or before the last day of the off-therapy interval. In a more specific embodiment, when chemotherapy or radiotherapy is administered during a treatment cycle consisting of at least one day of treatment (i.e., chemotherapy or radiotherapy) followed by at least one week of off-therapy, the conditionally active protein or pharmaceutical composition is administered on one day of the off-therapy interval (the sixth day of the off-therapy interval). In other specific embodiments, when chemotherapy or radiotherapy is administered during a treatment cycle consisting of at least one day of treatment (i.e., chemotherapy or radiotherapy) followed by at least two weeks of off-therapy, the conditionally active protein or pharmaceutical composition is administered starting on the sixth day of the off-therapy interval and ending at least one or at least two days before the first day of the subsequent chemotherapy or radiotherapy treatment course.

[0460] In another embodiment for treating metastases, the conditionally active protein or pharmaceutical composition may be administered after a chemotherapy or radiotherapy regimen has been completed. In one specific embodiment, the conditionally active protein or pharmaceutical composition is administered for one or more days within a treatment window of no more than 14 days (i.e., a senescent cell scavenger course) after chemotherapy or radiotherapy has been completed.

[0461] The methods described herein can also be used to inhibit, delay, or slow the progression of metastatic cancer in any type of tumor described in the medical field. Types of cancer (tumors) include: adrenocortical carcinoma, pediatric adrenocortical carcinoma, HIV-related cancers, anal cancer, appendix cancer, basal cell carcinoma, pediatric basal cell carcinoma, bladder cancer, pediatric bladder cancer, bone cancer, brain tumors, pediatric astrocytoma, pediatric brainstem glioma, pediatric atypical malformations / rhabdoid tumors of the central nervous system, embryonal tumors of the central nervous system in children, germ cell tumors of the central nervous system in children, craniopharyngioma / brain tumors in children, ependymoma / brain tumors in children, breast cancer, pediatric bronchial tumors, carcinoid tumors, pediatric carcinoid tumors, and gastric tumors. Intestinal carcinoid tumors, primary cancers of unknown cause, primary childhood cancers of unknown cause, pediatric 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 glioma, ocular cancer, malignant fibrous histiocytoma of bone, gallbladder cancer, gastric cancer, childhood gastric cancer, gastrointestinal stromal tumors (GIST). Children's gastrointestinal stromal tumors, children's extracranial germ cell tumors, external germ cell tumors, gestational trophoblastic tumors, gliomas, head and neck cancer, children's head and neck cancer, hepatocellular carcinoma (liver cancer), hypopharyngeal cancer, kidney cancer, renal cell carcinoma, nephroblastoma, children's kidney tumors, Langerhans cell histiocytosis, laryngeal cancer, children's 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 liver cancer in children, lobular carcinoma in situ (LCIS), lung cancer, non-small cell lung cancer, small cell lung cancer, lymphoma, HIV-associated 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 neck carcinoma, midline tract carcinoma involving the NUT gene.Carcinoma, oral cancer, multiple endocrine tumor syndrome in children, mycosis fungoides, myelodysplastic syndrome, myelodysplastic tumor, myeloma, multiple myeloma, nasal cavity cancer, nasopharyngeal carcinoma, pediatric nasopharyngeal carcinoma, neuroblastoma, oral cancer, pediatric oral cancer, oropharyngeal cancer, ovarian cancer, pediatric ovarian cancer, epithelial ovarian cancer, low-grade malignant potential tumor ovarian cancer, pancreatic cancer, pediatric pancreatic cancer, pancreatic neuroendocrine tumor (islet cell tumor), pediatric papilloma, paraganglioma, sinus cancer, parathyroid cancer, penile cancer, pharyngeal cancer, pheochromocytoma, pituitary adenoma, plasma cell tumor, pediatric pleuropneumonia Leukoblastoma, prostate cancer, rectal cancer, renal pelvis transitional cell carcinoma, retinoblastoma, salivary gland cancer, childhood salivary gland cancer, Ewing sarcoma family 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, pharyngeal cancer, thymoma and thymic carcinoma, childhood thymoma and thymic carcinoma, thyroid cancer, childhood thyroid cancer, ureteral transitional cell carcinoma, urethral cancer, endometrial cancer, vaginal cancer, vulvar cancer, Waldenström macroglobulinemia.

[0462] Side effects of chemotherapy and radiation therapy In another implementation, the disease or condition associated with senescent cells is a side effect of chemotherapy or radiation therapy. Examples of chemotherapeutic agents that induce senescence in non-cancerous cells include anthracyclines (e.g., doxorubicin, daunorubicin); paclitaxel (e.g., paclitaxel); gemcitabine; pomalidomide; and lenalidomide. One or more senescent cell scavengers administered as described herein can be used to treat and / or prevent (i.e., reduce the likelihood of occurrence) side effects of chemotherapy or radiation therapy. Removal or destruction of senescent cells can mitigate acute toxicity, including acute toxicity from chemotherapy or radiation therapy, including energy imbalances. Acute toxic side effects include, but are not limited to, gastrointestinal toxicities (e.g., nausea, vomiting, constipation, anorexia, diarrhea), peripheral neuropathy, fatigue, malaise, reduced physical activity, hematologic toxicities (e.g., anemia), hepatotoxicity, alopecia (hair loss), pain, infection, mucositis, fluid retention, skin toxicities (e.g., rash, dermatitis, hyperpigmentation, urticaria, photosensitivity, nail changes), oral problems (e.g., stomatitis), gum or throat problems, or any toxic side effects caused by chemotherapy or radiation therapy. For example, the methods described herein can alleviate toxic side effects caused by radiation therapy or chemotherapy (see, for example, the website of the National Cancer Institute). Therefore, in some embodiments, this document provides methods for mitigating (reducing, inhibiting, or preventing the occurrence (i.e., reducing the likelihood of occurrence)) the acute toxicity or severity of toxic side effects (i.e., harmful side effects) of chemotherapy or radiation therapy or both in a treated individual, wherein the method comprises administering to the individual an agent that selectively kills, removes, or destroys senescent cells or promotes the selective destruction of senescent cells.

