US 2016.0033516A1 (19) United States (12) Patent Application Publication (10) Pub. No.: US 2016/0033516 A1 Hunter et al. (43) Pub. Date: Feb. 4, 2016
(54) USE OF POLYCLONAL AND MONOCLONAL Publication Classification ANTIBODES SPECIFICFOR 3-PHOSPHOHISTONE (51) Int. Cl. GOIN33/574 (2006.01) (71) Applicant: SALK INSTITUTE FOR GOIN33/68 (2006.01) BIOLOGICAL STUDIES, La Jolla, CA GOIN 33/573 (2006.01) (US) (52) U.S. C. CPC ...... G0IN33/57496 (2013.01); G0IN33/573 (72) Inventors: Tony Hunter, Del Mar, CA (US); (2013.01); G0IN33/6854 (2013.01); G0IN Stephen Rush Fuhs, San Diego, CA 2800/52 (2013.01) (US); Jill Meisenhelder, Vista, CA (US) (57) ABSTRACT (73) Assignee: SALKINSTITUTE FOR Isolated monoclonal antibodies and antigen binding frag BIOLOGICAL STUDIES, La Jolla, CA ments are disclosed herein that specifically bind polypeptides (US) comprising a histidine phosphorylated at N3 (3-pHis). Nucleic acids encoding these antibodies, vectors including (21) Appl. No.: 14/789,811 these nucleic acids, and host cells transformed with these vectors and nucleic acids are also disclosed. Methods are also Filed: Jul. 1, 2015 disclosed for using these antibodies, such as for detection of (22) polypeptides comprising a histidine phosphorylated at N3 (3-pHis), detection of a tumor, monitoring the effectiveness Related U.S. Application Data of therapeutic agent, and identifying antibiotics. In some (60) Provisional application No. 62/031,796, filed on Jul. embodiments, the methods can be used to investigate signal 31, 2014. transduction pathways. Patent Application Publication Feb. 4, 2016 Sheet 1 of 16 US 2016/0033516 A1
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USE OF POLYCLONAL AND MONOCLONAL acids 32, 145 (January, 2007); McAllister et al., Biochemical ANTIBODIES SPECIFC FOR Society transactions 41, 1072 (August, 2013)). NME1 and 3-PHOSPHOHISTONE the closely related NME2 catalyze transfer of phosphate from ATP onto NDPs through a 1-pHis enzyme intermediate. The CROSS REFERENCE TO RELATED 3-pHis isomer has been shown to be more thermodynamically APPLICATIONS stable (Attwood et al., Amino acids 32, 145 (January, 2007)) than 1-pHis and may be more prevalent. 3-pHis is used by 0001. This claims the benefit of U.S. Application No. bacterial histidine kinases that autophosphorylate to initiate 62/031,796, filed Jul. 31, 2014, which is incorporated by phosphotransfer cascades and it also plays an important role reference herein. as an enzymatic intermediate for phospholipase D as well as ACKNOWLEDGMENT OF GOVERNMENT several key metabolic enzymes including; phosphoglycerate SUPPORT mutase (PGAM), succinyl-CoA synthetase (SCS), ATP-cit rate lyase (ACLY) (see, for example, Bond et al., J. Biol. 0002 This invention was made with government support Chem. 276, 3247 (2001)). under grant no. 5 RO 1 CA082683-15 awarded by the 0006. There is a need for the development of specific, National Institutes of Health and grant no.5 T32 CA009370 monoclonal antibodies (mAbs) for detection of pHis that can 31 from the National Institutes of Health. The government has be used to detect and functionally evaluate novel sites of certain rights in the invention. protein phosphorylation. These antibodies can be used, for example, to investigate signal transduction pathways. FIELD 0003. This relates to the field of antibodies, specifically to SUMMARY the use of antibodies that specifically bind a polypeptide that 0007 Uses of monoclonal antibodies, as well as antigen includes a histidine phosphorylated at N3 (3-pHis), such as binding fragments thereof, are disclosed herein that specifi for the identification of antibiotics and detecting the presence cally bind polypeptides including a histidine phosphorylated of a tumor in a Subject. at N3 (3-pHis). In some embodiments, the antibody includes a heavy chain variable region and a light chain variable BACKGROUND region, wherein the heavy chain variable region comprises a 0004. The majority of intracellular proteins are phospho H-CDR1, a H-CDR2, and a H-CDR3, wherein the antibody rylated at any given time, and, while nine of the 20 amino or antigenbinding fragment includes one of: a) the H-CDR1, acids can be phosphorylated, the current focus has been on the H-CDR2, and the H-CDR3 of the heavy chain variable serine (Ser), threonine (Thr), and tyrosine (Tyr) phosphory region of the amino acid sequence set forth as SEQID NO: 1; lation despite pHis having been first identified over 50 years b) the H-CDR1, the H-CDR2, and the H-CDR3 of the heavy ago (Boyer, J. Biol. Chem., 3306 (1962)). These OH-contain chain variable region of the amino acid sequence set forth as ing amino acids form acid-stable, phosphoester (P -O) SEQ ID NO: 2; c) the H-CDR1, the H-CDR2, and the bonds upon phosphorylation (Attwood, et al., Amino acids H-CDR3 of the heavy chain variable region of the amino acid 32, 145 (January, 2007)). Histidine (His) forms a heat and sequence set forth as SEQID NO:3: or d) the H-CDR1, the acid-labile phosphoramidate (P N) bond when phosphory H-CDR2, and the H-CDR3 of the heavy chain variable region lated. Phosphospecific antibodies have enabled the routine of the amino acid sequence set forth as SEQ ID NO: 4, study of phosphoesterprotein phosphorylation, and the use of wherein the monoclonal antibody specifically binds a MS-proteomics has identified over 200,000 non-redundant polypeptide comprising a histidine phosphorylated at N3 sites of phosphorylation (Hornbeck et al., Nucl.acids res 40, (3-pHis). In additional embodiments, the light chain variable D261 (January, 2012)). The lack of specific antibodies to region of the monoclonal antibody or antigen binding frag study pHis and the relative instability of the P N bond under ment includes a L-CDR1, a L-CDR2, and a L-CDR3, wherein typical conditions used for proteomics have made it impos the antibody or antigen binding fragment includes one of: a) sible to determine the prevalence of pHis, although it has been the L-CDR1, the L-CDR2, and the L-CDR3 of the light chain estimated that up to 6% of phosphorylation in eukaryotes variable region of the amino acid sequence set forth as SEQ occurs on His (Matthews, Pharmac. Ther: 67,232 (1995)). ID NO: 5; b) the L-CDR1, the L-CDR2, and the L-CDR3 of Thus, it is possible that phosphohistidine (pHis) could be the light chain variable region of the amino acid sequence set more abundant than phosphotyrosine (pTyr), which, despite forth as SEQID NO: 6; c) the L-CDR1, the L-CDR2, and the its importance, comprises ~1% of all known phosphorylation L-CDR3 of the light chain variable region of the amino acid sites (Hunter and Sefton, Proc. Natl. Acad. Sci. USA 77, 1311 sequence set forth as SEQID NO: 7; or d) the L-CDR1, the (Mar. 1, 1980, 1980); Olsen et al., Cell 127. 635 (Nov. 3, L-CDR2, and the L-CDR3 of the light chain variable region 2006)). Since current biochemical and proteomic technolo of the amino acid sequence set forth as SEQID NO: 8. gies have been optimized for preservation, enrichment and 0008. In some embodiments, the antibodies include a detection of the phosphoester amino acids (pSer, pThr and heavy chain variable region and a light chain variable region, pTyr), pHis has remained invisible. wherein the heavy chain variable region comprises a 0005 pHis is unique among phosphoamino acids in that H-CDR1, a H-CDR2, and a H-CDR3, wherein: a) the two distinct, biologically relevant isomers occur. The imida H-CDR1, the H-CDR2, and the H-CDR3 comprise amino Zole side chain of His contains two nitrogen atoms (N1 and acids 21-28, 45-52, and 88-97 of SEQID NO: 1, respectively: N3) that can both be phosphorylated to generate two bio b) the H-CDR1, the H-CDR2, and the H-CDR3 comprise chemically distinct isomers: 1-phosphohistidine (1-pHis) or amino acids 21-28, 46-52, and 91-101 of SEQ ID NO: 2, 3-phosphohistidine (3-pHis) (FIG. 1A) which are also respectively; c) the H-CDR1, the H-CDR2, and the H-CDR3 referred to as tele-phosphohistidine (r-pHis) and proS-phos comprise amino acids 24-31,49-55,94-104 of SEQIDNO:3, phohistidine (L-pHis) respectively (Attwood et al., Amino respectfully; or d) the H-CDR1, the H-CDR2, and the US 2016/0033516 A1 Feb. 4, 2016
H-CDR3 comprise amino acids 24-31, 49-55,94-104 of SEQ (lane 1) or 3 hr post-IPTG at 30°C. (lane 2) were run along ID NO: 4, respectively. In additional embodiments, the light side purified PGAM (lane 3) after cleavage of the GST tag chain variable region comprises a L-CDR1, a L-CDR2, and a (FIG. 2E). Purified PGAM was auto-phosphorylated in vitro L-CDR3, wherein: a) the L-CDR1, the L-CDR2, and the by incubation with 2,3-DPG for 10 min. at 30° C. Reactions L-CDR3 comprise amino acids 28-22, 51-53, and 90-102 of were stopped by addition of 5xpH 8.8 sample buffer and SEQ ID NO: 5, respectively; b) the L-CDR1, the L-CDR2, treated with or without heating to 95°C. for 10 min. Samples and the L-CDR3 comprise amino acids 27-34, 52-54, 91-103 were immediately analyzed by SDS-PAGE and immunoblot of SEQID NO: 6, respectively; c) the L-CDR1, the L-CDR2, ted with antisera from 7303 as in FIG. 2C. (FIG. 2F) 3-pHis and the L-CDR3 comprise amino acids 27-34, 52-54, and isoform specificity. Recombinant NME1 and PGAM were 91-109 of SEQID NO: 7, respectively; ord) the L-CDR1, the auto-phosphorylated in vitro by incubation with ATP or 2.3- L-CDR2, and the L-CDR3 comprise amino acids 27-33, DPG respectively and blotted with 3-pHis antisera (7303). 51-53 and 90-102 of SEQID NO: 8, respectively. (FIG. 2G) Phospho-PGAM spot blots. In vitro phosphoryla 0009. In further embodiments, methods are disclosed for tion of PGAM was performed as in FIG.3E except reactions using the antibodies, such as for detection of a polypeptide were stopped with addition of 2% SDS rather than sample including a histidine phosphorylated at N3 (3-pHis). In some buffer. Reactions were treated with or without heating to 95° embodiments methods are disclosed for detecting the pres C. for 10 min, diluted 1:5 and spotted directly on nitrocellu ence of a tumor and/or determining if a subject with a tumor lose instead of SDS-PAGE as a more rapid and convenient will respond to a biologic or chemotherapeutic agent. In other method that was developed to screen potential hybridoma embodiments, methods are disclosed for identifying an anti clones for ability to produce anti-3-pHis antibodies. A repre biotic. sentative immunoblot with anti-3-pHis (mAb MC39-4) is 0010. The foregoing and other features of the disclosure shown. will become more apparent from the following detailed (0013 FIGS. 3A-3E. Affinity purification of anti-1-pHis description, which proceeds with reference to the accompa and anti-3-pHis antibodies (FIG. 3A) Structures of the PEG nying figures. linker pTza peptide libraries used for affinity purification are shown covalently linked to agarose beads (Sulfolink coupling BRIEF DESCRIPTION OF THE DRAWINGS resin, Pierce) via a thioether bond with an N-terminal Cys 0011 FIGS. 1A-1D. Non-hydrolyzable phosphohistidine residue. The agarose-linked pTza libraries were used in affin analogues and their incorporation into peptide libraries. (FIG. ity columns to purify pHis antibodies from rabbit serum. 1A) Structure of histidine and the two pHis isomers: 1-phos (FIG. 3B) Fractions from the PEG-1-pTza affinity column phohistidine (1-pHis) and 3-phosphohistidine (3-pHis). including; input (IN), flow through (FT), washes (W1, W2, (FIG. 1B) Structures of the three synthetic peptide libraries W3 and W4), 10 ul column material (col) and the elution used in this study in which either His or a stable pHis mimetic fractions (E1 to E12) were analyzed by SDS-PAGE and Coo (1-pTza or 3-pTza) is flanked by randomized, neutral amino massie staining. (FIG. 3C) Western blotting of in vitro phos acids (alanine A and glycine IG)). Each library is com phorylated NME1 (5 or 200 ng) was performed with PEG-1- posed of 2-256 unique peptides, is acylated at the N-termi pTza column elution fractions E3 to E11 at a 1:200 dilution. nus, amidated at the C-terminus, and contains L-cysteine (FIG. 3D) Fractions from the PEG-3-pTza affinity column (Cys) for chemical ligation to KLH (Ac-Cys.G/A.G/A.G/A. were analyzed as described in B. (FIG.3E) Western blotting G/A.X.G/AG/AG/A.G/A-CONH2 (SEQID NO:9)). (FIG. of in vitro phosphorylated PGAM (5 or 200 ng) was per 1C) MS analysis was performed on all three of the peptide formed with PEG-3-pTza column elution fractions E3 to E11 libraries. The results from analysis of the 3-pTza library is at a 1:200 dilution. PGAM is the only band visible. shown. (FIG. 1D) The peptide libraries were conjugated to 0014 FIGS. 4A-4F. Characterization of anti-1-pHis and the carrier protein keyhole limpet hemocyanin (KLH). Three anti-3-pHis antibodies using peptide dot blot arrays. Syn rabbits were immunized with the 3-pTza library (7302, 7303 thetic peptide dot blot arrays consisting of the His, 1-pTza or and 7304) and three rabbits were immunized with the 1-pTza 3-pTza libraries (FIG. 1B), a pTyr (NCK) peptide and pep library (7305,7306 and 7307). tides with either His, 1-pTza or 3-pTza incorporated into 0012 FIGS. 2A-2G. Dot blot screening of 3-pHis antisera nonapeptides of defined sequences (based on the pHis protein and development of PGAM in vitro phosphorylation assays. substrates: ACLY, NME1/2, histone H4, KCa3.1 and GNB1) (FIG. 2A) Dot blot screening of 3-pHis Antisera. (FIG. 2B) were spotted on nitrocellulose and probed with; (FIG. 4A) Crystal structure of PGAM co-crystallized with its phosphate affinity-purified polyclonal 3-pHis (7303-E6 (elution fraction donor 2,3-diphosphoglycerate (2,3-DPG). (FIG. 2C) GST #6)) or 1-pHis (7305-E6 (elution fraction #6)) antibodies or PGAM fusion protein was auto-phosphorylated in vitro by (FIG. 4B) anti-1-pHis mAbs (7305-SC1-1, SC50-3 and addition of increasing concentrations of 2.3-DPG. Reactions SC50-11). Peptide layouts, sequences and their sources are were stopped by addition of 5xpH 8.8 sample buffer and shown. (FIG. 4C) 3-pTza peptide dot blot characterization of treated with or without heating to 95°C. for 10 min. Samples anti-3-pHis mAbs. Peptide layouts, sequences and their were analyzed immediately by a modified SDS-PAGE Sources are shown. A partially-deprotected, mono-ethyl ester method in which gels were run and transferred at 4°C. and a version of the ACLY-based pTza peptide (AGAG-mono-Et pH 8.8 stacking gel was used. Immunoblotting with anti-sera 3-pTza-AGAG) was included. (FIGS. 4D-4F) Synthetic pTyr from the three immunized rabbits (7302, 7303 and 7304) peptide dot blots. Peptides based on Nck, Eck and Fak were revealed that phospho-PGAM could only be detected by anti synthesized with or without a pTyr residue (there is no sera from rabbit 7303. PGAM phosphorylation was abolished unphosphorylated Fak peptide) and spotted on nitrocellulose by heating the samples prior to SDS-PAGE. Mutation of the membranes in order to screen for pTyr crossreactivity of catalytic His residue (H11) also abolished phosphorylation of anti-pHis antibodies. Membranes were probed with; (FIG. PGAM. (FIG. 2D) Purification of recombinant PGAM from 4D) anti-pTyr mAb 4G10. (FIG. 4E) anti-3-pHis mAbs E. coli. Crude E. coli lysates from cells pre-IPTG induction (7303-MC39, 7304-MC44 and 7304-MC56) or (FIG. 4F) US 2016/0033516 A1 Feb. 4, 2016
Anti-1-pHis mAbs (7305-SC1-1 and 7305-SC50-3). Anti anti-ATP synthase antibodies (a mitochondrial enzyme) to pHis mAbs were used at a concentration of 0.5ug/ml. It was check for co-localization with mitochondria. concluded that 3-pHis antibodies do not crossreact with pTyr. (0019 FIGS. 9A-9D. Immunoaffinity purification using 0015 FIGS. 5A-5C. Mammalian and cancer cell lysate immobilized anti-1-pHis mAb SC 1-1. A stably transfected blotting and pTyrcross-reactivity. (FIG.5A) Src-transformed HEK 293 cell line, FLAG-NME1, was used to prepare whole and non-transformed fibroblast cell lines (Psrc 11 and pancre cell lysates for immunoaffinity purification of histidine phos atic stellate cells (PaSCs) respectively) were analyzed by phorylated proteins. (FIG. 9A). Immunoblotting of 1-pHis Western blotting. Cells were pre-treated with 1 mM ortho mAb column fractions was performed using 1-pHis mAb vanadate for 30 min prior to lysis. The major 1-pHis (NME1/ SC 1-1 and anti-NME1/2 antibodies. Identical 1-pHis mAb 2) and 3-pHis (SCS and ACLY) bands detected are indicated. column fractions were immunoblotted with; (FIG. 9B) There is no detectable cross-reactivity of the pHis antibodies 3-pHis mAbs SC39-4 and SC44-8 (FIG.9C) anti-tubulin and with pTyr. (FIG. 5B) A representative Western blot of pan anti-Rab5 antibodies, and (FIG.9D) anti-3-pHis mAb SC56 creatic cancer cell line lysates with an anti-1-pHis mAb. (FIG. 2. The IN and FT fractions were treated with and without 5C) provides Western blot of the FLAG-NME1 stable 293 heating to 95°C. for 15 min. (Abbreviations: IN, input; FT, cells with an anti-3-pHis. flow through; W1-W4, washes, E1-E6, elutions). 0016 FIGS. 6A-6G. 3-pHis hybridoma subclone screen 0020 FIGS. 10A-10B. Immunoaffinity purification using ing and antibody characterization. (FIG. 6A) High through immobilized anti-3-pHis mAb SC39-6. A stably transfected put, slot blot screening of 3-pHis hybridoma cell Supernatants HEK 293 cell line, FLAG-NME1, was used to prepare whole using lysates from E. coli transformed with GST-PGAM. The cell lysates for immunoaffinity purification of histidine phos four best multiclonal (MC) anti-3-pHis hybridoma cell lines phorylated proteins. (FIG. 10A). Immunoblotting of 3-pHis were identified (MC39, MC44, MC56 and MC60). Subclon mAb column fractions was performed using 3-pHis mAb ing of these cell lines was performed to obtain anti-3pHis SC39-6 and anti-PGAM antibodies. Identical 3-pHis mAb monoclonal cell lines. This resulted in 12 different, 3-pHis column fractions were immunoblotted with; (FIG. 10B) positive subclones (SC) for MC39 (SC39-1 to -12) and MC44 3-pHis mAb SC44-8 and ACLY. The IN and FT fractions (SC44-1 to -12), 3 different subclones for MC56 (SC56-2, -10 were treated with and without heating to 95°C. for 15 min. and -12) and 9 different subclones for MC60. Hybridoma cell (Abbreviations: IN, input; FT, flow through; W1-W4, washes, supernatants from these subclones were normalized to 0.5 El-E6, elutions). ug/mL IgG and incubated with PVDF membranes (using a (0021 FIGS. 11A-11K. 1-pHis mAbs Negatively Stain BioRad slot blotting apparatus) transferred from preparative Macrophage Phagosomes and 3-pHis mAbs Stain Cen gels that were loaded with crude E. coli lysates. (FIG. 6B) trosomes and Spindle Poles in HeLa Cells. (FIG. 11A) HeLa Representative immunoblots blots from E. coli lysates, iden cells were fixed with PFA and stained with 1-pHis mAb tical to those used in FIG. 6A, were treated with and without SC 1-1. White arrows indicate acidic compartments. (FIG. heating to 95°C. for 10 min. All of the detected bands were 11B) Macrophages were fed Dextran-AF488 and labeled heat-sensitive, indicating the mabs are 3-pHis specific and with LysoTracker(R) for 60 min prior to fixation with PFA and that there are many 3-pHis containing proteins present in the staining with 1-pHis mAb SC 1-1 was detected by Cy5 con E. coli lysates. (FIGS. 6C-6E) Mammalian cell lysates were jugated secondary antibodies. Bar, 10um. (FIG. 11C) Mac probed with an anti-3-pHis multiclonal mAb (MC44) that rophages were incubated with Dextran-AF488 for 60 min and specifically recognized SCS in E. coli lysates but not PGAM. staining with mAb SC 1-1 was detected by Cy5-conjugated This mAb appears to have a sequence bias that is similar to the secondary antibodies. (FIG. 11D) Macrophages were labeled A/G peptide libraries used as immunogens. SCS is highly with LysoTracker(R) for 60 min prior to fixation and mAb conserved from bacteria to humans. This mAb also detects the SC 1-1 staining was detected by AF-488 conjugated second 3-pHis residue in ACLY (H760) and a number of other pro ary antibodies. (FIG. 11E) Co-staining of macrophages with teins in mammalian cell lysates. The amino acid sequences mAb SC 1-1 and Phalloidin-TRITC. (FIGS. 11 F-11K) HeLa surrounding the pHis residues in ACLY, SCS and PGAM are cells were fixed with: PFA (FIGS. 11 F-11G), or pre-perme shown for comparison. (FIG. 6F) A stable 293 cell line abilized with 0.5% Triton X-100 and fixed with PFA (FIGS. expressing FLAG-NME1 was blotted with the anti-3-pHis 11 I and 11K) or methanol (FIGS. 11H and 11J) and stained mAb SC39-5 and an anti-PGAM antibodies. SC39-5 detected with 3-pHis mAb SC39-4 alone (FIGS. 11 F-11G) or co a strong, heat sensitive band that corresponds to 3-pHis phos stained with Aurora A (FIG. 11H), Y-tubulin (FIG. 11I) or pho-PGAM. NME1 was not detected, indicating the 3-pHis C-tubulin (FIGS. 11J-11K) antibodies. (FIG. 11F) Metaphase mAbs are isomer specific (i.e., do not cross-react with 1-pHis) cells are shown in an expanded view in the right panel. (FIG. (FIG. 6G). The same lysates from FIG. 6F were blotted with 11G) From left to right, interphase, an early prophase and the anti-3-pHis mAb SC44-11 and a number of heat-sensitive anaphase cells. (FIGS. 11H-11K) Cells in metaphase, bands are detected, including the known 3-pHis proteins SCS prometaphase and telophase are shown. White arrows indi and ACLY. cate centrosomes and spindle poles and grey arrows indicate midbodies in telophase cells. Nuclei were visualized with 0017 FIGS. 7A-7B. Mass spectra and phosphorylation DAPI. Size Bar, 20 pum. See also FIG. 12. site assignment of in vitro phosphorylated NME1 and (0022 FIGS. 12A-12S. pHis mAb Immunofluorescence PGAM. (FIG. 7A) 5ug of purified NME1 was incubated at Staining of Macrophages and HeLa Cells with Negative Con RT with 1 mM ATP. (FIG. 7B) 5ug of purified PGAM was trols. 3-pHis mAbs Stain Cytoplasmic and Nuclear Structures incubated at 30° C. for 10 min with 1 mM 2,3-DPG. Distinct from 1-pHis mAbs, Related to FIG.11. Macrophages 0018 FIGS. 8A-8B. Primary murine macrophages were were fixed with 4% PFA and co-stained with 3-pHis mAb isolated from bone marrow and fixed with paraformaldehyde. SC39-4 and antibodies to the organelle markers LC3 (au Macrophages were co-stained with the anti-3-pHis mAb tophagosomes FIG. 12A) and Rab5 (early endosomes FIG. SC39-4 (FIG.8A) or anti-3-pHis mAb SC44-8 (FIG.8B) and 12B). (FIGS. 12C-12E) U2OS cells were fixed with PFA US 2016/0033516 A1 Feb. 4, 2016
