WO2009025847A2 - Methods for diagnosis, prognosis and methods of treatment - Google Patents
Methods for diagnosis, prognosis and methods of treatment Download PDFInfo
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- WO2009025847A2 WO2009025847A2 PCT/US2008/009975 US2008009975W WO2009025847A2 WO 2009025847 A2 WO2009025847 A2 WO 2009025847A2 US 2008009975 W US2008009975 W US 2008009975W WO 2009025847 A2 WO2009025847 A2 WO 2009025847A2
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
- G01N33/57407—Specifically defined cancers
- G01N33/57426—Specifically defined cancers leukemia
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
- G01N33/5008—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
- G01N33/502—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics for testing non-proliferative effects
- G01N33/5041—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics for testing non-proliferative effects involving analysis of members of signalling pathways
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
- G01N33/5008—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
- G01N33/5044—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics involving specific cell types
- G01N33/5047—Cells of the immune system
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
- G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
- G01N2333/46—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans from vertebrates
- G01N2333/47—Assays involving proteins of known structure or function as defined in the subgroups
- G01N2333/4701—Details
- G01N2333/4703—Regulators; Modulating activity
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
- G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
- G01N2333/705—Assays involving receptors, cell surface antigens or cell surface determinants
- G01N2333/70503—Immunoglobulin superfamily, e.g. VCAMs, PECAM, LFA-3
- G01N2333/7051—T-cell receptor (TcR)-CD3 complex
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
- G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
- G01N2333/705—Assays involving receptors, cell surface antigens or cell surface determinants
- G01N2333/70503—Immunoglobulin superfamily, e.g. VCAMs, PECAM, LFA-3
- G01N2333/70514—CD4
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
- G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
- G01N2333/705—Assays involving receptors, cell surface antigens or cell surface determinants
- G01N2333/70503—Immunoglobulin superfamily, e.g. VCAMs, PECAM, LFA-3
- G01N2333/70517—CD8
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
- G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
- G01N2333/705—Assays involving receptors, cell surface antigens or cell surface determinants
- G01N2333/70596—Molecules with a "CD"-designation not provided for elsewhere in G01N2333/705
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
- G01N2333/90—Enzymes; Proenzymes
- G01N2333/91—Transferases (2.)
- G01N2333/912—Transferases (2.) transferring phosphorus containing groups, e.g. kinases (2.7)
Definitions
- a method for classifying a cell comprising contacting the cell with an inhibitor, determining the presence or absence of a change in activation level of an activatable element in the cell, and classifying the cell based on the presence or absence of the change in the activation level of the activatable element.
- the change in activation level of an activatable element is an increase in the activation level of an activatable element.
- the activatable element is a protein subject to phosphorylation or dephosphorylation.
- the invention provides a method for classifying a cell by contacting the cell with an inhibitor, determining the activation levels of a plurality of activatable elements in the cell, and classifying the cell based on the activation level
- the inhibitor is a kinase or phosphatase inhibitor, such as adaphostin, AG 490, AG 825, AG 957, AG 1024, aloisme, aloisine A, alsterpaullone, aminogenistein, API-2, apigenm, arctigenin, AY-22989, BAY 61-3606, bisindolylmaleimide IX, chelerythnne, 10- [4'-(N,N-Diethylamino)butyl]-2-chlorophenoxazine hydrochloride, dasatinib, 2-Dimethylamino-4,5,6,7-tetrabromo-lH- benzimidazole,
- the classification m cludes classifying the cell as a cell that is correlated with a clinical outcome
- the clinical outcome is the prognosis and/or diagnosis of a condition
- the clinical outcome is the presence or absence of a neoplastic or a hematopoietic condition, such as Non-Hodgkin Lymphoma, Hodgkin or other lymphomas, acute or chrome leukemias, polycythemias, thrombocythemias, multiple myeloma or plasma cell disorders, e g , amyloidosis and Waldenstrom's macroglobulinemia, myelodysplastic disorders, myeloproliferative disorders, myelofibrosis, or atypical immune lymphoproliferations
- the neoplastic or hematopoietic condition is non-B lmeage denved, such as acute myeloid leukemia (AML), Chronic Myeloid
- classifying the cell based on activation levels of activatable element mcludes classifying the cell as a cell that is correlated to a patient response to a treatment, such as complete response, partial response, nodular partial response, no response, progressive disease, stable disease, relapse or adverse reaction
- the method may further comp ⁇ se determining a method of treatment, e g , chemotherapy, biological therapy, radiation therapy, bone marrow transplantation, Peripheral stem cell transplantation, umbilical cord blood transplantation, autologous stem cell transplantation, allogeneic stem cell transplantation, syngeneic stem cell transplantation, surgery, mduction therapy, maintenance therapy, watchful waiting, or holistic/alternative therapy
- the classifying of the cell based on activation level m cludes classifying the cell as a cell that is correlated with mimmal residual disease or emerging resistance
- the activation level of the plurality of activatable elements in the cell is selected from the group consisting of cleavage by extracellular or intracellular protease exposure, novel hetero-ohgomer formation, glycosylation level, phosphorylation level, acetylation level, methylation level, biotinylation level, glutamylation level, glycylation level, hydroxylation level, isomenzation level, prenylation level, mynstoylation level, lipoylation level, phosphopantetheinylation level, sulfation level, ISGylation level, nitrosylation level, palmitoylation level, SUMOylation level, ubiquitination level, neddylation level, citrullination
- PARP ion channels
- molecular transporters molecular chaperones
- metabolic enzymes vesicular transport proteins
- hydroxylases isomerases
- transferases deacetylases
- methylases demethylases
- proteases esterases
- hydrolases DNA binding proteins or transcription factors.
- the protein is selected from the group consisting of PI3-Kinase (p85, pi 10a, pi 10b, pi 1Od), Jakl, Jak2, SOCs, Rac, Rho, Cdc42, Ras-GAP, Vav, Tiam, Sos, DbI, Nek, Gab, PRK, SHPl, and SHP2, SHIPl, SHIP2, sSHIP, PTEN, She, Grb2, PDKl, SGK, Aktl, Akt2, Akt3, TSC1,2, Rheb, mTor, 4EBP-1, p70S6Kinase, S6, LKB-I, AMPK, PFK, Acetyl-CoAa Carboxylase, DokS, Rafs, Mos, Tpl2, MEK1/2, MLK3, TAK, DLK, MKK3/6, MEKK 1,4, MLK3, ASKl, MKK4/7, SAPK/JNK1,2,3, p38s, Erkl, PI3
- the protein selected from the group consisting of Erk, Syk, Zap70, Lck, Btk, BLNK, CbI, PLC ⁇ 2, Akt, ReIA, p38, S6. In some embodiments the protein is S6.
- the protein is selected from the group consisting of HER receptors, PDGF receptors, Kit receptor, FGF receptors, Eph receptors, Trk receptors, IGF receptors, Insulin receptor, Met receptor, Ret, VEGF receptors, TIEl, TIE2, FAK, Jakl, Jak2, Jak3, Tyk2, Src, Lyn, Fyn, Lck, Fgr, Yes, Csk, AbI, Btk, ZAP70, Syk, IRAKs, cRaf, ARaf, BRAF, Mos, Lim kinase, ILK, TpI, ALK, TGF ⁇ receptors, BMP receptors, MEKKs, ASK, MLKs, DLK, PAKs, Mek 1, Mek 2, MKK3/6, MKK4/7, ASKl.Cot, NDC, Bub, Myt 1, Weel, Casein kinases, PDKl, SGKl, SGK2, SGK
- SAPK/JNK1,2,3, p38s PKR, DNA-PK, ATM, ATR, Receptor protein tyrosine phosphatases (RPTPs), LAR phosphatase, CD45, Non receptor tyrosine phosphatases (NPRTPs), SHPs, MAP kinase phosphatases (MKPs), Dual Specificity phosphatases (DUSPs), CDC25 phosphatases, Low molecular weight tyrosine phosphatase, Eyes absent (EYA) tyrosine phosphatases, Slingshot phosphatases (SSH), serine phosphatases, PP2A, PP2B, PP2C, PPl, PP5, inositol phosphatases, PTEN, SHIPs, myotubularins, phosphoinositide kinases, phospholipases, prostaglandin synthases, 5-lipoxygenase, sphingosine kinases,
- JHDM2A UTX, VHL, WT-I, p53, Hdm, PTEN, ubiquitin proteases, urokinase-type plasminogen activator (uPA) and uPA receptor (uPAR) system, cathepsins, metalloproteinases, esterases, hydrolases, separase, potassium channels, sodium channels, , multi-drug resistance proteins, P-Gycoprotein, nucleoside transporters, , Ets, Elk, SMADs, ReI-A (p65-NFKB), CREB 1 NFAT, ATF-2, AFT, Myc, Fos, SpI, Egr-1, T-bet, ⁇ -catemn, HIFs, FOXOs, E2Fs, SRFs, TCFs, Egr-1, ⁇ -catemn, FOXO STATl, STAT 3, STAT 4, STAT 5, STAT 6, p53, WT-I, HMGA,
- the inhibitor is a kinase or phosphatase inhibitor, such as adaphostin, AG 490, AG 825, AG 957, AG 1024, aloisine, aloisine A, alsterpaullone, aminogenistein, API-2, apigenin, arctigemn, AY-22989, BAY 61-3606, bisindolylmaleimide IX, chelerythnne, 10-[4'-(N,N-Diethylamino)butyl]-2-chlorophenoxazine hydrochlo ⁇ de, dasatinib, 2-Dimethylamino-4,5,6,7-tetrabromo-lH-benzimidazole, 5,7-Dimethoxy-3-(4-pyridinyl)quinohne dihydrochlo ⁇ de, edelfosine, ellagic acid, enzastau ⁇ n, ER 27319 maleate, er
- the invention provides a method of determining a tonic signaling status of a cell by subjecting the cell to a modulator, determining the activation level of an activatable element that participates in a tonic signaling pathway in the cell, and determining the status of a tome signaling pathway in the cell from the activation level
- a condition of an individual is determined based on tonic signaling status of a cell
- the condition is a neoplastic and/or hematopoietic condition
- the neoplastic or hematopoietic condition is selected from the group consistmg of Non-Hodgkin Lymphoma, Hodgkin or other lymphomas, acute or chronic leukemias, polycythemias, thrombocythemias, multiple myeloma and plasma cell disorders, e g , amyloidosis and Waldenstrom's macroglobulmemia, myelodysplastic
- the correlation is determining the individual's response to a treatment, e g , normal responder, hyper responder, poor responder, having emerging resistance, non-compliant, and adverse reaction
- the correlation mcludes classifying the cell as minimal residual disease or emerging resistance
- the correlation may further mclude determining a method of treatment, such as chemotherapy, biological therapy, radiation therapy, bone marrow transplantation, Pe ⁇ pheral stem cell transplantation, umbilical cord blood transplantation, autologous stem cell transplantation, allogeneic stem cell transplantation, syngeneic stem cell transplantation, surgery, induction therapy, maintenance therapy, or watchful waitmg
- the invention provides a method of correlating an activation level of a B-lymphocyte lineage de ⁇ ved cell with a neoplastic or hematopoietic condition m an individual by subjectmg the B- lymphocyte lmeage de ⁇ ved cell from the individual to
- the modulator to which the cell is subjected is an activator or an inhibitor
- the modulator is, e g , a growth factor, cytokine, adhesion molecule modulator, hormone, small molecule, polynucleotide, antibodies, natural compounds, lactones, chemotherapeutic agents, immune modulator, carbohydrate, proteases, ions, reactive oxygen species, or radiation
- the modulator is a B cell receptor modulator, e g , a B cell receptor activator such as a cross-linker of the B cell receptor complex or the B-cell co-receptor complex
- the cross-linker is an antibody, or molecular binding entity
- the cross-linker is an antibody, such as a multivalent antibody.
- the antibody is a monovalent, bivalent, or multivalent antibody made more multivalent by attachment to a solid surface or tethered on a nanoparticle surface to increase the local valency of the epitope binding domain.
- the cross-linker is a molecular binding entity, such as an entity that acts upon or binds the B cell receptor complex via carbohydrates or an epitope in the complex.
- the molecular binding entity is a monovalent, bivalent, or multivalent binding entity that is made more multivalent by attachment to a solid surface or tethered on a nanoparticle surface to increase the local valency of the epitope binding domain.
- cross- linking includes binding of an antibody or molecular binding entity to the cell and then causing its crosslinking via interaction of the cell with a solid surface that causes crosslinking of the BCR complex via antibody or molecular binding entity-.
- the crosslinker is selected from the group consisting of F(ab)2 IgM, , IgG, IgD, polyclonal BCR antibodies, monoclonal BCR antibodies, Fc receptor derived binding elements and/or a combination thereof.
- the Ig may be derived from a species selected from the group consisting of mouse, goat, rabbit, pig, rat, horse, cow, shark, chicken, or llama.
- the crosslinker is F(ab)2 IgM, Polyclonal IgM antibodies, Monoclonal IgM antibodies, Biotinylated F(ab)2 IgCM, Biotinylated Polyclonal IgM antibodies, Biotinylated Monoclonal IgM antibodies and/or a combination thereof.
- the modulator to which the cell is subjected is an inhibitor of a cellular factor or a plurality of factors that participates in a signaling cascade in the cell.
- the inhibitor is a kinase or phosphatase inhibitor, such as adaphostin, AG 490, AG 825, AG 957, AG 1024, aloisine, aloisine A, alsterpaullone, aminogenistein, API-2, apigenin, arctigenin, AY-22989, BAY 61-3606, bisindolylmaleimide IX, chelerythrine, 10-[4'-(N,N-Diethylamino)butyl]-2-chlorophenoxazine hydrochloride, dasatinib, 2- Dimethylamino-4,5,6,7-tetrabromo-lH-benzimidazole, 5,7-Dimethoxy-3-(4-
- the phosphatase inhibitor is H 2 O 2 .
- the cell is further subjected to a second modulator, e g , the cell may be subjected to a B cell receptor activator and a phosphatase inhibitor, such as F(ab) 2 IgM or biotinylated F(ab) 2 IgM and a phosphatase inhibitor (e g H 2 O 2 )
- the activation level of the plurality of activatable elements in the cell is selected from the group consisting of cleavage by extracellular or intracellular protease exposure, novel hetero-ohgomer formation, glycosylation level, phosphorylation level, acetylation level, methylation level, biotinylation level, glutamylation level, glycylation level, hydroxylation level, lsomenzation level, prenylation level, mynstoylation level, lipoylation level, phosphopantetheinylation level, sulfation level, ISGylation level, mtrosylation level, palmitoylation level, SUMOylation level, ubiquitination level, neddylation level, citrullination level, deamidation level, disulfide bond formation level, proteolytic cleavage level, translocation level, changes m protein turnover, multi-protein complex level, oxidation level, multi-hpid complex,
- the protem is selected from the group consisting of HER receptors, PDGF receptors, Kit receptor, FGF receptors, Eph receptors, Trk receptors, IGF receptors, Insulin receptor, Met receptor, Ret, VEGF receptors, TIEl, TIE2, FAK, Jakl, Jak2, Jak3, Tyk2, Src, Lyn, Fyn, Lck, Fgr, Yes, Csk, AbI, Btk, ZAP70, Syk, IRAKs, cRaf, ARaf, BRAF, Mos, Lim kinase, ILK, TpI, ALK, TGF ⁇ receptors, BMP receptors, MEKKs, ASK, MLKs, DLK, PAKs, Mek 1, Mek 2, MKK3/6, MKK4/7, ASKl.Cot, NDC, Bub, Myt 1, Weel, Casern kinases, PDKl, SGKl, SGK2,
- the methods for classifying a cell further compnse determining the level of an additional intracellular marker and/or a cell surface marker In some embodiments the methods for classifying a cell compnse determining the level of an additional intracellular marker In some embodiments the intracelluar marker is a
- the methods of the invention for prognosis, diagnosis, or determination of treatment further compnse determining the level of an additionalserum marker
- the serum marker compnses a protein
- the serum marker is a cytokine, growth factor, chemokine, soluble receptor, small compound, or phamaceutical drug
- the serum marker compnses a component or product of a pathogen or parasite
- the serum marker is selected from a group consisting of beta-2-microglobulin, calcitonin, thymidine kinase and ferritin
- the invention provides a method of correlating an activation level of B-lymphocyte lineage denved cells with a neoplastic or hematopoietic condition in an individual by subjecting the B-lymphocyte lineage denved cell from the individual to a modulator, determining the activation levels of a plurality of activatable elements in the B-lymphocyte lmeage derived cell, and identifying a pattern of the activation levels of the plurality of activatable elements in the cell that correlates with the neoplastic condition
- the activatable element is selected from the group consisting of elements selected from the group consisting of Erk, Syk, Zap70, Lck, Btk, BLNK, CbI, PLC ⁇ 2, Akt, ReIA, p38, S6
- the activatable element is selected from the group consisting of CbI, PLO ⁇ 2, and S6
- the activatable element is S6 In some embodiments,
- the neoplastic or hematopoietic condition is non-B lineage denved, such as acute myeloid leukemia (AML), Chrome Myeloid Leukemia (CML), non-B cell acute lymphocytic leukemia (ALL), non-B cell lymphomas, myelodysplastic disorders, myeloproliferative disorders, myelofibrosis, polycythemias, thrombocythemias, and non-B atypical immune lymphoproliferations
- the neoplastic or hematopoietic condition is a B-CeIl or B cell lmeage denved disorder, such as B- CeIl or B cell lmeage denved disorder is selected from the group consisting of Chronic Lymphocytic Leuk
- the clinical outcome is the staging or gradmg of a neoplastic or hematopoietic condition, such as aggressive, indolent, benign, refractory, Roman Numeral staging, TNM Staging, Rai staging, Binet staging, WHO classification, FAB classification, IPSS score, WPSS score, limited stage, extensive stage, staging accordmg to cellular markers such as ZAP70, IgV H mutational status and CD38, occult, including information that may inform on time to progression, progression free survival, overall survival, or event-free survival [0036] In some embodiments, the correlation is determining the individual's response to a specific treatment, e.g., normal responder, hyper responder, poor responder, having emerging resistance, non-compliant, and adverse reaction.