[0463] Conditioned active proteins or pharmaceutical compositions for treating or reducing the likelihood or severity of chemotherapy or radiotherapy side effects can be administered via the same course of treatment as described above for treating / preventing metastasis. As described for treating or preventing metastasis (i.e., reducing the likelihood of metastasis), the conditioned active protein or pharmaceutical composition may be administered during the interval between chemotherapy or radiotherapy cessation or upon completion of the chemotherapy or radiotherapy treatment regimen.

[0464] In more specific embodiments, acute toxicity is an acute toxicity that includes energy imbalance and may include one or more of weight loss, endocrine changes (e.g., hormonal imbalance, changes in hormone signaling), and changes in body composition. In some embodiments, acute toxicity involving energy imbalance involves a decline or reduction in an individual's ability to perform physical activities, as indicated by a decrease or reduction in energy expenditure compared to individuals not receiving drug treatment. As a non-limiting example, such acute toxicity involving energy imbalance includes low physical activity. In other specific embodiments, energy imbalance includes fatigue or discomfort.

[0465] In one embodiment, the chemotherapy side effect treated or prevented (i.e., reduced in likelihood of occurrence) by the conditionally active protein or pharmaceutical composition is cardiotoxicity. Individuals with cancer treated with anthracyclines (e.g., doxorubicin, daunorubicin) can be treated with one or more senescent cell scavengers described herein that can reduce, mitigate, or decrease the cardiotoxicity of anthracyclines. As is well understood in the medical field, due to the cardiotoxicity associated with anthracyclines, even if the cancer responds to the drug, the maximum acceptable lifetime dose for an individual is limited. Administration of one or more conditionally active proteins can reduce cardiotoxicity, allowing for the administration of additional amounts of anthracyclines to an individual, thereby improving the prognosis associated with cancer disease. In one embodiment, cardiotoxicity is caused by the administration of anthracyclines (e.g., doxorubicin). Doxorubicin is an anthracycline topoisomerase approved for the treatment of ovarian cancer patients who have failed platinum-based therapy; Kaposi's sarcoma patients who have failed (or are intolerant to) primary systemic chemotherapy; or in combination with bortezomib for multiple myeloma patients who have not previously received bortezomib or have received at least one prior treatment. If the total lifetime dose given to the patient exceeds 550 mg / m² 2 Doxorubicin may cause myocardial damage, leading to congestive heart failure. Cardiotoxicity can occur even at lower doses if the patient is also receiving mediastinal irradiation or another cardiotoxic drug. See the drug information leaflet (e.g., doxorubicin liposomes (doxil), doxorubicin).

[0466] In other embodiments, the conditionally active proteins or pharmaceutical compositions described herein may be used in the methods provided herein to alleviate chronic or long-term side effects. Chronic toxic side effects are often caused by repeated administration of chemotherapy or radiation over a prolonged period. Some toxic effects (also known as late toxic effects) appear long after treatment and are caused by damage to organs or systems caused by the treatment. Organ dysfunction (e.g., neurological, pulmonary, cardiovascular, and endocrine dysfunction) has been observed in patients who received cancer treatment in childhood (see, for example, Hudson et al.). JAMA(vol. 309, pp. 2371-81, 2013). Without being bound by any particular theory, it is possible to reduce the likelihood of chronic side effects, or to reduce or decrease the severity of chronic side effects, or to delay the onset of chronic side effects, by disrupting senescent cells, especially normal cells induced to age by chemotherapy or radiation therapy. Chronic and / or late-onset toxic side effects occurring in individuals receiving chemotherapy or radiation therapy include (non-limiting examples) cardiomyopathy, congestive heart disease, inflammation, early menopause, osteoporosis, infertility, cognitive impairment, peripheral neuropathy, secondary cancers, cataracts and other vision problems, hearing loss, chronic fatigue, decreased lung capacity, and lung disease.

[0467] Furthermore, by administering conditionally active proteins or pharmaceutical compositions to kill or remove senescent cells in individuals with cancer, sensitivity to chemotherapy or radiotherapy can be enhanced in a clinically or statistically significant manner compared to the absence of such administration. In other words, when conditionally active proteins or pharmaceutical compositions are administered to individuals treated with chemotherapy or radiotherapy, respectively, the development of chemotherapy or radiotherapy resistance can be inhibited.