co-stained with 3-pHis mAb SC39-4 and antibodies to Y-tu - Continued bulin and C-tubulin. White arrows indicate centrosomes and AKLGSGNPVAIWGPGTLVTVSSGOPKAPSWFPLAPCCGDTPSSTV spindle poles and grey arrows indicate midbodies intelophase cells. (FIGS. 12F-12J) 1-pHis mAb SC 1-1 negative controls. TLGCLWKGYLPEPWTWTWNSGTLTNGWRTFPAWRESSGLYSLNSW Macrophages were stained with mAb SC 1-1 that was pre GKVTSSSOPVTCNVAHPATNTKVDKTV incubated with or without the immunizing peptide libraries CDR1 21–28; CDR2 46-52; CDR3 91-101; VH 1-112 for 30 minat RT with gentle agitation: (FIG. 12F) no peptide, SEQ ID NO: 3 is the amino acid sequence of the (FIG. 12G) His peptide library, (FIG. 12H) 1-pTza peptide heavy chain of mAb SC.56-2. library or (FIG. 12I) 3-pTza peptide library. (J) Slides with SVKESEGGLIKPGGILTLTCTASGFSLSSYGFSWVROAPGKGLEH PFA fixed macrophages were treated with or without boiling in citrate buffer for 10 min prior to staining with mAb SC 1-1. IGYLHANGRAYYATWAKSRSTITRNTNLNTWTLQLTSLTAADTAT (FIGS. 12K-12O) 3-pHis mAb SC39-4 negative controls. Macrophages were stained with mAb SC39-4 that was pre YFCAKIGSWSDWAIWGPGTLVTVSSGOPKAPSVFPLAPCCGDTPS incubated with or without the immunizing peptide libraries: STVTLGCLVKGYLPEPWTWTWNSGTLTNGVRTFPSVROSSGLYSL (FIG. 12K) no peptide, (FIG. 12L) His peptide library, (FIG. 12M) 1-pTza peptide library or (FIG. 12N) 3-pTza peptide SSVVSVTSSSOPVTCNVAHPATNTKVDKTV library. (FIG. 12O) Slides with PFA fixed macrophages were CDR1 24-31; CDR2 49-55; CDR3 94 - 104, WH 1-115 treated with or without boiling in citrate buffer for 10 min SEQ ID NO: 4 is the amino acid sequence of the prior to staining with 3-pHis mAb SC39-4. (FIGS. 12P-12S) heavy chain of mAb SC60-2 pTZablocking peptide negative controls in HeLa cells. HeLa SWKESEGGLFKPTDTLTLTCTWSGFSLTTYGFSWWRQAPGKGLEW cells were fixed with PFA and stained with mAb SC 1-1 IGYWRSDGRYYTSWAKSRSTLTRNTNLNTWTLIMTSLTWADTAT (FIGS. 12P-12Q) or mAb SC39-4 (FIGS. 12R-12S) that was pre-incubated with or without the 1-pTza or 3-pTza peptide YFCAKIGSGTGVAIWGPGTLVTVSSGOPKAPSWFPLAPCCGDTPS libraries respectively for 30 min at RT with gentle agitation. STVTLGCLVKGYLPEPWTWTWNSGTLTNGVRTFPSVROSSGLYSL Nuclei were visualized with DAPI. Size bars (FIGS. 12A 12O)10 um, (FIGS. 12P-12S) 20 um. SSVVSVTSSSOPVTCNVAHPATNTKVDKTV CDR1 24-31; CDR2 49-55; CDR3 94-104; VH 1-115 SEQUENCES SEQ ID NO: 5 is the amino acid sequence of the 0023 The nucleic and amino acid sequences listed are light chain of mAb SC39-4. shown using standard letter abbreviations for nucleotide AQFWMTOTPASWEAWWGGTWTIKCQASRDTGDGLIWYQQKPGQPP bases, and three letter code for amino acids, as defined in 37 KRLIYKASTWASGWPSRFKGRGSGTDFTLTISDLECADAATYYCH C.F.R. 1.822. Only one strand of each nucleic acid sequence is shown, but the complementary Strand is understood as SNFYNRWTYGNAFGGGTEVVVKGDPWAPTVLIFPPAADQVATGTV included by any reference to the displayed strand. The Sequence Listing is submitted as an ASCII text file 7158 TIVCVANKYFPDWTVTWEVDGTTOTTGIENSKTPQNSADCTYNLS 93022-02 Sequence Listing, Jul. 1, 2015, 39.1 KB, which STLTLTSTOYNSHKEYTCKVTOGTTSVVOSFNRGDC is incorporated by reference herein. CDR1 28-33; CDR2 51-53; CDR3 90-102, VH 1-113 0024. The amino acid sequences for antibodies are pro SEQ ID NO: 6 is the amino acid sequence of the vided below. In the following Fab fragment sequences (V- light chain of mAb SC44-8. C1 or V-C1), the heavy and light chain variable domains DPVMTPTPSFTSAAVGGTVTINCOSSQSWWRNKNLAWYOOKPGOP (V, and V) are underlined with the CDRs highlighted in bold. The heavy and light chain constant domains (C1 and PKRLIYAIATLDSGWPSRFSGSGSGTQFTLTISDWQCDDAATYYC C1) are in plain capital letters. Exemplary locations of the WGHGSENDAYYAFGGGTEWWWKGDPWAPTWLIFPPSADLWATGT CDRs (as identified by IMGT) are listed below each sequence. The program available at www.IMGT.org was used VTIVCVANKYFPDWTVTWEVDGTTOTTGIENSKTPQNSADCTYNL to align the sequences and ID the CDRs. SSTLTLTSTOYNSHKEYTCKVTOGTTSV WOSFNRGDC CDR1 27-34; CDR2 52-54; CDR3 91-103; WH 1-114 SEQ ID NO: 1 is the amino acid sequence of the SEQ ID NO: 7 is the amino acid sequence of the heavy chain of mAb SC39 - 4. light chain of mAb SC 56-2. ESGGRLWTPGGSLTLTCTWSGFSLSRYNMGWWRQAPGKGLEWIGW DPWMTQTPSSTSAAWGGTWTINCQSSESIYNNKNLAWYQQKPGQS
PFRGSKYATWATGRFTISRTSTTWDLRMTGLTAADTATYFCWR PRRLIYSISTLASGVSSRFKGSGSGTOFTLTISDVOCDDAATYYC
SSDGFDLWGPGTLVTVSSGOPKAPSWFPLAPCCGDTPSSTWTLGC WGYYYSGGYYYSGSAAYYAFGGGTEWWWKGDPWAPTWLIFPPSAD
LVKGYLPEPWTVTWNSGTLTNGVRTFPSVROSSGLYSLSSVVSVT LVATGTVTIVCVANKYFPDWTVTWEVDGTTOTTGIENSKTPQNSA
SSSOPVTCNVAHPATNTKVDKTV DCTYNLSSTLTLTSTOYNSHKEYTCKVTOGTTSVVOSFNRGDC CDR 1: 21-28; CDR2 45-52, CDR3 88-97; WH 1-108 CDR1 27-34 : CDR2 52-54; CDR3 91-109 : WH 1-12O SEQ ID NO: 2 is the amino acid sequence of the SEQ ID NO: 8 is the amino acid sequence of the heavy chain of mAb SC44-8. light chain of mAb SC60-2. ESGRGLVOPGGSLTLTCTASGFSIDSYGFSWVROAPGKGLEHIGY DGVMTPTPASASAGVGGTVTINCOSSQSIYKKYIAWYOOKPGOPP
TAGGRAFYASWAKSRSTITRNTNENTWTLKMTSLTAADTATYFC KRLIYSTSTLASGVSSRFKGSGSGTQFTL TISDWQCDDWATYYCW US 2016/0033516 A1 Feb. 4, 2016
- Continued Small molecules, organic compounds, inorganic compounds, GYYTNDAYYSFGGGTEWWWKGDPWAPTWLIFPPSADLWATGTW or other molecules of interest. In some embodiments, the agent is a polypeptide agent (such as an antibody), or a phar TIVCVANKYFPDWTVTWEVDGTTOTTGIENSKTPQNSADCTYNLS maceutical compound. The skilled artisan will understand STLTLTSTOYNSHKEYTCKVTOGTTSVVOSFNRGDC that particular agents may be useful to achieve more than one CDR1 27-33; CDR2 51-53; CDR3 90-102; WH 1-113 result. 0025 SEQ ID NO: 9 is the amino acid sequence of a 0035 Amino acid substitution: The replacement of one synthetic polypeptides. amino acid in peptide with a different amino acid. 0026 SEQID NOs: 10-54 are the amino acid sequences of 0036 Amplification: A technique that increases the num pHis substrates. ber of copies of a nucleic acid molecule (such as an RNA or 0027 SEQID NOs: 55-59 are the amino acid sequences of DNA). An example of amplification is the polymerase chain synthetic polypeptides. reaction, in which a biological sample is contacted with a pair 0028 SEQID NOs: 60-88 are the nucleic acid sequences of oligonucleotide primers, under conditions that allow for of primers. the hybridization of the primers to a nucleic acid template in 0029 SEQID NOs: 89-94 are the amino acid sequences of the sample. The primers are extended under suitable condi synthetic polypeptides. tions, dissociated from the template, and then re-annealed, extended, and dissociated to amplify the number of copies of DETAILED DESCRIPTION the nucleic acid. The product of amplification can be charac 0030 Stable pHis mimetics were incorporated into degen terized by electrophoresis, restriction endonuclease cleavage erate peptide libraries to immunize rabbits and develop the patterns, oligonucleotide hybridization or ligation, and/or anti-3-pHis mAbs that constitute defined reagents with infi nucleic acid sequencing using standard techniques. Other nite Supply. Several novel screening assays were developed to examples of amplification include Strand displacement characterize these mAbs and it was demonstrated that they amplification, as disclosed in U.S. Pat. No. 5,744,311; tran lack pTyr cross-reactivity and appear to detect pHis in a Scription-free isothermal amplification, as disclosed in U.S. sequence-independent manner. Multiple rabbit hybridoma Pat. No. 6,033,881; repair chain reaction amplification, as cell lines have been established for each pHis isomer and disclosed in PCT Publication No.WO 90/01069; ligase chain sequencing of the IgG heavy and light chain variable regions reaction amplification, as disclosed in European Patent Pub (V, and V.) revealed the distinct complementarity determin lication EP-A-320 308; gap filling ligase chain reaction ing regions (CDRs). Antibodies were isolated that specifi amplification, as disclosed in U.S. Pat. No. 5,427,930; and cally bind polypeptides including a histidine phosphorylated NASBATM RNA transcription-free amplification, as dis at N3 (3-pHis). Nucleic acids encoding these antibodies, vec closed in U.S. Pat. No. 6,025,134. tors including these nucleic acids, host cells transformed with 0037 Animal: Living multi-cellular vertebrate organisms, these vectors are disclosed herein. a category that includes, for example, mammals and birds. 0031. These antibodies are of use in detecting phosphory The term mammal includes both human and non-human lation of polypeptides. In specific, non-limiting examples, the mammals. Similarly, the term “subject' includes both human antibodies can be used to detect polypeptides phosphorylated and Veterinary Subjects. in a signal transduction pathway. 0038 Antibody: A polypeptide substantially encoded by an immunoglobulin gene or immunoglobulin genes, or anti TERMS gen binding fragments thereof, which specifically binds and 0032 Unless otherwise noted, technical terms are used recognizes an analyte (antigen) Such as one or more phospho according to conventional usage. Definitions of common rylated polypeptides, such as one or more polypeptides that terms in molecular biology may be found in Benjamin Lewin, includes a phosphorylated histidine, for example a histidine Genes V, published by Oxford University Press, 1994 (ISBN phosphorylated at N3. Immunoglobulin genes include the 0-19-854287-9); Kendrew et al. (eds.). The Encyclopedia of kappa, lambda, alpha, gamma, delta, epsilon and mu constant Molecular Biology, published by Blackwell Science Ltd., region genes, as well as the myriad immunoglobulin variable 1994 (ISBN 0-632-02182-9); and Robert A. Meyers (ed.), region genes. Molecular Biology and Biotechnology: a Comprehensive 0039 Antibodies exist, for example, as intact immunoglo Desk Reference, published by VCH Publishers, Inc., 1995 bulins and as a number of well characterized fragments pro (ISBN 1-56081-569-8). Unless otherwise explained, all tech duced by digestion with various peptidases. Fabs, FVs, sclvs nical and Scientific terms used herein have the same meaning that specifically bind to a phosphorylated polypeptide. Such as commonly understood by one of ordinary skill in the art to as a polypeptide that includes a phosphorylated histidine, for which this disclosure belongs. example a histidine phosphorylated at N3, are specific bind 0033 Administration: The introduction of a composition ing agents. A scFv protein is a fusion protein in which a light into a Subject by a chosen route. Administration can be local chain variable region of an immunoglobulin and a heavy or systemic. For example, if the chosen route is intravenous, chain variable region of an immunoglobulin are bound by a the composition is administered by introducing the composi linker, while indsFVs, the chains have been mutated to intro tion into a vein of the subject. duce a disulfide bond to stabilize the association of the chains. 0034 Agent: Any substance or any combination of sub The term also includes genetically engineered forms such as stances that is useful for achieving an end or result, for chimeric antibodies and heteroconjugate antibodies such as example, a Substance or combination of substances useful for bispecific antibodies. See also, Pierce Catalog and Hand inhibiting phosphorylation or for identifying the role of phos book, 1994-1995 (Pierce Chemical Co., Rockford, Ill.); phorylation in a biological process. Agents include, and are Kuby. Immunology, 3" Ed., W.H. Freeman & Co., New York, not limited to, proteins, nucleic acid molecules, compounds, 1997. US 2016/0033516 A1 Feb. 4, 2016
0040 Antibody fragments include, but are not limited to, from residue 26 to residue 32. Thus “CDR-H1’, as used the following: (1) Fab, the fragment which contains a herein, comprises residues 26 to 35, as described by a com monovalent antigen-binding fragment of an antibody mol bination of the Kabat numbering system and Chothias topo ecule produced by digestion of whole antibody with the logical loop definition. The CDRs of the light chain variable enzyme papain to yield an intact light chain and a portion of domain are located at residues 24-34 (CDR-L1), residues one heavy chain; (2) Fab', the fragment of an antibody mol 50-56 (CDR-L2) and residues 89-97 (CDR-L3) according to ecule obtained by treating whole antibody with pepsin, fol the Kabat numbering system. Lefranc, et al. (“IMGT unique lowed by reduction, to yield an intact light chain and a portion numbering for immunoglobulin and T cell receptor variable of the heavy chain; two Fab' fragments are obtained per domains and Ig Superfamily V-like domains. Dev. Comp. antibody molecule; (3) (Fab'), the fragment of the antibody Immunol., 27:55-77, 2003) discloses the “IMGT' numbering obtained by treating whole antibody with the enzyme pepsin scheme for CDRs. The Kabat database is now maintained without subsequent reduction; (4) F(ab'), a dimer of two Fab' online. The sequences of the framework regions of different fragments held together by two disulfide bonds; (5) Fv, a light or heavy chains are relatively conserved within a spe genetically engineered fragment containing the variable cies. The framework region of an antibody, that is the com region of the light chain and the variable region of the heavy bined framework regions of the constituent light and heavy chain expressed as two chains; and (6) single chain antibody chains, serves to position and align the CDRS in three-dimen (SCA), a genetically engineered molecule containing the sional space. variable region of the light chain, the variable region of the 0045. The CDRs of each chain are typically referred to as heavy chain, linked by a suitable polypeptide linker as a CDR1, CDR2, and CDR3 (from the N-terminus to C-termi genetically fused single chain molecule. nus), and are also typically identified by the chain in which 0041 Antigen binding fragments of an antibody can be the particular CDR is located. Thus, a VCDR3 is located in produced by the modification of whole antibodies or those the variable domain of the heavy chain of the antibody in synthesized denovo using recombinant DNA methodologies. which it is found, whereas a V, CDR1 is the CDR1 from the In some examples, the term antibody includes the amino acid variable domain of the light chain of the antibody in which it sequences of one or more of the CDRs from the antibody is found. Light chain CDRs are sometimes referred to as CDR grafted onto a scaffold. L1, CDRL2, and CDRL3. Heavy chain CDRs are sometimes 0.042 Typically, a naturally occurring immunoglobulin referred to as CDR H1, CDR H2, and CDR H3. has heavy (H) chains and light (L) chains interconnected by 0046 References to “V” or “VH’ refer to the variable disulfide bonds. There are two types of light chain, lambda (W) region of an immunoglobulin heavy chain, including that of and kappa (K). There are five main heavy chain classes (or an antibody fragment, such as Fv, scFv, dsEv or Fab. Refer isotypes) which determine the functional activity of an anti ences to “V,” or “VL refer to the variable region of an body molecule: IgM, Ig|D, IgG, IgA and IgE. The disclosed immunoglobulin light chain, including that of an Fv, ScPv, antibodies can be class Switched. dsFv or Fab. 0043. Each heavy and light chain contains a constant 0047. A "monoclonal antibody' is an antibody produced region and a variable region, (the regions are also known as by a single clone of B-lymphocytes or by a cell into which the “domains'). In several embodiments, the heavy and the light light and heavy chain genes of a single antibody have been chain variable domains combine to specifically bind the anti transfected, or by a single cloned immunoglobulin. Mono gen. In additional embodiments, only the heavy chain vari clonal antibodies are produced by methods known to those of able domain is required. For example, naturally occurring skill in the art, for instance by making hybrid antibody-form camelid antibodies consisting of a heavy chain only are func ing cells from a fusion of myeloma cells with immune spleen tional and stable in the absence of light chain (see, e.g., cells. These fused cells and their progeny are termed “hybri Hamers-Casterman et al., Nature, 363:446-448, 1993; Sheriff domas. In some embodiments, monoclonal antibodies can et al., Nat. Struct. Biol. 3:733–736, 1996). Light and heavy be humanized monoclonal antibodies. In some embodiments, chain variable domains contain a “framework” region inter monoclonal antibodies can be chimeric antibodies. In some rupted by three hypervariable regions, also called “comple examples monoclonal antibodies are isolated from a Subject. mentarity-determining regions” or “CDRs (see, e.g., Kabat The amino acid sequences of such isolated monoclonal anti et al., Sequences of Proteins of Immunological Interest, U.S. bodies can be determined. Department of Health and Human Services, 1991). The 0048 A“humanized' antibody is an antibody including a sequences of the framework regions of different light or human framework region and one or more CDRS from a heavy chains are relatively conserved within a species. The non-human (such as a chimpanzee, mouse, rat, or synthetic) framework region of an antibody, that is the combined frame immunoglobulin. The non-human antibody providing the work regions of the constituent light and heavy chains, serves CDRs is termed a "donor,” and the human antibody providing to position and align the CDRS in three-dimensional space. the framework is termed an “acceptor.” In one embodiment, 0044) The CDRs are primarily responsible for antigen all the CDRs are from the donor antibody in a humanized binding. The extent of the framework region and CDRs have antibody. Constant regions need not be present, but if they are, been defined (see, Kabat et al., Sequences of Proteins of they must be substantially identical to human antibody con Immunological Interest, U.S. Department of Health and stant regions, such as at least about 85-90%, such as about Human Services, 1991, which is hereby incorporated by ref 95% or more identical. Hence, all parts of a humanized anti erence in its entirety). The CDRs of the heavy chain variable body, except possibly the CDRs, are substantially identical to domain are located at residues 31-35 (CDR-H1), residues corresponding parts of natural human antibody sequences. A 50-65 (CDR-H2) and residues 95-102 (CDR-H3) according "humanized antibody’ can include a humanized light chain to the Kabat numbering system. However, according to and a humanized heavy chain. A humanized antibody binds to Chothia (Chothia, C. and Lesk, A. M. J. Mol. Biol., 196, the same antigen as the donor antibody that provides the 901-917 (1987)), the loop equivalent to CDR-H1 extends CDRs. The acceptor framework of a humanized antibody US 2016/0033516 A1 Feb. 4, 2016
may have a limited number of Substitutions by amino acids Apoptosis is a result of programmed cell death. According to taken from the donor framework. Humanized or other mono characteristic morphological and biochemical features, apo clonal antibodies can have additional conservative amino acid ptosis is characterized by shrinkage of the cell, dramatic Substitutions which have Substantially no effect on antigen reorganization of the cell nucleus, cell membrane and cell binding or other immunoglobulin functions. Humanized metabolism, active membrane blebbing, and ultimate frag immunoglobulins can be constructed by means of genetic mentation of the cell into membrane-enclosed vesicles (apo engineering (for example, see U.S. Pat. No. 5,585,089). ptotic bodies). The nuclear events of apoptosis begin with 0049. A “chimeric' antibody is an antibody which collapse of the chromatin against the nuclear periphery and includes sequences from two different antibodies, which typi into one or a few large clumps within the nucleus. Nuclear cally are of different species. In some examples, a chimeric features include chromatin aggregation followed by DNA antibody includes one or more CDRs and/or framework fragmentation (a specific marker of apoptotic process) after regions from one chimpanzee antibody and CDRS and/or activation of endonucleases resulting in multiples Subunits of framework regions from another chimpanzee antibody. In DNA of an approximately 180 base pairs. The cellular events Some embodiments, a chimeric antibody comprises heavy include cytoplasmic condensation and partition of the cyto and light chain variable regions derived from a first species plasm and nucleus into membrane bound-vesicles which con and heavy and light chain constant regions derived from a tain ribosomes, intact mitochondria and nuclear material second species. In some embodiments, the variable and con which are Surrounded by an intact cellular membrane (a spe stant regions of the light chain are derived from a first species cific marker of apoptotic process when compared with necro while the variable region of the heavy chain is derived from sis, the other non physiological cell death process). the first species and the constant region of the heavy chain is 0054 Binding affinity: Affinity of an antibody or antigen derived from a second species. In some embodiments, the first binding fragment thereof for an antigen. In one embodiment, species is non-human and includes, but is not limited to, a affinity is calculated by a modification of the Scatchard rabbit. In additional embodiments, the second species method described by Frankel et al., Mol. Immunol., 16:101 includes, but is not limited to, humans, non-human primate, 106, 1979. In another embodiment, binding affinity is mea mouse Or rat. Sured by an antigen/antibody dissociation rate. In yet another 0050 Antigen: A compound, composition, or substance embodiment, a high binding affinity is measured by a com that can stimulate the production of antibodies or a T cell petition radioimmunoassay. In several examples, a high bind response in an animal, including compositions that are ing affinity is at least about 1x10 M. In other embodiments, injected or absorbed into an animal. An antigen reacts with a high binding affinity is at least about 1.5x10, at least about the products of specific humoral or cellular immunity, includ 2.0x10, at least about 2.5x10, at least about 3.0x10, at ing those induced by heterologous antigens. Such as the dis least about 3.5x10, at least about 4.0x10, at least about closed antigens. “Epitope' or “antigenic determinant” refers 4.5x10, or at least about 5.0x10 M. to the region of an antigen to which Band/or T cells respond. 0055 Cancer: A malignant neoplasm that has undergone In one embodiment, T cells respond to the epitope, when the characteristic anaplasia with loss of differentiation, increase epitope is presented in conjunction with an MHC molecule. rate of growth, invasion of Surrounding tissue, and is capable Epitopes can be formed both from contiguous amino acids or of metastasis. For example, thyroid cancer is a malignant noncontiguous amino acids juxtaposed by tertiary folding of neoplasm that arises in or from thyroid tissue, and breast a protein. Epitopes formed from contiguous amino acids are cancer is a malignant neoplasm that arises in or from breast typically retained on exposure to denaturing solvents whereas tissue (Such as a ductal carcinoma). Residual cancer is cancer epitopes formed by tertiary folding are typically lost on treat that remains in a subject after any form of treatment given to ment with denaturing solvents. An epitope typically includes the subject to reduce or eradicate thyroid cancer. Metastatic at least 3, and more usually, at least 5, about 9, or about 8-10 cancer is a cancer at one or more sites in the body other than amino acids in a unique spatial conformation. An epitope can the site of origin of the original (primary) cancer from which be phosphorylated. Methods of determining spatial confor the metastatic cancer is derived. Cancer includes, but is not mation of epitopes include, for example, X-ray crystallogra limited to, Solid tumors and hematologic malignancies. phy and nuclear magnetic resonance. 0056 Chemotherapy; chemotherapeutic agents: As used 0051 Examples of antigens include, but are not limited to, herein, any chemical agent with therapeutic usefulness in the peptides, lipids, polysaccharides, and nucleic acids contain treatment of diseases characterized by abnormal cell growth. ing antigenic determinants, such as those recognized by an Such diseases include tumors, neoplasms, and cancer as well immune cell. In some examples, antigens include peptides as diseases characterized by hyperplastic growth Such as pso derived from a pathogen of interest or a cell type of interest, riasis. In one embodiment, a chemotherapeutic agent is an Such as a tumor cell. Exemplary pathogens include bacteria, agent of use intreating neoplasms such as Solid tumors. In one fungi, viruses and parasites. In some embodiments, an anti embodiment, a chemotherapeutic agent is radioactive mol gen is a phosphorylated polypeptide. ecule. One of skill in the art can readily identify a chemo 0052 A“target epitope' is a specific epitope on an antigen therapeutic agent of use (e.g. see Slapak and Kufe, Principles that specifically binds an antibody of interest, such as a mono of Cancer Therapy, Chapter 86 in Harrison's Principles of clonal antibody. In some examples, a target epitope includes Internal Medicine, 14th edition: Perry et al., Chemotherapy, the amino acid residues that contact the antibody of interest, Ch. 17 in Abeloff, Clinical Oncology 2" ed., (C) 2000 Such that the target epitope can be selected by the amino acid Churchill Livingstone, Inc.; Baltzer L., Berkery R. (eds): residues determined to be in contact with the antibody. In Oncology Pocket Guide to Chemotherapy, 2nd ed. St. Louis, Some embodiments, the target epitope includes a phosphory Mosby-Year Book, 1995: Fischer D S. Knobf M. F. Durivage lated histidine. H J (eds): The Cancer Chemotherapy Handbook, 4th ed. St. 0053 Apoptotic cells: Non-dividing, non-viable cells that Louis, Mosby-Year Book, 1993). Chemotherapeutic agents can be distinguished from necrotic cells (other dead cells). include those known by those skilled in the art, including but US 2016/0033516 A1 Feb. 4, 2016