- a specific treatment e.g., normal responder, hyper responder, poor responder, having emerging resistance, non-compliant, and adverse reaction.
- the correlation includes classifying the cell as minimal residual disease or emerging resistance.
- the correlation may further include determining a method of treatment, such as chemotherapy, biological therapy, targeted therapy, radiation therapy, bone marrow transplantation, Peripheral stem cell transplantation, umbilical cord blood transplantation, autologous stem cell transplantation, allogeneic stem cell transplantation, syngeneic stem cell transplantation, surgery, induction therapy, maintenance therapy, or watchful waiting.
- the correlation may further include determination of the appropriate dosage or timing of a given treatment.
- the modulator to which the cell is subjected is an activator or an inhibitor.
- the modulator is, e.g., a growth factor, cytokine, adhesion molecule modulator, hormone, small molecule, polynucleotide, antibodies, natural compounds, lactones, chemotherapeutic agents, immune modulator, carbohydrate, proteases, ions, reactive oxygen species, or radiation.
- the modulator is an antibody e.g. anti- CD20 (Rituxan), anti-CD22 (epratuzumab), anti-CD23
- the modulator is a B cell receptor complex modulator, e.g., anti-CD20, which recognizes a component of the B cell receptor co-complex, or a B cell receptor activator such as a cross-linker of the B cell receptor complex or the B-cell co-receptor complex.
- the cross-linker is an antibody, or molecular binding entity. In some embodiments, the cross-linker is an antibody, such as a multivalent antibody.
- the antibody is a monovalent, bivalent, or multivalent antibody made more multivalent by attachment to a solid surface or tethered on a nanoparticle surface to increase the local valency of the epitope binding domain.
- the cross-linker is a molecular binding entity, such as an entity that acts upon or binds the B cell receptor complex via carbohydrates or an epitope in the complex.
- the molecular binding entity is a monovalent, bivalent, or multivalent binding entity that is made more multivalent by attachment to a solid surface or tethered on a nanoparticle surface to increase the local valency of the epitope binding domain.
- cross-linking includes binding of an antibody or molecular binding entity to the cell and then causing its crosslihking via interaction of the cell with a solid surface that causes crosslinking of the BCR complex via antibody or molecular binding entity.
- the crosslinker is selected from the group consisting of F(ab)2 IgM, , IgG, IgD, polyclonal BCR antibodies, monoclonal BCR antibodies, Fc receptor derived binding elements and/or a combination thereof.
- the Ig may be derived from a species selected from the group consisting of mouse, goat, rabbit, pig, rat, horse, cow, shark, chicken, or llama.
- the crosslinker is F(ab)2 IgM, Polyclonal IgM antibodies, Monoclonal IgM antibodies, Biotinylated F(ab)2 IgCM, Biotinylated Polyclonal IgM antibodies, Biotinylated Monoclonal IgM antibodies and/or a combination thereof.
- the modulator to which the cell is subjected is an inhibitor of a cellular factor or a plurality of factors that participates in a signaling cascade in the cell.
- the inhibitor is a kinase or phosphatase inhibitor, such as adaphostin, AG 490, AG 825, AG 957, AG 1024, aloisine, aloisine A, alsterpaullone, aminogenistein, API-2, apigenin, arctigenin, AY-22989, BAY 61-3606, bisindolylmaleimide IX, chelerythrine, 10-[4'-(N,N-Diethylamino)butyl]-2-chlorophenoxa2ine hydrochloride, dasatinib, 2- Dimethylamino-4,5,6,7-tetrabromo-lH-benzimidazole, 5,7-Dimethoxy-3-(4-
- the cell is further subjected to a second modulator, e g , the cell may be subjected to a B cell receptor activator and a kinase inhibitor or a phosphatase inhibitor, such as F(ab) 2 IgM or biotinylated F(ab) 2 IgM and H 2 O 2
- the activation level of the activatable element m the cell is selected from the group consistmg of cleavage by extracellular or intracellular protease exposure, novel hetero-oligomer formation, glycosylation level, phosphorylation level, acetylation level, methylation level, biotinylation level, glutamylation level, glycylation level, hydroxylation level, lsome ⁇ zation level, prenylation level, my ⁇ stoylation level, lipoylation level, phosphopantetheinylation level, sulfation level,
- the modulator is an activator or an inhibitor
- the modulators are mdependently selected from the group consisting of growth factor, cytokine, adhesion molecule modulator, hormone, small molecule, polynucleotide, antibodies, natural compounds, lactones, chemotherapeutic agents, immune modulator, carbohydrate, proteases, ions, reactive oxygen species, and radiation
- at least one modulator is a B cell receptor modulator
- the B cell receptor modulator is a B cell receptor activator, such as Rituxan or a cross-linker of the B cell receptor complex or the B-cell co-receptor complex
- the modulator is PMA, BAFF, April, SDFIa, SCF, CD40L, IGF-I, Imiquimod, polyCpG, fludarabine, cyclophosphamide, chlorambucil IL-7, IL-6, lL-10, IL-
- the cell population is a hematopoietic-de ⁇ ved cell population
- the hematopoietically de ⁇ ved cell population is selected from the group consisting of plunpotent hematopoietic stem cells, B-lymphocyte lmeage progenitor or de ⁇ ved cells, T-lymphocyte lineage progenitor or denved cells, NK cell lineage progenitor or de ⁇ ved cells, granulocyte lineage progenitor or denved cells, monocyte lmeage progenitor or de ⁇ ved cells, megakaryocyte lmeage progenitor or denved cells and erythroid lmeage progemtor or de ⁇ ved cells
- the hematopoietic denved cell population is a B-lymphocyte lmeage progemtor and denved cell population, e g , an early pro-B cell population, late pro-B cell
- the classification mcludes classifying the cell population as a cell population that is correlated with a clinical outcome
- the clinical outcome is the predicted respose to a specific therapy, or the prognosis and/or diagnosis of a condition
- the cluneal outcome is the presence or absence of a neoplastic or a hematopoietic condition, such as Non-Hodgkin Lymphoma, Hodgkin or other lymphomas, acute or chrome leukemias, polycythemias, thrombocythemias, multiple myeloma or plasma cell disorders, e g , amyloidosis and Waldenstrom's macroglobulmemia, myelodysplastic disorders, myeloproliferative disorders, myelofibrosis, or atypical immune lymphoprohferations
- the neoplastic or hematopoietic condition is non-B lineage denved
- the clinical outcome is the staging or grading of a neoplastic or hematopoietic condition
- staging in methods provided by the mvention mclude aggressive, indolent, benign, refractory, Roman Numeral staging, TNM Staging, Rai staging, Bmet staging, WHO classification, FAB classification, IPSS score, WPSS score, limited stage, extensive stage, staging according to cellular markers such as ZAP70, Ig V H mutation status and CD38, occult, including information that may inform on time to progression, progression free survival, overall survival, or event-free survival
- the classifying of the cell population based on activation level includes classifying the cell population as a cell population that is correlated to a patient response to a treatment, such as complete response, partial response, nodular partial response, no response, progressive disease, stable disease, relapse or adverse reaction
- the method may further comprise determining a method of treatment, e g , chemotherapy, biological therapy, targeted therapy, radiation therapy, bone marrow transplantation, Penpheral stem cell transplantation, umbilical cord blood transplantation, autologous stem cell transplantation, allogeneic stem cell transplantation, syngeneic stem cell transplantation, surgery, induction therapy, maintenance therapy, watchful waiting, or holistic/alternative therapy
- the classifying of the cell population based on activation level includes classifying the cell population as a cell population that is correlated with minimal residual disease or emerging resistance
- Figure 1 depicts histograms showing the activation of p-Erk and p-Syk/pZap70 in Ramos cells following F(ab) 2 IgM stimulation
- Figure 2 depicts histograms showmg the phosphorylation of BLNK, CbI, PLC ⁇ 2, Lck, and p38 m Ramos
- Figure 3 depicts histograms showing increased phosphorylation of Erk m Pheresed CLL Samples and m the Ramos cell lines following PMA activation
- Figure 4 depicts a histogram showmg the activation of Erk in Pheresed CLL Samples and Ramos cells following activation with mcreasmg amounts of F(ab) 2 IgM for 15 Mm
- Figure 5 depicts contour plots showing increased phosphorylation of Erk and Syk/Zap 70 in CLL Samples following treatment with PMA or F(ab) 2 IgM
- Figure 6 depicts contour plots showing phosphorylation of Erk and Syk/Zap70 in CLL samples following treatment with H 2 O 2 alone or in combination with F(ab) 2 IgM
- Figure 7 depicts histograms showmg the phosphorylation of Erk and Syk/Zap70 m healthy B cells following treatment with H 2 O 2
- Figure 8 depicts contour plots showing the phosphorylation of Erk and Syk/ Zap70 m CLL cells upon treatment with H 2 O 2 and F(ab) 2 IgM
- Figure 9 depicts contour plots showing Erk and Syk in CLL samples following activation with PMA or
- Figure 10 depicts contour plots showing the phosphorylation of Erk and Syk/ Zap70 following treatment with H 2 O 2 alone or in combination with F(ab) 2 IgM
- Figure 11 depicts contour plots comparing CLL samples with low and high Frequency ZAP70 the phosphorylation or Erk and Syk/ Zap70 following treatment with H 2 O 2 alone or in combination with F(ab) 2 IgM
- Figure 12 depicts contour plots showing Syk/Zap70 and Erk phosphorylation after treatment F(ab) 2 IgM and H 2 O 2 at different times in CLL samples
- Figure 13 depicts contour plots showing Syk/Zap70 and Erk phosphorylation after treatment with F(ab) 2 IgM and H 2 O 2 in CLL samples
- Figure 14 depicts histograms showing the kinetics of signaling mediated by F(ab) 2 IgM and H 2 O 2 m CLL
- Figure 15 depicts contour plots showing the influence of %ZAP70 on BLNK phosphorylation following treatment with H 2 O 2 alone or in combination with F(ab) 2 IgM
- Figure 16 depicts histograms showing the influence of ZAP70 status on BLNK phosphorylation
- Figure 17 depicts histograms showing the kinetics of phosphorylation of pPLC ⁇ 2, prpS6, and pCbl after B- Cell Receptor crosslinking by F(ab) 2 IgM Alone in CD20+/CD5+ population of CLL samples
- Figure 18 depicts histograms showing the kinetics of phosphorylation of pPLC ⁇ 2, ⁇ rpS6, and pCbl after B- CeIl Receptor crosslinking by F(ab) 2 IgM and H 2 O 2 m CD20+/CD5+ population in CLL samples
- Figure 19 depicts histograms showing the kinetics of phosphorylation of pPLC ⁇ 2, prpS6, and pCbl after B- CeIl Receptor crosslinking by F(ab) 2 IgM Alone m CD20+/CD5+ population in CLL samples
- Figure 20 depicts histograms showing the kinetics of phosphorylation of pPLC ⁇ 2, prpS6, and pCbl after B- CeIl Receptor crosslinking by F(ab) 2 lgM and H 2 O 2 in CD20+/CD5+ population in CLL samples
- Figure 21 depicts contour plots showing the phosphoryl
- Figure 23 depicts histograms showing phosphorylation of Syk, Erk and BLNK in response to F(ab) 2 IgM over time in the CD20+/CD5+ population of CLL samples
- Figure 24 depicts histograms showing the kinetics of phosphorylation of pPLC ⁇ 2, prpS6, and pCbl after H 2 O 2 treatment m CD20+/CD5+ population of CLL samples
- Figure 25 depicts histograms showing the kinetics of phosphorylation of pPLC ⁇ 2, prpS6, and pCbl after H 2 O 2 treatment in CD20+/CD5+ population of CLL Samples
- Figure 26 depicts a heat- map showing intracellular responses of CLL patient peripheral blood samples to BCR and/or H 2 O 2 stimulation
- CLL indicates that the sample was taken from a patient diagnosed with CLL CON mdicates that the sample was taken from a healthy subject
- the patient sample numbers are indicated at the top of the heat map and each column represents a single patient
- the shaded squares within the column represent a phospho-protein node
- the different shades represent the degree of phosphorylation of each node (see scale at top of figure)
- Bright white located to the far nght of the scale) represents the greatest increase (i e + 3 0 Log Change)
- Black located m the center of the scale
- dark grey located to the far left of the scale
- m phosphorylation status i e - 3 0 Log Change
- Figure 28 depicts the lower portion of the heat map shown in Figure 26 further illustrating the two patient clusters, 10/22 patients (top left white framed box) and 11/22 patients (bottom nght white framed box), that are distinguished by H 2 O 2 treatment
- the cartoon on the nght of the figure depicts the B cell receptor signaling pathway
- this mvention is directed to methods and compositions for diagnosis, prognosis and to methods of treatment
- the physiological status of cells present in a sample e g clinical sample
- a sample e g clinical sample
- patient selection for therapy e g
- momtor treatment e g
- modify therapeutic regimens e.g
- therapeutic regimens can be individualized and tailored according to the data obtained pnor to, and at different times over the course of treatment, thereby providing a regimen that is individually approp ⁇ ate
- the present mvention is directed to methods for classifying a sample de ⁇ ved from an individual having or suspected of havmg a condition, e g , a neoplastic or a hematopoietic condition The mvention allows for identification of prognostically and therapeutically relevant subgroups of conditions and prediction of the c
- the mvention is directed to methods for classifying a cell by contacting the cell with an inhibitor, determining the presence or absence of an increase in activation level of an activatable element in the cell, and classifying the cell based on the presence or absence of the increase m the activation of the activatable element
- the invention is directed to methods of determining the presence or absence of a condition in an individual by subjectmg a cell from the individual to a modulator and an inhibitor, determining the activation level of an activatable element m the cell, and determining the presence or absence of the condition based on the activation level upon treatment with a modulator and an inhibitor
- the invention is directed to methods for classifying a cell by contacting the cell with an inhibitor, determining the presence or absence of a change in activation level of an activatable element in the cell, and classifying the cell based on the presence or absence of the change m the activation of the activatable element In some embodiments the change
- the invention is directed to methods of determining a phenotypic profile of a population of cells by exposmg the population of cells in separate cultures to a plurality of modulators, wherein at least one of the modulators is an inhibitor, determining the presence or absence of an increase m activation level of an activatable element in the cell population from each of the separate culture and classifying the cell population based on the presence or absence of the increase in the activation of the activatable element from populations of cells m each separate culture [0089] In some embodiments, the invention is directed to methods of classifying a cell population by contacting the cell population with at least one modulator, where the modulator is F(ab)2 IgM, H 2 O 2 , PMA, BAFF, Ap ⁇ l, SDFIa, CD40L, IGF-I, Imiquimod, polyCpG, IL-7, IL-6, IL-IO, IL-27, IL-4, IL-2, IL-3, thapsig
- a method for classifying a cell comprises contacting the cell with an inhibitor, determining the presence or absence of a change in an activation level of at least one activatable element in said cell, and classifying said cell based on said presence or absence of said change in the activation level of said at least one activatable element
- the change is an increase In some embodiments the change is a decrease
- the method of classifying a cell further comprises determining the level of an intracellular marker, cell surface marker or any combination thereof
- a cell may be classified by a change in activation level of an activatable element and also by the level of one or more cell surface markers
- a cell may be classified by a change in activation level of an activatable element and by the level of an intracellular marker Combinations may also be used
- Serum markers are also useful in methods of diagnosis, prognosis, determmg treatments effects and/or choosmg a treatment
- One or more cell surface markers may also be used m the method of the invention in addition to intracellular markers (e g phospho-proteins)
- the method compnses determining the level of a plurality of cell surface markers
- Cell surface markers may include any cell surface molecule that is detected by flow cytometry
- the cell surface marker is a human leukocyte differentiation antigen
- the human leukocyte differentiation antigen is selected from the list CDl, CD2, CD3, CD4, CD5, CD8, CDlO, CD14, CD19, CD20, CD22, CD23, CD40, CD52, CDlOO, CD280, CD281, CD282, CD283, CD284, and CD289
- the human leukocyte differentiation antigen is selected from the list comprising CDl though CD300
- the cell surface marker is any cell surface receptor or hgand Examples of cell surface hgands and receptors include, but are not limited to, members of the TNF superfamily, lnterleukins, hormones,
- the mvention provides methods, including methods to determine the physiological status of a cell, e g , by determining the activation level of an activatable element upon contact with one or more modulators.