[0468] Age-related diseases and disorders Conditionally active proteins or pharmaceutical compositions can also be used to treat or prevent (i.e., reduce the likelihood of occurrence) age-related diseases or disorders that occur as part of the natural aging process or when an individual is exposed to aging inducers or factors (e.g., radiation, chemotherapy, smoking, high-fat / high-sugar diets, other environmental factors). Age-related diseases or disorders or age-sensitivity traits may be associated with stimuli that induce aging. The efficacy of the treatments described herein can be demonstrated by reducing the number of symptoms of age-related conditions or age-sensitivity traits associated with aging-inducing stimuli, reducing the severity of one or more symptoms, or delaying the progression of age-related conditions or age-sensitivity traits associated with aging-inducing stimuli. In other specific embodiments, prevention of age-related conditions or age-sensitivity traits associated with aging-inducing stimuli refers to preventing (i.e., reducing the likelihood of occurrence) or delaying the onset of age-related conditions or age-sensitivity traits associated with aging-inducing stimuli or the recurrence of one or more age-related conditions or age-sensitivity traits associated with aging-inducing stimuli.

[0469] Age-related diseases or conditions include, for example, kidney dysfunction, kyphosis, herniated discs, frailty, hair loss, hearing loss, vision loss (blindness or impaired vision), muscle fatigue, skin conditions, skin nevi, diabetes, metabolic syndrome, and sarcopenia. Vision loss refers to the loss of vision in an individual who previously had it. Various scales have been developed to describe visual acuity based on visual acuity and the degree of vision loss. Age-related diseases and conditions also include skin conditions, such as, but not limited to, treatment of one or more of the following: wrinkles, including fine lines; pigmentation; scars; keloids; dermatitis; psoriasis; eczema (including seborrheic dermatitis); rosacea; vitiligo; ichthyosis vulgaris; dermatomyositis; and actinic keratosis.

[0470] Frailty has been defined as a clinically identifiable state of increased vulnerability resulting from a decline in age-related reserves and function across multiple physiological systems, impairing an individual's ability to cope with daily or acute stressors. In some implementations, aging and age-related diseases and disorders can be treated or prevented (i.e., reduced in likelihood) by administering conditionally active proteins or pharmaceutical compositions. Conditionally active proteins or pharmaceutical compositions can inhibit the senescence of adult stem cells or inhibit the accumulation of senescent adult stem cells, kill senescent adult stem cells, or promote their removal. See, for example, Park et al. J. Clin. Invest ., vol. 113, pp. 175-79, 2004 and Sousa-Victor, Nature , vol. 506, pp. 316-21, 2014, which describes the importance of preventing stem cell senescence in order to maintain tissue regeneration capacity.

[0471] The effectiveness of conditionally active proteins or pharmaceutical compositions in treating the diseases or conditions associated with senescent cells described herein can be readily determined by those skilled in the art of medicine and clinical practice. For example, diagnostic methods known to those skilled in the art for a specific disease or condition, including one or any combination of the following, can be used to monitor an individual's health status and the effectiveness of the senescent cell scavenger: physical examination, patient self-assessment, assessment and monitoring of clinical symptoms, performance of analytical tests and methods including clinical laboratory tests, physical tests, and exploratory procedures. The effects of the treatments described herein can be analyzed using techniques known in the art, such as comparing the symptoms of patients with a specific disease or condition or at risk of developing such a disease who receive conditionally active proteins or pharmaceutical compositions with the symptoms of patients who do not receive conditionally active proteins or pharmaceutical compositions or who receive placebo.

[0472] The effectiveness of a conditionally active protein or pharmaceutical composition may include beneficial or desired clinical effects, including but not limited to the reduction, decrease, or alleviation of symptoms caused by or associated with the disease to be treated; a decrease in the incidence of symptoms; an improvement in quality of life; a longer disease-free state (i.e., a reduced likelihood or predisposition to the occurrence of symptoms that form the basis for diagnosis of the disease); a reduction in the severity of the disease; a stable disease state (i.e., no exacerbation); a delay or slowing of disease progression; an improvement or mitigation of the disease state; remission (whether local or total), whether detectable or undetectable; and / or overall survival. The effectiveness of a conditionally active protein or pharmaceutical composition may also refer to a prolonged survival compared to the expected survival of an individual without the conditionally active protein or pharmaceutical composition.

[0473] Subjects, patients, or individuals requiring treatment with the conditionally active proteins or pharmaceutical compositions described herein may be humans or non-human primates or other animals (i.e., for veterinary use) who already exhibit symptoms of or are at risk of developing diseases or conditions associated with senescent cells. Non-human animals that may be treated include mammals, such as non-human primates (e.g., monkeys, chimpanzees, gorillas, etc.), rodents (e.g., rats, mice, gerbils, hamsters, ferrets, rabbits), rabbits, suidae (e.g., pigs, miniature pigs), horses, dogs, cats, cattle, elephants, bears, and other domesticated, farm, and zoo animals.

[0474] Example Examples 1-9 for the preparation of conditionally active proteins are described in WO 2016 / 138071.

[0475] Example 10: Activity of conditionally active antibodies in different buffer solutions The activities of conditionally active antibodies derived from two different monoclonal antibodies (mAb 048-01 and mAb 048-02 as parental antibodies) were measured in two different buffer solutions. Figure 4 The two buffers are phosphate-buffered saline (condition IV) and Krebs buffer (condition I). Six conditionally active antibodies are derived from mAb 048-01: 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. Three conditionally active antibodies are derived from mAb 048-02: CAB Hit 048-07, CAB Hit 048-08, and CAB Hit 048-09.