not limited to: 5-fluorouracil (5-FU), azathioprine, cyclo ecule or label is further joined to a lipid or other molecule to phosphamide, antimetabolites (such as Fludarabine), antine a protein or peptide to increase its half-life in the body. oplastics (such as Etoposide, Doxorubicin, methotrexate, and 0062 Contacting: Placement in direct physical associa Vincristine), carboplatin, cis-platinum and the taxanes. Such tion; includes both in Solid and liquid form, which can take as taxol. Rapamycin has also been used as a chemotherapeu place either in vivo or in vitro. Contacting includes contact tic. between one molecule and another molecule, for example the 0057 Chemotherapy includes treatment with biological amino acid on the Surface of one polypeptide, such as an molecules Such as cytokines, for example, an interleukin (IL), antigen, that contacts another polypeptide. Such as an anti Such as IL-2, or another factor. Such as a tumor necrosis factor body. Contacting can also include contacting a cell for (TNF). Chemotherapy can also include treatment with example by placing an antibody in direct physical association nucleic acids, such as immunostimulatory nucleic acids, see with a cell. for example, PCT Publication No. WO 2011/109422. 0063 Control: A reference standard. In some embodi 0058 Clonal variant: Any sequence, which differs by one ments, the control is a sample obtained from a healthy patient. or more nucleotides or amino acids, in presence of V region In other embodiments, the control is a tissue sample obtained with identical mutations compared to the germline, identical from a patient diagnosed with a disease of interest, such as VDJ or VJ gene usage, and identical D and J length. The cancer, that serves as a positive control. In still other embodi 'germline' sequence is intended to be the sequence coding ments, the control is a historical control or standard reference for the antibody/immunoglobulin (or of any fragment value or range of values (such as a previously tested control thereof) deprived of mutations, for example somatic muta sample, such as a group of patients with known prognosis or tions. The percentage of homology represents an indication of outcome, or group of samples that represent baseline or nor the mutational events which any type of heavy chain portion mal values). undergoes after contact with an antigen. 0064. A difference between a test sample and a control can be an increase or conversely a decrease. The difference can be 0059 Cognate Response Regulator: A component of a a qualitative difference or a quantitative difference, for two-component signal transduction system. The response example a statistically significant difference. Suitable statis regulator usually has a two-domain structure, with a con tical analyses are well known in the art, and include, but are served N-terminal regulatory domain, also called a receiver not limited to, Students T test and ANOVA assays. In some domain, and a variable C-terminal effector domain. The regu examples, a difference is an increase or decrease, relative to a latory domain contains a conserved aspartate residue that control, of at least about 5%, Such as at least about 10%, at receives the phosphoryl group from a phosphorylated histi least about 20%, at least about 30%, at least about 40%, at dine kinase. The phosphorylation of the regulatory domain least about 50%, at least about 60%, at least about 70%, at brings about the conformational change, which leads to the least about 80%, at least about 90%, at least about 95%, at activation of the effector domain that usually functions as the least about 96%, at least about 97%, at least about 98%, at DNA-binding domain. Depending on the presence or absence least about 99%, at least about 100%, at least about 150%, at of an environmental signal, the activity of a histidine kinase is least about 200%, at least about 250%, at least about 300%, at regulated, and this in turn controls the cellular abundance of least about 350%, at least about 400%, at least about 500%, or the phosphorylated response regulator that forms the phos greater than 500%. phorelay couple with the histidine kinase. 0065 Cytokine: Proteins made by cells that affect the 0060 Computer readable media: Any medium or media, behavior of other cells, such as lymphocytes. In one embodi which can be read and accessed directly by a computer, so that ment, a cytokine is a chemokine, a molecule that affects the media is Suitable for use in a computer system. Such cellular trafficking. Specific non-limiting examples of cytok media include, but are not limited to: magnetic storage media ines are IL-2, IFNY, IL-6, and IL-10. Such as floppy discs, hard disc storage medium and magnetic 0.066 Detectable marker: A detectable molecule (also tape; optical storage media Such as optical discs or CD-ROM; known as a label) that is conjugated directly or indirectly to a electrical storage media such as RAM and ROM; and hybrids second molecule. Such as an antibody, to facilitate detection of these categories such as magnetic/optical storage media. of the second molecule. For example, the detectable marker 0061 Conjugate: A complex of two molecules linked can be capable of detection by ELISA, spectrophotometry, together, for example, linked together by a covalent bond. In flow cytometry, microscopy or diagnostic imaging tech one embodiment, an antibody is linked to an effector mol niques (such as CT scans, MRIs, ultrasound, fiberoptic ecule; for example, an antibody that specifically binds to a examination, and laparoscopic examination). Non-limiting phosphorylated polypeptide. Such as a polypeptide that examples of detectable markers include fluorophores, fluo includes a phosphorylated histidine, for example a histidine rescent proteins, chemiluminescent agents, enzymatic link phosphorylated at N3, covalently linked to an effector mol ages, radioactive isotopes and heavy metals or compounds ecule or to a label. The linkage can be by chemical or recom (for example Super paramagnetic iron oxide nanocrystals for binant means. In one embodiment, the linkage is chemical, detection by MRI). In one example, a “labeled antibody' wherein a reaction between the antibody moiety and the refers to incorporation of another molecule in the antibody. effector molecule has produced a covalent bond formed For example, the label is a detectable marker, such as the between the two molecules to form one molecule. A peptide incorporation of a radiolabeled amino acid or attachment to a linker (short peptide sequence) can optionally be included polypeptide of biotinyl moieties that can be detected by between the antibody and the effector molecule. Because marked avidin (for example, Streptavidin containing a fluo conjugates can be prepared from two molecules with separate rescent marker or enzymatic activity that can be detected by functionalities, such as an antibody and an effector molecule, optical or colorimetric methods). Various methods of labeling they are also sometimes referred to as “chimeric molecules.” polypeptides and glycoproteins are known in the art and may In one embodiment, an antibody linked to an effector mol be used. Examples of labels for polypeptides include, but are US 2016/0033516 A1 Feb. 4, 2016 not limited to, the following: radioisotopes or radionuclides tailpiece, and may or may not be bound by the J chain. For (such as S or ''I), fluorescent labels (such as fluorescein IgG, the Fc region comprises immunoglobulin domains isothiocyanate (FITC), rhodamine, lanthanide phosphors), Cgamma2 and Cgamma3 (Cy2 and Cy3) and the lower part of enzymatic labels (such as horseradish peroxidase, beta-galac the hinge between Cgammal (CY 1) and Cy2. Although the tosidase, luciferase, alkaline phosphatase), chemilumines boundaries of the Fc region may vary, the human IgG heavy cent markers, biotinyl groups, predetermined polypeptide chain Fc region is usually defined to comprise residues C226 epitopes recognized by a secondary reporter (such as a leu or P230 to its carboxyl-terminus, wherein the numbering is cine Zipper pair sequences, binding sites for secondary anti according to the EU index as in Kabat. For IgA, the Fc region bodies, metal binding domains, epitope tags), or magnetic comprises immunoglobulin domains Calpha2 and Calpha3 agents, such as gadolinium chelates. In some embodiments, (CO2 and CO3) and the lower part of the hinge between labels are attached by spacer arms of various lengths to reduce Calpha1 (CC.1) and CO2. Encompassed within the definition potential steric hindrance. Methods for using detectable of the Fc region are functionally equivalent analogs and vari markers and guidance in the choice of detectable markers ants of the Fc region. A functionally equivalent analog of the appropriate for various purposes are discussed for example in Fc region may be a variant Fc region, comprising one or more Sambrook et al. (Molecular Cloning: A Laboratory Manual, amino acid modifications relative to the wild-type or naturally Cold Spring Harbor, N. Y., 1989) and Ausubel et al. (In existing Fc region. Variant Fc regions will possess at least Current Protocols in Molecular Biology, John Wiley & Sons, 50% homology with a naturally existing Fc region, such as New York, 1998). about 80%, and about 90%, or at least about 95% homology. 0067 Detecting, Determining or Measuring: To identify Functionally equivalent analogs of the Fc region may com the existence, presence, or fact of Something. General meth prise one or more amino acid residues added to or deleted ods of detecting are known to the skilled artisan (see, for from the N- or C-termini of the protein, such as no more than example, U.S. Pat. No. 7,635,476) and may be supplemented 30 or no more than 10 additions and/or deletions. Function with the protocols and reagents disclosed herein. For ally equivalent analogs of the Fc region include Fc regions example, included herein are methods of detecting a cell that operably linked to a fusion partner. Functionally equivalent expresses a phosphorylated polypeptide. Such as a polypep analogs of the Fc region must comprise the majority of all of tide that includes a phosphorylated histidine, for example a the Ig domains that compose Fc region as defined above; for histidine phosphorylated at N3. These terms refer to measur example IgG and IgA Fc regions as defined herein must ing a quantity or quantitating a target molecule in the sample, comprise the majority of the sequence encoding CH and the either absolutely or relatively. Generally, detecting, measur majority of the sequence encoding CH. Thus, the CH ing or determining a biological molecule requires performing domain on its own, or the CH domain on its own, are not an assay, Such as mass spectrometry, and not simple observa considered Fc region. The Fc region may refer to this region tion. in isolation, or this region in the context of an Fc fusion 0068 Diagnostic: Identifying the presence or nature of a polypeptide. pathologic condition. Diagnostic methods differ in their sen 0072 Fluorophore: A chemical compound, which when sitivity and specificity. The 'sensitivity of a diagnostic assay excited by exposure to a particular stimulus, Such as a defined is the percentage of diseased individuals who test positive wavelength of light, emits light (fluoresces), for example at a (percent of true positives). The “specificity of a diagnostic different wavelength (such as a longer wavelength of light). assay is 1 minus the false positive rate, where the false posi 0073. Fluorophores are part of the larger class of lumines tive rate is defined as the proportion of those without the cent compounds. Luminescent compounds include chemilu disease who test positive. While a particular diagnostic minescent molecules, which do not require a particular wave method may not provide a definitive diagnosis of a condition, length of light to luminesce, but rather use a chemical Source it suffices if the method provides a positive indication that of energy. Therefore, the use of chemiluminescent molecules aids in diagnosis. “Prognostic' is the probability of develop (such as aequorin) can eliminate the need for an external ment (for example severity) of a pathologic condition. In Source of electromagnetic radiation, Such as a laser. Some examples prognostic is the probability that a subject 0074 Examples of particular fluorophores that can be will respond favorably to a treatment agent. used in the methods and for attachment to antibodies are 0069 Effector molecule: The portion of a chimeric mol provided in U.S. Pat. No. 5,866.366 to Nazarenko et al., such ecule that is intended to have a desired effectonacell to which aS 4-acetamido-4-isothiocyanatoStilbene-2.2"disulfonic the chimeric molecule is targeted. Effector molecule is also acid, acridine and derivatives such as acridine and acridine known as an effector moiety, therapeutic agent, or diagnostic isothiocyanate, 5-(2-aminoethyl)aminonaphthalene-1-sul agent, or similar terms. fonic acid (EDANS), 4-amino-N-3-vinylsulfonyl)phenyl 0070 Framework Region: Amino acid sequences inter naphthalimide-3.5 disulfonate (Lucifer Yellow VS), N-(4- posed between CDRs. The term includes variable light and anilino-1-naphthyl)maleimide, anthranilamide, Brilliant variable heavy framework regions. The framework regions Yellow, coumarin and derivatives such as coumarin, 7-amino serve to hold the CDRs in an appropriate orientation for 4-methylcoumarin (AMC, Coumarin 120), 7-amino-4-trif antigen binding. luoromethylcouluarin (Coumaran 151); cyanosine; 4,6-di 0071 Fc polypeptide: The polypeptide comprising the aminidino-2-phenylindole (DAPI); 5'5"-dibromopyrogallol constant region of an antibody excluding the first constant sulfonephthalein (Bromopyrogallol Red): 7-diethylamino-3- region immunoglobulin domain. Fc region generally refers to (4'-isothiocyanatophenyl)-4-methylcoumarin; the last two constant region immunoglobulin domains of IgA, diethylenetriamine pentaacetate; 4,4'-diisothiocyanatodihy IgD, and IgG, and the last three constant region immunoglo dro-stilbene-2,2'-disulfonic acid; 4,4'-diisothiocyanatostil bulin domains of IgE and IgM. An Fc region may also include bene-2,2'-disulfonic acid; 5-dimethylaminonaphthalene-1- part or all of the flexible hinge N-terminal to these domains. sulfonyl chloride (DNS, dansyl chloride); For IgA and IgM, an Fc region may or may not comprise the 4-dimethylaminophenylaZophenyl-4-isothiocyanate US 2016/0033516 A1 Feb. 4, 2016
(DABITC); eosin and derivatives such as eosin and eosin -continued isothiocyanate; erythrosin and derivatives such as erythrosin B and erythrosin isothiocyanate; ethidium; fluorescein and Clostridium paraputrificum Clostridium perfingens derivatives such as 5-carboxyfluorescein (FAM), 5-(4.6- Clostridium phytofermenians dichlorotriazin-2-yl)aminofluorescein (DTAF), 27'- Clostridium piliforme dimethoxy-45'-dichloro-6-carboxyfluorescein (JOE), fluo Clostridium ragsdalei rescein, fluorescein isothiocyanate (FITC), and QFITC Cliostridium ranostin Clostridium septicum (XRITC); fluorescamine: IR144: IR 1446; Malachite Green Cliostridium sordei isothiocyanate: 4-methylumbelliferone; ortho cresolphtha Clostridium sporogenes lein: nitrotyrosine; pararosaniline; Phenol Red; B-phyco Cliostridium Stickiandi erythrin; o-phthaldialdehyde; pyrene and derivatives Such as Cliostridium tertium Cliostridium tetani pyrene, pyrene butyrate and Succinimidyl 1-pyrene butyrate; Clostridium thermosaccharolyticum Reactive Red 4 (CibacronTM Brilliant Red 3B-A); rhodamine Clostridium tyrobutyricum and derivatives such as 6-carboxy-X-rhodamine (ROX), Corynebacterium 6-carboxyrhodamine (R6G), lissamine rhodamine B sulfonyl Corynebacterium bovis Corynebacterium diphtheriae chloride, rhodamine (Rhod), rhodamine B, rhodamine 123, Corynebacterium granulosum rhodamine X isothiocyanate, sulforhodamine B, sulfor Corynebacterium jeikeium hodamine 101 and sulfonyl chloride derivative of sulfor Corynebacterium minutissimum hodamine 101 (Texas Red); N.N.N',N'-tetramethyl-6-carbox Corynebacterium renale Enterococcits yrhodamine (TAMRA); tetramethyl rhodamine; tetramethyl Lactobacillales rhodamine isothiocyanate (TRITC); riboflavin; rosolic acid Listeria and terbium chelate derivatives: LightCycler Red 640; Cy5.5; Nocardia and Cy56-carboxyfluorescein; 5-carboxyfluorescein Nocardia asteroides Nocardia brasiiensis (5-FAM); boron dipyrromethene difluoride (BODIPY); N.N., Propionibacterium acnes N',N'-tetramethyl-6-carboxyrhodamine (TAMRA); acridine, Rhodococci is equi stilbene, -6-carboxy-fluorescein (HEX), TET (Tetramethyl Sarcina fluorescein), 6-carboxy-X-rhodamine (ROX), Texas Red, Solobacterium moorei Staphylococcits 2.7'-dimethoxy-4',5'-dichloro-6-carboxyfluorescein (JOE), Staphylococcusatiretts Cy3, Cy5, VICR) (Applied Biosystems), LC Red 640, LC Red Staphylococcus capitis 705, Yakima yellow amongst others. Staphylococci is caprae Staphylococci is epidermidis 0075 Other suitable fluorophores include those known to Staphylococciis haemolyticals those skilled in the art, for example those available from Staphylococcus hominis Molecular Probes (Eugene, Oreg.). In particular examples, a Staphylococci is lugdunensis fluorophore is used as a donor fluorophore or as an acceptor Staphylococci is miscae fluorophore. Staphylococci is nepalensis Staphylococci is peitenkoferi 0076 Gram-positive bacteria: Bacteria that stain dark blue Staphylococci is saprophyticus or violet during Gram staining, and have a thick peptidogly Staphylococci is succinus can layer. Exemplary Gram-positive bacteria that can be used Staphylococciis warneri Staphylococci is xylost is in the disclosed methods include: Strangles Streptococci is Streptococci is agaiaciae Actinobacteria Streptococci is anginostis Actinomyces Streptococcus bovis Actinomyces israeli Streptococci is canis Bacillales Streptococcusiniae Bacilius Streptococci is lactarius Cliostridium Streptococci is mitis Clostridium acetobutyllicum Streptococci is mutans Cliostridium aerotoierans Streptococci is oralis Clostridium argentinense Streptococci is parasanguinis Cliostridium barati Streptococci is peroris Clostridium beijerinckii Streptococci is pneumoniae Clostridium bifermenians Streptococci is pyogenes Cliostridium bointinum Streptococci is ratti Clostridium butyricum Streptococci is Saivarius Cliostridium cadaveris Streptococci is sanguinis Clostridium cellulolyticum Streptococci is sobrinus Cliostridium chauvoei Streptococci is stiis Clostridium clostridioforme Streptococci is Saivarius thermophilus Cliostridium coicanis Streptococci is liberis Clostridium difficile Cliostridium esteriheticum Streptococcus vestibularis Clostridium fallax Streptococcus viridans Clostridium formicaceticum Clostridium histolyticum Cliostridium innocuum Clostridium kluyveri 0077 Gram-negative bacteria: Bacteria that loose or do Clostridium iungdahli not retain dark blue or violet stain during Gram staining, but Clostridium novyi instead are colored by a counterstain, Such as Safranin, and appear pink or ed. Gram-negative bacteria have a thin pepti US 2016/0033516 A1 Feb. 4, 2016 11 doglycan layer. Exemplary Gram-negative bacteria that can (Matthews, 1995). Although pHis