- the invention provides methods, mcludmg methods to classify a cell accordmg to the status of an activatable element in a cellular pathway
- the information can be used in prognosis and diagnosis, including susceptibility to disease(s), status of a diseased state and response to changes, in the environment, such as the passage of tune, treatment with drugs or other modalities
- the physiological status of the cells provided in a sample e g clinical sample
- the cells can also be classified as to their ability to respond to therapeutic agents and treatments
- One or more cells, or samples containing one or more cells can be isolated from body samples, such as, but not limited to, smears, sputum, biopsies, secretions, cerebro
- serum such as fetal bovine serum, bovine serum, human serum, porcine serum, horse serum, or goat serum.
- serum When serum is present in the media it could be present at a level ranging from 0.0001 % to 100%.
- serum is present in the media at a level ranging from .0001% to 90%.
- serum is present in the media at a level ranging from 0.01% to 30%.
- serum is present in the media at 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 %.
- serum is present in the media at any suitable level.
- the cell is a hematopoietic cell.
- hematopoietic cells include but are not limited to pluripotent hematopoietic stem cells, B-lymphocyte lineage progenitor or derived cells, T-lymphocyte lineage progenitor or derived cells, NK cell lineage progenitor or derived cells, granulocyte lineage progenitor or derived cells, monocyte lineage progenitor or derived cells, megakaryocyte lineage progenitor or derived cells and erythroid lineage progenitor or derived cells.
- the cells used in the present invention are taken from a patient.
- Cells used in the present invention can be purified from whole blood by any suitable method.
- the term "patient” or "individual” as used herein includes humans as well as other mammals.
- the methods generally involve determining the status of an activatable element.
- the methods also involve determining the status of a plurality of activatable elements.
- the invention provides a method of classifying a cell by determining the presence or absence of a change in activation level of an activatable element in the cell upon treatment with one or more modulators, and classifying the cell based on the presence or absence of the change in the activation of the activatable element.
- the change is a decrease.
- the change is an increase.
- the activation level of the activatable element is determined by contacting the cell with a binding element that is specific for an activation state of the activatable element.
- a cell is classified according to the activation level of a plurality of activatable elements after the cell have been subjected to a modulator.
- the activation levels of a plurality of activatable elements are determined by contacting a cell with a plurality of binding element, where each binding element is specific for an activation state of an activatable element.
- the classification of a cell according to the status of an activatable element can comprise classifying the cell as a cell that is correlated with a clinical outcome.
- the clinical outcome is the prognosis and/or diagnosis of a condition.
- the clinical outcome is the presence or absence of a neoplastic or a hematopoietic condition such as Non-Hodgkin Lymphoma, Hodgkin or other lymphomas, acute or chronic leukemias, polycythemias, thrombocythemias, multiple myeloma or plasma cell disorders, e.g., amyloidosis and Waldenstrom's macroglobulinemia, myelodysplastic disorders, myeloproliferative disorders, myelofibrosis, or atypical immune lymphoproliferations.
- the neoplastic or hematopoietic condition is non-B lineage derived, such as Acute myeloid leukemia (AML), Chronic Myeloid Leukemia (CML), non-B cell Acute lymphocytic leukemia (ALL ), non-B cell lymphomas, myelodysplastic disorders, myeloproliferative disorders, myelofibrosis, polycythemias, thrombocythemias, or non-B atypical immune lymphoproliferations, Chronic Lymphocytic Leukemia (CLL), B lymphocyte lineage leukemia, B lymphocyte lineage lymphoma, Multiple Myeloma, or plasma cell disorders, e.g., amyloidosis or Waldenstrom's macroglobulinemia.
- AML Acute myeloid leukemia
- CML Chronic Myeloid Leukemia
- ALL non-B cell Acute lymphocytic leukemia
- non-B cell lymphomas myelodys
- the clinical outcome is the presence or absence of a neoplastic or a hematopoietic condition, such as Chronic Lymphocytic Leukemia (CLL), B lymphocyte lineage leukemia, B lymphocyte lineage lymphoma, Multiple Myeloma, acute lymphoblastic leukemia (ALL), B-cell pro- lymphocytic leukemia, precursor B lymphoblastic leukemia, hairy cell leukemia or plasma cell disorders, e g , amyloidosis or Waldenstrom's macroglobulinemia, B cell lymphomas including but not limited to diffuse large B cell lymphoma, follicular lymphoma, mucosa associated lymphatic tissue lymphoma, small cell lymphocytic lymphoma, mantle cell lymphoma and marginal zone lymphoma
- the condition is CLL
- the clinical outcome is the staging or grading of a neoplastic or hematopoietic condition
- the classification of a cell accordmg to the status of an activatable element can compnse selectmg a method of treatment
- Example of methods of treatments include, but are not limited to, chemotherapy, biological therapy, radiation therapy, bone marrow transplantation, Penpheral stem cell transplantation, umbilical cord blood transplantation, autologous stem cell transplantation, allogeneic stem cell transplantation, syngeneic stem cell transplantation, surgery, mduction therapy, maintenance therapy, watchful waiting, and holistic/alternative therapy
- Modulators m clude compounds or conditions capable of impacting cellular signaling networks
- a modulator can be an activator or an inhibitor
- Modulators can take the form of a wide variety of environmental inputs Examples of modulators mclude but are not limited to growth factors, cytokines, chemokines, soluble receptors, Toll-like receptor ligands, pathogens, parasites, components of pathogens or parasites, adhesion molecule modulators, pharmaceutical compounds, drugs, hormones, small molecules, poly
- the phosphatase inhibitor is H 2 O 2 .
- the methods of the invention provide methods for determining the presence or absence of a condition in an individual by subjecting a cell from the individual to a modulator and an inhibitor, determining the activation level of an activatable element in the cell, and determining the presence or absence of a condition based on the activation level.
- the activation level of a plurality of activatable elements in the cell is determined.
- the inhibitor can be an inhibitor as described herein.
- the inhibitor is a phosphatase inhibitor.
- the inhibitor is H 2 O 2 .
- the modulator can be any modulator described herein.
- the modulator is a B cell receptor modulator.
- the B cell receptor modulator is a B cell receptor activator.
- An example of B cell receptor activator is a cross-linker of the B cell receptor complex or the B-cell co-receptor complex.
- cross-linker is an antibody or molecular binding entity.
- the cross-linker is an antibody.
- the antibody is a multivalent antibody.
- the antibody is a monovalent, bivalent, or multivalent antibody made more multivalent by attachment to a solid surface or tethered on a nanoparticle surface to increase the local valency of the epitope binding domain.
- the cross-linker can be a molecular binding entity.
- the molecular binding entity acts upon or binds the B cell receptor complex via carbohydrates or an epitope in the complex.
- the molecular is a monovalent, bivalent, or multivalent is made more multivalent by attachment to a solid surface or tethered on a nanoparticle surface to increase the local valency of the epitope binding domain.
- the cross-linking of the B cell receptor complex or the B-cell co-receptor complex can comp ⁇ se binding of an antibody or molecular binding entity to the cell and then causing its crosshnking via interaction of the cell with a solid surface that causes crosslinking of the BCR complex via antibody or molecular bmding entity
- the crosslinker can be F(ab)2 IgM, IgG, IgD, polyclonal BCR antibodies, monoclonal BCR antibodies, Fc receptor de ⁇ ved binding elements and/or a combination thereof
- the Ig can be de ⁇ ved from a species selected from the group consisting of mouse, goat, rabbit, pig, rat, horse, cow, shark, chicken, llama or human
- the Ig or bmdmg element can be fully human or partially human and can be generated by any suitable method known in the art
- the crosslinker is F(ab)2 IgM, Polyclonal IgM antibodies, Monoclonal I
- the methods of the mvention provides for the use of more than one modulator
- the methods of the invention utilize a B cell receptor activator and a phosphatase inhibitor
- the methods of the invention utilize F(ab)2IgM or biotinylated F(ab)2IgM and H 2 O 2
- the methods of the mvention provides for methods of classifying a cell population by exposing the cell population m separate cultures to a plurality of modulators and determining the status of activatable elements in the cell populations
- the status of a plurality of activatable elements m the cell population is determined
- at least one of the modulators of the plurality of modulators is an inhibitor
- the modulator can be any modulators described herem
- the modulator is selected from the group consisting of F(ab)2 IgM, H 2 O 2 , PMA, BAFF, Apnl, SDFIa,
- the methods of the mvention provide for determining a phenotypic profile of a population of cells by exposmg the population of cells in separate cultures to a plurality of modulators, wherem at least one of the modulators is an inhibitor, determining the presence or absence of a change m activation level of an activatable element m the cell population from each of the separate cultures and classifying the cell population based on the presence or absence of the change in the activation of the activatable element from each of the separate cultures
- the change is a decrease
- the change is an increase
- the modulator is selected from the group consisting of F(ab)2 IgM, Rituxan, Alemtuzumab, anti CD22 (epratuzumab), anti-CD23 (lumiliximab), Campath, H 2 O 2 , PMA, BAFF, April, SDFIa, CD40L, IGF-I, Imiquimod, polyCpG, flu
- the mvention provides methods for correlating and/or classifying an activation state of a CLL cell with a cluneal outcome in an individual by subjectmg the CLL cell from the individual to a modulator, wherein the CLL cell expresses B-CeIl receptor (BCR), determining the activation levels of a plurality of activatable elements, and identifying a pattern of the activation levels of the plurality of activatable elements to determine the presence or absence of an alteration m signaling proximal to the BCR, wherein the presence of the alteration is indicative of a clinical outcome
- the activation levels of a plurality of activatable elements are determined by contacting the cell with a plurality of bmdmg elements, where each bindmg element is specific for an activation state of an activatable element
- the clinical outcome can be any clinical outcome descnbed herein [00121]
- the methods of the mvention provide methods for determining tonic signal
- cells e g normal non-transformed cells other than the cells associated with a condition (e g cancer cells) can be used to make clinical decisions That is that cells, other than cells associated with a condition (e g cancer cells), are in fact reflective of the condition process
- Normal cells e g healthy cells or non-transformed cells
- m assigning a nsk group predictmg an mcreased nsk of relapse, predictmg an mcreased nsk of developmg secondary complications, choosing a therapy for an individual, predictmg response to a therapy for an individual, determining the efficacy of a therapy m an individual, and/or determining the prognosis for an individual
- cells other than cells associated with a condition e g cancer cells
- infiltrating immune cells can determine the outcome of the disease
- infiltrating immune cells can determine the outcome of the disease
- the methods of the mvention are applicable to any condition m an individual mvolvmg, indicated by, and/or ansing from, in whole or m part, altered physiological status m a cell
- physiological status includes mechanical, physical, and biochemical functions in a cell
- the physiological status of a cell is determined by measuring charactenstics of cellular components of a cellular pathway
- Cellular pathways are well known m the art
- the cellular pathway is a signaling pathway
- Signaling pathways are also well known in the art (see, e g , Hunter T , Cell (2000) 100(1) 113-27, Cell Signaling Technology, Inc , 2002 Catalogue, Pathway Diagrams pgs 232-253)
- a condition mvolvmg or charactenzed by altered physiological status may be readily identified, for example, by determining the state in a cell of one or more activatable elements, as taught herein
- the condition is a neoplastic or hematopoietic condition
- the neoplastic or hematopoietic condition is selected from the group consisting of Non-Hodgkm Lymphoma, Hodgkin or other lymphomas, acute or chrome leukemias, polycythemias, thrombocythemias, multiple myeloma and plasma cell disorders, mcludmg amyloidosis and Waldenstrom's macroglobuhnemia, myelodysplastic disorders, myeloproliferative disorders, myelofibrosis, and atypical immune lymphoproliferations
- the neoplastic or hematopoietic condition is non-B lineage de ⁇ ved Examples of non- B lmeage de ⁇ ved neoplastic or hematopoietic condition include, but are not limited to, Acute myeloid leuk
- the neoplastic or hematopoietic condition is a B-CeIl or B cell lmeage de ⁇ ved disorder
- B-CeIl or B cell lmeage de ⁇ ved neoplastic or hematopoietic condition mclude but are not limited to Chronic Lymphocytic Leukemia (CLL), B lymphocyte lmeage leukemia, B lymphocyte lineage lymphoma, Multiple Myeloma, and plasma cell disorders, including amyloidosis and Waldenstrom's macroglobuhnemia
- CLL Chronic Lymphocytic Leukemia
- B lymphocyte lmeage leukemia B lymphocyte lineage lymphoma
- Multiple Myeloma Multiple Myeloma
- plasma cell disorders including amyloidosis and Waldenstrom's macroglobuhnemia
- the condition is CLL
- CLL is defined by a monoclonal B cell population that co-expresses CD5 with CD 19 and CD23 or CD5 with CD20 and CD23 and by surface immunoglobulin expression
- CLL is defined by a monoclonal B cell population that co- expresses CD5 with CD 19 and CD23 or CD5 with CD20 and CD23 and dim surface immunoglobulin expression
- Other conditions within the scope of the present mvention include, but are not limited to, cancers such as gliomas, lung cancer, colon cancer and prostate cancer Specific signaling pathway alterations have been desc ⁇ bed for many cancers, including loss of PTEN and resultmg activation of Akt signaling m prostate cancer (Whang Y E Proc Natl Acad Sci USA Apr 28, 1998,95(9) 5246-50), increased IGF-I expression in prostate cancer (Schaefer et al , Science October 9 1998, 282 199a), EGFR over expression and
- the present invention is directed to methods for classifying one or more cells m a sample de ⁇ ved from an individual having or suspected of havmg condition
- the mvention allows for identification of prognostically and therapeutically relevant subgroups of the conditions and prediction of the clinical course of an individual
- the mvention provides method of classifying a cell accordmg to the activation level of one or more activatable element m a cell from an individual havmg or suspected of havmg condition
- the classification m cludes classifying the cell as a cell that is correlated with a clinical outcome The clinical outcome can be the prognosis and/or diagnosis of a condition, and/or stagmg or gradmg of a condition
- the classifying of the cell cludes classifying the cell as a cell that is correlated to a patient response to a treatment
- the classifying of the cell m cludes classifying the cell as a patient response to a treatment
- the methods and compositions of the invention may be employed to examine and profile the status of any activatable element in a cellular pathway, or collections of such activatable elements Single or multiple distinct pathways may be profiled (sequentially or simultaneously), or subsets of activatable elements within a single pathway or across multiple pathways may be examined (again, sequentially or simultaneously)
- activation events can find use in the present invention
- the basic requirement is that the activation results in a change in the activatable protein that is detectable by some indication (termed an "activation state indicator"), preferably by altered binding of a labeled bmdmg element or by changes in detectable biological activities (e g , the activated state has an enzymatic activity which can be measured and compared to a lack of activity m the non-activated state)
- detectable events or moieties between two or more activation states (e g "off' and "on")
- the activation state of an individual activatable element is either in the on or off state
- an individual phosphorylatable site on a protem can activate or deactivate the protem
- activation levels can exist as a distribution and that an activation level of a particular element used to classify a cell may be a particular point on the distribution but more typically may be a portion of the distribution.
- expression levels of intracellular or extracellular biomolecues, e.g , proteins can be used alone or in combination with activation states of activatable elements to classify cells
- additional cellular elements e.g., biomolecules or molecular complexes such as RNA, DNA, carbohydrates, metabolites, and the like, may be used in conjunction with activatable states or expression levels m the classification of cells encompassed here
- other characteristics that affect the status of a cellular constituent may also be used to classify a cell.
- Examples include the translocation of biomolecules or changes m their turnover rates and the formation and disassociation of complexes of biomolecule.