[0476] This study indicates that the selectivity of conditionally active antibodies (the ratio of activity in a test at pH 6.0 to that in a test at pH 7.4) is affected by the buffer used in the test. The conditionally active antibody derived from wild-type mAb 048-02 showed significantly higher selectivity in Krebs buffer than in phosphate buffer. Figure 4 ).

[0477] Example 11: Selectivity of conditionally active antibodies and bicarbonate In Example 10, higher selectivity of the conditionally active antibody was observed in Krebs buffer (condition I) than in phosphate buffer (condition IV). This involved identifying the component in the Krebs buffer that contributed most significantly to the higher selectivity observed in Example 10. The selectivity of the conditionally active antibody was retested in a buffer derived from Krebs buffer, each time subtracting one component from the various components of the Krebs buffer. Figure 5 (Left bar chart). When using intact Krebs buffer, the conditionally active antibody exhibits high selectivity, with an activity ratio of approximately 8 at pH 6.0 / 7.4. Subtracting component AF from the Krebs buffer does not result in a loss of selectivity, although subtracting components C and D reduces selectivity. However, subtracting component G (bicarbonate) from the Krebs buffer completely eliminates selectivity. See also... Figure 5 This suggests that bicarbonate is at least partly responsible for the high selectivity of conditionally active antibodies in Krebs buffer.

[0478] The selectivity of the same conditionally active antibody was then measured in phosphate-buffered saline (condition IV), which does not contain bicarbonate. It was observed that the selectivity of the conditionally active antibody was completely lost in phosphate-buffered saline. When bicarbonate was added to the phosphate-buffered saline, the selectivity of the conditionally active antibody recovered to the level observed in Krebs buffer. This confirms that the selectivity of this conditionally active antibody requires bicarbonate.

[0479] Example 12: Inhibition of binding by bicarbonate at pH 7.4 This embodiment measured the physiological concentrations of bicarbonate (approximately 20 mM) at pH 7.4 with different concentrations of bicarbonate (ranging from 0 to approximately 20 mM). Figure 6 The binding activity of three conditionally active antibodies (CAB Hit A, CAB Hit B, and CAB Hit C) in a buffer solution was measured. It was observed that the binding activity of all three conditionally active antibodies at pH 7.4 decreased in a dose-dependent manner as the bicarbonate concentration increased from 0 to physiological concentration. Figure 6On the other hand, the binding activity of wild-type antibodies is not affected by bicarbonate. This study suggests that the selectivity of conditionally active antibodies in the presence of bicarbonate may be at least partly due to the loss of binding activity of conditionally active antibodies at pH 7.4 caused by the interaction with bicarbonate.

[0480] Example 13: Induction of Senescent Cells Cell seeding: Seed cells into 6-well plates as follows: at a concentration of 1.0 × 10⁶ cells / well. 5 Cells were seeded with MDA-MB468(P10) and MDA-MB231(Px) at a concentration of 2.0 × 10⁶ cells / year. 5 One cell line was seeded with MCF-7(Px) as a blank control, and treated with 2 mL of culture medium per well. The cells were cultured overnight.

[0481] • MCF-7 is an ERα+ cell line. Palbociclib has antiproliferative activity in this cell line, inhibiting cell growth and inducing senescent cells.

[0482] • MDA-MB231 is an ERα- cell line. Papositories in this cell line exhibit antiproliferative activity, inhibiting cell growth and inducing senescent cells.

[0483] • MDA-MB468 is another ERα- cell line. Papositories in this cell line do not exhibit antiproliferative activity, do not inhibit cell growth, and do not induce senescent cells.

[0484] Preparation of Paboseside solution: 25 mg of Paboseside hydroxyethyl sulfonate (PD-0332991, Selechchem, catalog number: S1579, batch number: 4, 25 mg) was added to 0.5 mL of H₂O to form a Paboseside solution with a concentration of 87.15 mM as a stock solution. 2.3 μL of the stock solution was mixed with 198 μL of H₂O to form a 1 mM Paboseside solution.

[0485] Inducing senescent cells: 2 μL of 1 mM Pabosipy solution was added to 2 mL of culture medium to obtain a final concentration of 1 μM Pabosipy, which was used to treat cultured cells (MCF-7, MDA-MB231, and MDA-MB468). The cultured cells were treated with this medium for 7 days in an attempt to induce senescent cells.

[0486] Senescent cells were detected by FAC (B-gal and antibody co-staining): After 7 days of treatment with Pabosipi, cells were co-stained with SA-B-gal fluorescent substrate (C12FDG) and a set of antibodies, and Zombie NIR live / dead dye was used.

[0487] 1. Wash cells twice with PBS and then with Detachin. TM Cells are separated using a cell separation solution.

[0488] 2. Terminate Detachin using DMEM TM The reaction was performed and the cells were counted.

[0489] 3. In PBS, add 2 mM C12FDG (final concentration 33 μM) and antibody (5 μL / 1×10⁻⁶). 6 (1 cell) and Zombie NIR dye (1:1000) were stained on ice for 1 hour.

[0490] 4. Wash the cells twice with PBS and fix them with 4% PFA for 10 minutes at room temperature.

[0491] 5. Wash with PBS and collect FAC in 100 μL PBS.

[0492] 6. Use FITC-PE-APC / Cy7.