signaling is commonly be used in the disclosed methods include: used in bacteria, the question remains whether or not pHis plays a role in eukaryotic signaling. There is growing evi dence implicating putative mammalian histidine kinases Acetic acid bacteria Fusobacterium necrophorum Acinetobacter battmannii Fusobacterium nucleatum NME1 and NME2 in cancer and tumor metastasis (Andolfo et Agrobacterium timefaciens Fusobacterium polymorphium al., 2011; Boissan et al., 2010; Thakur et al., 2011; Tso et al., Anaerobiospirilium Haemophilus haemolyticus 2013; Zhao et al., 2013). In fact, NME1 (AKA Nm23-H1 or Bacteroides Haemophilus influenzae nucleoside diphosphate kinase NDPK) was the first candi Bacteroides fragilis Helicobacter date metastasis Suppressor gene identified (Steegetal., 1988). Bdeliovibrio Helicobacter pylori Brachyspira Klebsiella pneumoniae I0081 Host cells: Cells in which a vector can be propagated Cardiobacterium hominis Legionelia and its DNA expressed, for example a disclosed antibody can Coxieia burnetii Legionella pneumophila be expressed in a host cell. The cell may be prokaryotic or Cyanobacteria Leptotrichia buccalis Cytophaga Megamonas eukaryotic. The term also includes any progeny of the Subject Dialister Megasphaera host cell. It is understood that all progeny may not be identical Enterobacter Moraxeia to the parental cell since there may be mutations that occur Enterobacter cloacae Moraxeia bovis during replication. However, Such progeny are included when Enterobacteriaceae Moraxeiia catarrhais the term "host cell' is used. Escherichia Moraxeia Osioensis Escherichia coi Morganelia morgani I0082 IgG: A polypeptide belonging to the class or isotype Pseudomonas genome database Negativicutes of antibodies that are substantially encoded by a recognized Rickettsia rickettsii Neisseria gonorrhoeae immunoglobulin gamma gene. In humans, this class com Saimoneiia Neisseria meningitidis Saimoneiia enterica Neisseria sicca prises IgG, IgG, IgGs, and IgG. In mice, this class com Saimoneiia enterica enterica Pectiniatus prises IgG, IgG2a, IgG2b, IgGs. Selenomonadales Propionispora I0083. Immune complex: The binding of antibody to a Serraia marcesCens Proteobacteria Shigella Proteus mirabilis soluble antigen forms an immune complex. The formation of Spirochaeta Proteus penneri an immune complex can be detected through conventional Spirochaetaceae Pseudomonas methods known to the skilled artisan, for instance immuno Sporomisa Pseudomonas aeruginosa histochemistry, immunoprecipitation, flow cytometry, immu Stenotrophomonas Streptococci isgordonii nofluorescence microscopy, ELISA, immunoblotting (for Vampirococcus example, Western blot), magnetic resonance imaging, CT Verminephrobacter scans, X-ray and affinity chromatography. Immunological Vibrio choierae binding properties of selected antibodies may be quantified Wolbachia Zymophilus using methods well known in the art. I0084. Immunoadhesin: A molecular fusion of a protein with the Fc region of an immunoglobulin, wherein the immu 0078 Histidine kinase: An enzyme that phosphorylates noglobulin retains specific properties, such as Fc receptor histidine, using the reaction: binding and increased half-life. An Fc fusion combines the Fc ATP+polypeptide L-histidinees ADP+polypeptide region of an immunoglobulin with a fusion partner, which in N-phospho-L-histidine. general can be any protein, polypeptide, peptide, or Small molecule. In one example, an immunoadhesin includes the In this reaction, the two substrates are ATP and a polypeptide hinge, CH, and CH domains of the immunoglobulingamma including a histidine, and the two products are ADP and a 1 heavy chain constant region. In another example, the immu polypeptide including N-phospho-L-histidine, with phos noadhesin includes the CH2, and CH domains of an IgG. phate linked either to the N1 or the N3 position. Histidine I0085. Immunoassay: A biochemical test that measures the kinases are present, for example, in bacterial cells and mam presence or concentration of a Substance in a sample, Such as malian cells. a biological sample, for example a serum sample obtained 0079 A large family of histidine kinases and downstream from a Subject, using the reaction of an antibody to its cognate signaling proteins, known as two-component regulatory sys antigen, for example the specific binding of an antibody to a tems, are widely employed by bacteria to link extracellular as a polypeptide that includes a phosphorylated histidine, for signals with transcription and chemotaxis. A bacterial histi example a histidine phosphorylated at N3. The presence of dine kinase is composed of several domains starting with a antigen and/or the amount of antigen present can be mea short N-terminal cytoplasmic portion connected to an extra Sured. The phosphorylation state of the antigen can also be cellular sensing domain via a transmembrane C. helix. A sec measured. In some examples, the amount of a polypeptide ond transmembrane C. helix connects the extracellular that includes a phosphorylated histidine, for example a histi domain to the C-terminal cytoplasmic catalytic domain. His dine phosphorylated at N3 is measured. tidine kinases are known to serve roles in many different 0086 Measuring the quantity of antigen (Such as a phos signal transduction pathways. The cytoplasmic domaintends phorylated polypeptide) can be achieved by a variety of meth to have high sequence homology and contains several well ods. One of the most common is to label either the antigen or known motifs, including the H, N, G1, F, and G2 boxes, which antibody with a detectable label. In some examples an anti the extracellular sensing domain is not well conserved. Sev body that specifically binds a polypeptide that includes a eral crystal structures of a histidine kinase are available, see phosphorylated histidine, for example a histidine phosphory PDB Accession Nos. 1 POZ, 2CMN, 2GJ3, 2HJE, 2J48, lated at N3 is labeled. Methods for labeling and guidance in 2O9B, 2O9C, 2R78, and 2R8R. the choice of labels appropriate for various purposes are 0080. Similar phosphotransfer cascades function in plants discussed for example in Sambrook et al. (Molecular Clon to regulate processes such as ripening and circadian rhythms ing: A Laboratory Manual, Cold Spring Harbor, N.Y., 1989) US 2016/0033516 A1 Feb. 4, 2016
Ausubel et al. (In Current Protocols in Molecular Biology, sample. The elements or molecules are identified by correlat John Wiley & Sons, New York, 1998), and Harlow & Lane, ing known masses by the identified masses. “Time-of-flight (Antibodies, A Laboratory Manual, Cold Spring Harbor Pub mass spectrometry” (TOFMS) is a method of mass spectrom lications, New York, 1988). etry in which an ion’s mass-to-charge ratio is determined via 0087 Immunologically reactive conditions: Includes ref a time measurement. Ions are accelerated by an electric field erence to conditions which allow an antibody raised against a of known strength. This acceleration results in an ion having particular epitope to bind to that epitope to a detectably the same kinetic energy as any other ion that has the same greater degree than, and/or to the Substantial exclusion of charge. The Velocity of the ion depends on the mass-to-charge binding to Substantially all other epitopes. Immunologically ratio. The time that it subsequently takes for the particle to reactive conditions are dependent upon the format of the reach a detector at a known distance is measured. This time antibody binding reaction and typically are those utilized in will depend on the mass-to-charge ratio of the particle immunoassay protocols or those conditions encountered in (heavier particles reach lower speeds). From this time and the vivo. See Harlow & Lane, Supra, for a description of immu known experimental parameters one can find the mass-to noassay formats and conditions. The immunologically reac charge ratio of the ion. “Liquid chromatography-mass spec tive conditions employed in the methods are “physiological trometry” or “LC-MS is a chemistry technique that com conditions” which include reference to conditions (e.g., tem bines the physical separation capabilities of liquid perature, osmolarity, pH) that are typical inside a living mam chromatography (or HPLC) with the mass analysis capabili mal or a mammalian cell. While it is recognized that some ties of mass spectrometry. Liquid chromatography mass spec organs are subject to extreme conditions, the intra-organismal trometry (LC-MS) separates compounds chromatographi and intracellular environment normally lies around pH 7 (e.g., from pH 6.0 to pH 8.0, more typically pH 6.5 to 7.5), cally before they are introduced to the ion source and mass contains water as the predominant solvent, and exists at a spectrometer. It differs from gas chromatography (GC-MS) temperature above 0° C. and below 50° C. Osmolarity is in that the mobile phase is liquid, usually a mixture of water within the range that is supportive of cell viability and pro and organic solvents, instead of gas and the ions fragments. liferation. Most commonly, an electrospray ionization source is used in 0088 Isolated: An "isolated' biological component (such LC-MS. as a cell, for example a B-cell, a nucleic acid, peptide, protein, 0092. Mean and Standard Deviation: The arithmetic mean heavy chain domain orantibody) has been Substantially sepa is the “standard’ average, often simply called the “mean’. rated, produced apart from, or purified away from other bio logical components in the cell of the organism in which the component naturally occurs, such as, other chromosomal and 1 - extrachromosomal DNA and RNA, and proteins. Nucleic it. acids and polypeptides which have been "isolated thus include nucleic acids and proteins purified by standard puri fication methods. The term also embraces nucleic acids and polypeptides prepared by recombinant expression in a host The mean is the arithmetic average of a set of values. cell as well as chemically synthesized nucleic acids. In some 0093. The standard deviation (represented by the symbol examples an antibody, such as an antibody that specifically sigma, O) shows how much variation or “dispersion' exists binds phosphorylated polypeptide, Such as a polypeptide that from the mean. The standard deviation of a random variable, includes a phosphorylated histidine, for example a histidine statistical population, data set, or probability distribution is phosphorylated at N3 can be isolated. the square root of its variance. The standard deviation is 0089. K. The dissociation constant for a given interac commonly used to measure confidence in statistical conclu tion, such as a polypeptide ligand interaction or an antibody sions. Generally, twice the standard deviation is about the antigen interaction. For example, for the bimolecular interac radius of a 95 percent confidence interval. Effects that fall far tion of an antibody (Such as any of the antibodies disclosed outside the range of Standard deviation are generally consid herein) and an antigen (Such as phosphorylated polypeptide) ered statistically significant. One of skill in the art can readily it is the concentration of the individual components of the calculate the mean and the standard deviation from a popu bimolecular interaction divided by the concentration of the lation of values. complex. 0094. Nucleic acid: A polymer composed of nucleotide 0090 Label: A detectable compound or composition that units (ribonucleotides, deoxyribonucleotides, related natu is conjugated directly or indirectly to another molecule. Such rally occurring structural variants, and synthetic non-natu as an antibody or a protein, to facilitate detection of that rally occurring analogs thereof) linked via phosphodiester molecule. Specific, non-limiting examples of labels include bonds, related naturally occurring structural variants, and fluorescent tags, enzymatic linkages, and radioactive iso synthetic non-naturally occurring analogs thereof. Thus, the topes. In some examples, a disclosed antibody is labeled. term includes nucleotide polymers in which the nucleotides 0091 Mass Spectrometry: A process used to separate and and the linkages between them include non-naturally occur identify molecules based on their mass. Mass spectrometry ring synthetic analogs, such as, for example and without ionizes chemical compounds to generate charged molecules limitation, phosphorothioates, phosphoramidates, methyl or molecule fragments and measures their mass-to-charge phosphonates, chiral-methylphosphonates, 2-O-methyl ribo ratios. In a typical MS procedure, as sample is ionized. The nucleotides, peptide-nucleic acids (PNAS), and the like. Such ions are separated according to their mass-to-charge ratio, polynucleotides can be synthesized, for example, using an and the ions are dynamically detected by some mechanism automated DNA synthesizer. The term "oligonucleotide' capable of detecting energetic charged particles. The signal is typically refers to short polynucleotides, generally no greater processed into the spectra of the masses of the particles of that than about 50 nucleotides. It will be understood that when a US 2016/0033516 A1 Feb. 4, 2016 nucleotide sequence is represented by a DNA sequence (i.e., parison algorithm, test and reference sequences are entered A, T.G, C), this also includes an RNA sequence (i.e., A., U.G., into a computer, Subsequence coordinates are designated, if C) in which “U” replaces “T” necessary, and sequence algorithm program parameters are 0.095 Conventional notation is used herein to describe designated. Default program parameters are used. Methods of nucleotide sequences: the left-hand end of a single-stranded alignment of sequences for comparison are well known in the nucleotide sequence is the 5'-end; the left-hand direction of a art. Optimal alignment of sequences for comparison can be double-stranded nucleotide sequence is referred to as the conducted, e.g., by the local homology algorithm of Smith & 5'-direction. The direction of 5' to 3' addition of nucleotides to Waterman, Adv. Appl. Math. 2:482, 1981, by the homology nascent RNA transcripts is referred to as the transcription alignment algorithm of Needleman & Wunsch, J. Mol. Biol. direction. The DNA strand having the same sequence as an 48:443, 1970, by the search for similarity method of Pearson mRNA is referred to as the “coding strand;” sequences on the & Lipman, Proc. Natl. Acad. Sci. USA 85:2444, 1988, by DNA strand having the same sequence as an mRNA tran computerized implementations of these algorithms (GAP, scribed from that DNA and which are located 5' to the 5'-end BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics of the RNA transcript are referred to as “upstream Software Package, Genetics Computer Group, 575 Science sequences: sequences on the DNA strand having the same Dr. Madison, Wis.), or by manual alignment and visual sequence as the RNA and which are 3' to the 3' end of the inspection (see, e.g., Current Protocols in Molecular Biology coding RNA transcript are referred to as “downstream (Ausubel et al., eds 1995 supplement)). sequences.” 0102 One example of a useful algorithm is PILEUP. 0096 “cDNA refers to a DNA that is complementary or PILEUP uses a simplification of the progressive alignment identical to an mRNA, in either single stranded or double method of Feng & Doolittle, J. Mol. Evol. 35:351-360, 1987. stranded form. The method used is similar to the method described by Hig 0097 “Encoding refers to the inherent property of spe gins & Sharp, CABIOS 5:151-153, 1989. Using PILEUP, a cific sequences of nucleotides in a polynucleotide. Such as a reference sequence is compared to other test sequences to gene, a cDNA, or an mRNA, to serve as templates for Syn determine the percent sequence identity relationship using thesis of other polymers and macromolecules in biological the following parameters: default gap weight (3.00), default processes having either a defined sequence of nucleotides gap length weight (0.10), and weighted end gaps. PILEUP (i.e., rRNA, tRNA and mRNA) or a defined sequence of can be obtained from the GCG sequence analysis software amino acids and the biological properties resulting therefrom. package, e.g., version 7.0 (Devereaux et al., Nuc. Acids Res. Thus, a gene encodes a protein if transcription and translation 12:387-395, 1984. of mRNA produced by that gene produces the protein in a cell 0103) Another example of algorithms that are suitable for or other biological system. Both the coding Strand, the nucle determining percent sequence identity and sequence similar otide sequence of which is identical to the mRNA sequence ity are the BLAST and the BLAST 2.0 algorithm, which are and is usually provided in sequence listings, and non-coding described in Altschul et al., J. Mol. Biol. 215:403-410, 1990 Strand, used as the template for transcription, of a gene or and Altschulet al., Nucleic Acids Res. 25:3389-3402, 1977. cDNA can be referred to as encoding the protein or other Software for performing BLAST analyses is publicly avail product of that gene or cDNA. Unless otherwise specified, a able through the National Center for Biotechnology Informa “nucleotide sequence encoding an amino acid sequence' tion (ncbi.nlm.nih.gov). The BLASTN program (for nucle includes all nucleotide sequences that are degenerate versions otide sequences) uses as defaults a word length (W) of 11, of each other and that encode the same amino acid sequence. alignments (B) of 50, expectation (E) of 10, M-5, N=-4, and Nucleotide sequences that encode proteins and RNA may a comparison of both strands. The BLASTP program (for include introns. amino acid sequences) uses as defaults a word length (W) of 0.098 “Recombinant nucleic acid' refers to a nucleic acid 3, and expectation (E) of 10, and the BLOSUM62 scoring having nucleotide sequences that are not naturally joined matrix (see Henikoff & Henikoff, Proc. Natl. Acad. Sci. USA together. This includes nucleic acid vectors comprising an 89:10915, 1989). An oligonucleotide is a linear polynucle amplified or assembled nucleic acid which can be used to otide sequence of up to about 100 nucleotide bases in length. transform a suitable host cell. A host cell that comprises the 0104 ClustalW is a program that aligns three or more recombinant nucleic acid is referred to as a “recombinant host sequences in a computationally efficient manner. Aligning cell. The gene is then expressed in the recombinant host cell multiple sequences highlights areas of similarity which may to produce, e.g., a “recombinant polypeptide. A recombinant be associated with specific features that have been more nucleic acid may serve a non-coding function (e.g., promoter, highly conserved than other regions. Thus, this program can origin of replication, ribosome-binding site, etc.) as well. classify sequences for phylogenetic analysis, which aims to 0099. A first sequence is an “antisense' with respect to a model the substitutions that have occurred over evolution and second sequence if a polynucleotide whose sequence is the derive the evolutionary relationships between sequences. The first sequence specifically hybridizes with a polynucleotide ClustalW multiple sequence alignment web form is available whose sequence is the second sequence. on the internet from EMBL-EBI (ebi.ac.uk/Tools/msa/clust 0100 Terms used to describe sequence relationships alw2/), see also Larkin et al., Bioinformatics 2007 23(21): between two or more nucleotide sequences or amino acid 2947-2948. sequences include “reference sequence.” “selected from.” 0105. A polynucleotide or nucleic acid sequence refers to “comparison window. “identical.’ “percentage of sequence a polymeric form of nucleotide at least 10 bases in length. A identity.” “substantially identical.” “complementary,” and recombinant polynucleotide includes a polynucleotide that is “substantially complementary.” not immediately contiguous with both of the coding 0101 For sequence comparison of nucleic acid sequences, sequences with which it is immediately contiguous (one on typically one sequence acts as a reference sequence, to which the 5' end and one on the 3' end) in the naturally occurring test sequences are compared. When using a sequence com genome of the organism from which it is derived. The term US 2016/0033516 A1 Feb. 4, 2016