- Such complexes can include multi-protein complexes, multi-lipid complexes, homo- or hetero-dimers or oligomers, and combinations thereof.
- Other characteristics include proteolytic cleavage, e.g. from exposure of a cell to an extracellular protease or from the intracellular proteolytic cleavage of a biomolecule.
- Additional elements may also be used to classify a cell, such as the expression level of extracellular or intracellular markers, nuclear antigens, enzymatic activity, protem expression and localization, cell cycle analysis, chromosomal analysis, cell volume, and morphological characteristics like granularity and size of nucleus or other distinguishing characteristics.
- B cells can be further subdivided based on the expression of cell surface markers such as CD45, CD5, CD19, CD20, CD22, CD23, CD27, CD37, CD40, CD52, CD79, CD38, CD96, major histocompatability antigen (MHC) Class lor MHC Class 2
- predefined classes of cells can be classified based upon shared characteristics that may include inclusion in one or more additional predefined class or the presence of extracellular and/or intracellular markers, a similar gene expression profile, mutational status, epigenetic silencing, nuclear antigens, enzymatic activity, protein expression and localization, cell cycle analysis, chromosomal analysis, cell volume, and morphological characteristics like granularity and size of nucleus or other distinguishing characteristics.
- the physiological status of one or more cells is determined by examining and profiling the activation level of one or more activatable elements in a cellular pathway
- a cell is classified according to the activation level of a plurality of activatable elements.
- a hematopoietic cell is classified according to the activation levels of a plurality of activatable elements. In some embodiments, the activation level of one or more activatable elements of a hematopoietic cell is correlated with a condition.
- the activation level of one or more activatable elements of a hematopoietic cell is correlated with a neoplastic or hematopoietic condition as described herein
- hematopoietic cells include but are not limited to plu ⁇ potent hematopoietic stem cells, myeloid progenitors, B-lymphocyte lineage progenitor or derived cells, T-lymphocyte lineage progemtor or derived cells, NK cell lmeage progenitor or denved cells, granulocyte lineage progenitor or denved cells, monocyte lineage progenitor or derived cells, megakaryocyte lmeage progenitor or derived cells and erythroid lmeage progenitor or denved cells
- the hematopoietic cell is a B-lymphocyte lineage progenitor or derived cell as described herem.
- the activation level of one or more activatable elements in single cells within the sample is determined.
- Cellular constituents that may mclude activatable elements include without limitation, proteins, carbohydrates, lipids, nucleic acids and metabolites
- the activatable element may be a portion of the cellular constituent, for example, an amino acid residue in a protem that may undergo phosphorylation, or it may be the cellular constituent itself, for example, a protein that is activated by translocation from one part of the cell to another, change in conformation (due to, e g , change in pH or ion concentration), by proteolytic cleavage, and the like
- a change occurs to the activatable element, such as covalent modification of the activatable element (e g , binding of a molecule or group to the activatable element, including but not limited to, phosphorylation, acetylation, methylation, ubiquitination) or a conformational change
- Such changes generally contribute to changes
- Activation states of activatable elements may result from chemical additions or modifications of biomolecules and mclude biochemical processes such as glycosylation, phosphorylation, acetylation, methylation, biotinylation, glutamylation, glycylation, hydroxylation, isomenzation, prenylation, my ⁇ stoylation, hpoylation, phosphopantetheinylation, sulfation, ISGylation, mtrosylation, palmitoylation, SUMOylation, ubiquitination, neddylation, citrullination, amidation, and disulfide bond formation, disulfide bond reduction
- Other possible chemical additions or modifications of biomolecules include the formation of protein carbonyls, direct modifications of protem side chains, such as o-tyrosine, chloro-, mtrotyrosine, and dityrosme, and protem adducts derived from reactions with carbohydrate and lipid de ⁇ v
- proteins that may mclude activatable elements mclude, but are not limited to kinases, phosphatases, lipid signaling molecules, adaptor/scaffold proteins, cytokines, cytokine regulators, ubiquitination enzymes, adhesion molecules, cytoskeletal/contractile proteins, heterotnme ⁇ c G proteins, small molecular weight GTPases, guanine nucleotide exchange factors, GTPase activating proteins, caspases, proteins involved m apoptosis (e g PARP), cell cycle regulators, molecular chaperones, metabolic enzymes, vesicular transport proteins, hydroxylases, lsomerases, deacetylases, methylases, demethylases, tumor suppressor genes, proteases, ion channels, molecular transporters, transcription factors/DNA binding factors, regulators of transcription, and regulators of translation Examples of activatable elements, activation states and methods of determining the activation level of
- the protem is selected from the group consisting of HER receptors, PDGF receptors, Kit receptor, FGF receptors, Eph receptors, Trk receptors, IGF receptors, Insulin receptor, Met receptor, Ret, VEGF receptors, TIEl, TIE2, FAK, Jakl, Jak2, Jak3, Tyk2, Src, Lyn, Fyn, Lck, Fgr, Yes, Csk, AbI, Btk, ZAP70, Syk, IRAKs, cRaf, ARaf, BRAF, Mos, Lim kinase, ILK, TpI, ALK, TGF ⁇ receptors, BMP receptors, MEKKs, ASK, MLKs, DLK, PAKs, Mek 1, Mek 2, MKK3/6, MKK4/7, ASKl, Cot, NIK, Bub, Myt 1, Weel, Casern kinases, PDKl, SGKl, SGK2,
- methods and compositions are provided for the classification of a cell according to the activation level of an activatable element, e g , in a cellular pathway wherein the classification comp ⁇ ses classifying the cell as a cell that is correlated with minimal residual disease or emerging resistance
- methods and compositions are provided for the classification of a cell accordmg to the activation level of an activatable element, e g , in a cellular pathway wherem the classification comp ⁇ ses selecting a method of treatment
- Example of methods of treatments include, but are not limited to, chemotherapy, biological therapy, radiation therapy, bone marrow transplantation, Peripheral stem cell transplantation, umbilical cord blood transplantation, autologous stem cell transplantation, allogeneic stem cell transplantation, syngeneic stem cell transplantation, surgery, induction therapy, maintenance therapy, and watchful waiting
- the methods of the invention involve determining the activation levels of an activatable element m a plurality of single cells in a sample
- the methods of the mvention are employed to determine the status of an activatable element in a signaling pathway
- a cell is classified, as desc ⁇ bed herein, accordmg to the activation level of one or more activatable elements m one or more signaling pathways
- Signaling pathways and their members have been extensively desc ⁇ bed See (Hunter T Cell (2000) 100(1) 13-27)
- Exemplary signaling pathways include the following pathways and their members The MAP kinase pathway mcludmg Ras, Raf, MEK, ERK and elk, the PI3K/Akt pathway mcludmg PI-3-kinase, PDKl , Akt and Bad, the NF- ⁇ B pathway including
- the correlated activatable elements bemg assayed are members of the MAP kinase, Akt, NFkB, WNT, STAT and/or PKC signaling pathways
- the methods of the mvention also comprise the methods, signaling pathways and signaling molecules disclosed in US 61/085,789 which is hereby incorporated by reference in its entirety
- the methods of the mvention are employed to determine the status of a signaling protein in a signaling pathway known m the art mcludmg those descnbed herem
- Exemplary types of signaling proteins within the scope of the present mvention include, but are not limited to, kinases, kinase substrates (i e phosphorylated substrates), phosphatases, phosphatase substrates, binding proteins (such as 14-3-3), receptor ligands and receptors (cell surface receptor tyrosine kinases and nuclear receptors)) Kmases and protem binding domains, for example, have been well desc ⁇ bed (see, e g , Cell Signaling Technology, Inc , 2002 Catalogue "The Human Protem Kmases" and "Protem Interaction Domains” pgs 254-279)
- Exemplary signaling proteins m include, but are not limited to, kmases, HER receptors, PDGF receptors, Kit receptor,
- the protein is selected from the group consisting of PI3-Kinase (p85, pi 10a, pi 10b, pi 1Od), Jakl, Jak2, SOCs, Rac, Rho, Cdc42, Ras-GAP, Vav, Tiam, Sos, DbI, Nek, Gab, PRK, SHPl, and SHP2, SHIPl, SHIP2, sSHIP, PTEN, She, Grb2, PDKl, SGK, Aktl, Akt2, Akt3, TSC1,2, Rheb, mTor, 4EBP-1, p70S6Kinase, S6, LKB-I, AMPK, PFK, Acetyl-CoAa Carboxylase, DokS, Rafs, Mos, Tpl2, MEK1/2, MLK3, TAK, DLK, MKK3/6, MEKK1.4, MLK3, ASKl, MKK4/7, SAPK/JNK1,2,3, p38s,
- MAP kinase pathway In some embodiments, the methods of the mvention are employed to determine the status of an activatable element in the MAP kinase pathway
- the MAP Kinase pathway is a signal transduction pathway that couples intracellular responses to the binding of growth factors to cell surface receptors This pathway is very complex and mcludes many protein components In many cell types, activation of this pathway promotes cell division
- Receptor-linked tyrosine kinases such as the epidermal growth factor receptor (EGFR) are activated by extracellular ligands Binding of epidermal growth factor (EGF) to the EGFR activates the tyrosine kinase activity of the cytoplasmic domain of the receptor The EGFR becomes phosphorylated on tyrosines Docking proteins such as GRB2 contain SH2 domains that bmd to the phosphotyrosines of the activated receptor GRB2 bmds to the guanine nucleotide exchange factor SOS by way of an SH3 domain of GRB2 When the GRB2-SOS complex docks to phosphorylated EGFR, SOS becomes activated Activated SOS promotes the removal of GDP from Ras Ras can then bmd GTP and become active Other small G proteins can be activated in a similar way, but are not discussed further here Activated Ras activates the protein kinase activity of RAF kinase,
- RAF, MEK and MAPK are all mitogen-activated kinases, as is MNK MAPK was originally called "extracellular signal-regulated kinases” (ERKs) and microtubule-associated protem kinase (MAPK)
- ERKs extracellular signal-regulated kinases
- MAPK microtubule-associated protem kinase
- One of the first proteins known to be phosphorylated by ERK was a microtubule-associated protem
- MAPK mitogen-activated protem kinase
- the se ⁇ es of kinases from RAF to MEK to MAPK is an example of a protem kinase cascade
- Such series of kinases provide opportunities for feedback regulation and signal amplification RAS is activated m a wide range of cancers (see Cell Signaling Technology, Inc Catolog, supra at pages 231-279 and Hunter T, supra and references therein)
- Pl$K/ AkI pathway the methods of the mvention are employed to determine the status of an activatable element m a PI3K/Akt pathway
- the PI3K/Akt pathway plays a role m effecting alterations in abroad range of cellular functions in response to extracellular signals
- a downstream effector of PI3K is the serine-threonine kinase Akt which m response to PI3K activation, phosphorylates and regulates the activity of a number of targets including kinases, transcription factors and other regulatory molecules
- the serine / threonine kinase Akt functions intracellularly as a nodal pomt for a constellation of convergmg upstream signaling pathways, which involve stimulation of receptor tyrosine kinases such as IGF-IR, HER2 / Neu, VEGF-R, PDGF-R), and an assembly of membrane-localized complexes of receptor-PI3K
- PKC pathway is associated with cell proliferation, differentiation, and apoptosis
- PKC pathway is associated with cell proliferation, differentiation, and apoptosis
- At least eleven closely related PKC isozymes have been reported that differ in then- structure, biochemical properties, tissue distribution, subcellular localization, and substrate specificity They are classified as conventional ( ⁇ , ⁇ l, ⁇ 2, ⁇ ), novel ( ⁇ , ⁇ , ⁇ , ⁇ , ⁇ ), and atypical ( ⁇ , ⁇ ) isozymes
- Conventional PKC isozymes are Ca2+-dependent, while novel and atypical isozymes do not require Ca2+ for their activation
- All PKC isozymes, with the exception of ⁇ and ⁇ , are activated by diacylglycerol (DAG) PKC isozymes negatively or positively regulate c ⁇ tical cell cycle transitions, including cell cycle entry and exit and the Gl and G2 checkpoints Al
- DAG diacylglycerol
- methods and compositions are provided for the classification of a cell according to the status of an activatable element in a signaling pathway wherein the classification compnses classifying a cell as a cell that is correlated to a patient response to a treatment
- the patient response is selected from the group consisting of complete response, partial response, nodular partial response, no response, progressive disease, stable disease and adverse reaction
- methods and compositions are provided for the classification of a cell according to the status of an activatable element in a signaling pathway wherem the classification compnses classifying the cell as a cell that is correlated with minimal residual disease or emerging resistance
- methods and compositions are provided for the classification of a cell according to the status of an activatable element m a signaling pathway wherem the classification compnses selecting a method of treatment
- Example of methods of treatments include, but are not limited to, chemotherapy, biological therapy, radiation therapy, bone marrow transplantation, Penpheral stem cell transplantation, umbilical cord blood transplantation, autologous stem cell transplantation, allogeneic stem cell transplantation, syngeneic stem cell transplantation, surgery, induction therapy, maintenance therapy, watchful waitmg, and holistic/alternative therapy
- the mvention is not limited to presently elucidated signaling pathways and signal transduction proteins, and encompasses signaling pathways and proteins subsequently identified
- B. B-CeIl Receptor Pathway the methods and compositions of the invention may be employed to examine and profile the status of any activatable element in B-CeIl Receptor (BCR) signaling, or collections of such activatable elements in a B-lymphocyte lineage progemtor or de ⁇ ved cell
- BCR B-CeIl Receptor
- the physiological status of one or more B-lymphocyte lineage progemtor or de ⁇ ved cell is determined by examining and profiling the status of one or more activatable element m BCR signaling
- a B-lymphocyte lmeage progenitor or de ⁇ ved cell is classified, as descnbed herein, accordmg to the activation level of one or more activatable elements in BCR signaling
- Examples of B-lymphocyte lmeage de ⁇ ved cell m include, but are not limited to, B-lymphocyte lmeage early pro-B cell, late pro-B cell, large pre-B
- BCR cross-linking triggers phosphorylation of tyrosines within the ITAM motif domains of Ig ⁇ and Ig ⁇ by Src family member tyrosine kinases (e g , Lyn, Lck, BIk, Fyn)
- Src family member tyrosine kinases e g , Lyn, Lck, BIk, Fyn
- the phosphorylated ITAMs of Ig ⁇ recruit and enhance phosphorylation of Syk (directly) and Btk (via Syk)
- BCR cross-linking also brings together numerous regulator and adapter molecules (e g , SLP-65/BLNK, Grb2, CD22, SHP-I) and compartmentalizes the BCR m lipid rafts with coreceptors CD19 and CD21
- the enzymes phospholipase-C ⁇ 2 (PLC ⁇ 2) and PI3K propagate BCR signaling
- PLC ⁇ 2 activation generates calcium flux,
- the classifying of the B-lymphocyte lineage progemtor or denved cells based on activation of an activatable element m BCR pathway includes classifying the cell as a cell that is correlated with minimal residual disease or emerging resistance
- the methods and compositions of the invention may be employed to determine the status of a tome signaling pathway in a cell
- the methods and compositions of the invention may be employed to examine and profile the status of any activatable element in a tonic signaling pathway, or collections of such activatable elements in a cell
- the physiological status of a cell is determined by examining and profiling the status of one or more activatable elements in a tonic signaling pathway
- a cell is classified, as descnbed herein, according to the status of one or more activatable elements in a tome signaling pathway
- the term "tonic signaling" includes antigen-independent signaling, mdependent basal signaling and non-induced or hgand-independent signaling [00177] Without intending to be limited to any theory, recent evidence supports the notion that in most signal transduction systems regulated by cellular receptors some basal level of signaling occurs continuously in a hgand- mdependent manner,
- the clinical outcome is the staging or gradmg of a neoplastic or hematopoietic condition
- staging mclude, but are not limited to, aggressive, mdolent, benign, refractory, Roman Numeral staging, TNM Staging, Rai staging, Bmet staging, WHO classification, FAB classification, IPSS score, WPSS score, limited stage, extensive stage, staging accordmg to cellular markers such as ZAP70, IgV H mutational status and CD38, including information that may inform on time to progression, progression free survival, overall survival, or event-free survival
- the invention provides methods for classifying a CLL cell based on an alteration in signaling proximal to the BCR that is indicative of the presence of tome signaling The presence of the alteration is indicative of a cluneal outcome, where the clinical outcome is as descnbed herein
- methods and compositions are provided for the classification of a cell accordmg to the status of an activatable element m a tonic signaling pathway wherein the classification compnses classifying a cell as a cell that is correlated to a patient response to a treatment
- the patient response is selected from the group consistmg of complete response, partial response, nodular partial response, no response, progressive disease, stable disease and adverse reaction
- methods and compositions are provided for the classification of a cell accordmg to the status of an activatable element in a tome signaling pathway wherein the classification compnses classifying the cell as a cell that is correlated with mimmal residual disease or emergmg resistance
- methods and compositions are provided for the classification of a cell accordmg to the status of an activatable element m a tonic signaling pathway wherein the classification compnses selectmg a method of treatment
- Example of methods of treatments mclude, but are not limited to, chemotherapy, biological therapy, radiation therapy, bone marrow transplantation, Penpheral stem cell transplantation, umbilical cord blood transplantation, autologous stem cell transplantation, allogeneic stem cell transplantation, syngeneic stem cell transplantation, surgery, mduction therapy, maintenance therapy, watchful waiting, and holistic/alternative therapy Binding Element
- the activation level of an activatable element is determined by contacting a cell with a binding element that is specific for an activation state of the activatable element.