[0493] 7. Use the following antibodies to co-stain cells to detect the expression of the corresponding antigens: a) PE anti-human CD54 clone HCD54, 200ug / mL, isotype (Iso): Ms IgG1. Biolegend, catalog number: 322707, lot number: B232865, 5μL / 10 6 cell b) PE anti-human CD73 clone AD2, isotype: Ms IgG1. Biolegend, catalog number: 344004, lot number: B216193, 5μL / 10 6 cell c) PE anti-human CD261 (DR4, TRAIL-R1) clone DJR1, 200ug / mL, isotype: Ms IgG1. Biolegend, catalog number: 307205, batch number: B189821, 5μL / 10 6 cell d) PE anti-human CD95 (Fas) clone DX2, 100 μg / mL, isotype: Ms IgG1. Biolegend, catalog number: 305607, lot number: B203942, 5 μL / 10 6 cell e) PE anti-human CD39 clone A1, 50 μg / mL, isotype: Ms IgG1, Biolegend, catalog number: 328208, batch number: B199643, 5 μL / 10 6 cell f) PE anti-human adhesion protein 4 (Nectin4), isotype: Ms IgG1. R&D Systems, catalog number: FAB2659P, batch number: AAAO0217031, 5μL / 10 6 cell g) PE-isotype mouse anti-IgG1, k:clone MOPC-21, 0.2 mg / mL. Biolegend, catalog number: 400112, batch number: B220359, 5 μL / 10 6 cell 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 for FACS analysis. SA-B-gal (senescence-associated B-gal) staining was also performed as a control using the CBA-230 kit from Cell Biolabs.

[0494] Following paboseib treatment, cell lines (MCF-7, MDA-MB231, and MDA-MB468 cells) were observed under a microscope. Furthermore, the target profiles expressed in the cell lines after paboseib treatment were analyzed using corresponding antibody staining. The targets plotted were target 1 (CD54), target 2 (CD73), target 3 (CD261), target 4 (CD95), target 5 (CD39), and target 6 (adhesionin 4).

[0495] MCF-7 cells responded to pabosipi treatment, and pabosipi induced the cells to become senescent. Figures 9A to 9B MCF-7 cells form clusters that possess the extracellular environment of senescent cells. Figure 9B FACS analysis clearly showed that cells treated with Pabosipi (senescent cells) differed from untreated cells (non-senescent cells). Figure 9C The target levels were found to be different in cells treated with Pabosipi (senescent cells) compared to untreated cells (non-senescent cells). Figure 9D The differences are as follows. Specifically, targets 1, 2, and 6 are more abundantly expressed in senescent cells, with target 2 showing the greatest increase in expression level.

[0496] Similarly, MDA-MB231 cells also responded to pabosesib treatment, which induced the cells to become senescent. Figures 10A to 10B MDA-MB231 cells also form clusters with an extracellular environment. Figure 10B FACS analysis clearly showed that cells treated with Pabosipi (senescent cells) were different from untreated cells (non-senescent cells). Figure 10CThe study found that the target levels in Pabosipi-treated cells (senescent cells) were significantly lower than those in untreated cells (non-senescent cells). Figure 10D The differences are as follows. Specifically, compared with untreated non-senescent cells, target 1 and target 2 showed significantly higher expression levels in senescent cells.

[0497] The control group (MDA-MB468 cells) did not respond to Pabosipi treatment, therefore this treatment did not induce the control cells to become senescent cells. Figures 11A to 11B FACS and target level analysis did not show any significant difference between treated and untreated cells.

[0498] Example 14: Paposippi treatment and β-galactosidase staining of MDA-MB231 cells MDA-MB231 cells were fed at a rate of 1×10⁻⁶. 5 Cells / well were seeded in 6-well plates and cultured overnight. The cultured cells were divided into two batches: one batch was treated with 1 μM Pabosipi hydroxyethyl sulfonate for 7 days, and the other batch remained untreated. Both batches were harvested by isolating the cells from the wells.

[0499] Harvested cells were stained on ice for 1 hour in PBS buffer with β-galactosidase (B-gal) substrate (FITC), target antibody (anti-CD73 antibody), and live / dead dye (APC / Cy7). B-gal staining was performed using a kit from Cell Signaling Technologies, catalog number 9860S. The stained MDA-MB231 cells were observed under a microscope. Figure 12A The results showed that there were almost no senescent cells in the untreated MDA-MB231 cells, as no cell clusters were observed. Figure 12B MDA-MB231 cells treated with Pabosipi are shown. Some cells were induced to form clusters of senescent cells, and the extracellular environment was also present.

[0500] 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.

[0501] Most untreated cells were B-gal negative, although they were sorted by FACS and separated based on their CD73 activity. Figure 14A B-gal positive cells were present in significantly smaller numbers, although they were also sorted by FACS and isolated based on their CD73 activity. Figure 14C Conversely, cells treated with Pabosipi had approximately the same number of B-gal negative cells and B-gal positive cells. Figure 14Band 14D Similarly, treated cells, whether B-gal negative or B-gal positive, were sorted by FACS and separated according to their CD73 activity. Figure 14B and 14D ).

[0502] FACS sorting results of MDA-MB231 cells are summarized as follows: Figures 15A to 15B middle. Figure 15A The image shows untreated cells, compared with treated cells containing a large number of senescent cells and higher CD73 activity. Figure 15B In contrast, the number of senescent cells in untreated cells was much smaller and the CD73 activity of the cells was at a much lower level.