therefore includes, for example, a recombinant DNA which is 0116 5) Isoleucine (I), Leucine (L), Methionine (M), incorporated into a vector, into an autonomously replicating Valine (V); and plasmid or virus; or into the genomic DNA of a prokaryote or 0.117 6) Phenylalanine (F), Tyrosine (Y), Tryptophan eukaryote, or which exists as a separate molecule (e.g., a (W). cDNA) independent of other sequences. The nucleotides can 0118. A “polypeptide including a histidine phosphory be ribonucleotides, deoxyribonucleotides, or modified forms lated at N3 has one or more histidine residues phosphory of either nucleotide. The term includes single- and double lated at N3 in its amino acid sequence. The polypeptide can stranded forms of DNA. also have histidine residues phosphorylated at N1 and/or 0106 Pharmaceutically acceptable carriers: The pharma unphosphorylated histidines in its amino acid sequence, pro ceutically acceptable carriers of use are conventional. Rem vided the polypeptide has at least one histidine phosphory ington's Pharmaceutical Sciences, by E. W. Martin, Mack lated at N3 is present in the amino acid sequence of the Publishing Co., Easton, Pa., 19th Edition, 1995, describes polypeptide. In some embodiments, a polypeptide including a compositions and formulations Suitable for pharmaceutical histidine phosphorylated at N3, can have 1, 2, 3, 4, 5, 6, 7, 8, delivery of the antibodies herein disclosed. 9, 10 or more histidines phosphorylated at N3 in its amino 0107. In general, the nature of the carrier will depend on acid sequence. the particular mode of administration being employed. For instance, parenteral formulations usually comprise injectable 0119 Preventing or treating a disease: “Preventing a dis fluids that include pharmaceutically and physiologically ease refers to inhibiting the full development of a disease, for acceptable fluids, which include, but are not limited to, water, example in a person who is known to have a predisposition to physiological saline, balanced salt Solutions, aqueous dex a disease Such as a tumor. An example of a person with a trose, glycerol or the like as a vehicle. For Solid compositions known predisposition is someone with a history of breast (e.g., powder, pill, tablet, or capsule forms), conventional cancer in the family, or who has been exposed to factors that non-toxic Solid carriers can include, for example, pharmaceu predispose the Subject to a condition, Such as melanoma. tical grades of mannitol, lactose, starch, or magnesium Stear “Treatment” refers to a therapeutic intervention that amelio ate. In addition to biologically neutral carriers, pharmaceuti rates a sign or symptom of a disease or pathological condition cal compositions to be administered can contain minor after it has begun to develop. In several embodiments, treat amounts of non-toxic auxiliary Substances, such as wetting or ment refers to a reduction in the size or Volume of a tumor, a emulsifying agents, preservatives, and pH buffering agents decrease in the number and/or size of metastases, a decrease and the like, for example sodium acetate or sorbitan mono in a symptom of the tumor, or combinations thereof. laurate. I0120 Purified: The term purified does not require absolute 0108) Pharmaceutical agent: A chemical compound or purity; rather, it is intended as a relative term. Thus, for composition capable of inducing a desired therapeutic or example, a purified peptide preparation is one in which the prophylactic effect when properly administered to a subject peptide or protein (such as an antibody) is more enriched than or a cell. In some examples a pharmaceutical agent includes the peptide or protein is in its natural environment within a one or more of the disclosed antibodies. cell. In one embodiment, a preparation is purified Such that 0109 Phosphorylation: The addition of a phosphate the protein or peptide represents at least 50%, 60%, 70%, (PO) group to a polypeptide or other organic molecule. 80%, 90%. 95%, 96%, 97%, 98%, or 99% of the total peptide Phosphorylation of proteins plays a significant role in a num or protein content of the preparation. ber of biological processes. The reversible phosphorylation 0121 Quantitating: Determining or measuring a quantity of proteins is an important regulatory mechanism that occurs (such as a relative quantity) of a molecule or the activity of a in both prokaryotic and eukaryotic organisms. In vivo, serine molecule. Such as the quantity of a polypeptide that includes phosphorylation is the most common type of phosphoryla phosphorylated histidine, for example a histidine phosphory tion, followed by threonine phosphorylation. Tyrosine, aspar lated at N3 present in a sample. tate and histidine are also phosphorylated. Phosphorylation 0.122 Recombinant: A recombinant nucleic acid is one of polypeptides can be detected by antibodies, electrophore that has a sequence that is not naturally occurring or has a sis, such as SDS-PAGE, and mass spectrometry. sequence that is made by an artificial combination of two 0110 Polypeptide: Any chain of amino acids, regardless otherwise separated segments of sequence. This artificial of length or posttranslational modification (e.g., glycosyla combination is often accomplished by chemical synthesis or, tion orphosphorylation). In one embodiment, the polypeptide more commonly, by the artificial manipulation of isolated includes a phosphorylated histidine, for example a histidine segments of nucleic acids, e.g., by genetic engineering tech phosphorylated at N3. In one embodiment, the polypeptide is niques. a disclosed antibody or a fragment thereof. I0123. Sample: A biological sample obtained from a sub 0111. A “residue' refers to an amino acid or amino acid ject, such as a human or other primate or mammal, which mimetic incorporated in a polypeptide by an amide bond or contains for example nucleic acids and/or proteins. As used amide bond mimetic. A polypeptide has an amino terminal herein, biological samples include all clinical samples that (N-terminal) end and a carboxy terminal end. Conservative include polypeptides, such as those obtained from Subjects, amino acid Substitution tables providing functionally similar including, but not limited to, cells, tissues, and bodily fluids, amino acids are well known to one of ordinary skill in the art. such as: blood; derivatives and fractions of blood, such as The following six groups are examples of amino acids that are serum; extracted galls; biopsied or Surgically removed tissue, considered to be conservative substitutions for one another: including tissues that are, for example, unfixed, frozen, fixed 0112 1) Alanine (A), Serine (S). Threonine (T): in formalin and/or embedded in paraffin, tears; milk; skin 0113. 2) Aspartic acid (D), Glutamic acid (E): scrapes; Surface washings; urine; sputum; cerebrospinal 0114 3) Asparagine (N), Glutamine (Q); fluid; prostate fluid; pus; or bone marrow aspirates. In par 0115 4) Arginine (R), Lysine (K); ticular embodiments, the biological sample is obtained from US 2016/0033516 A1 Feb. 4, 2016
a Subject, such as in the form of a blood sample, Such as serum achieve a particular level of Stringency will vary, depending sample. In one example, the sample is a tumor sample. on the nature of the nucleic acids being hybridized. For 0.124 Sequence identity: The similarity between amino example, the length, degree of complementarity, nucleotide acid sequences is expressed interms of the similarity between sequence composition (for example, GC v. AT content), and the sequences, otherwise referred to as sequence identity. nucleic acid type (for example, RNA versus DNA) of the Sequence identity is frequently measured interms of percent hybridizing regions of the nucleic acids can be considered in age identity (or similarity or homology); the higher the per selecting hybridization conditions. An additional consider centage, the more similar the two sequences are. Homologs or ation is whether one of the nucleic acids is immobilized, for variants of a polypeptide will possess a relatively high degree example, on a filter. of sequence identity when aligned using standard methods. I0128. A specific example of progressively higher strin 0.125 Methods of alignment of polypeptide sequences for gency conditions is as follows: 2xSSC/0.1% SDS at about comparison are well known in the art. Various programs and room temperature (hybridization conditions); 0.2xSSC/0.1% alignment algorithms are described in: Smith and Waterman, SDS at about room temperature (low stringency conditions); Adv. Appl. Math. 2:482, 1981; Needleman and Wunsch, J. 0.2xSSC/0.1% SDS at about 42° C. (moderate stringency Mol. Biol. 48:443, 1970: Pearson and Lipman, Proc. Natl. conditions); and 0.1xSSC at about 68° C. (high stringency Acad. Sci. U.S.A. 85:2444, 1988: Higgins and Sharp, Gene conditions). One of skill in the art can readily determine 73:237, 1988; Higgins and Sharp, CABIOS 5:151, 1989: variations on these conditions (e.g., Molecular Cloning. A Corpet et al., Nucleic Acids Research 16:10881, 1988; and Laboratory Manual, 2nd ed., vol. 1-3, ed. Sambrook et al., Pearson and Lipman, Proc. Natl. Acad. Sci. U.S.A. 85:2444, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, 1988. Altschul et al., Nature Genet. 6: 119, 1994, presents a N.Y., 1989). Washing can be carried out using only one of detailed consideration of sequence alignment methods and these conditions, e.g., high Stringency conditions, or each of homology calculations. The NCBI Basic Local Alignment the conditions can be used, e.g., for 10-15 minutes each, in the Search Tool (BLAST) (Altschulet al., J. Mol. Biol. 215:403, order listed above, repeating any or all of the steps listed. 1990) is available from several sources, including the However, as mentioned above, optimal conditions will vary, National Center for Biotechnology Information (NCBI, depending on the particular hybridization reaction involved, Bethesda, Md.) and on the internet (along with a description and can be determined empirically. of how to determine sequence identity using this program). I0129. Signal Transduction: A process that occurs when an 0126 Homologs and variants of a V, or a V of an anti extracellular molecule activates a receptor on the Surface of a body that specifically binds a polypeptide are typically char cell. The receptor triggers biochemical events inside the cells, acterized by possession of at least about 75%, for example at leading to a biological response. These biological responses least about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, can be, for example, changes in cell metabolism, phenotype, 96%, 97%, 98% or 99% sequence identity counted over the differentiation, proliferation, and/or gene expression. Signal full length alignment with the amino acid sequence of inter transduction can involve phosphorylation of the receptor or est. Proteins with even greater similarity to the reference polypeptides within the cell. sequences will show increasing percentage identities when 0.130 Specifically bind: When referring to an antibody, assessed by this method, such as at least 80%, at least 85%, at refers to a binding reaction which determines the presence of least 90%, at least 95%, at least 98%, or at least 99% sequence a target protein, peptide, orphosphorylated polypeptide in the identity. When less than the entire sequence is being com presence of a heterogeneous population of proteins and other pared for sequence identity, homologs and variants will typi biologics. Thus, under designated conditions, an antibody cally possess at least 80% sequence identity over short win binds preferentially to a particular target protein or peptide dows of 10-20 amino acids, and may possess sequence (such as phosphorylated form of the polypeptide, Such as a identities of at least 85% or at least 90% or 95% depending on polypeptide that includes a phosphorylated histidine, for their similarity to the reference sequence. One of skill in the example a histidine phosphorylated at N3) and do not bind in art will appreciate that these sequence identity ranges are a significant amount to (1) other polypeptides or proteins provided for guidance only; it is entirely possible that present in the sample, does not bind the unphosphorylated strongly significant homologs could be obtained that fall out form of the polypeptide, and/or does not bind the polypeptide side of the ranges provided. Thus, in Some examples a heavy including a different phosphorylated amino acid and/or does chain of an antibody or antigen binding fragment thereof has not bind the polypeptide include a histidine phosphorylated at at least 90%, at least 91%, at least 92%, at least 93%, at least N1/N2. In a specific example, an antibody that specifically 94%, at least 95%, at least 96%, at least 97%, at least 98% or binds a polypeptide comprising a histidine phosphorylated at at least 99% sequence identity to any of SEQID NOS: 1,2,3, N3 does not bind in a significant amount to other polypeptides or 4, wherein the variant specifically binds a polypeptide or proteins present in the sample, the unphosphorylated form phosphorylated at a histidine, specifically a histidine phos of the polypeptide, the polypeptide including a different phorylated at N3. In some examples a light chain of an anti phosphorylated amino acid and the polypeptide include a body or antigen binding fragment thereof has at least 90%, at histidine phosphorylated at N1/N2. Specific binding can be least 91%, at least 92%, at least 93%, at least 94%, at least determined by methods known in the art. With reference to an 95%, at least 96%, at least 97%, at least 98% or at least 99% antibody antigen complex, specific binding of the antigen and sequence identity to any of SEQID NOS: 5, 6, 7 or 8, wherein antibody has a K of less than about 107 Molar, such as less the variant specifically binds a polypeptide phosphorylated at than about 107 Molar, 10 Molar, 10 Molar, or even less a histidine, specifically a histidine phosphorylated at N3. than about 10' Molar. 0127. Nucleic acids that “selectively hybridize” or “selec 0131 Standard: A substance or solution of a substance of tively bind do so under moderately or highly stringent con known amount, purity or concentration that is useful as a ditions that excludes non-related nucleotide sequences. In control. A standard can also be a known value or concentra nucleic acid hybridization reactions, the conditions used to tion of a particular substance. A standard can be compared US 2016/0033516 A1 Feb. 4, 2016
(such as by spectrometric, chromatographic, spectrophoto CNS tumors (such as a glioma, astrocytoma, medulloblas metric, or statistical analysis) to an unknown sample (of the toma, craniopharyrgioma, ependymoma, pinealoma, heman same or similar substance) to determine the presence of the gioblastoma, acoustic neuroma, oligodendroglioma, menin Substance in the sample and/or determine the amount, purity gioma, melanoma, neuroblastoma and retinoblastoma). In or concentration of the unknown sample. In one embodiment, several examples, a tumor is melanoma, lung cancer, lym a standard can be the amount of a polypeptide including a phoma breast cancer or colon cancer. histidine phosphorylated at N3 in a sample from a subject that 0.137 Under conditions sufficient for: A phrase that is used does not have a particular condition, Such as a tumor. to describe any environment that permits the desired activity. 0132) Therapeutic agent: Used in a generic sense, it 0.138 Vector: A nucleic acid molecule as introduced into a includes treating agents, prophylactic agents, and replace host cell, thereby producing a transformed host cell. A vector ment agents. may include nucleic acid sequences that permit it to replicate 0133. Therapeutically effective amount or effective in a host cell. Such as an origin of replication. A vector may amount: A quantity of a specific Substance, such as an anti also include one or more selectable marker genes and other body, sufficient to achieve a desired effect in a subject being genetic elements known in the art. treated. In several embodiments, a therapeutically effective (0.139. The singular terms “a,” “an,” and “the include plu amount is the amount necessary to reduce a sign or symptom ral referents unless context clearly indicates otherwise. Simi of a disorder. When administered to a subject, a dosage will larly, the word 'or' is intended to include “and” unless the generally be used that will achieve target tissue concentra context clearly indicates otherwise. It is further to be under tions that has been shown to achieve a desired in vitro effect. stood that all base sizes oramino acid sizes, and all molecular 0134 Tumor: An abnormal growth of cells, which can be weight or molecular mass values, given for nucleic acids or benign or malignant. Cancer is a malignant tumor, which is polypeptides are approximate, and are provided for descrip characterized by abnormal or uncontrolled cell growth. Other tion. Although methods and materials similar or equivalent to features often associated with malignancy include metastasis, those described herein can be used in the practice or testing of interference with the normal functioning of neighboring this disclosure, suitable methods and materials are described cells, release of cytokines or other secretory products at below. The term “comprises' means “includes.” All publica abnormal levels and Suppression or aggravation of inflamma tions, patent applications, patents, and other references men tory or immunological response, invasion of Surrounding or tioned herein are incorporated by reference in their entirety. distant tissues or organs, such as lymph nodes, etc. "Meta In case of conflict, the present specification, including expla static disease' refers to cancer cells that have left the original nations of terms, will control. In addition, the materials, tumor site and migrate to other parts of the body for example methods, and examples are illustrative only and not intended via the bloodstream or lymph system. to be limiting. 0135 The amount of a tumor in an individual is the “tumor burden' which can be measured as the number, volume, or Monoclonal Antibodies that Specifically Bind weight of the tumor. A tumor that does not metastasize is Phosphorylated Histidine referred to as “benign.” A tumor that invades the surrounding 0140 Isolated monoclonal antibodies (mAb) and antigen tissue and/or can metastasize is referred to as “malignant.” binding fragments thereof are disclosed herein that specifi Examples of hematological tumors include leukemias, cally bind a polypeptide including a phosphorylated histi including acute leukemias (such as 11q23-positive acute leu dine, specifically a histidine phosphorylated at N3. In some kemia, acute lymphocytic leukemia, acute myelocytic leuke embodiments, the monoclonal antibodies specifically bind a mia, acute myelogenous leukemia and myeloblastic, promy polypeptide including a histidine phosphorylated at N3 elocytic, myelomonocytic, monocytic and erythroleukemia), (3-pHis) with an equilibrium constant (K) of 1 nM or less. In chronic leukemias (such as chronic myelocytic (granulo several embodiments, the monoclonal antibodies and antigen cytic) leukemia, chronic myelogenous leukemia, and chronic binding fragments bind a polypeptide including a histidine lymphocytic leukemia), polycythemia Vera, lymphoma, phosphorylated at N3, with a binding affinity of 1x10M, at Hodgkin’s disease, non-Hodgkin’s lymphoma (indolent and least about 1.5x10M, at least about 2x10M, at least about high grade forms), multiple myeloma, Waldenstrom's mac 3x10M, at least about 4x10M, at least about 5x10M, at roglobulinemia, heavy chain disease, myelodysplastic Syn least about 6x10M, at least about 7x10 M, at least about drome, hairy cell leukemia and myelodysplasia. 8x10M, at least about 9x10M, or at least about 1x10' 0.136 Examples of solid tumors, such as sarcomas and M carcinomas, include fibrosarcoma, myxosarcoma, liposar 0.141. The structure of histidine is shown below (arrow coma, chondrosarcoma, osteogenic sarcoma, and other sar shows the N3 position): comas, synovioma, mesothelioma, Ewings tumor, leiomyo sarcoma, rhabdomyosarcoma, colon carcinoma, lymphoid malignancy, pancreatic cancer, breast cancer (including basal O breast carcinoma, ductal carcinoma and lobular breast carci noma), lung cancers, ovarian cancer, prostate cancer, hepato N OH cellular carcinoma, squamous cell carcinoma, basal cell car cinoma, adenocarcinoma, Sweat gland carcinoma, medullary ( thyroid carcinoma, papillary thyroid carcinoma, pheochro {, , mocytomas Sebaceous gland carcinoma, papillary carcinoma, papillary adenocarcinomas, medullary carcinoma, bron The monoclonal antibodies disclosed herein bind to a chogenic carcinoma, renal cell carcinoma, hepatoma, bile polypeptide including a histidine phosphorylated at N3 duct carcinoma, choriocarcinoma, Wilms tumor, cervical regardless of the amino acid sequence of the polypeptide. In cancer, testicular tumor, Seminoma, bladder carcinoma, and Some embodiments, the monoclonal antibodies disclosed US 2016/0033516 A1 Feb. 4, 2016 herein can specifically bind to a polypeptide that includes a by disulfide bonds. Each heavy chain is comprised of a heavy histidine phosphorylated at N3 that is present in any amino chain variable region (VH) and aheavy chain constant region. acid sequence. Thus, any amino acid sequence can be specifi The heavy chain constant region is comprised of three cally bound by the mab, provided the amino acid sequence domains, CH1, CH2 and CH3. Each light chain is comprised includes histidine phosphorylated at N3. of a light chain variable region (VL) and a light chain constant 0142. In some embodiments, the monoclonal antibody can region. The light chain constant region is comprised of one bind more than one polypeptide including a histidine phos domain, CL. The VH and VL regions can be further subdi phorylated at N3, wherein the amino acid sequences of the vided into regions of hyperVariability, called complementar polypeptide differ. However, all the polypeptides must ity determining regions (CDR), interspersed with regions that include a histidine phosphorylated at N3. Thus, the antibody are more conserved, called framework regions (FWR). Each specifically binds to these polypeptides. In specific examples, VH and VL is composed of three CDRs and four FWRs, the amino acid sequence of the polypeptide is not critical for arranged from amino-terminus to carboxy-terminus in the binding of the monoclonal antibody. In additional embodi following order: FWR1, CDR1, FWR2, CDR2, FWR3, ments, the antibody specifically binds one or more polypep CDR3, FWR4. tides including a histidine phosphorylated at N3, but does not 0.147. In several embodiments, the monoclonal antibodies bind the polypeptides when histidine is not phosphorylated at include a heavy chain comprising a heavy chain complemen N3. tarity determining region (HCDR)1, a HCDR2 and an 0143. In further embodiments, the monoclonal antibody HCDR3, and a light chain comprising a light chain comple can bind a polypeptide with a specified amino acid sequence mentarity determining region (LCDR)1, LCDR2 and (“X”) including a histidine phosphorylated at N3, but does LCDR3. In some embodiments, the antibodies include a vari not bind the polypeptide with the specified amino acid able heavy (V) and a variable light (V) chain. In several sequence (“X”) when the polypeptide does not include a embodiments, the antibody or antigen binding fragment histidine phosphorylated at N3. The monoclonal antibody can thereof includes heavy and light chain variable regions also bind a polypeptide with a different amino acid sequence including the HCDR1, HCDR2, and HCDR3, and LCDR1, (“Y”) including a histidine phosphorylated at N3, but does LCDR2, and LCDR3, respectively, of one of the SC39-4, not bind the polypeptide with the specified amino acid SC44-8, SC56-2, or SC60-2 antibodies. sequence (“Y”) when the polypeptide does not include a 0.148. The discussion of monoclonal antibodies below histidine phosphorylated at N3. refers to isolated monoclonal antibodies that include heavy 0144. The monoclonal antibody can be of any isotype. The and light chain variable domains including at least one monoclonal antibody can be, for example, an IgM or an IgG complementarity determining region (CDR). Such as a antibody, such as IgG or an IgG. The class of an antibody CDR1, CDR2 and CDR3. The person of ordinary skill in the that specifically binds a polypeptide phosphorylated at a his art will understand that various CDR numbering schemes tidine, specifically a histidine phosphorylated at N1 or N3. (such as the Kabat, Chothia or IMGT numbering schemes) can be Switched with another. In