- the term "Binding element” includes any molecule, e.g., peptide, nucleic acid, small organic molecule which is capable of detecting an activation state of an activatable element over another activation state of the activatable element.
- the binding element is a peptide, polypeptide, oligopeptide or a protein.
- the peptide, polypeptide, oligopeptide or protein may be made up of naturally occurring amino acids and peptide bonds, or synthetic peptidomimetic structures.
- amino acid or “peptide residue”, as used herein include both naturally occurring and synthetic amino acids.
- homo-phenylalanine, citrulline and noreleucine are considered amino acids for the purposes of the invention.
- the side chains may be in either the (R) or the (S) configuration.
- the amino acids are in the (S) or L-conf ⁇ guration. If non-naturally occurring side chains are used, non-amino acid substituents may be used, for example to prevent or retard in vivo degradation.
- Proteins including non-naturally occurring amino acids may be synthesized or in some cases, made recombinantly; see van Hest et al, FEBS Lett 428:(l-2) 68-70 May 22, 1998 and Tang et al., Abstr. Pap Am. Chem. S218: U138 Part 2 Aug. 22, 1999, both of which are expressly incorporated by reference herein.
- Methods of the present invention may be used to detect any particular activatable element in a sample that is antigenically detectable and antigenically distinguishable from other activatable element which is present in the sample.
- the activation state-specific antibodies of the present invention can be used in the present methods to identify distinct signaling cascades of a subset or subpopulation of complex cell populations; and the ordering of protein activation (e.g., kinase activation) in potential signaling hierarchies.
- protein activation e.g., kinase activation
- the expression and phosphorylation of one or more polypeptides are detected and quantified using methods of the present invention.
- the expression and phosphorylation of one or more polypeptides that are cellular components of a cellular pathway are detected and quantified using methods of the present invention.
- the term "activation state-specific antibody” or “activation state antibody” or grammatical equivalents thereof refer to an antibody that specifically binds to a corresponding and specific antigen.
- the corresponding and specific antigen is a specific form of an activatable element.
- the binding of the activation state-specific antibody is indicative of a specific activation state of a specific activatable element.
- the binding element is an antibody.
- the binding element is an activation state-specific antibody.
- the binding element is an phospho-specific antibody.
- antibody includes full length antibodies and antibody fragments, and may refer to a natural antibody from any organism, an engineered antibody, or an antibody generated recombinantly for experimental, therapeutic, or other purposes as further defined below. Examples of antibody fragments, as are known in the art, such as Fab, Fab', F(ab')2, Fv, scFv, or other antigen-binding subsequences of antibodies, either produced by the modification of whole antibodies or those synthesized de novo using recombinant DNA technologies.
- Fab fragment antigen-binding subsequences of antibodies
- antibody comprises monoclonal and polyclonal antibodies. Antibodies can be antagonists, agonists, neutralizing, inhibitory, or stimulatory.
- the antibodies of the present invention may be nonhuman, chimeric, humanized, or fully human.
- Chimeric antibodies comprise the variable region of a nonhuman antibody, for example VH and VL domains of mouse or rat origin, operably linked to the constant region of a human antibody (see for example U.S. Pat. No. 4,816,567).
- the antibodies of the present invention are humanized.
- humanized antibody as used herein is meant an antibody comprising a human framework region (FR) and one or more complementarity determining regions (CDR's) from a non-human (usually mouse or rat) antibody.
- the non-human antibody providing the CDR's is called the “donor” and the human immunoglobulin providing the framework is called the “acceptor”.
- Humanization relies principally on the grafting of donor CDRs onto acceptor (human) VL and VH frameworks (Winter U.S. Pat. No. 5,225,539). This strategy is referred to as "CDR grafting". "Backmutation" of selected acceptor framework residues to the corresponding donor residues is often required to regain affinity that is lost in the initial grafted construct (U.S.
- the humanized antibody optimally also will comprise at least a portion of an immunoglobulin constant region, typically that of a human immunoglobulin, and thus will typically comprise a human Fc region.
- humanized murine monoclonal antibodies are also known in the art, for example antibodies binding human protein C (O'Connor et al., 1998, Protein Eng 11:321-8), interleukin 2 receptor (Queen et al., 1989, Proc Natl Acad Sci, USA 86: 10029-33), and human epidermal growth factor receptor 2 (Carter et al., 1992, Proc Natl. Acad Sci USA 89:4285-9).
- the antibodies of the present invention may be fully human, that is the sequences of the antibodies are completely or substantially human.
- a number of methods are known in the art for generating fully human antibodies, including the use of transgenic mice (Bruggemann et al.,
- aglycosylated antibody as used herein is meant an antibody that lacks carbohydrate attached at position 297 of the Fc region, wherein numbering is according to the EU system as in Kabat
- the aglycosylated antibody may be a deglycosylated antibody, which is an antibody for which the Fc carbohydrate has been removed, for example chemically or enzymatically.
- the aglycosylated antibody may be a nonglycosylated or unglycosylated antibody, that is an antibody that was expressed without Fc carbohydrate, for example by mutation of one or residues that encode the glycosylation pattern or by expression in an organism that does not attach carbohydrates to proteins, for example bacteria.
- activation state specific antibodies can be used to detect kinase activity, however additional means for determining kinase activation are provided by the present invention.
- substrates that are specifically recognized by protein kinases and phosphorylated thereby are known.
- Antibodies that specifically bind to such phosphorylated substrates but do not bind to such non-phosphorylated substrates may be used to determine the presence of activated kinase in a sample.
- an element activation profile is determined using a multiplicity of activation state antibodies that have been immobilized.
- Antibodies may be non-diffi ⁇ sibly bound to an insoluble support having isolated sample-receiving areas (e.g. a microliter plate, an array, etc.).
- the insoluble supports may be made of any composition to which the compositions can be bound, is readily separated from soluble material, and is otherwise compatible with the overall method of screening.
- the surface of such supports may be solid or porous and of any convenient shape. Examples of suitable insoluble supports include microtiter plates, arrays, membranes, and beads.
- Microtiter plates and arrays are especially convenient because a large number of assays can be earned out simultaneously, using small amounts of reagents and samples. In some cases magnetic beads and the like are included.
- the particular manner of binding of the composition is not crucial so long as it is compatible with the reagents and overall methods of the invention, maintains the activity of the composition and is non-diffusible.
- Methods of binding include the use of antibodies (which do not ste ⁇ cally block either the ligand bmding site or activation sequence when the protein is bound to the support), direct binding to "sticky" or iomc supports, chemical cross-linking, the synthesis of the antibody on the surface, etc. Following binding of the antibody, excess unbound matenal is removed by washing. The sample receiving areas may then be blocked through mcubation with bovine serum albumin (BSA), casein or other innocuous protein or other moiety
- BSA bovine serum albumin
- an activated isoform of an activatable element is distinguishable from the antigenicity of non-activated isoform of an activatable element or from the antigenicity of an isoform of a different activation state.
- an activated isoform of an element possesses an epitope that is absent in a non- activated isoform of an element, or vice versa.
- this difference is due to covalent addition of moieties to an element, such as phosphate moieties, or due to a structural change in an element, as through protein cleavage, or due to an otherwise mduced conformational change in an element which causes the element to present the same sequence in an antigenically distinguishable way.
- such a conformational change causes an activated isoform of an element to present at least one epitope that is not present in a non-activated isoform, or to not present at least one epitope that is presented by a non-activated isoform of the element.
- the epitopes for the distinguishing antibodies are centered around the active site of the element, although as is known m the art, conformational changes in one area of an element may cause alterations in different areas of the element as well.
- proteins that can be analyzed with the methods described herein include, but are not limited to, kinases, HER receptors, PDGF receptors, Kit receptor, FGF receptors, Eph receptors, Trk receptors, IGF receptors, Insulin receptor, Met receptor, Ret, VEGF receptors, TIEl, TIE2, FAK, Jakl, Jak2, JaB, Tyk2, Src, Lyn, Fyn, Lck, Fgr, Yes, Csk, AbI, Btk, ZAP70, Syk, IRAKs, cRaf, ARaf, BRAF, Mos, Lim kinase, ILK, TpI, ALK, TGF ⁇ receptors, BMP receptors, MEKKs, ASK, MLKs, DLK, PAKs, Mek 1, Mek 2, MKK3/6, MKK4/7, ASKl.Cot, NIK, Bub, Myt 1, Weel, Casein kinases, PDKl, SGK
- Slmgshot phosphatases SSH
- serine phosphatases PP2A, PP2B, PP2C, PPl, PP5, inositol phosphatases, PTEN, SHIPs, myotubularins, lipid signaling, phosphoinositide kinases, phospholipases, prostaglandin synthases, 5- lipoxygenase, sphingosine kinases, sphingomyelinases, adaptor/scaffold proteins, She, Grb2, BLNK, LAT, B cell adaptor for PI3-kinase (BCAP), SLAP, Dok, KSR, MyD88, Crk, CrkL, GAD, Nek, Grb2 associated binder (GAB), Fas associated death domain (FADD), TRADD, TRAF2, RIP, T-CeIl leukemia family, cytokines, IL-2, IL-4, IL-8, IL-6, mterferon
- an epitope-recognizing fragment of an activation state antibody rather than the whole antibody is used in some embodiments, the epitope-recognizing fragment is immobilized in some embodiments, the antibody light chain that recognizes an epitope is used A recombinant nucleic acid encoding a light chain gene product that recogmzes an epitope may be used to produce such an antibody fragment by recombinant means well known in the art
- Non-activation state antibodies may also be used in the present mvention
- non- activation state antibodies bind to epitopes in both activated and non-activated forms of an element Such antibodies may be used to determine the amount of non-activated plus activated element in a sample
- non-activation state antibodies bmd to epitopes present in non-activated forms of an element but absent in activated forms of an element Such antibodies may be used to determine the amount of non-activated element in a sample
- Both types of non-activation state antibodies may be used to determine if a change in the amount of activation state element, for example from samples before and after treatment with a candidate bioactive agent as descnbed herem, coincide with changes in the amount of non-activation state element
- such antibodies can be used to determine whether an increase in activated element is due to activation of non-activation state element, or due to increased expression of the element, or both [00199]
- antibodies are immobilized using beads analogous to
- Attachment of a multiplicity of activation state specific antibodies to beads may be done by methods known in the art and/or descnbed herein.
- Such conjugated beads may be contacted with sample, preferably cell extract, under conditions that allow for a multiplicity of activated elements, if present, to bind to the multiplicity of immobilized antibodies.
- a second multiplicity of antibodies comprising non-activation state antibodies which are uniquely labeled may be added to the immobilized activation state specific antibody-activated element complex and the beads may be sorted by FACS on the basis of the presence of each label, wherein the presence of label indicates binding of corresponding second antibody and the presence of corresponding activated element.
- aromatic amino acids of protein binding elements may be replaced with D- or L-naphylalanine, D- or L-phenylglycine, D- or L-2-thieneylalanine, D- or L-I-, 2-, 3- or 4- pyreneylalamne, D- or L-3-thieneylalamne, D- or L-(2-py ⁇ dinyl)-alamne, D- or L-(3-pyridinyl)-alanine, D- or L-(2- pyrazinyl)-alanine, D- or L-(4-isopropyl)-phenylglycine, D-(trifluoromethyl)-phenylglycine, D-(tnfluoromethyl)- phenylalamne, D-p-fluorophenylalanine, D- or L-p-biphenylphenylalanine, D- or L-p- me
- Acidic ammo acids can be substituted with non-carboxylate ammo acids while maintaining a negative charge, and derivatives or analogs thereof, such as the non-limiting examples of (phosphono)alanine, glycine, leucme, isoleucine, threonine, or serine; or sulfated (e g , --SO3H) threonine, serine, or tyrosine.
- (phosphono)alanine glycine, leucme, isoleucine, threonine, or serine
- sulfated e g , --SO3H
- alkyl refers to a branched or unbranched saturated hydrocarbon group of 1 to 24 carbon atoms, such as methyl, ethyl, n-propyl, isoptopyl, n-butyl, lsobutyl, t-butyl, octyl, decyl, tetradecyl, hexadecyl, eicosyl, tetracisyl and the like.
- Alkyl includes heteroalkyl, with atoms of nitrogen, oxygen and sulfur
- alkyl groups herem contain 1 to 12 carbon atoms.
- Basic ammo acids may be substituted with alkyl groups at any position of the naturally occurring amino acids lysine, arginine, ornithine, citrulhne, or (guanidino)-acetic acid, or other (guanidino)alkyl-acetic acids, where "alkyl" is define as above.
- Nitrile de ⁇ vatives e.g., containing the CN-moiety in place of COOH
- methionine sulfoxide may be substituted for methionine.
- any amide linkage in any of the polypeptides may be replaced by a ketomethylene moiety.
- denvatives are expected to have the property of increased stability to degradation by enzymes, and therefore possess advantages for the formulation of compounds which may have increased in vivo half lives, as administered by oral, intravenous, intramuscular, intraperitoneal, topical, rectal, intraocular, or other routes.
- Additional ammo acid modifications of amino acids of variant polypeptides of to the present invention may include the following: Cysteinyl residues may be reacted with alpha-haloacetates (and corresponding amines), such as 2-chloroacetic acid or chloroacetamide, to give carboxymethyl or carboxyamidomethyl de ⁇ vatives.
- alpha-haloacetates such as 2-chloroacetic acid or chloroacetamide
- Cysteinyl residues may also be derivatized by reaction with compounds such as bromotrifluoroacetone, alpha-bromo-beta-(5- imidozoyl)propionic acid, chloroacetyl phosphate, N-alkylmaleimides, 3-nitro-2-pyridyl disulfide, methyl 2-pyridyl disulfide, p-chloromercunbenzoate, 2-chloromercuri-4-nitrophenol, or chloro-7-nitrobenzo-2-oxa-l,3-diazole.