[0503] Example 15: Papository treatment and β-galactosidase staining of MDA-MB468 cells As described in Example 14 for MDA-MD231 cells, MDA-MB468 cells were cultured, stained, and harvested. The stained MDA-MB468 cells were observed under a microscope. Figure 13A Untreated MDA-MB468 cells are shown. Figure 13B MDA-MB468 cells treated with Pabosipi are shown. No obvious senescent cells (cell clusters) were observed after treatment. The morphology of untreated and treated cells appeared similar under a microscope.

[0504] Untreated and stained cells treated with Pabosipi were washed with PBS and fixed with 4% paraformaldehyde for 10 minutes at room temperature. The fixed cells were used for FACS cell sorting.

[0505] Most untreated cells were B-gal negative, although they were sorted by FACS and separated based on their CD73 activity. Figure 16A The separation was not as clear as the MDA-MB231 cells in Example 14. B-gal positive cells were present in significantly smaller numbers, although they were also sorted by FACS and separated based on their CD73 activity. Figure 16C Similarly, the treated cells were mostly B-gal negative. Figure 16B and 16D Similarly, the treated cells, whether B-gal negative or B-gal positive, were sorted by FACS and separated according to their CD73 activity, although not as clearly as the MDA-MB231 cells in Example 14. Figure 14B and 14D ).

[0506] FACS sorting results of MDA-MB468 cells are summarized as follows: Figures 17A to 17B In the study, treated and untreated cells showed similar numbers of senescent cells and CD73 activity levels. These results indicate that Pabosebi treatment does not induce a large number of senescent cells.

[0507] Example 16: Expression of CD73 in MDA-MB231 and MDA-MB468 cells Measure the CD73 expression level in MDA-MB231 and MDA-MB468 cells after Pabosipi treatment. Figure 18 In MDA-MB231 cells, pabosesib treatment significantly increased the expression level of CD73. Figure 18 The two columns on the left side of the middle section). Compared with untreated MDA-MB231 cells, untreated MDA-MB468 cells had a lower CD73 expression level ( Figure 18 The first and third columns in the image). Furthermore, Pabosipi treatment did not significantly increase the expression level of CD73 in MDA-MB468 cells (the first and third columns in the image). Figure 18 (The two pillars on the right side of the middle).

[0508] Example 17: Pabosipi treatment and β-galactosidase staining of MCF-7, T47D, and MDA-MB468 cells Except for treating the cells with 3 µM Pabosibi hydroxyethyl sulfonate for 6 days, the culture, staining, and harvesting of MCF-7 cells, T47D cells, and MDA-MB468 cells were similar to the procedures in Examples 13-15.

[0509] Test cells MCF-7 (ATCC, catalog number HTB-22) T47D (ATCC, catalog number HTB-133) MDA-MB468 (ATCC, catalog number HTB-132) Test chemicals Papositories (PD-0332991) Hydroxyethanesulfonate (Selleck Chemicals, Catalog No. S1579) Age-related β-galactosidase assay 1) Cell plating: Placing cells at a density of 1x10⁻⁶ cells / cm². 5 10 cells / well ...

Claims

1. A method for preparing a conditionally active senescent cell clearance antibody or antibody fragment that binds to a target associated with senescent cells from a parent antibody or antibody fragment that binds to a target associated with senescent cells, the method comprising the following steps: (i) Using one or more evolution techniques to evolve the DNA encoding the parental antibody or antibody fragment to produce mutant DNA; (ii) Express the mutant DNA to obtain a mutant antibody or antibody fragment; (iii) The mutant antibody or antibody fragment is subjected to an extracellular binding activity test with the target under the conditions of the senescent cells, and the binding activity test with the target is performed under normal physiological conditions. and (iv) Select from the mutant antibodies or antibody fragments tested in step (iii) a conditionally active senescent cell clearance antibody or antibody fragment exhibiting at least one of the following properties: (a) The binding activity to the target is reduced in the test under the normal physiological conditions compared with the binding activity of the parent antibody or antibody fragment to the target in the same test. Compared to the binding activity of the conditionally active senescent cell clearance antibody or antibody fragment to the same target in the test under the normal physiological conditions, the binding activity to the target is increased in the test under the extracellular conditions of the senescent cells. as well as (b) The binding activity to the target is reduced in the test under the normal physiological conditions compared with the binding activity of the parent antibody or antibody fragment to the target in the same test. Compared to the same binding activity to the target in the test under extracellular conditions of the parent antibody or antibody fragment in the senescent cells, the binding activity to the target is increased in the test under extracellular conditions of the senescent cells. The targets are selected from the following groups: 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, LaminB1, LANCL1, LCMT2, LPHN1, MADCAM1, MAG, MA P3K14, MAPK, MEF2C, miR22, MMP3, MTHFD2, NAIP, NAPG, NCKAP1, Connectin 4, NNMT, NOTCH3, NTAL, OPG, OSBPL3, p16, p16INK4a, p19, p21, p53, PAI1, PARK2, PF N1, PGM, PLD3, PMS2, POU5F1, PPP1A, PPP1CB, PRKRA, PRPF19, PRTG, RAC1, RAPGEF1, RET, Smurf2, STX4, VAMP3, VIT, VPS26A, WEE1, YAP1, YH2AX and YWHAE.