one aspect, a nucleic acid can be used to determine CDR positions. The amino acid molecule encoding V, or V is isolated using methods well sequence and the CDR positions of the heavy and light chain known in the art, Such that it does not include any nucleic acid of the SC39-4, SC44-8, SC56-2 and SC60-2 monoclonal sequences encoding the constant region of the light or heavy antibodies according to the IMGT are provided herein. The chain, respectively. The nucleic acid molecule encoding V, or person of skill in the art will readily understand use of various V is then operatively linked to a nucleic acid sequence CDR numbering schemes when referencing particular amino encoding a C, or C from a different class of immunoglobulin acids of the antibodies disclosed herein. molecule. This can be achieved using a vector or nucleic acid 0149. In some embodiments, disclosed is an isolated molecule that comprises a C or C chain, as known in the art. monoclonal antibody or antigen binding fragment thereof, For example, an antibody that specifically binds a polypep including a heavy chain variable region and a light chain tide phosphorylated at a histidine, specifically a histidine variable region, wherein the heavy chain variable region phosphorylated at N1 or N3 that was originally IgM may be includes a H-CDR1, a H-CDR2, and a H-CDR3, wherein the class Switched to an IgG. Class Switching can be used to antibody or antigen binding fragment includes: a) the convert one IgG Subclass to another, Such as from IgG to H-CDR1, the H-CDR2, and the H-CDR3 of the heavy chain IgG2. variable domain of the amino acid sequence set forth as SEQ 0145 The monoclonal antibodies disclosed herein can be ID NO: 1; b) the H-CDR1, the H-CDR2, and the H-CDR3 of rabbit antibodies and can include a rabbit framework region. the heavy chain variable domain of the amino acid sequence In some embodiments, the monoclonal antibodies are human set forth as SEQID NO: 2; c) the H-CDR1, the H-CDR2, and ized, and thus include one or more human framework regions. the H-CDR3 of the heavy chain variable domain of the amino Exemplary framework regions are disclosed, for example, in acid sequence set forth as SEQID NO:3: or d) the H-CDR1, PCT Publication No. WO 2011/038290 and U.S. Patent the H-CDR2, and the H-CDR3 of the heavy chain variable Application No. 2012/0244166A1, which are incorporated domain of the amino acid sequence set forth as SEQID NO: by reference herein. In some embodiments, the monoclonal 4, wherein the monoclonal antibody specifically binds a antibodies disclosed herein are chimericantibodies. In some polypeptide including a histidine phosphorylated at N3 embodiments, the monoclonal antibodies include rabbit and (3-pHis). In additional embodiments, disclosed is an isolated human regions. monoclonal antibody or antigenbinding fragment of claim 1, 0146 In some embodiments, the monoclonal antibody wherein the light chain variable domain includes a L-CDR1. includes both a heavy chain variable domain and a light chain a L-CDR2, and a L-CDR3, wherein the antibody or antigen variable domain. Naturally-occurring antibodies are immu binding fragment includes: a) the L-CDR1, the L-CDR2, and noglobulin molecules comprised of four polypeptide chains, the L-CDR3 of the light chain variable domain of the amino two heavy (H) chains and two light (L) chains inter-connected acid sequence set forth as SEQID NO: 5; b) the L-CDR1, the US 2016/0033516 A1 Feb. 4, 2016
L-CDR2, and the L-CDR3 of the light chain variable domain including a LCDR1, LCDR2, and LCDR3 including amino of the amino acid sequence set forth as SEQID NO: 6; c) the acids 27-34, 52-54,91-103 of SEQID NO: 6, respectively. In L-CDR1, the L-CDR2, and the L-CDR3 of the light chain further embodiments, the antibody includes a heavy chain variable domain of the amino acid sequence set forth as SEQ variable region including a HCDR1, HCDR2, and HCDR3 ID NO: 7; or d) the L-CDR1, the L-CDR2, and the L-CDR3 including amino 24-31, 49-55, 94-104 of SEQ ID NO: 3, of the light chain variable domain of the amino acid sequence respectively, and a light chain variable region including a set forth as SEQID NO: 8. In additional embodiments, dis LCDR1, LCDR2, and LCDR3 including amino acids 27-34, closed is an isolated monoclonal antibody or antigen binding 52-54, and 91-109 of SEQ ID NO: 7, respectively. In more fragment, including a) the H-CDR1, H-CDR2, and H-CDR3 embodiments, the antibody includes a heavy chain variable of the amino acid sequence set forth as SEQID NO: 1, and the region including a HCDR1, HCDR2, and HCDR3 including L-CDR1, L-CDR2, and L-CDR3 of the amino acid sequence amino acids 24-31, 49-55,94-104 of SEQID NO: 4, respec set forth as SEQ ID NO: 5; b) the H-CDR1, H-CDR2, and tively, and a light chain variable region including a LCDR1. H-CDR3 of the amino acid sequence set forth as SEQID NO: LCDR2, and LCDR3 including amino acids 27-33, 51-53 and 2 and the L-CDR1, L-CDR2, and L-CDR3 of the amino acid 90-102 of SEQID NO: 8, respectively. sequence set forth as SEQ ID NO: 6; c) the H-CDR1, 0153. In further embodiments, the antibody includes a H-CDR2, and H-CDR3 of the amino acid sequence set forth heavy chain variable region including an amino acid as SEQID NO:3 and the L-CDR1, L-CDR2, and L-CDR2 of sequence at least 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence set forth as SEQID NO: 7; or d) the the amino acid sequence set forth as one of a) amino acids H-CDR1, H-CDR2, and H-CDR3 of the amino acid sequence 1-108 of SEQID NO:1; b) amino acids 1-112 of SEQID NO: set forth as SEQID NO: 4, and the L-CDR1, L-CDR2, and 2; c) amino acid 1-115 of SEQID NO:3, or d) amino acids L-CDR3 of the amino acid sequence set forth as SEQID NO: 1-115 of SEQID NO: 4. In more embodiments, the antibody 8 includes a light chain variable region including an amino acid 0150. For example, in some embodiments, the antibody sequence at least 95%, 96%, 97%, 98%, or 99% identical to includes a heavy chain variable region including a HCDR1, the amino acid sequence set forth as one of a) amino acids HCDR2, and/or HCDR3 including amino acids 21-28, 45-52, 1-113 of SEQID NO:5; b) amino acids 1-114 of SEQID NO: and 88-97 of SEQID NO: 1, respectively. In further embodi 6; c) amino acid 1-120 of SEQID NO: 7; or d) amino acids ments, the antibody includes a heavy chain variable region 1-113 of SEQID NO: 8. Thus, the antibody can includea) a including a HCDR1, HCDR2, and/or HCDR3 including heavy chain variable region including an amino acid amino acids 21-28, 46-52, and 91-101 of SEQ ID NO: 2, sequence at least 95%, 96%, 97%, 98%, or 99% identical to respectively. In additional embodiments, the antibody the amino acid sequence set forth as amino acids 1-108 of includes a heavy chain variable region including a HCDR1, SEQID NO: 1 and a light chain variable region including an HCDR2, and/or HCDR3 including amino acids 24-31, 49-55, amino acid sequence at least 95%, 96%, 97%, 98%, or 99% 94-104 of SEQID NO:3, respectively. In more embodiments, identical to the amino acid sequence set forth as amino acids the antibody includes aheavy chain variable region including amino acids 1-113 of SEQIDNO: 5; b) aheavy chain variable a HCDR1, HCDR2, and/or HCDR3 including amino acids region including an amino acid sequence at least 95%, 96%, 24-31, 49-55, 94-104 of SEQ ID NO: 4, respectively. The 97%, 98%, or 99% identical to the amino acid sequence set antibody specifically binds a polypeptide comprising a histi forth as amino acids 1-112 of SEQID NO: 2 and a light chain dine phosphorylated at N3. variable region including an amino acid sequence at least 0151. In some embodiments, the antibody includes a light 95%, 96%, 97%, 98%, or 99% identical to the amino acid chain variable region including a LCDR1, LCDR2, and/or sequence set forth as amino acids amino acids 1-114 of SEQ LCDR3 including amino acids 28-22, 51-53, and 90-102 of ID NO: 6; c) aheavy chain variable region including an amino SEQ ID NO: 5, respectively. In further embodiments, the acid sequence at least 95%,96%.97%.98%, or 99% identical antibody includes a light chain variable region including a to the amino acid sequence set forth as amino acids 1-115 of LCDR1, LCDR2, and/or LCDR3 including amino acids SEQID NO:3 and a light chain variable region including an 27–34, 52-54, 91-103 of SEQ ID NO: 6, respectively. In amino acid sequence at least 95%, 96%, 97%, 98%, or 99% additional embodiments, the antibody includes a light chain identical to the amino acid sequence set forth as amino acids variable region including a LCDR1, LCDR2, and/or LCDR3 amino acids 1-120 of SEQ ID NO: 7; or d) a heavy chain including amino acids 27-34, 52-54, and 91-109 of SEQID variable region including an amino acid sequence at least NO: 7, respectively. In more embodiments, the antibody 95%, 96%, 97%, 98%, or 99% identical to the amino acid includes a light chain variable region including a LCDR1. sequence set forth as amino acids 1-115 of SEQID NO. 4 and LCDR2, and/or LCDR3 including amino acids 27-33, 51-53 a light chain variable region including an amino acid and 90-102 of SEQ ID NO: 8, respectively. The antibody sequence at least 95%, 96%, 97%, 98%, or 99% identical to specifically binds a polypeptide including a histidine phos the amino acid sequence set forth as amino acids amino acids phorylated at N3. 1-113 of SEQ ID NO: 8. The antibody specifically binds a 0152. In some embodiments, the antibody includes a polypeptide including a histidine phosphorylated at N3. heavy chain variable region including a HCDR1, HCDR2, 0154) In additional embodiments, the antibody includes a and HCDR3 including amino acids 28, 45-52, and 88-97 of heavy chain variable region that includes a) amino acids SEQID NO: 1, respectively, and a light chain variable region 1-108 of SEQID NO:1; b) amino acids 1-112 of SEQID NO: including a LCDR1, LCDR2, and LCDR3 including amino 2; c) amino acid 1-115 of SEQID NO:3, or d) amino acids acids 28-22,51-53, and 90-102 of SEQIDNO:5 respectively. 1-115 of SEQID NO: 4. In specific non-limiting examples, In additional embodiments, the antibody includes a heavy the heavy chain variable region can be paired with any light chain variable region including a HCDR1, HCDR2, and chain variable region, provided the antibody specifically HCDR3 including amino acids 21-28, 46-52, and 91-101 of binds a polypeptide including a histidine phosphorylated at SEQID NO: 2, respectively, and a light chain variable region N3. US 2016/0033516 A1 Feb. 4, 2016
0155. In some embodiments, the antibody includes a light 0.163 4) Arginine (R), Lysine (K); chain variable region that includes a) amino acids 1-113 of 0.164 5) Isoleucine (I), Leucine (L), Methionine (M), SEQ ID NO: 5; b) amino acids 1-114 of SEQID NO: 6; c) Valine (V); and amino acid 1-120 of SEQID NO: 7; ord) amino acids 1-113 0.165 6) Phenylalanine (F), Tyrosine (Y), Tryptophan of SEQID NO:8. In specific non-limiting examples, the light (W). chain variable region can be paired with any heavy chain 0166 Additionally, to increase binding affinity of the anti variable region, provided the antibody specifically binds a body, the V, and V segments can be randomly mutated, such polypeptide including a histidine phosphorylated at N3. as within H-CDR3 region or the L-CDR3 region, in a process 0156 Thus, in specific non-limiting examples, the mono analogous to the in vivo Somatic mutation process responsible clonal antibody includes a) a heavy chain variable region for affinity maturation of antibodies during a natural immune including amino acids 1-108 of SEQ ID NO: 1 and a light response. Thus, in vitro affinity maturation can be accom chain variable region including amino acids 1-113 of SEQID plished by amplifying V and V, regions using PCR primers NO: 5; b) a heavy chain variable region including amino acids complementary to the H-CDR3 or L-CDR3, respectively. In 1-112 of SEQ ID NO: 2 and a light chain variable region this process, the primers have been “spiked with a random including amino acids 1-114 of SEQ ID NO: 6; c) a heavy mixture of the four nucleotide bases at certain positions such chain variable region including amino acids 1-115 of SEQID that the resultant PCR products encode V and V segments NO:3 and a light chain variable region including amino acids into which random mutations have been introduced into the 1-120 of SEQID NO: 7, or d) a heavy chain variable region V and/or V, CDR3 regions. These randomly mutated V. including amino acids 1-115 of SEQID NO. 4 and light chain and V, segments can be tested to determine the binding affin variable region including amino acids 1-113 of SEQID NO: ity. 8. 0.167 Random mutagenesis of the V, and V segments 0157. In some embodiments, an antibody that specifically could also be used to alteran undesired sequence dependence binds a polypeptide including a histidine phosphorylated at for any particular mAb for binding to phosphohistidine. N3, as disclosed herein, includes up to 10 amino acid substi H-CDR2 has been shown to be particularly important for tutions (such as up to 1,2,3,4, 5, 6, 7, 8, or up to 9 amino acid recognition of phosphoamino acids by pSer, pThr and pTyr substitutions) in the framework regions of the heavy chain of specific mAbs (Koerber et al., Nature Biotechnology 31:10 the antibody, or the light chain of the antibody, or the heavy 2013). Mutagenesis of H-CDR2 can be used to engineer and light chains of the antibody. The antibody specifically antibodies with improved sequence-independence and affin binds a polypeptide including a histidine phosphorylated at ity for histidine phosphorylated at N3. N3 0168 Chimericantibodies are also provided. The antibod 0158. In several embodiments, the constant region of the ies can include any Suitable framework region, such as (but antibody includes one or more amino acid Substitutions to not limited to) a human framework region. Human frame optimize half-life of the antibody. The half-life of IgG Abs is work regions, and mutations that can be made in a human in serum regulated by the neonatal Fc receptor (FcRn). Thus, antibody framework regions, are known in the art (see, for in several embodiments, the antibody includes an amino acid example, in U.S. Pat. No. 5,585,089, which is incorporated substitution that increases binding to the FcRn. Several such herein by reference). Alternatively, a heterologous frame substitutions are known to the person of ordinary skill in the work region, such as, but not limited to a mouse framework art, such as Substitutions at IgG constant regions T250O and region, can be included in the heavy or light chain of the M428L (see, e.g., Hinton et al., J Immunol., 176:346-356, antibodies. (See, for example, Jones et al., Nature 321:522, 2006); M428L and N434S (see, e.g., Zalevsky, et al., Nature 1986; Riechmann et al., Nature 332:323, 1988; Verhoeyenet Biotechnology, 28:157-159, 2010): N434A (see, e.g., Petkova al., Science 239:1534, 1988; Carter et al., Proc. Natl. Acad. et al., Int. Immunol., 18:1759-1769, 2006): T307A, E380A, Sci. U.S.A. 89:4285, 1992; Sandhu, Crit. Rev. Biotech. and N434A (see, e.g., Petkova et al., Int. Immunol., 18:1759 12:437, 1992; and Singeret al., J. Immunol. 150:2844, 1993.) 1769, 2006); and M252Y, S254T, and T256E (see, e.g., 0169. The antibodies or antigen binding fragments dis Dall’Acqua et al., J. Biol. Chem., 281:23514-23524, 2006). closed hereincan be derivatized or linked to another molecule The antibody can also be an immunoadhesin. (such as another peptide or protein). In general, the antibodies 0159. One of skill will realize that conservative variants of or portion thereof is derivatized such that the binding to the antibodies can be produced. Such conservative variants polypeptides including a histidine phosphorylated at N3 is employed in antigen binding fragments, such as dsFV frag not affected adversely by the derivatization or labeling. For ments or in ScFv fragments, will retain critical amino acid example, the antibody can be functionally linked (by chemi residues necessary for correct folding and stabilizing between cal coupling, genetic fusion, noncovalent association or oth the V and the V, regions, and will retain the charge charac erwise) to one or more other molecular entities, such as teristics of the residues in order to preserve the low pland low another antibody (for example, a bi-specific antibody or a toxicity of the molecules. Amino acid substitutions (such as at diabody), a detectable marker, an effector molecule, or a most one, at most two, at most three, at most four, or at most protein or peptide that can mediate association of the anti five amino acid Substitutions) can be made in the V or the V, body or antibody portion with another molecule (such as a regions to increase yield. Conservative amino acid substitu streptavidin core region or a polyhistidine tag). tion tables providing functionally similar amino acids are 0170. One type of derivatized antibody is produced by well known to one of ordinary skill in the art. The following crosslinking two or more antibodies (of the same type or of six groups are examples of amino acids that are considered to different types, such as to create bispecific or multispecific be conservative substitutions for one another: antibodies). Suitable crosslinkers include those that are het 0160 1) Alanine (A), Serine (S). Threonine (T): erobifunctional, having two distinctly reactive groups sepa 0161) 2) Aspartic acid (D), Glutamic acid (E): rated by an appropriate spacer (Such as m-maleimidobenzoyl 0162 3) Asparagine (N), Glutamine (Q); N-hydroxysuccinimide ester) or homobifunctional (such as US 2016/0033516 A1 Feb. 4, 2016 20 disuccinimidyl suberate). Such linkers are available from 0180 Methods of making these fragments are known in Pierce Chemical Company, Rockford, Ill. Thus, bivalent and the art (see for example, Harlow and Lane, Antibodies. A multivalent antibodies can be produced, such as including Laboratory Manual, Cold Spring Harbor Laboratory, New more than one monoclonal antibody or antigen binding from York, 1988). of antibody that specifically bind polypeptides including a 0181. In some embodiments, the antigen binding frag histidine phosphorylated at N3. In some examples, the dis ments are FV antibodies, which are typically about 25 kDa closed antibodies are oligomers of antibodies, such as dimers, and contain a complete antigen-binding site with three CDRS trimers, tetramers, pentamers, hexamers, septamers, octo per each heavy chain and each light chain. To produce these mers and so on. In some examples, the antibodies are dimers antibodies, the V and the V, can be expressed from two or pentamers. individual nucleic acid constructs in a host cell. If the V and 0171 The monoclonal antibodies disclosed herein can be the V, are expressed non-contiguously, the chains of the FV of any isotype. The monoclonal antibody can be, for example, antibody are typically held together by noncovalent interac an IgM or an IgG antibody. Such as IgG, IgG, IgG or an tions. However, these chains tend to dissociate upon dilution, IgG. The class of an antibody that specifically binds a so methods have been developed to crosslink the chains polypeptide including a histidine phosphorylated at N3 can through glutaraldehyde, intermolecular disulfides, or a pep be switched with another (for example, IgG can be switched tide linker. Thus, in one example, the Fv can be a disulfide to IgM), according to well-known procedures. For example, a stabilized Fv (dsEv), wherein the heavy chain variable region nucleic acid molecule encoding the V, or V of a disclosed and the light chain variable region are chemically linked by antibody can be operatively linked to a nucleic acid sequence disulfide bonds. encoding a C or C from a different class of immunoglobulin 0182. In an additional examples, the Fv fragments include molecule. This can be achieved using a vector or nucleic acid V, and V, chains connected by a peptide linker. These single molecule that comprises a C or C chain, as known in the art. chain antigen binding proteins (ScFV) are prepared by con For example, an antibody that specifically binds a polypep structing a structural gene including DNA sequences encod tide including a histidine phosphorylated at N3 that was origi ing the V and V, domains connected by an oligonucleotide. nally IgG, may be class Switched to an IgM. Class Switching The structural gene is inserted into an expression vector, can be used to convert one IgG subclass to another, Such as which is Subsequently introduced into a host cell Such as E. from IgG to IgG, IgGs, or IgG4. coli. The recombinant host cells synthesize a single polypep 0172 Antigen binding fragments of the antibodies that tide chain with a linker peptide bridging the two V domains. specifically bind to polypeptides including a histidine phos Methods for producing sclvs are known in the art (see Whit phorylated at N3 are also encompassed by the present disclo low et al., Methods: a Companion to Methods in Enzymology, Sure, such as single-domain antibodies (for example, VH Vol.2, page97, 1991; Bird et al., Science 242:423, 1988: U.S. domain antibodies), Fab, F(ab'), and Fv. These antigenbind Pat. No. 4,946,778; Pack et al., Bio/Technology 11:1271, ing fragments retain the ability to specifically bind polypep 1993; and Sandhu, Supra). Dimers of a single chain antibody tides including a histidine phosphorylated at N3. These frag (ScPV), are also contemplated. ments include: 0183 Antigen binding fragments can be prepared by pro teolytic hydrolysis of the antibody or by expression in E. coli 0173 (1) Fab, the fragment which contains a monovalent of DNA encoding the fragment. Antigen binding fragments antigen-binding fragment of an antibody molecule, can be can be obtained by pepsin or papain digestion of whole anti produced by digestion of whole antibody with the enzyme bodies by conventional methods. For example, antigen bind papainto yield an intact light chain and a portion of one heavy ing fragments can be produced by enzymatic cleavage of chain; antibodies with pepsin to provide a 5S fragment denoted 0.174 (2) Fab', the fragment of an antibody molecule can F(ab'). This fragment can be further cleaved using a thiol be obtained by treating whole antibody with pepsin, followed reducing agent, and optionally a blocking group for the Sulf by reduction, to yield an intact light chain and a portion of the hydryl groups resulting from cleavage of disulfide linkages, heavy chain; two Fab' fragments are obtained per antibody to produce 3.5S Fab' monovalent fragments. Alternatively, an molecule; enzymatic cleavage using pepsin produces two monovalent 0175 (3) (Fab'), the fragment of the antibody that can be Fab' fragments and an Fc fragment directly (see U.S. Pat. No. obtained by treating whole antibody with the enzyme pepsin 4,036,945 and U.S. Pat. No. 4,331,647, and references con without subsequent reduction; F(ab') is a dimer of two Fab' tained therein; Nisonhoff et al., Arch. Biochem. Biophys. fragments held together by two disulfide bonds; 89:230, 1960; Porter, Biochem. J. 73:119, 1959; Edelman et 0176 (4) Fv, a genetically engineered fragment contain al., Methods in Enzymology, Vol. 1, page 422, Academic ing the variable region of the light chain and the variable Press, 1967; and Coligan et al. at sections 2.8.1-2.8.10 and region of the heavy chain expressed as two chains; 2.10.1-2.10.4). 