- compounds such as bromotrifluoroacetone, alpha-bromo-beta-(5- imidozoyl)propionic acid, chloroacetyl phosphate, N-alkylmaleimides, 3-nitro-2-pyridyl disulfide, methyl 2-pyridyl disulfide, p-chloromercunbenzoate, 2-chloromercuri-4-
- Histidyl residues may be denvatized by reaction with compounds such as diethylprocarbonate e g , at pH 5 5-7 0 because this agent is relatively specific for the histidyl side chain, and para-bromophenacyl bromide may also be used, e g , where the reaction is preferably performed in 0 IM sodium cacodylate at pH 6 0
- Lysinyl and amino terminal residues may be reacted with compounds such as succinic or other carboxylic acid anhydrides Denvatization with these agents is expected to have the effect of reversing the charge of the lysinyl residues
- Suitable reagents for denvatizmg alpha-amino-containing residues include compounds such as lmidoesters, e g , as methyl picolinimidate, py ⁇ doxal phosphate, py ⁇ doxal, chloroborohyd ⁇ de, t ⁇ mtrobenzenesulfomc acid, O-methyhsourea, 2,4 pentanedione, and transammase-catalyzed reaction with glyoxylate
- Arginyl residues may be modified by reaction with one or several conventional reagents, among them phenylglyoxal, 2,3-butanedione, 1 ,2-cyclohexanedione, and ninhydnn accordmg to known method steps Denvatization of arginine residues requires that the reaction be performed in alkaline conditions because of the high pKa of the guanidine functional group Furthermore, these reagents may react with
- N-acetylimidizol and tetramtromethane may be used to form O-acetyl tyrosyl species and 3-nitro denvatives, respectively
- Carboxyl side groups (aspartyl or glutamyl) may be selectively modified by reaction with carbodiimides (R' ⁇ N-C-N ⁇ R') such as l-cyclohexyl-3-(2-morpholmy- l-(4-ethyl) carbodiimide or l-ethyl-3-(4- azonia-4,4-dimethylpentyl) carbodiimide
- aspartyl and glutamyl residues may be converted to asparaginyl and glutaminyl residues by reaction with ammonium ions
- the activation state-specific binding element is a peptide comp ⁇ smg a recognition structure that binds to a target structure on an activatable protem
- phage display libraries see e g , Gururaja et al (2000) Chem Biol 7 515-27, Houimel et al , (2001) Eur J Immunol 31 3535-45, Cochran et al (200I) J Am Chem Soc 123 625-32, Houimel et al (2001) Int J Cancer 92 748-55, each incorporated here
- the binding element is a nucleic acid
- nucleic acid include nucleic acid analogs, for example, phosphoramide (Beaucage et al , (1993) Tetrahedron 49(10) 1925 and references therein, Letsmger, J (1970) Org Chem 35 3800, Sp ⁇ nzl et al , (1977) Eur J Biochem 81 579, Letsinger et al , (1986) Nucl Acids Res 14 3487, Sawai et al, (1984) Chem Lett 805, Letsinger et al , (1988) J Am Chem Soc
- nucleic acid analogs may find use m the present invention
- mixtures of naturally occurring nucleic acids and analogs can be made
- mixtures of different nucleic acid analogs, and mixtures of naturally occurring nucleic acids and analogs may be made
- peptide nucleic acids (PNA) which includes peptide nucleic acid analogs are used
- PNA peptide nucleic acids
- the nucleic acids may be smgle stranded or double stranded, as specified, or contain portions of both double stranded or single stranded sequence
- the nucleic acid may be DNA, both genomic and cDNA, RNA or a hyb ⁇ d, where the nucleic acid contains any combination of deoxy ⁇ bo- and ⁇ bo-nucleotides, and any combmation of bases, mcludmg uracil, adenine, thymine, cytosine, guanine, mosine, xathamne hypoxathanine, isocytosine, isoguanine, etc
- the binding element is a synthetic compound Any numbers of techniques are available for the random and directed synthesis of a wide variety of organic compounds and biomolecules, including expression of randomized oligonucleotides See for example WO 94/24314, hereby expressly incorporated by reference, which discusses methods for generatmg new compounds, including random chemistry methods as well as enzymatic methods [00216] Alternatively, some embodiments utilize natural compounds, as binding elements, in the form of bactenal, fungal, plant and animal extracts that are available or readily produced
- the binding element is a small organic compound Bmdmg elements can be synthesized from a senes of substrates that can be chemically modified "Chemically modified” herem includes traditional chemical reactions as well as enzymatic reactions
- substrates generally include, but are not limited to, alkyl groups (including alkanes, alkenes, alkynes and heteroalkyl), aryl groups (mcludmg arenes and heteroaryl), alcohols, ethers, amines, aldehydes, ketones, acids, esters, amides, cyclic compounds, heterocyclic compounds (mcludmg purines, py ⁇ midmes, benzodiazepine, beta-lactams, tetracylines, cephalosporins, and carbohydrates), steroids (mcludmg estrogens, androgens, cortisone, ecodysone, etc ), alkaloids (mcludmg ergots, vinca, curare, pyrollizd
- the bmdmg element is a carbohydrate
- carbohydrate is meant to mclude any compound with the general formula (CFkO) n
- carbohydrates are di-, hi- and oligosaccharides, as well polysaccharides such as glycogen, cellulose, and starches
- the bmdmg element is a lipid
- the term lipid herem is meant to mclude any water insoluble organic molecule that is soluble m nonpolar organic solvents
- lipids are steroids, such as cholesterol, and phospholipids such as sphingomeylin
- label is meant a molecule that can be directly (i e , a primary label) or indirectly (l e , a secondary label) detected, for example a label can be visualized and/or measured or otherwise identified so that its presence or absence can be known
- a compound can be directly or indirectly conjugated to a label which provides a detectable signal, e g radioisotopes, fluorescers, enzymes, antibodies, particles such as magnetic particles, chemiluminescers, or specific bmdmg molecules, etc
- Specific binding molecules include pairs, such as biotm and streptavidin, digoxin and antidigoxin etc
- labels include, but are not limited to, optical fluorescent and chromogemc dyes mcludmg labels, label enzymes and radioisotopes
- one or more binding elements are umquely label
- a first activation state antibody recogmzing a first activated element comprises a first label
- second activation state antibody recogmzmg a second activated element comp ⁇ ses a second label, wherein the first and second labels are detectable and distinguishable, making the first antibody and the second antibody uniquely labeled
- labels fall into four classes a) isotopic labels, which may be radioactive or heavy isotopes, b) magnetic, electrical, thermal labels, c) colored, optical labels mcludmg luminescent, phosphorous and fluorescent dyes or moieties; and d) binding partners. Labels can also include enzymes (horseradish peroxidase, etc.) and magnetic particles.
- the detection label is a primary label.
- a primary label is one that can be directly detected, such as a fluorophore.
- Labels include optical labels such as fluorescent dyes or moieties.
- Fluorophores can be either "small molecule” fluors, or proteinaceous fluors (e.g. green fluorescent proteins and all variants thereof).
- Suitable fluorescent labels include, but are not limited to, fluorescein, rhodamine, tetramethylrhodamine, eosin, erythrosin, coumarin, methyl-coumarins, pyrene, Malacite green, stilbene, Lucifer Yellow, Cascade BlueTM, Texas Red, IAEDANS, EDANS, BODIPY FL, LC Red 640, Cy 5, Cy 5.5, LC Red 705 and Oregon green.
- Suitable optical dyes are described in the 1996 Molecular Probes Handbook by Richard P. Haugland, hereby expressly incorporated by reference.
- Suitable fluorescent labels also include, but are not limited to, green fluorescent protein (GFP; Chalfie, et al., Science 263(5148):802-805 (Feb. 11, 1994); and EGFP; Clontech-Genbank Accession Number U55762 ), blue fluorescent protein (BFP; 1. Quantum Biotechnologies, Inc. 1801 de Maisonneuve Blvd. West, 8th Floor, Montreal (Quebec) Canada H3H 1 J9; 2. Stauber, R. H. Biotechniques 24(3):462-471 (1998); 3. Heim, R. and Tsien, R. Y. Curr. Biol. 6:178-182 (1996)), enhanced yellow fluorescent protein (EYFP; 1. Clontech Laboratories, Inc., 1020 East Meadow Circle, Palo Alto, Calif. 94303), luciferase (Ichiki, et al., J. Immunol.
- labels for use in the present invention include: Alexa-Fluor dyes (Alexa Fluor 350, Alexa Fluor 430, Alexa Fluor 488, Alexa Fluor 546, Alexa Fluor 568, Alexa Fluor 594, Alexa Fluor 633, Alexa Fluor 660, Alexa Fluor 680), Cascade Blue, Cascade Yellow and R-phycoerythrin (PE) (Molecular Probes) (Eugene, Oreg.), FITC, Rhodamine, and Texas Red (Pierce, Rockford, 111.), Cy5, Cy5.5, Cy7 (Amersham Life Science, Pittsburgh, Pa.).
- Alexa-Fluor dyes Alexa Fluor 350, Alexa Fluor 430, Alexa Fluor 488, Alexa Fluor 546, Alexa Fluor 568, Alexa Fluor 594, Alexa Fluor 633, Alexa Fluor 660, Alexa Fluor 680
- Cascade Blue Cascade Yellow and R-phycoerythrin (PE
- Tandem conjugate protocols for Cy5PE, Cy5.5PE, Cy7PE, Cy5.5APC, Cy7APC are known in the art. Quantitation of fluorescent probe conjugation may be assessed to determine degree of labeling and protocols including dye spectral properties are also well known in the art.
- the fluorescent label is conjugated to an aminodextran linker which is conjugated to a binding element or antibody. Additional labels listed in and are available through the on-line and hard copy catalogues of BD Biosciences, Beckman Coulter, AnaSpec, Invitrogen, Cell Signaling Technology, Millipore, eBioscience, Caltag, Santa Cruz Biotech, Abeam and Sigma, the contents of which are incorporated herein by reference.
- the fluorescent label is a GFP and, more preferably, a Renilla, Ptilosarcus, or Aequorea species of GFP.
- a secondary detectable label is used.
- a secondary label is one that is indirectly detected; for example, a secondary label can bind or react with a primary label for detection, can act on an additional product to generate a primary label (e.g. enzymes), etc.
- Secondary labels include, but are not limited to, one of a binding partner pair; chemically modifiable moieties; nuclease inhibitors, enzymes such as horseradish peroxidase, alkaline phosphatases, luciferases, etc.
- the secondary label is a binding partner pair.
- the label may be a hapten or antigen, which will bind its binding partner.
- suitable binding partner pairs include, but are not limited to: antigens (such as proteins (including peptides) and small molecules) and antibodies (including fragments thereof (F Abs, etc.)); proteins and small molecules, including biotin/streptavidin; enzymes and substrates or inhibitors, other protein-protein interacting pairs, receptor-hgands, and carbohydrates and their binding partners Nucleic acid—nucleic acid binding proteins pairs are also useful Binding partner pairs include, but are not limited to, biotin (or lmino-biotin) and streptavidin, digeoxinin and Abs, and ProhnxTM reagents
- the binding partner pan- comp ⁇ ses an antigen and an antibody that will specifically bind to the antigen
- specifically bind herein is meant that the partners bmd with specificity sufficient to differentiate between die pair and other components or contaminants of the system The binding should be sufficient to remain bound under the conditions of the assay, including wash steps to remove non-specific binding
- the dissociation constants of the pair will be less than about 10 "4 to 10 9 M ', with less than about 10 5 to 10 9 M ' being preferred and less than about 10 7 to 10 ' M ' bemg particularly preferred
- the secondary label is a chemically modifiable moiety
- labels comprising reactive functional groups are incorporated into the molecule to be labeled The functional group can then be subsequently labeled (e g either before or after the assay) with a primary label
- Suitable functional groups include, but are not limited to, ammo groups, carboxy groups, maleimide groups, ox
- each lable is distmct and distinguishable from other labels
- labeled antibodies are used for functional analysis of activatable proteins m cells In performing such analysis several areas of the expe ⁇ ment are considered (1) identification of the proper combination of antibody cocktails for the stains (2), identification of the sequential procedure for die staining usmg the antigens (i e , the activatable protein) and antibody clones of interest, and (3) thorough evaluation of cell culture conditions' effect on cell stimulation
- Antigen clone selection is of particular importance for surface antigens of human cells, as different antibody clones yield different result and do not stain similarly in different protocols Selection of cell types and optimization
- Quantum dot labeled antibodies can be used alone or they can be employed m conjunction with organic fluorochrome-conjugated antibodies to increase the total number of labels available As the number of labeled antibodies mcrease so does the ability for subtyping known cell populations Additionally, activation state-specific antibodies can be labeled using chelated or caged lanthanides as disclosed by Erkki, J et al Lanthanide chelates as new fluorochrome labels for cytochemistry J Histochemistry Cytochemistry, 36 1449-1451, 1988, and U S Patent No 7,018850, entitled Salicylamide-Landianide Complexes for Use as Luminescent Markers Other methods of
- FRET fluorescence resonance energy transfer
- FRET Fluorescence Activated FRET
- a FRET pair consists of a donor fluorophore and an acceptor fluorophore
- the fluorescence emission spectrum of the donor and the fluorescence absorption spectrum of the acceptor must overlap, and the two molecules must be in close proximity
- the distance between donor and acceptor at which 50% of donors are deactivated (transfer energy to the acceptor) is defined by the Forster radius (Ro), which is typically 10- 100
- Ro Forster radius
- FRET pairs (donor/acceptor) useful m the invention include, but are not limited to, EDANS/fluorescein, IAEDANS/fluorescem, fluorescein/tetramethylrhodamine, fluorescein/LC Red 640, fluorescein/Cy 5, fluorescein/Cy 5 5 and fluorescein/LC Red 705
- a fluorescent donor molecule and a non-fluorescent acceptor molecule may be employed in this application.
- fluorescent emission of the donor will increase when quencher is displaced from close proximity to the donor and fluorescent emission will decrease when the quencher is brought into close proximity to the donor
- Useful quenchers include, but are not limited to, TAMRA, DABCYL, QSY 7 and QSY 33
- Useful fluorescent donor/quencher pairs mclude, but are not limited to EDANS/DABCYL, Texas Red/D ABCYL, BODIPY/D ABCYL, Lucifer yellow/D ABCYL, couma ⁇ n/D ABCYL and fluorescein/QSY 7 dye
- changes m the degree of FRET are determined as a function of the change m the ratio of the amount of fluorescence from the donor and acceptor moieties, a process referred to as "ratioing"
- ratioing Changes m the absolute amount of substrate, excitation intensity, and turbidity or other background absorbances m the sample at the excitation wavelength affect the intensities of fluorescence from both the donor and acceptor approximately m parallel Therefore the ratio of the two emission intensities is a more robust and preferred measure of cleavage than either intensity alone
- the ratio-metric fluorescent reporter system descnbed herein has significant advantages over existing reporters for protem integration analysis, as it allows sensitive detection and isolation of both expressmg and non- expressing single living cells
- the assay system uses a non-toxic, non-polar fluorescent substrate that is easily loaded and then trapped intracellularly Modification of the fluorescent substrate by a cognate protein yields a fluorescent emission shift as substrate is converted to product
- the reporter readout is ratiomet ⁇ c it is unique among reporter protein assays m that it controls for variables such as the amount of substrate loaded into individual cells
- the stable, easily detected, intracellular readout eliminates the need for establishing clonal cell lines p ⁇ or to expression analysis
- This system and other analogous flow sorting systems can be used to isolate cells having a particular receptor element clustering and/or activation profile from pools of millions of viable cells
- the methods and composition of the present mvention may also make use of label enzymes
- label enzyme is meant an enzyme that may be reacted in the presence of a label enzyme substrate that
- radioisotope any radioactive molecule Suitable radioisotopes for use m the invention include, but are not limited to 14 C, 3 H, 32 P, 33 P, 35 S, 125 I, and 131 I
- labels may be indirectly detected, that is, the tag is a partner of a binding pair
- partner of a binding pair is meant one of a first and a second moiety, wherein the first and the second moiety have a specific binding affinity for each other Suitable binding pairs for use in the invention mclude, but are not limited to, antigens/antibodies (for example, digoxigenin/anti-digoxigenin, dinitrophenyl (DNP)/anti-DNP, dansyl-X-anti- dansyl, Fluorescein/anti-fluorescein, lucifer yellow/anti-lucifer yellow, and rhodamine anti-rho
- DNP dinitrophenyl
- dansyl-X-anti- dansyl dansyl-X-anti- dansy
- surface substrate binding molecule or "attachment tag” and grammatical equivalents thereof is meant a molecule have binding affinity for a specific surface substrate, which substrate is generally a member of a binding pair applied, incorporated or otherwise attached to a surface
- Suitable surface substrate binding molecules and their surface substrates include, but are not limited to poly-histidine (poly-his) or poly-histidine-glycine (poly-his-gly) tags and Nickel substrate, the Glutathione-S Transferase tag and its antibody substrate (available from Pierce Chemical), the flu HA tag polypeptide and its antibody 12CA5 substrate [Field et al , MoI Cell Biol , 8 2159-2165 (1988)], the c-myc tag and the 8F9, 3C7, 6E10, G4, B7 and 9E10 antibody substrates thereto [Evan et al , Molecular and Cellular Biology, 5 3610-3616 (1985)], and the Herpes Simplex virus glycoprotein D (gD
- tag-components of the invention can be made in vanous ways, dependmg largely upon the form of the tag Components of the invention and tags are preferably attached by a covalent bond
- tag-polypeptides by recombinant means when the tag is also a polypeptide is descnbed below
- Production of tag-labeled proteins is well known in the art and kits for such production are commercially available (for example, from Kodak and Sigma)
- Examples of tag labeled proteins include, but are not limited to, a Flag-polypeptide and His-polypeptide Methods for the production and use of tag-labeled proteins are found, for example, m Winston et al , Genes and Devel 13 270-283 (1999), incorporated herein in its entirety, as well as product handbooks provided with the above-mentioned kits
- Biotinylation of target molecules and substrates is well known, for example, a large number of
- a coupling moiety may be synthesized directly onto a component of the invention and contains at least one functional group to facilitate attachment of the tag.
- the coupling moiety may have at least two functional groups, which are used to attach a functionalized component to a functionalized tag, for example.
- the linker is a polymer.
- covalent attachment is accomplished either directly, or through the use of coupling moieties from the component or tag to the polymer.
- the covalent attachment is direct, that is, no linker is used.
- the component preferably contains a functional group such as a carboxylic acid that is used for direct attachment to the functionalized tag. It should be understood that the component and tag may be attached in a variety of ways, including those listed above.
- the tag is attached to the amino or carboxy terminus of the polypeptide.