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

3. The method according to claim 1, wherein the ratio of the binding activity of the conditionally active senescent cell clearance antibody or antibody fragment to the target in the test under extracellular conditions of the senescent cells to the binding activity of the conditionally active senescent cell clearance antibody or antibody fragment to the target in the test under normal physiological conditions is at least about 2:

1.

4. The method of claim 1, wherein the extracellular conditions of the senescent cells are a pH ranging from about 5.5 to about 7.0, and the normal physiological conditions are a pH ranging from about 7.2 to about 7.

8.

5. The method according to claim 1, wherein the extracellular conditions of the senescent cells are selected from the group consisting of: The concentration of deoxynucleotides was lower than the normal physiological concentration of the same type of deoxynucleotide; The NAD+ / NADH ratio is lower than the normal physiological NAD+ / NADH ratio; The concentration of at least one redox homeostatic metabolite selected from the group consisting of taurine, cysteine ​​sulfinic acid, cysteine-glutathione disulfide, γ-glutamyl alanine, γ-glutamyl methionine, pyridoxate, γ-glutamyl glutamine, and alanine is increased relative to the normal physiological concentration of the same redox homeostatic metabolite. The concentration of thymidine was lower than the normal physiological concentration of thymidine. The concentration of at least one dipeptide selected from the group consisting of glycyl isoleucine, glycylvaline, glycylleucine, isoleucylglycine and valineglycine is lower than the normal physiological concentration of the same dipeptide. The concentration of at least one fatty acid selected from the group consisting of linoleic acid, di-homo-linoleic acid and 10-heptadecenoic acid is lower than the normal physiological concentration of said fatty acid; the concentration of at least one phospholipid metabolite selected from the group consisting of 2-hydroxypalmitic acid, 2-hydroxystearic acid, 3-hydroxydecanoic acid, 3-hydroxyoctanoic acid and glycerophosphocholine is higher than the normal physiological concentration of said phospholipid metabolite. The concentration of at least one amino acid metabolite selected from the group consisting of alanine, C-glycosyltryptophan, kynurenine, dimethylarginine, and ornithine is increased relative to the normal physiological concentration of said amino acid metabolite; The concentration of phenylpyruvic acid is lower than the normal physiological concentration of phenylpyruvic acid; An increase in the concentration of at least one metabolite selected from the group consisting of fumaric acid, malonic acid, eicosapentaenoic acid, and citric acid relative to the normal physiological concentration of said metabolite; and The ratio of glycerophosphate choline to phosphate choline is increased relative to the normal physiological ratio.

6. The method of claim 1, wherein the extracellular conditions of the senescent cells are a first pH ranging from about 5.5 to about 7.0, and the normal physiological conditions are a second pH ranging from about 7.2 to about 7.8, and One or more tests are performed in a test solution containing at least one substance with a molecular weight less than 900 a.m. and a pKa that differs from the first pH by at most 4 pH units.

7. The method of claim 1, wherein the extracellular conditions of the senescent cells are a first pH ranging from about 5.5 to about 7.0, and the normal physiological conditions are a second pH ranging from about 7.2 to about 7.

8. One or more tests are performed in a test solution containing at least one substance with a molecular weight less than 900 a.m., and The substance has a pKa between the first pH and the second pH.

8. The method of claim 1, wherein the extracellular conditions of the senescent cells are a first pH ranging from about 5.5 to about 7.0, and the normal physiological conditions are a second pH ranging from about 7.2 to about 7.8, and The test is performed in a test solution containing at least one substance selected from the group consisting of histidine, histamine, hydrogen adenosine diphosphate, hydrogen adenosine triphosphate, citrate, bicarbonate, acetate, lactate, disulfide, hydrogen sulfide, ammonium, and dihydrogen phosphate.

9. The method of claim 1, wherein the selection step (iv) comprises selecting a conditionally active senescent cell clearance antibody or antibody fragment exhibiting the following characteristics: (a) decreased binding activity to the target in a test under normal physiological conditions compared to the homologous binding activity of the parent antibody or antibody fragment to the target in the same test; and increased binding activity to the target in a test under extracellular conditions of senescent cells compared to the homologous binding activity of the conditionally active senescent cell clearance antibody or antibody fragment to the target in a test under normal physiological conditions.

10. The method of claim 1, wherein the method further comprises the step of conjugating the conditionally active senescent cell clearing antibody to a masking portion via a connector, wherein the masking portion is identified by screening a diverse peptide library to find peptides that bind to one or more variable regions of the conditionally active senescent cell clearing antibody.

11. The method of claim 10, wherein the masking portion reduces the activity of the conditionally active antibody in binding to the target by at least 50%.

12. The method of claim 1, further comprising the step of conjugating the conditionally active senescent cell-clearing antibody or antibody fragment selected in step (iv) to a cytotoxic drug, a cell growth inhibitor, or an antiproliferative drug via a connector.

13. The method of claim 12, wherein the adapter comprises a cleavage site capable of being cleaved by a protease in the extracellular environment of the senescent cell.

14. The method of claim 1, further comprising the step of conjugating the conditionally active senescent cell-clearing antibody or antibody fragment selected in step (iv) to an agent selected from the group consisting of a toxic agent, a radioactive agent, or a D-reverse peptide.