0.184 Other methods of cleaving antibodies, such as sepa 0177 (5) Single chain antibody (such as scFv), a geneti ration of heavy chains to form monovalent light-heavy chain cally engineered molecule containing the variable region of fragments, further cleavage of fragments, or other enzymatic, the light chain, the variable region of the heavy chain, linked chemical, or genetic techniques may also be used, so long as by a suitable polypeptide linker as a genetically fused single the fragments bind to the antigen that is recognized by the chain molecule: intact antibody. Fab' fragments can also be generated by clon 0.178 (6) A dimer of a single chain antibody (sclfV), ing the two chains into expression vectors with an IgK secre defined as a dimer of a scFV (also known as a “mini-anti tion signal and co-expressing them in 293F cells. body'); and 0185. In some cases, antigen binding fragments can be 0179 (7) VHsingle-domain antibody, an antigen binding prepared by proteolytic hydrolysis of the antibody or by fragment consisting of the heavy chain variable domain. expressionina host cell (such as E. coli) of DNA encoding the US 2016/0033516 A1 Feb. 4, 2016 fragment. Antigen binding fragments can be obtained by disulfide linkage to cysteine) or to the alpha carbonamino and pepsin or papain digestion of whole antibodies by conven carboxyl groups of the terminal amino acids. tional methods. For example, antigen binding fragments can 0189 Additionally, in several embodiments, the linker can be produced by enzymatic cleavage of antibodies with pepsin include a spacer element, which, when present, increases the to provide a 5S fragment denoted F(ab'). This fragment can size of the linker such that the distance between the effector be further cleaved using a thiol reducing agent, and optionally molecule or the detectable marker and the antibody or antigen a blocking group for the Sulfhydryl groups resulting from binding fragment is increased. Exemplary spacers are known cleavage of disulfide linkages, to produce 3.5S Fab' monova to the person of ordinary skill, and include those listed in U.S. lent fragments. Alternatively, an enzymatic cleavage using Pat. Nos. 7,964,5667,498,298, 6,884,869, 6,323,315, 6,239, pepsin produces two monovalent Fab' fragments and an Fc 104, 6,034,065, 5,780,588, 5,665,860, 5,663,149, 5,635,483, fragment directly (see U.S. Pat. No. 4,036,945 and U.S. Pat. 5,599,902, 5,554,725, 5,530,097, 5,521,284, 5,504,191, No. 4,331,647). 5,410,024, 5,138,036, 5,076,973, 4,986,988, 4,978,744, 0186. Other methods of cleaving antibodies, such as sepa 4,879,278, 4,816,444, and 4,486,414, as well as U.S. Pat. Pub. ration of heavy chains to form monovalent light-heavy chain Nos. 20110212088 and 20110070248, each of which is incor fragments, further cleavage of fragments, or other enzymatic, porated by reference in its entirety. chemical, or genetic techniques may also be used, so long as 0190. A monoclonal antibody that specifically binds a the fragments bind to the antigen that is recognized by the polypeptide including a histidine phosphorylated at N3 (or intact antibody. antigen binding fragment thereof) can be conjugated with a detectable marker; for example, a detectable marker capable Conjugates of detection by ELISA, spectrophotometry, flow cytometry, microscopy or diagnostic imaging techniques (such as com 0187 Monoclonal antibodies, or antigen binding frag puted tomography (CT), computed axial tomography (CAT) ments thereof, that specifically bind polypeptides including a scans, magnetic resonance imaging (MRI), nuclear magnetic histidine phosphorylated at N3, can be conjugated to an resonance imaging NMRI), magnetic resonance tomography agent, such as an effector molecule or detectable marker, (MTR), ultrasound, fiberoptic examination, and laparoscopic using any number of means known to those of skill in the art. examination). Specific, non-limiting examples of detectable Both covalent and noncovalent attachment means may be markers include fluorophores, chemiluminescent agents, used. Conjugates include, but are not limited to, molecules in enzymatic linkages, radioactive isotopes and heavy metals or which there is a covalent linkage of an effector molecule or a compounds (for example Super paramagnetic iron oxide detectable marker to an antibody orantigenbinding fragment nanocrystals for detection by MRI). For example, useful that specifically binds a polypeptide including a histidine detectable markers include fluorescent compounds, including phosphorylated at N3. One of skill in the art will appreciate fluorescein, fluorescein isothiocyanate, rhodamine, 5-dim that various effector molecules and detectable markers can be ethylamine-1-napthalenesulfonyl chloride, phycoerythrin, used, including (but not limited to) radioactive agents such as lanthanide phosphors and the like. Bioluminescent markers 'I, ‘P, H and S and other detectable labels, enzymes, are also of use. Such as luciferase, Green fluorescent protein target moieties, drugs and ligands, etc. (GFP), Yellow fluorescent protein (YFP). 0188 Effector molecules and detectable markers can be 0191 Anantibody orantigenbinding fragment can also be linked to an antibody or antigen binding fragment of interest conjugated with enzymes that are useful for detection, such as using any number of means known to those of skill in the art. horseradish peroxidase, B-galactosidase, luciferase, alkaline Both covalent and noncovalent attachment means may be phosphatase, glucose oxidase and the like. When an antibody used. The procedure for attaching an effector molecule or or antigen binding fragment is conjugated with a detectable detectable marker to an antibody orantigenbinding fragment enzyme, it can be detected by adding additional reagents that varies according to the chemical structure of the effector. the enzyme uses to produce a reaction product that can be Polypeptides typically contain a variety of functional groups; discerned. For example, when the agent horseradish peroxi such as carboxylic acid (COOH), free amine ( NH) or dase is present the addition of hydrogen peroxide and diami sulfhydryl ( SH) groups, which are available for reaction nobenzidine leads to a colored reaction product, which is with a Suitable functional group on an antibody to result in the visually detectable. An antibody or antigenbinding fragment binding of the effector molecule or detectable marker. Alter may also be conjugated with biotin, and detected through natively, the antibody or antigen binding fragment is deriva indirect measurement of avidin or streptavidin binding. It tized to expose or attach additional reactive functional should be noted that the avidin itself can be conjugated with groups. The derivatization may involve attachment of any of an enzyme or a fluorescent label. a number of known linker molecules such as those available 0.192 An antibody or antigen binding fragment may be from Pierce Chemical Company, Rockford, Ill. The linker can conjugated with a paramagnetic agent, such as gadolinium. be any molecule used to join the antibody or antigen binding Paramagnetic agents such as Superparamagnetic iron oxide fragment to the effector molecule or detectable marker. The are also of use as labels. Antibodies can also be conjugated linker is capable of forming covalent bonds to the antibody (or with lanthanides (such as europium and dysprosium), and antigen binding fragment) and to the effector molecule or manganese. An antibody or antigen binding fragment may detectable marker. Suitable linkers are well knownto those of also be labeled with a predetermined polypeptide epitopes skill in the art and include, but are not limited to, straight or recognized by a secondary reporter (Such as leucine Zipper branched-chain carbon linkers, heterocyclic carbon linkers, pair sequences, binding sites for secondary antibodies, metal or peptide linkers. Where the antibody or antigen binding binding domains, epitope tags). fragment and the effector molecule or detectable marker are 0193 An antibody or antigen binding fragment can be polypeptides, the linkers may be joined to the constituent conjugated with a radiolabeled amino acid. Examples of amino acids through their side groups (such as through a labels for polypeptides include, but are not limited to, the US 2016/0033516 A1 Feb. 4, 2016 22 following radioisotopes or radionucleotides: H, C, N, ally equivalent nucleic acids, such as nucleic acids which 35S, 90Y. 99Tc, 11 In, 125I, 131I. differ in sequence but which encode the same effector mol 0194 Means of detecting such detectable markers are well ecule, detectable marker or antibody or antigenbinding frag known to those of skill in the art. Thus, for example, radiola ment Sequence. bels may be detected using photographic film or Scintillation 0199 Nucleic acid sequences encoding the antibodies that counters, fluorescent markers may be detected using a pho specifically bind polypeptides including a histidine phospho todetector to detect emitted illumination. Enzymatic labels rylated at N3 can be prepared by any suitable method includ are typically detected by providing the enzyme with a Sub ing, for example, cloning of appropriate sequences or by strate and detecting the reaction product produced by the direct chemical synthesis by methods such as the phosphot action of the enzyme on the substrate, and colorimetric labels riester method of Narang et al., Meth. Enzymol. 68:90-99, are detected by simply visualizing the colored label. 1979; the phosphodiester method of Brown et al., Meth. Enzy 0.195 An antibody orantigenbinding fragment can also be mol. 68:109-151, 1979; the diethylphosphoramidite method derivatized with a chemical group Such as polyethylene gly of Beaucage et al., Tetra. Lett. 22:1859-1862, 1981; the solid col (PEG), a methyl or ethyl group, or a carbohydrate group. phase phosphoramidite triester method described by Beau These groups may be useful to improve the biological char cage & Caruthers, Tetra. Letts. 22(20): 1859-1862, 1981, for acteristics of the antibody or antigen binding fragment, Such example, using an automated synthesizer as described in, for as to increase serum half-life or to increase tissue binding. example, Needham-VanDevanter et al., Nucl. Acids Res. 0196. The average number of effector molecule or detect 12:6159-6168, 1984; and, the solid support method of U.S. able marker moieties per antibody or antigen binding frag Pat. No. 4.458,066. Chemical synthesis produces a single ment in a conjugate can range, for example, from 1 to 20 stranded oligonucleotide. This can be converted into double moieties per antibody or antigen binding fragment. For some stranded DNA by hybridization with a complementary conjugates, the average number of effector molecule or sequence, or by polymerization with a DNA polymerase detectable marker moieties per antibody or antigen binding using the single strand as a template. fragment may be limited by the number of attachment sites on 0200 Exemplary nucleic acids including sequences the antibody or antigen binding fragment. For example, encoding an antibody that specifically binds a polypeptide where the attachment is a cysteine thiol, an antibody or anti including a histidine phosphorylated at N3 (or antigen bind gen binding fragment may have only one or several cysteine ing fragment thereof) can be prepared by cloning techniques. thiol groups, or may have only one or several Sufficiently Examples of appropriate cloning and sequencing techniques, reactive thiol groups through which a linker may be attached. and instructions sufficient to direct persons of skill through In certain embodiments, the average number of effector mol cloning are found in Sambrook et al., Supra, Berger and Kim ecule or detectable marker moieties per antibody or antigen mel (eds.), Supra, and Ausubel, Supra. Product information binding fragment in a conjugate range from 1 to about 8; from from manufacturers of biological reagents and experimental about 2 to about 6: from about 3 to about 5; from about 3 to equipment also provide useful information. Such manufac about 4; from about 3.1 to about 3.9; from about 3.2 to about turers include the SIGMA Chemical Company (Saint Louis, 3.8; from about 3.2 to about 3.7; from about 3.2 to about 3.6: Mo.), R&D Systems (Minneapolis, Minn.), Pharmacia Amer from about 3.3 to about 3.8; or from about 3.3 to about 3.7. sham (Piscataway, N.J.), CLONTECH Laboratories, Inc. See, for example, U.S. Pat. No. 7,498,298, incorporated by (Palo Alto, Calif.), Chem Genes Corp., Aldrich Chemical reference herein in its entirety. The average number of effec Company (Milwaukee, Wis.), Glen Research, Inc., GIBCO tor molecule or detectable marker moieties per antibody or BRL Life Technologies, Inc. (Gaithersburg, Md.), Fluka antigen binding fragment in preparations of conjugates may Chemica-Biochemika Analytika (Fluka Chemie AG, Buchs, be characterized by conventional means Such as mass spec Switzerland), Invitrogen (San Diego, Calif.), and Applied troscopy and, ELISA assay. Biosystems (Foster City, Calif.), as well as many other com 0197) The loading (for example, effector molecule/anti mercial Sources known to one of skill. body ratio) of an conjugate may be controlled in different 0201 Nucleic acids can also be prepared by amplification ways, for example, by: (i) limiting the molar excess of effec methods. Amplification methods include polymerase chain tor molecule-linker intermediate or linker reagent relative to reaction (PCR), the ligase chain reaction (LCR), the tran antibody, (ii) limiting the conjugation reaction time or tem Scription-based amplification system (TAS), the self-sus perature, (iii) partial or limiting reductive conditions for cys tained sequence replication system (3SR). A wide variety of teine thiol modification, (iv) engineering by recombinant cloning methods, host cells, and in vitro amplification meth techniques the amino acid sequence of the antibody Such that odologies are well known to persons of skill the number and position of cysteine residues is modified for 0202 In one example, an antibody orantigenbinding frag control of the number or position of linker-effector molecule attachments (such as thioMab or thioFab prepared as dis ment of use is prepared by inserting the cDNA which encodes closed in W02006/03448, incorporated by reference herein in a variable region from an antibody into a vector which its entirety. includes the cDNA encoding an effector molecule or detect able marker, such as an enzyme or label. The insertion is made so that the variable region and the effector molecule or detect Nucleotides, Expression Vectors and Host Cells able marker are read inframe so that one continuous polypep 0198 Nucleic acids encoding the amino acid sequences of tide is produced. Thus, the encoded polypeptide contains a antibodies that specifically bind polypeptides including a his functional Fv region and a functional effector molecule or tidine phosphorylated at N3 are provided. Nucleic acid mol detectable marker region. In one embodiment, cDNA encod ecules encoding these antibodies can readily be produced by ing an enzyme is ligated to a scv So that the enzyme is one of skill in the art, using the amino acid sequences pro located at the carboxyl terminus of the sclv. In several vided herein, and the genetic code. In addition, one of skill examples, cDNA encoding a horseradish peroxidase or alka can readily construct a variety of clones containing function line phosphatase, or a polypeptide marker of interest is ligated US 2016/0033516 A1 Feb. 4, 2016 to a scFv So that the enzyme (or polypeptide marker) is can also be performed after forming a protoplast of the host located at the amino terminus of the Sclv. In another example, cell if desired, or by electroporation. the label is located at the amino terminus of the scEv. In a 0207. When the host is a eukaryote, such methods of trans further example, cDNA encoding the protein or polypeptide fection of DNA as calcium phosphate coprecipitates, conven marker is ligated to a heavy chain variable region of an anti tional mechanical procedures such as microinjection, elec body or antigen binding fragment, so that the enzyme or troporation, insertion of a plasmid encased in liposomes, or polypeptide marker is located at the carboxyl terminus of the virus vectors may be used. Eukaryotic cells can also be heavy chain variable region. The heavy chain-variable region cotransformed with polynucleotide sequences encoding the can Subsequently be ligated to a light chain variable region of antibody, labeled antibody, or antigen binding fragment the antibody or antigen binding fragment using disulfide thereof, and a second foreign DNA molecule encoding a bonds. In a yet another example, cDNA encoding an enzyme selectable phenotype. Such as the herpes simplex thymidine or a polypeptide marker is ligated to a light chain variable kinase gene. Another method is to use a eukaryotic viral region of an antibody orantigenbinding fragment, so that the vector, such as simian virus 40 (SV40) or bovine papilloma enzyme or polypeptide marker is located at the carboxyl virus, to transiently infect or transform eukaryotic cells and terminus of the light chain variable region. The light chain express the protein (see for example, Eukaryotic Viral Vec variable region can Subsequently be ligated to a heavy chain tors, Cold Spring Harbor Laboratory, Gluzman ed., 1982). variable region of the antibody or antigen binding fragment One of skill in the art can readily use expression systems such using disulfide bonds. as plasmids and vectors of use in producing proteins in cells 0203 Once the nucleic acids encoding the conjugate, anti including higher eukaryotic cells such as the COS, CHO, body, or fragment thereof, are isolated and cloned, the protein HeLa, 293, and myeloma cell lines. can be expressed in a recombinantly engineered cell Such as 0208 Isolation and purification of recombinantly bacteria, plant, yeast, insect and mammalian cells using a expressed polypeptide can be carried out by conventional suitable expression vector. One or more DNA sequences means including preparative chromatography and immuno encoding the antibody or fragment thereof can be expressed logical separations. Once expressed, the conjugate, antibody, in vitro by DNA transfer into a suitable host cell. The cell may or antigen binding fragment thereof, can be purified accord be prokaryotic or eukaryotic. The term also includes any ing to standard procedures of the art, including ammonium progeny of the subject host cell. It is understood that all Sulfate precipitation, affinity columns, column chromatogra progeny may not be identical to the parental cell since there phy, and the like (see, generally, R. Scopes, Protein Purifica may be mutations that occur during replication. Methods of tion, Springer-Verlag, N.Y., 1982). Substantially pure com stable transfer, meaning that the foreign DNA is continuously positions of at least about 90 to 95% homogeneity are maintained in the host, are known in the art. disclosed herein, and 98 to 99% or more homogeneity can be 0204 Polynucleotide sequences encoding the antibody or used for pharmaceutical purposes. Once purified, partially or antigen binding fragment or conjugate thereof, can be opera to homogeneity as desired, if to be used therapeutically, the tively linked to expression control sequences. An expression polypeptides should be substantially free of endotoxin. control sequence operatively linked to a coding sequence is 0209 Methods for expression of single chain antibodies ligated Such that expression of the coding sequence is and refolding to an appropriate active form, including single achieved under conditions compatible with the expression chain antibodies, from bacteria such as E. coli have been control sequences. The expression control sequences include, described and are well-known and are applicable to the anti but are not limited to appropriate promoters, enhancers, tran bodies disclosed herein. See, Buchner et al., Anal. Biochem. Scription terminators, a start codon (i.e., ATG) in front of a 205:263-270, 1992: Pluckthun, Biotechnology 9:545, 1991; protein-encoding gene, splicing signal for introns, mainte Huse et al., Science 246:1275, 1989 and Ward et al., Nature nance of the correct reading frame of that gene to permit 341:544, 1989, all incorporated by reference herein. Often, proper translation of mRNA, and stop codons. functional heterologous proteins from E. coli or other bacteria are isolated from inclusion bodies and require solubilization 0205 The polynucleotide sequences encoding the anti using strong denaturants, and Subsequent refolding. During body, orantigenbinding fragment or conjugate thereof can be the solubilization step, as is well known in the art, a reducing inserted into an expression vector including, but not limited to agent must be present to separate disulfide bonds. An exem a plasmid, virus or other vehicle that can be manipulated to plary buffer with a reducing agent is: 0.1 M Tris pH 8, 6 M allow insertion or incorporation of sequences and can be guanidine, 2 mM EDTA, 0.3 M DTE (dithioerythritol). expressed in either prokaryotes or eukaryotes. Hosts can Reoxidation of the disulfide bonds can occur in the presence include microbial, yeast, insect and mammalian organisms. of low molecular weight thiol reagents in reduced and oxi Methods of expressing DNA sequences having eukaryotic or dized form, as described in Saxena et al., Biochemistry, 9: viral sequences in prokaryotes are well known in the art. 5015-5021, 1970, incorporated by reference herein, and espe Biologically functional viral and plasmid DNA vectors cially as described by Buchner et al., supra. Renaturation is capable of expression and replication in a host are known in typically accomplished by dilution (for example, 100-fold) of the art. the denatured and reduced protein into refolding buffer. An 0206 Transformation of a host cell with recombinant exemplary buffer is 0.1 M Tris, pH 8.0, 0.5 ML-arginine, 8 DNA may be carried out by conventional techniques as are mM oxidized glutathione (GSSG), and 2 mM EDTA. well known to those skilled in the art. Where the host is 0210. As a modification to the two chain antibody purifi prokaryotic, such as E. coli, competent cells which are cation protocol, the heavy and light chain regions are sepa capable of DNA uptake can be prepared from cells harvested rately solubilized and reduced and then combined in the after exponential growth phase and Subsequently treated by refolding solution. An exemplary yield is obtained when the CaCl method using procedures well known in the art. these two proteins are mixed in a molar ratio such that a 5 fold Alternatively, MgCl2 or RbCl can be used. Transformation molar excess of one protein over the other is not exceeded. US 2016/0033516 A1 Feb. 4, 2016 24