- the tag is functionalized to facilitate covalent attachment, as is generally outlined above.
- tags are commercially available which contain functional groups, including, but not limited to, isothiocyanate groups, amino groups, haloacetyl groups, maleimides, succinimidyl esters, and sulfonyl halides, all of which may be used to covalently attach the tag to a second molecule, as is described herein.
- the choice of the functional group of the tag will depend on the site of attachment to either a linker, as outlined above or a component of the invention.
- amino modified or hydrazine modified tags will be used for coupling via carbodiimide chemistry, for example using 1- ethyl-3-(3-dimethylaminopro ⁇ yl)-carbodiimi- de (EDAC) as is known in the art (see Set 9 and Set 11 of the Molecular Probes Catalog, supra; see also the Pierce 1994 Catalog and Handbook, pages T-155 to T-200, both of which are hereby incorporated by reference).
- the carbodiimide is first attached to the tag, such as is commercially available for many of the tags described herein.
- An alternative activation state indicator useful with the instant invention is one that allows for the detection of activation by indicating the result of such activation.
- phosphorylation of a substrate can be used to detect the activation of the kinase responsible for phosphorylating that substrate.
- cleavage of a substrate can be used as an indicator of the activation of a protease responsible for such cleavage. Methods are well known in the art that allow coupling of such indications to detectable signals, such as the labels and tags described above in connection with binding elements. For example, cleavage of a substrate can result in the removal of a quenching moiety and thus allowing for a detectable signal being produced from a previously quenched label.
- the methods and composition utilize a modulator.
- a modulator can be an activator, an inhibitor or a compound capable of impacting a cellular pathway.
- Modulators can take the form of environmental cues and inputs.
- Modulation can be performed in a variety of environments. In some embodiments, cells are exposed to a modulator immediately after collection. In some embodiments where there is a mixed population of cells, purification of cells is performed after modulation. In some embodiments, whole blood is collected to which a modulator is added. In some embodiments, cells are modulated after processing for single cells or purified fractions of single cells.
- whole blood can be collected and processed for an enriched fraction of lymphocytes that is then exposed to a modulator
- Modulation can mclude exposing cells to more than one modulator
- cells are exposed to at least 2, 3, 4, 5, 6, 7, 8, 9, or 10 modulators
- the media is a growth media
- the growth media is a complex media that may include serum
- the growth media comprises serum
- the serum is selected from the group consisting of fetal bovme serum, bovine serum, human serum, porcme serum, horse serum, and goat serum
- the serum level ranges from 0 0001% to 30 %
- any suitable amount of serum is used
- the growth media is a chemically defined minimal media and is without serum
- cells are cultured in a differentiating media [00264] Modulators
- Modulators can act directly on the activatable elements or indirectly through the interaction with one or more intermediary biomolecule
- Indirect modulation includes alterations of gene expression wherem the expressed gene product is the activatable element or is a modulator of the activatable element
- modulators produce different activation states dependmg on the concentration of the modulator, duration of exposure or whether they are used in combmation or sequentially with other modulators
- the modulator is selected from the group consisting of growth factor, cytokine, adhesion molecule modulator, drugs, hormone, small molecule, polynucleotide, antibodies, natural compounds, lactones, chemotherapeutic agents, immune modulator, carbohydrate, proteases, ions, reactive oxygen species, peptides, and protein fragments, either alone or in the context of cells, cells themselves, viruses, and biological and non-biological complexes (e g beads, plates, viral envelopes, antigen presentation molecules such as major histocompatibility complex)
- biological and non-biological complexes e
- the modulator is an activator In some embodiments the modulator is an inhibitor In some embodiments, cells are exposed to one or more modulator In some embodiments, cells are exposed to at least 2, 3, 4, 5, 6, 7, 8, 9, or 10 modulators In some embodiments, cells are exposed to at least two modulators, wherem one modulator is an activator and one modulator is an inhibitor In some embodiments, cells are exposed to at least 2, 3, 4, 5, 6, 7, 8, 9, or 10 modulators, where at least one of the modulators is an inhibitor
- the modulator is a B cell receptor modulator
- the B cell receptor modulator is a B cell receptor activator
- An example of B cell receptor activator is a cross-linker of the B cell receptor complex or the B-cell co-receptor complex
- cross-linker is an antibody or molecular bindmg entity
- the cross-linker is an antibody
- the antibody is a multivalent antibody
- the antibody is a monovalent, bivalent, or multivalent antibody made more multivalent by attachment to a solid surface or tethered on a nanoparticle surface to increase the local valency of the epitope bindmg domain
- the cross-linker is a molecular binding entity
- the molecular bmdmg entity acts upon or bmds the B cell receptor complex via carbohydrates or an epitope in the complex
- the molecular is a monovalent, bivalent, or multivalent is made more multivalent by attachment to a solid surface or tethered on a nanoparticle surface to mcrease the local valency of the epitope bmdmg domain
- the cross-linking of the B cell receptor complex or the B-cell co-receptor complex compnses bmdmg of an antibody or molecular bmdmg entity to the cell and then causing its crosslinkmg via mteraction of the cell with a solid surface that causes crosslinkmg of the BCR complex via antibody or molecular bmdmg entity
- the crosslinker is F(ab)2 Ig
- the phosphatase inhibitor is H 2 O 2
- H 2 O 2 is administered as an inhibitor In some embodiments H 2 O 2 is administered at between 0 01 and 50 mM In some embodiments H 2 O 2 is administered at between 0 1 and 10 mM In some embodiments H 2 O 2 is administered at between 1 and 10 mM In some embodiments H 2 O 2 is administered at between 1 and 5 mM In some embodiments H 2 O 2 is administered at 0 5, 1 , 1 5, 2, 2 5, 3, 3 5, 4, 4 5, 5, 5 5, 6, 6 5, 7, 7 5, 8, 8 5, 9, 9 5 or 10 mM In certain embodiments, H 2 O 2 is administered at 3 0 mM In certain embodiments, H 2 O 2 is administered at 3 3 mM In some embodiments the duration of exposure OfH 2 O 2 is between 0 01 and 360 minutes In some embodiments the duration of exposure OfH 2 O 2 is between 0 1 and 240 minutes In some embodiments the duration of exposure OfH 2 O 2 is between 0 5 and 180 minutes In some embodiments the duration of exposure OfH 2 O 2 is
- a population of cells can be exposed to one or more, all or a combmation of the following combination of modulators (i) F(ab)2 IgM, (ii) Rituxan,, (in) Campath, (iv) H 2 O 2 (v) PMA, (vi) BAFF, (vu) Apnl, (via) SDFIa, (ix) CD40L, (x) IGF-I, (xi) Imiquimod, (x ⁇ ) polyCpG, (x
- the detection of the status of the one or more activatable elements can be carried out by a person, such as a technician in the laboratory. Alternatively, the detection of the status of the one or more activatable elements can be carried out using automated systems. In either case, the detection of the status of the one or more activatable elements for use according to the methods of this invention is performed according to standard techniques and protocols well-established in the art.
- One or more activatable elements can be detected and/or quantified by any method that detect and/or quantitates the presence of the activatable element of interest.
- Such methods may include radioimmunoassay (RIA) or enzyme linked immunoabsorbance assay (ELISA), immunohistochemistry, immunofluorescent histochemistry with or without confocal microscopy, reversed phase assays, homogeneous enzyme immunoassays, and related non- enzymatic techniques, Western blots, whole cell staining , immunoelectronmicroscopy, nucleic acid amplification, gene array, protein array, mass spectrometry, patch clamp, 2-dimensional gel electrophoresis, differential display gel electrophoresis, microsphere-based multiplex protein assays, label-free cellular assays and flow cytometry, etc.
- RIA radioimmunoassay
- ELISA enzyme linked immunoabsorbance assay
- immunohistochemistry immunofluorescent histochemistry with or without confocal
- U.S. Pat. No. 4,568,649 describes ligand detection systems, which employ scintillation counting. These techniques are particularly useful for modified protein parameters. Cell readouts for proteins and other cell determinants can be obtained using fluorescent or otherwise tagged reporter molecules. Flow cytometry methods are useful for measuring intracellular parameters.
- the present invention provides methods for determining an activatable element's activation profile for a single cell.
- the methods may comprise analyzing cells by flow cytometry on the basis of the activation level of at least two activatable elements.
- Binding elements e.g. activation state-specific antibodies
- non- binding elements systems as described above can be used in any system described herein.
- flow cytometric systems are used or systems dedicated to high throughput screening, e.g. 96 well or greater microtiter plates.
- Methods of performing assays on fluorescent materials are well known in the art and are described in, e.g., Lakowicz, J. R., Principles of Fluorescence Spectroscopy, New York: Plenum Press (1983); Herman, B., Resonance energy transfer microscopy, in: Fluorescence Microscopy of Living Cells in Culture, Part B, Methods in Cell Biology, vol. 30, ed. Taylor, D. L. & Wang, Y.-L., San Diego: Academic Press (1989), pp. 219-243; Turro, N. J., Modern Molecular Photochemistry, Menlo Park: Benjamin/Cummings Publishing Col, Inc. (1978), pp. 296-361.
- Fluorescence in a sample can be measured using a fluorimeter.
- excitation radiation from an excitation source having a first wavelength, passes through excitation optics.
- the excitation optics cause the excitation radiation to excite the sample.
- fluorescent proteins in the sample emit radiation that has a wavelength that is different from the excitation wavelength.
- Collection optics then collect the emission from the sample.
- the device can include a temperature controller to maintain the sample at a specific temperature while it is being scanned.
- a multi-axis translation stage moves a microtiter plate holding a plurality of samples in order to position different wells to be exposed.
- the multi-axis translation stage, temperature controller, auto-focusing feature, and electronics associated with imaging and data collection can be managed by an appropriately programmed digital computer.
- the computer also can transform the data collected during the assay into another format for presentation.
- known robotic systems and components can be used.
- Other methods of detecting fluorescence may also be used, e g , Quantum dot methods (see, e g , Goldman et al .
- flow cytometry involves the passage of individual cells through the path of a laser beam
- the scattering the beam and excitation of any fluorescent molecules attached to, or found within, the cell is detected by photomultipher tubes to create a readable output, e g size, granularity, or fluorescent intensity
- the detectmg, sorting, or isolating step of the methods of the present mvention can entail fluorescence- activated cell sorting (FACS) techniques, where FACS is used to select cells from the population containing a particular surface marker, or the selection step can entail the use of magnetically responsive particles as retrievable supports for target cell capture and/or background removal
- FACS fluorescence- activated cell sorting
- a FACS cell sorter e g a FACSVantageTM Cell Sorter, Becton Dickinson Immunocytometry Systems, San Jose, Calif
- a FACS cell sorter e g a FACSVantageTM Cell Sorter, Becton Dickinson Immunocytometry Systems, San Jose, Calif
- the change is a decrease
- the change is an mcrease
- the cells are first contacted with fluorescent-labeled activation state-specific binding elements (e g antibodies) directed against specific activation state of specific activatable elements
- the amount of bound binding element on each cell can be measured by passing droplets containing the cells through the cell sorter By imparting an electromagnetic charge to droplets containing the positive cells, the cells can be separated from other cells The positively selected cells can then be harvested in stenle collection vessels
- the present invention provides a valuable method of determining the presence of cellular subsets within cellular populations Ideally, signal transduction pathways are evaluated in homogeneous cell populations to ensure that variances in signaling between cells do not qualitatively nor quantitatively mask signal transduction events and alterations therein As the ultimate homogeneous system is the smgle cell, the present invention allows the individual evaluation of cells to allow true differences to be identified m a significant way
- the invention provides methods of distinguishing cellular subsets within a larger cellular population As outlined herein, these cellular subsets often exhibit altered biological characteristics (e g activation levels, altered response to modulators) as compared to other subsets within the population
- the methods of the invention allow the identification of subsets of cells from a population such as primary cell populations, e g penpheral blood mononuclear cells that exhibit altered responses (e g response associated with presence of a condition) as compared to other subsets
- this type of evaluation distinguishes between different activation states, altered responses to modulators, cell lineages, cell differentiation states, etc
- these methods provide for the identification of distinct signaling cascades for both artificial and stimulatory conditions m complex cell populations, such a peripheral blood mononuclear cells, or naive and memory lymphocytes
- a smgle cell suspension e g by enzymatic digestion with a suitable protease, collagenase, dispase, etc, and the like
- An approp ⁇ ate solution is used for dispersion or suspension
- Such solution will generally be a balanced salt solution, e g normal saline, PBS, Hanks balanced salt solution, etc , conveniently supplemented with fetal calf serum or other naturally occurring factors, in conjunction with an acceptable buffer at low concentration, generally from 5-25 mM
- Convement buffers include HEPES 1 phosphate buffers, lactate buffers, etc
- the cells may be fixed, e g with 3% paraformaldehyde, and are usually pe ⁇ neabihzed, e g with ice cold methanol, HEPES-buffered PBS containing 0 1% saponin, 3% BSA, covenng for 2 mm m acetone at -200C, and the like as known in
- Spectrochimica Acta Part B Atomic Spectroscopy, (2007), 62(3):188-195.).
- a chip analogous to a DNA chip can be used in the methods of the present invention.
- Arrayers and methods for spotting nucleic acid to a chip in a prefigured array are known.
- protein chips and methods for synthesis are known. These methods and materials may be adapted for the purpose of affixing activation state binding elements to a chip in a prefigured array.
- such a chip comprises a multiplicity of element activation state binding elements, and is used to determine an element activation state profile for elements present on the surface of a cell.
- a chip comprises a multiplicity of the "second set binding elements," in this case generally unlabeled.
- sample preferably cell extract
- a second multiplicity of binding elements comprising element activation state specific binding elements is used in the sandwich assay to simultaneously determine the presence of a multiplicity of activated elements in sample.
- each of the multiplicity of activation state-specific binding elements is uniquely labeled to facilitate detection.
- confocal microscopy can be used to detect activation profiles for individual cells.
- Confocal microscopy relies on the serial collection of light from spatially filtered individual specimen points, which is then electronically processed to render a magnified image of the specimen.
- the signal processing involved confocal microscopy has the additional capability of detecting labeled binding elements within single cells, accordingly in this embodiment the cells can be labeled with one or more binding elements.
- the binding elements used in connection with confocal microscopy are antibodies conjugated to fluorescent labels, however other binding elements, such as other proteins or nucleic acids are also possible.
- the methods and compositions of the instant invention can be used in conjunction with an "In-CeIl Western Assay.”
- an assay cells are initially grown in standard tissue culture flasks using standard tissue culture techniques. Once grown to optimum confluency, the growth media is removed and cells are washed and trypsinized. The cells can then be counted and volumes sufficient to transfer the appropriate number of cells are aliquoted into microwell plates (e.g., Nunc TM 96 Microwell TM plates). The individual wells are then grown to optimum confluency in complete media whereupon the media is replaced with serum- free media. At this point controls are untouched, but experimental wells are incubated with a modulator, e.g. EGF.
- a modulator e.g. EGF
- the detecting is by high pressure liquid chromatography (HPLC), for example, reverse phase HPLC, and in a further aspect, the detecting is by mass spectrometry.
- HPLC high pressure liquid chromatography
- These instruments can fit in a sterile laminar flow or fume hood, or are enclosed, self-contained systems, for cell culture growth and transformation in multi-well plates or tubes and for hazardous operations.
- the living cells may be grown under controlled growth conditions, with controls for temperature, humidity, and gas for time series of the live cell assays. Automated transformation of cells and automated colony pickers may facilitate rapid screening of desired cells.
- Flow cytometry or capillary electrophoresis formats can be used for individual capture of magnetic and other beads, particles, cells, and organisms.