15. The method of claim 14, wherein the amino acid sequence of the D-reverse peptide has at least 70% amino acid sequence identity with the reverse sequence of a fragment or full-length natural protein selected from the group consisting of FOXO4, AMPK, JNK, MST1, CK1, STAT3, p38, PRMT1 and ASK1.

16. The method of claim 14, wherein the D-reverse peptide comprises one or more functional domains selected from the group consisting of PPRRRQRRKKRG (SEQ ID NO:10), GALFLGFLGA AGSTMGAWSQ PKKKRKV (SEQ ID NO:11), KETWWETWWTEWSQPKKKRKV (SEQ ID NO:12), Ac-GLWRALWRLLRSLWRLLWRA-Cya (SEQ ID NO:13), LTLRKEPASE IAQSILEAYS QNGWANRRSG GKRP (SEQ ID NO:5), LTLRKEPASE IAQSILEAYSQNGWANRRSG GKRPPPRRRQ RRKKRG (SEQ ID NO:6), SEIAQSILEAYSQNGW (SEQ ID NO:7) and octargine.

17. A specific CD73-binding isolated polypeptide comprising a heavy chain variable region including three complementarity-determining regions (CDRs) having H1, H2, and H3 sequences, wherein: The H1 sequence is GFTFSSYAYS (SEQ ID NO: 52); The H2 sequence is AISGSGGRTYYADSVKG (SEQ ID NO: 53); and The H3 sequence is LGX1GRVDE (SEQ ID NO: 54); Where X1 is either Y or E, and The light chain variable region includes three complementarity-determining regions (CDRs) with L1, L2, and L3 sequences, wherein: The L1 sequence is SGSLSNIGRNPVN (SEQ ID NO: 47); The L2 sequence is X2DNLRLS (SEQ ID NO: 48); and The L3 sequence is ATWDDSHPGWT (SEQ ID NO: 51). Where X2 is L or D. The conditions are: X1 and X2 cannot be Y and L at the same time, or the combination of the heavy chain variable region and the light chain variable region is not SEQ ID NO: 45 and 43.

18. The isolated polypeptide according to claim 17, wherein the H3 sequence is selected from the group consisting of LGYGRVDE (SEQ ID NO: 55) and LGEGRVDE (SEQ ID NO: 56).

19. The isolated polypeptide according to claim 17, wherein the L3 sequence is selected from the group consisting of LDNLRLS (SEQ ID NO:49) and DDNLRLS (SEQ ID NO:50).

20. The isolated polypeptide according to claim 18, wherein the L3 sequence is selected from the group consisting of LDNLRLS (SEQ ID NO:49) and DDNLRLS (SEQ ID NO:50).

21. The isolated polypeptide according to claim 17, wherein the heavy chain variable region has a sequence selected from the group consisting of SEQ ID NO: 45 to 46.

22. The isolated polypeptide according to claim 17, wherein the light chain variable region has a sequence selected from the group consisting of SEQ ID NO: 43 to 44.

23. The isolated polypeptide according to claim 21, wherein the light chain variable region has a sequence selected from the group consisting of SEQ ID NO:43 to 44.

24. An isolated polypeptide that specifically binds to B7H4, comprising: The heavy chain variable region includes three complementarity-determining regions (CDRs) with H1, H2, and H3 sequences, wherein: The H1 sequence is GYTFTDRTIH (SEQ ID NO: 64); The H2 sequence is SIYPRDGSTKYNEKFKD (SEQ ID NO: 65); and The H3 sequence is SVGYAX3DY (SEQ ID NO: 66); Where X3 is F or D, and The light chain variable region includes three complementarity-determining regions (CDRs) with L1, L2, and L3 sequences, wherein: The L1 sequence is RVSEGIDNYGFTFIH (SEQ ID NO: 61); The L2 sequence is RASNLQS (SEQ ID NO: 62); and The L3 sequence is QQSDKDPFT (SEQ ID NO: 63). The condition is that X3 cannot be F, or the combination of the heavy chain variable region and the light chain variable region is not SEQ ID NO: 59 and 57.

25. The isolated polypeptide according to claim 24, wherein the H3 sequence is selected from the group consisting of SVGYAFDY (SEQ ID NO: 67) and SVGYADDY (SEQ ID NO: 68).

26. The isolated polypeptide according to claim 24, wherein the heavy chain variable region has a sequence selected from the group consisting of SEQ ID NO: 57 to 58.

27. The isolated polypeptide according to claim 24, wherein the light chain variable region has a sequence selected from the group consisting of SEQ ID NO: 59 to 60.

28. The isolated polypeptide according to claim 26, wherein the light chain variable region has a sequence selected from the group consisting of SEQ ID NO: 59 to 60.

29. The isolated polypeptide according to claim 24, comprising the heavy chain variable region of SEQ ID NO: 58 and the light chain variable region of SEQ ID NO:

60.

30. A method for treating a disease or disorder associated with senescent cells, comprising administering to an individual suffering from said disease or disorder a polypeptide of any one of claims 24 to 29 or a pharmaceutical composition comprising a polypeptide of any one of claims 24 to 29 to kill or remove senescent cells associated with said disease or disorder.

31. The method of claim 30, wherein the disease or disorder is selected from osteoarthritis, idiopathic pulmonary fibrosis, chronic obstructive pulmonary disease (COPD), and atherosclerosis.

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