- Flexible hardware and software allow instrument adaptability for multiple applications
- the software program modules allow creation, modification, and running of methods
- the system diagnostic modules allow instrument alignment, correct connections, and motor operations
- Customized tools, labware, and liquid, particle, cell and organism transfer patterns allow different applications to be performed
- Databases allow method and parameter storage Robotic and computer interfaces allow communication between instruments
- the methods of the invention include the use of liquid handling components
- the liquid handling systems can include robotic systems comprising any number of components
- any or all of the steps outlmed herein may be automated, thus, for example, the systems may be completely or partially automated
- components which can be used, including, but not limited to, one or more robotic arms, plate handlers for the positioning of microplates, automated lid or cap handlers to remove and replace lids for wells on non-cross contamination plates, tip assemblies for sample distribution with disposable tips, washable tip assemblies for sample distribution, 96 well loading blocks, cooled reagent racks, microtiter plate pipette positions (optionally cooled), stacking towers for plates and tips, and computer systems
- Fully robotic or microfluidic systems include automated liquid-, particle-, cell- and organism-handling including high throughput pipetting to perform all steps of screening applications
- This includes liquid, particle, cell, and organism manipulations such as aspiration, dispensmg, mixing, diluting, washing, accurate volumetric transfers, retrieving, and discarding of pipet tips, and repetitive pipetting of identical volumes for multiple deliveries from a single sample aspiration
- These manipulations are cross-contamination-free liquid, particle, cell, and organism transfers
- This instrument performs automated replication of microplate samples to filters, membranes, and/or daughter plates, high-density transfers, full-plate senal dilutions, and high capacity operation Additional examples of automation, automated sample collection and analysis are disclosed in US 61/048,657 which is hereby incorporated by reference in its entirety [00307]
- chemically de ⁇ vatized particles, plates, cartridges, tubes, magnetic particles, or other solid phase matrix with specificity to the assay components are used The binding surfaces of microplates, tubes,
- platforms for multi-well plates, multi-tubes, holders, cartridges, minitubes, deep- well plates, microfuge tubes, cryovials, square well plates, filters, chips, optic fibers, beads, and other solid-phase matrices or platform with various volumes are accommodated on an upgradeable modular platform for additional capacity
- This modular platform includes a variable speed orbital shaker, and multi-position work decks for source samples, sample and reagent dilution, assay plates, sample and reagent reservoirs, pipette tips, and an active wash station
- the methods of the mvention mclude the use of a plate reader
- thermocycler and thermoregulating systems are used for stabilizing the temperature of heat exchangers such as controlled blocks or platforms to provide accurate temperature control of incubating samples from O 0 C to 100° C
- interchangeable pipet heads single or multiple magnetic probes, affinity probes, or pipe
- the instrumentation will include a detector, which can be a wide variety of different detectors, depending on the labels and assay
- useful detectors include a microscope(s) with multiple channels of fluorescence, plate readers to provide fluorescent, ultraviolet and visible spectrophotomet ⁇ c detection with single and dual wavelength endpoint and kinetics capability, fluorescence resonance energy transfer (FRET), luminescence, quenching, two-photon excitation, and intensity redistribution, CCD cameras to capture and transform data and images into quantifiable formats, and a computer workstation
- the robotic apparatus mcludes a central processmg unit which communicates with a memory and a set of input/output devices (e g , keyboard, mouse, momtor, printer, etc ) through a bus Again, as outlined below, this may be m addition to or in place of the CPU for the multiplexing devices of the invention
- flow cytometry experiments are arrayed and the results are approximated as fold changes using a heat map to facilitate evaluation
- arrayed flow cytometry experiments simplify multidimensional flow cytometry data based on experimental design and observed differences between flow cytometry samples
- One common way of comparing changes m a set of flow cytometry samples is to overlay histograms of one parameter on the same plot
- Arrayed flow cytometry expe ⁇ ments ideally contain a reference sample against which experimental samples are compared This reference sample is placed in the first position of the array, and subsequent experimental samples follow the control in the sequence
- Reference samples can include normal and/or cells associated with a condition (e g tumor cells)
- p ⁇ or to analyzing of data the populations of interest and the method for characterizing these populations are determined For instance, there are at least two general ways of identifying populations for data analysis (i) "Outside-in" comparison of Parameter sets for individual samples or subset
- Each of these techniques capitalizes on the ability of flow cytometry to deliver large amounts of multiparameter data at the single cell level
- a condition e g neoplastic or hematopoetic condition
- a third "meta-level" of data exists because cells associated with a condition (e g cancer cells) are generally treated as a single entity and classified accordmg to historical techniques
- These techniques have included organ or tissue of o ⁇ gm, degree of differentiation, proliferation mdex, metastatic spread, and genetic or metabolic data regarding the patient
- the present invention uses variance mapping techniques for mappmg condition signaling space
- These methods represent a significant advance m the study of condition biology because it enables comparison of conditions independent of a putative normal control
- Traditional differential state analysis methods e g , DNA microarrays, subtractive Northern blotting
- they rely on multiple clusterings and re-clustermgs to group and then further stratify patient samples accordmg to phenotype
- variance mappmg of condition states compares condition samples first with themselves and then against the parent condition population
- activation states with the most diversity among conditions provide the core parameters m the differential state analysis Given a pool of diverse conditions, this technique allows a researcher to identify the molecular events that underlie differential condition pathology (e g , cancer responses to chemotherapy), as opposed to differences between conditions and a proposed normal control
- this technique allows a researcher to identify the molecular events that underlie differential condition pathology (e g , cancer responses to chemotherapy), as opposed to differences between conditions and a proposed normal control
- this technique allows a
- CLL serves as an example of the methods of the invention
- the data shown in Figures 26, 27 and 28 is a heat map comparing the activation states of multiple activatable elements in 22 CLL patients and 4 control patients
- This data demonstrates that B-cells from various CLL patients display distinguishable patterns of activatable elements as visualized by a heat map
- An inhibitor or inhibitor plus another modulator further define additional patterns of activatable elements that allow identification, classification and grouping of cryptic or aberrant hematopoetic populations (i e patient clustering)
- patient samples are indicated at the top of the heat map
- Each column represents a smgle patient CLL mdicates that the sample was obtained from a patient diagnosed with CLL CON mdicates that the sample was obtained from a control patient
- the heat map legend is indicated at the top of the figure and uses a shaded scale based on the log 10-fold mcrease, or decrease, in mean fluorescence intensity (MFI), relative to the unstimulated control (0 mm)
- the heat map defines the activation state of various activatable elements by denoting a change, or lack thereof, in the level of an activatable element revealed by the presence of an inhibitor and/or additional modulator
- the heat map can define the presence or absence of an increase m the activation level of a plurality of activatable elements m a cell upon contacting said cell with an inhibitor or a modulator Labels to the ⁇ ght of the heat map indicate the activatable element detected, e g a phospho-protem Labels to the ⁇ ght also indicate the modulator or inhibitor treatment for that row "US" indicates unstimulated or untreated
- Figure 28 illustrates a pattern of activation levels of a plurality of activatable elements in a cell
- Figure 28 further illustrates the identification of patient clustering groups (i e clustering groups)
- a patient clustering group is comprised of samples from patients that display similar or distmct patterns of activation levels m one or more activatable elements m response to one or more modulators
- kits Kits provided by the invention may comprise one or more of the state-specific bmdmg element descnbed herem, such as phospho-specific antibodies
- the kit comprises one or more of the phospho-specific antibodies specific for the proteins selected from the group consisting of PI3-Kmase (p85, pi 10a, pi 10b, pi 1Od), Jakl, Jak2, SOCs, Rac, Rho, Cdc42, Ras- GAP, Vav, Tiam, Sos, DbI, Nek, Gab, PRK, SHPl, and SHP2, SHIPl, SHIP2, sSHIP, PTEN, She, Grb2, PDKl, SGK, Aktl, Akt2, Akt3, TSC1,2, Rheb, mTor, 4EBP-1, p70S6Kinase, S6, LKB-I, AMPK, PFK, Acetyl-CoAa Carboxylase, Do
- Kits provided by the invention may comprise one or more of the modulators descnbed herem
- the kit compnses one or more modulators selected from the group consistmg of F(ab)2 IgM, H 2 O 2 , PMA, BAFF, Apnl, SDFIa, CD40L, IGF-I, Imiquimod, polyCpG, IL-7, IL-6, IL-IO, IL-27, IL-4, IL-2, IL-3, thapsigargin and a combination thereof
- the state-specific binding element of the invention can be conjugated to a solid support and to detectable groups directly or indirectly
- the reagents may also include ancillary agents such as buffering agents and stabilizing agents, e g , polysacchandes and the like
- the kit may further mclude, where necessary, other members of the signal-producing system of which system the detectable group is a member (e g , enzyme substrates), agents for reducing background interference in a test, control reagents, apparatus for conducting a test, and the like
- the kit may be packaged in any suitable manner, typically with all elements in a single container along with a sheet of printed instructions for carrying out the test
- kits enable the detection of activatable elements by sensitive cellular assay methods, such as IHC and flow cytometry, which are suitable for the clinical detection, prognosis, and screening of cells and tissue from patients, such as leukemia patients, having a disease involving altered pathway signaling
- kits may additionally compnse one or more therapeutic agents
- the kit may further compnse a software package for data analysis of the physiological status, which may include reference profiles for companson with the test profile
- kits may also include information, such as scientific literature references, package insert matenals, clinical tnal results, and/or summanes of these and the like, which mdicate or establish the activities and/or advantages of the composition, and/or which descnbe dosing, administration, side effects, drug interactions, or other information useful to the health care provider
- kits may also mclude instructions to access a database such as desc ⁇ bed in USSN 61/087,555 for selecting an antibody specific for the pathway of interest
- Kits desc ⁇ bed herein can be provided, marketed and/or promoted to health providers, including physicians, nurses, pharmacists, formulary officials, and the like Kits may
- the low-level signaling in CLL cells could be accounted for by either a defect in activ
- the concentration of the cells was adjusted as necessary to reduce crowding.
- Cells were incubated at 37 0 C for 2 hours.
- the final cell concentration, including additives and reagents e.g. stimulant, Fab fragment, H 2 O 2 , aqua, etc.
- the final cell concentration, including additives and reagents was 1.6 million/ml.
- Cells were aliquoted into wells.
- F(ab) 2 lgM alone, 3.3 mM H 2 O 2 alone orF(ab) 2 IgM and H 2 O 2 (H 2 O 2 added within 30 seconds of F(ab) 2 IgM) were added to the appropriate tubes and mixed by vortexing the samples. Cells were incubated with the different treatments for 15 minutes, unless a time course was performed.
- the cells were incubated with the different treatments for 5, 10, 30, 60, or 120 minutes. After incubation, the cells were fixed with 1.6% paraformaldehyde for 5 minutes at room temperature in the dark. Cells were then washed with 0.1% BSA/PBS (2 ml) and centrifuged at 2000 RPM for 5 minutes. The supernatant were decanted and pellet resuspended in 1 ml of 100% MeOH (methanol).
- Staining Tubes were washed with 2 ml of 0.4% BSA PBS (ice cold) and centrifuged at 2000 RPM for 10 minutes at 4 0 C. Cells were washed twice. Then cells were stained with fluorescent conjugated antibodies specific for CD20, CD3, CD4, CD8, CD5, p-Erk, p-BLNK, p-syk/Zap70, for 25 min. at RT. Cells were placed into 80 wells making a total of 2 plates. The cocktail mix used for the staining is described below: a. Cocktail
- CD3 pac blue 160 ⁇ l
- CD4 APC 80 ⁇ l
- CD8 PE Cy7 80 ⁇ l
- CD5 PE Cy5 400 ⁇ l
- CD20 PerCP Cy5.5 800 ⁇ l
- p-Erk 800 ⁇ l
- p-BLNK 800 ⁇ l
- p-Syk/ZAP70 800 ⁇ l b.
- Total antibody volume in the cocktail mix was 3920 ⁇ l. PBS was added to bring the total volume to 8.0 ml. Each well received 100 ⁇ l of this cocktail mix.
- the "CD20+” population was further gated to identify the mature B-CeIl (high CD5 expressing cells - CD5+) population by displaying them on the CD20 and CD5 axes. d. All the recorded events were also marked by the following intra-cellular markers p-Erk, p-
- a probability density estimate of log 10 of the fluorescence intensity value of each of the intra-cellular markers was computed for the cells in the CD20+ and CD5+ using the kernel density estimation function on the R- Statistics package (http://www.r-project.org/). The probability density estimates provided were then plotted to visualize the change in the phopho-levels of the various markers from the base-line.
- Figure 1 shows that both F(ab) 2 IgM and PMA activates p-Erk and p-Syk/pZap70 in Ramos cells.
- the large dashed line on the histograms represent the level of fluorescence of unstimulated and unstained cells referred herein after as autofluorescence.
- the thick solid line represents the level of fluorescence of unstimulated and stained cells referred hereinafter as background.
- the p-Erk and p-Syk fluorescence is above the autofluorescence and background signal indicating activation of those proteins upon stimulation.
- FIG. 2 shows that F(ab) 2 IgM (dotted line) also activates pBLNK, pCbl, ⁇ PLC ⁇ 2, pLck, p38 in Ramos cells.
- Ramos cells Ramos cells (Ramos cell lines) are used as positive control throughout the experiments performed herein.
- H 2 O 2 is a known inhibitor of phosphatases. Inhibition of phosphatases activates Erk and Syk as shown in Figure 6. Without intending to be limited to any theory, inhibition of phosphatases reveals strong tonic BCR signaling. This tonic signaling is not apparent by F(ab) 2 IgM alone ( Figure 4-5). F(ab) 2 IgM and H 2 O 2 reveals different kinetics subpopulation (heterogeneity) differences between CLL patients ( Figure 6).
- Figure 12 shows different kinetics of Syk and Erk phosphorylation by F(ab) 2 IgM and H 2 O 2 . Peak phosphorylation of Syk and BLNK occurred after 5 min of activation, whereas peak phosphorylation of Erk after 30 min.
- Figure 13 shows moderate activation of Syk/Zap70 and Erk by F(ab) 2 IgM and H 2 O 2 .
- Figure 14 shows the kinetics of signaling by F(ab) 2 IgM and H 2 O 2 .
- Figure 21 shows minimal activation of Syk/Zap70 and Erk by F(ab)2IgM alone over time.
- Figure 22 shows minimal activation of Syk/Zap70 and Erk in response to F(ab)2IgM alone over time.
- Figure 23 shows an F(ab)2 time course of CD20+/CD5+ population, without H 2 O 2 .
- Figure 15 and 16 show different levels of BLNK phosphorylation in response to an external stimulus in cells with different levels of ZAP70.
- Figures 17-21 show the kinetics of phosphorylation of PLC ⁇ 2, S6 and Cblin the CD20+/CD5+ cell population of CLL samples in response to B cell receptor crosslinking with and without peroxide.
- Figure 24 shows the kinetics OfH 2 O 2 treatment in CD20+/CD5+ population of CLL samples.
- Figure 25 shows the kinetics OfH 2 O 2 treatment in CD20+/CD5+ population of CLL samples.
- treatment with H 2 C> 2 reveals a patient clustering defined by the levels of p-PLC ⁇ 2, p- SyK/Zap-70, p-BLNK and p-Lck (bottom right boxed area) that are similar to those of the four control patients (bottom center box)
- Treatment with H 2 O 2 further reveals a patient clustering that is distinct from the controls (9 patients to the left of bottom boxed area)
- Modulation with H 2 O 2 and BCR crosslinking defines another patient clustering comprised of levels of p-BLNK, p-Syk and p-PLC ⁇ 2 (top left boxed area) that are similar to the control patients (top center box) and an abberant population of responders (10 patients to the nght of top boxed area)
- Example 3 Evaluation of Apoptosis Pathways in CLL Patient Samples [00353]
- Current therapeutic approaches for CLL involve fludarabine-based regimens combined with monoclonal antibodies such as ntuximab Fludarabine, a purine analog, inhibits DNA synthesis by interfering with ribonucleotide reductase and DNA polymerase
- Rituximab is a chimeric CD20 specific antibody and has mechanistically been shown to bind complement, induce antibody-dependent cellular cytotoxicity (ADCC) and, m some situations, ntuximab binding to CD20 inhibits proliferation and mduces cellular apoptosis (for a discussion of apoptosis see USSN 61/085,789)
- Cellular apoptosis in response to therapeutic agents can be measured by multiparameter flow cytometry usmg fluorophore-conjugated antibodies that recognize intracellular protem components or nodes of the apoptotic machinery
- nodes may include, but are not limited to, Caspase 3, Caspase 8, Cytochrome C, Poly ADP ⁇ bose polymerase (PARP), Bcl-2, BcI-X, p-Chk2, p-BAD
- Further information may be gathered by treating cells with a pan-caspase inhibitor Benzyloxycarbonyl-Val-Ala-Asp (OMe) fluoromethylketone (Z-VAD FMK) m order to reveal caspase-dependent and/or mdependent pathways
- the profile of how the apoptotic proteins respond to treatment with a therapeutic agent can be used to inform clinical decisions
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Also Published As
Publication number | Publication date |
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JP2010536371A (en) | 2010-12-02 |
WO2009025847A3 (en) | 2009-06-11 |
US9182385B2 (en) | 2015-11-10 |
EP2179037A4 (en) | 2010-12-22 |
CA2696402A1 (en) | 2009-02-26 |
US20160097770A1 (en) | 2016-04-07 |
EP2179037A2 (en) | 2010-04-28 |
US20090098594A1 (en) | 2009-04-16 |
CN101802182A (en) | 2010-08-11 |
US20120309029A1 (en) | 2012-12-06 |
AU2008289442A1 (en) | 2009-02-26 |
EP2860247A1 (en) | 2015-04-15 |
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