Excess oxidized glutathione or other oxidizing low molecular methods include contacting a biological sample with one or weight compounds can be added to the refolding Solution more of the conjugates, antibodies, or antigen binding frag after the redox-shuffling is completed. ments provided herein that specifically bind polypeptide 0211. In addition to recombinant methods, the antibodies, including a histidine phosphorylated at N3 to form an antigen binding fragments and conjugates thereof can be immune complex. The presence (or absence) of the immune constructed in whole or in part using standard peptide Syn complex is then detected. The presence of the immune com thesis. Solid phase synthesis of the polypeptides of less than plex indicates the presence of the polypeptide including a about 50 amino acids in length can be accomplished by histidine phosphorylated at N3. For example, an increase in attaching the C-terminal amino acid of the sequence to an the presence of the immune complex in the sample as com insoluble support followed by sequential addition of the pared to formation of the immune complex in a control remaining amino acids in the sequence. Techniques for Solid sample indicates the presence of the polypeptide including a phase synthesis are described by Barany & Merrifield, The histidine phosphorylated at N3. The amount of the immune Peptides. Analysis, Synthesis, Biology. Vol. 2: Special Meth complex can be quantitated. ods in Peptide Synthesis, Part A. pp. 3-284; Merrifield et al., 0216 A biological sample can be obtained from a mam J. Am. Chem. Soc. 85:2149-2156, 1963, and Stewart et al., malian Subject of interest, Such as human. The sample can be Solid Phase Peptide Synthesis, 2nd ed., Pierce Chem. Co., any sample, including, but not limited to, tissue from biop Rockford, Ill., 1984. Proteins of greater length may be syn sies, autopsies and pathology specimens. Biological samples thesized by condensation of the amino and carboxyl termini also include sections of tissues, for example, frozen sections of shorter fragments. Methods of forming peptide bonds by taken for histological purposes. In some embodiments, the activation of a carboxyl terminal end (such as by the use of the mammalian Subject is treated with a therapeutic agent of coupling reagent N,N'-dicylohexylcarbodimide) are well interest. The biological sample can also be an extract of cells known in the art. cultured in vitro. In some embodiments, cells are treated with an agent of interest to determine the effect of the agent on Methods of Detection phosphorylation of histidine. 0212 Methods are provided for detecting the presence of 0217. When using a control sample along with the test a polypeptide including a histidine phosphorylated at N3 in a sample, a complex is detected in both samples and any sta Subject. In some embodiments, the methods include contact tistically significant difference in the formation of complexes ing a cell from a subject with one or more of the antibodies between the samples is indicative of the presence of polypep disclosed herein to form an immune complex. The presence tide including histidine phosphorylated at N3 in the test (or absence) of the immune complex is then detected. The sample. presence of the immune complex indicates the presence of a 0218. In some examples of the disclosed methods, the histidine phosphorylated at N3 in the polypeptide. The detec antibody orantigenbinding fragment that specifically binds a tion methods can involve in vitro detection of the immune polypeptide including a histidine phosphorylated at N3 is complex. In some embodiments, the detection methods dis conjugated to a detectable marker. In additional examples, the tinguish the presence of histidine phosphorylated at N3 in the methods further include contacting a second antibody that polypeptide from histidine phosphorylated at N1. In addi specifically binds the antibody (or antigenbinding fragment) tional embodiments, the detection method distinguish the that specifically binds a polypeptide including a histidine presence of a histidine phosphorylated at N3 in the polypep phosphorylated at N3 for a sufficient amount of time to form tide from an unphosphorylated polypeptide. In additional an immune complex and detecting this immune complex. In embodiments, the methods are used to detect phosphorylated Some examples, the second antibody is conjugated to a detect proteins in a signal transduction pathway. In yet other able marker. An increase in the presence of this immune embodiments, the methods can be used to quantitate the complex in a biological sample compared to the presence of amount of a polypeptide including a histidine phosphorylated the immune complex in a control sample or other standard at N3 in a sample. detects the presence of a polypeptide including a histidine 0213 Examples of polypeptides including a histidine phosphorylated at N3 in the biological sample. phosphorylated at N3 are presented in the Tables 1 and 2 0219 Suitable detectable markers for the antibody or sec shown in Example 13. One or more of these polypeptides ondary antibody are described and known to the skilled arti including a histidine phosphorylated at N3 can be detected. san. For example, various enzymes, prosthetic groups, fluo Any combination of the polypeptides listed in Tables 1 and 2 rescent materials, luminescent materials, magnetic agents can be used in the present methods. In some embodiments, and radioactive materials. Non-limiting examples of Suitable any combination of the polypeptides listed in Table 2 can be enzymes include horseradish peroxidase, alkaline phos used in the present methods. In further embodiments, the phatase, beta-galactosidase, or acetylcholinesterase. Non method detects polypeptides that include only a histidine limiting examples of Suitable prosthetic group complexes phosphorylated at N3. In other embodiments, the method include streptavidin/biotin and avidin/biotin. Non-limiting detects polypeptides that include a histidine phosphorylated examples of suitable fluorescent materials include umbellif at N3 and a histidine phosphorylated at N1. erone, fluorescein, fluorescein isothiocyanate, rhodamine, 0214. In some embodiments, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 dichlorotriazinylamine fluorescein, dansyl chloride or phy of the listed polypeptides listed in Table 1 and/or Table 2 can coerythrin. A non-limiting exemplary luminescent material is be detected. In other embodiments, 5, 10, 15, 20, 25, 30, 35, luminol; a non-limiting exemplary a magnetic agent is gado 40, 45 or 50 of the polypeptides listed in Table 1 and/or Table linium, and non-limiting exemplary radioactive labels 2 can be detected. include II, II, S or H. 0215. In one embodiment, a biological sample is obtained, 0220. The antibodies can be used in immunohistochemi and the presence of a polypeptide including a histidine phos cal assays. These assays are well known to one of skill in the phorylated at N3 is assessed in vitro. For example, such art (see Harlow & Lane, Antibodies, A Laboratory Manual. US 2016/0033516 A1 Feb. 4, 2016
Cold Spring Harbor Publications, New York (1988), for a value/range of values expected for Such a sample). A signifi description of immunoassay formats. The assays can be, for cant increase or decrease in an amount can be evaluated using example, immunohistochemistry (IHC), immunofluores statistical methods known in the art, such as increase or cence (IF), immunoblotting (IB) and variations thereof decrease of at least 20%, at least 30%, at least 40%, at least including protein or peptide spot blots and slot blots, enzyme 50%, at least 75%, or at least 95%. linked immunosorbant assay ELISA), radioimmunoassay 0225. Similarly, assays that utilize two antibodies can be (RIA), Immune Radioimmunometric Assay (IRMA), used to detect a protein of interest that is phosphorylated at N3 Enzyme Immuno Assay (EIA), and CLIA (Chemiolumines in a sample. In this embodiment, the presence of polypeptides cent Immune Assay). including histidine phosphorylated at N3 is detected using the 0221. In one embodiment, the antibody or antigen binding methods disclosed above. In some embodiments, the amount fragment that specifically binds to a polypeptide including a of polypeptides including histidine phosphorylated at N3 can histidine phosphorylated at N3 is used to detect one or more be quantified. The presence of a polypeptide of interest is also phosphorylated polypeptides in a sample from a Subject. The detected using a second antibody that specifically binds the antibody or antigenbinding fragment can be directly labeled. polypeptide of interest. In some embodiments, the amount of In some embodiments, a biological sample from a subject is the polypeptide of interest can be quantified. In this manner, contacted with the antibody or antigen binding fragment and the presence of the polypeptide of interest, and whether it is the presence of an immune complex is detected. phosphorylated at N3, can be determined. Optionally, the 0222. In further embodiments, an additional sample is amount of the polypeptide of interest that is phosphorylated at obtained from the subject, such as following treatment with a N3, and the amount of the polypeptide of interest that is not therapeutic agent. After a sufficient amount of time has phosphorylated at N3 can be determined. elapsed, another sample is obtained. The antibody or antigen 0226. In some embodiments, the amount of polypeptides binding fragment that specifically binds to a polypeptide including histidine phosphorylated at N3, the amount of the including a histidine phosphorylated at N3 is used to detect specific polypeptide of interest, and/or the amount of the one or more phosphorylated polypeptides in the second polypeptide of interest that is phosphorylated at N3 is com sample. In some embodiments, a biological sample from a pared to a control. The control can be a control Subject, Such Subject is contacted with the antibody or antigen binding as a Subject not treated with a pharmaceutical agent, or a fragment and the presence of an immune complex is detected. Subject known to be healthy, or to another control (Such as a In some examples, an increase in the amount of the immune standard value or reference value). A significant increase or complex compared to a control. Such as in a sample taken decrease can be evaluated using statistical methods known in prior to the treatment, indicates that the treatment is not the art. effective. In other examples, a decrease in the immune com 0227. In some non-limiting examples, a sandwich ELISA plex compared to a control. Such as in a sample taken prior to can be used to detect the presence or determine the amount of the treatment, indicates that the treatment is effective. a protein in a sample. In this method, a Solid Surface is first 0223 The antibodies can also be used in screening assays, coated with an antibody or antigen binding fragment that wherein cells, optionally in a high through-put format, are specifically binds a polypeptide including a histidine phos contacted with one or more agents of interest. After a Suffi phorylated at N3, as disclosed herein. The test sample con cient amount of time has elapsed, a sample of the cells is taining proteins (such as, but not limited to, a blood, plasma, obtained. Extracts of the cells can be produced. The antibody serum, or urine sample), is then added and the antigen is or antigen binding fragment that specifically binds to allowed to react with the bound antibody. Any unbound anti polypeptides including a histidine phosphorylated at N3 is gen is washed away. A known amount of enzyme-labeled used to detect phosphorylated polypeptides in the sample. An protein-specific antibody is then allowed to react with the alteration in the binding of the antibody to the sample, as bound protein. Any excess unbound enzyme-linked antibody compared to a control sample (such as cells not contacted is washed away after the reaction. The substrate for the with the agent) or a standard value, indicates that the agent enzyme used in the assay is then added and the reaction affects phosphorylation. The assay can be used to identify between the substrate and the enzyme produces a color therapeutic agents. The assay can also be used to identify change. The amount of visual color change is a direct mea proteins that are phosphorylated in a signal transduction path Surement of specific enzyme-conjugated bound antibody, and way. consequently the quantity of the phosphorylated protein 0224 Detection of polypeptides including histidine phos present in the sample tested. phorylated at N3 can be achieved by immunoassay. In some 0228. In other non-limiting examples, a solid surface is embodiments, the presence of polypeptides including histi first coated with an antibody or antigenbinding fragment that dine phosphorylated at N3 is assessed in a sample from a specifically binds the polypeptide of interest. The test sample Subject of interest, Such as, but not limited to, a Subject with a containing proteins (such as, but not limited to, a blood, tumor. Optionally, the presence of polypeptides including plasma, serum, or urine sample), is then added and the antigen histidine phosphorylated at N3 also is assessed in a control is allowed to react with the bound antibody. Any unbound sample. In some embodiments, the amount of polypeptides antigen is washed away. A known amount of enzyme-labeled including histidine phosphorylated at N3 is quantified. The antibody orantigenbinding fragment that specifically binds a amount of polypeptides including histidine phosphorylated at polypeptide including a histidine phosphorylated at N3, as N3 in the sample from the subject of interest can be compared disclosed herein, is then allowed to react with the bound to levels of the polypeptides including histidine phosphory protein. Any excess unbound enzyme-linked antibody is lated at N3 found in the control. The amount of polypeptides washed away after the reaction. The substrate for the enzyme including histidine phosphorylated at N3 in the sample from used in the assay is then added and the reaction between the the Subject of interest can be compared to can also be com Substrate and the enzyme produces a color change. The pared to a standard value (such as a non-tumor sample, or a amount of visual color change is a direct measurement of US 2016/0033516 A1 Feb. 4, 2016 26 specific enzyme-conjugated bound antibody, and conse 0232 Another affinity technique is immunoprecipitation. quently the quantity of the phosphorylated protein present in The use of immunoprecipitation is known to one skilled in the the sample tested. art. See, for example, Molecular Cloning. A Laboratory Manual. 2d Edition, Maniatis, T. et al. eds. (1989) Cold 0229. In an alternative example, a protein can be assayed Spring Harbor Press and Antibodies, A Laboratory Manual, in a biological sample by a competition immunoassay utiliz Harlow, E. and Lane, D., eds. (1988) Cold Spring Harbor ing protein standards including a histidine phosphorylated at Press. An example of immunoprecipitation is the use of anti N3 labeled with a detectable substance and an unlabeled bodies coupled to beads. The antibodies coupled to the beads antibody or antigenbinding fragment that specifically binds a can bind directly to polypeptides including a histidine phos polypeptide including a histidine phosphorylated at N3. In phorylated at N3. A method of attaching antibodies to beads this assay, the biological sample (such as, but not limited to, a is disclosed and described in U.S. Pat. No. 5,011.912, incor blood, plasma, serum, or urine sample), the labeled protein porated herein by reference. For example, antibodies can be standards and the antibody that specifically binds the coupled to beads using a hydrazide linkage. Such methods are polypeptide including a histidine phosphorylated at N are generally described with respect to the use of the FLAGR) combined and the amount of labeled protein standard bound peptide in Brizzard et al., BioTechniques, Vol. 16, pg. 730 to the unlabeled antibody is determined. The amount of phos (1994). To accomplish separation using this affinity separa phorylated including a histidine phosphorylated at N3 in the tion technique, a sample is mixed with beads which are biological sample is inversely proportional to the amount of coupled to the antibody or antigen binding fragment. labeled standard bound to the antibody. Polypeptides including a histidine phosphorylated at N3 will 0230. In yet other embodiments, the antibodies described bind to the antibodies (or antigen binding fragment thereof) herein can be used in immunohistochemical assays, such as coupled to the beads, while polypeptides that do not include a on histological sections, including, but not limited to, a sec histidine phosphorylated at N3 will not bind. The polypep tion of a tumor or a fine needle aspirate of a tumor sample. tides bound to the beads can then be recovered by, for These assays are well known to one of skill in the art (see example, centrifugation and elution. Harlow & Lane, Antibodies, A Laboratory Manual, Cold 0233. Other methods of detection, identification, isola Spring Harbor Publications, New York (1988), for a descrip tion, capture, and/or purification of polypeptides are well tion of immunoassay formats). In these embodiments, a tissue known in the art, see for example, “Principles and Practice of section or cells from a sample of interest is contacted with a Immunoassay.” Price and Newman, eds., Stochton Press first antibody that specifically binds polypeptides including (1991), Molecular Cloning, A Laboratory Manual, 3rd Edi histidine phosphorylated at N3. In some embodiments, the tion, Sambrook et al. eds., Cold Spring Harbor Press (2001) monoclonal antibody or antigen-binding fragment thereof is and Antibodies, A Laboratory Manual. Harlow, E. and Lane, labeled. In other embodiments, the monoclonal antibody or D., eds. (1988) Cold Spring Harbor Press. Accordingly, meth antigen-binding fragment thereof is unlabeled. The method ods are provided for detecting, identifying, isolating, captur can also include contacting the biological sample with a sec ing or purifying a polypeptide from a sample, wherein the ond antibody that specifically binds the first monoclonal anti method includes contacting an antibody as disclosed herein body, wherein the second antibody is labeled. In additional with the sample to bind a polypeptide including a histidine embodiment, the method includes contact the tissue section phosphorylated at N3. The method also can include releasing or cells with a third antibody or antigen binding fragment the polypeptide, protein, protein fragment, or a portion thereof that specifically binds a polypeptide of interest. In thereof from the antibody. some embodiments, the third monoclonal antibody or anti 0234. In some embodiments, affinity purification can be gen-binding fragment thereof is labeled. In other embodi performed using the antibodies disclosed herein. The dis ments, the third monoclonal antibody or antigen-binding closed antibodies can be conjugated to resins, such as beads. fragment thereof is unlabeled. The method can also include A sample of interest that includes polypeptides is then passed contacting the biological sample with a fourth antibody that through the column, such that a polypeptide including a his specifically binds the third monoclonal antibody, wherein the tidine phosphorylated at N3 are bound to the column. The second antibody is labeled. bound polypeptides can then be eluted from the column, and 0231. The method can include contacting an antibody as optionally the polypeptides including a histidine phosphory described herein with a sample to bind the polypeptides lated at N3 can be quantitated. including a histidine phosphorylated at N3. The antibody can 0235. Following purification of proteins including a histi be either mobilized or immobilized and may be labeled or dine phosphorylated at N3, a detection method can be used to unlabeled. The process may further comprise releasing the identify these polypeptides. In some embodiments, in order to polypeptide, protein, protein fragment, or a portion thereof determine the identity of the eluted polypeptides, mass spec from the antibody. For example, the antibodies can be trometry can be performed. Mass spectrometry can also be coupled to biotin by a hydrazide linkage, and the fusion used to quantify peptides in a biological sample, for example peptides or proteins including a histidine phosphorylated at using isotopically labeled peptide standards. The application N3 can then be separated from peptides or proteins that do not of mass spectrometric techniques to identify proteins in bio include a histidine phosphorylated at N3 through the use of logical samples is known in the art and is described, for avidin or streptavidin attached to magnetic beads. When the example, in Akhilesh et al., Nature, 405:837-846, 2000; Dutt sample is placed in a magnetic field only the peptides or et al., Curr. Opin. Biotechnol., 11:176-179, 2000; Gygiet al., proteins including the histidine phosphorylated at N3 will Curr. Opin. Chem. Biol. 4 (5): 489-94, 2000; Gygi et al., bind to the magnetic beads via the linkage between the anti Anal. Chem., 72 (6): 1112–8, 2000; and Anderson et al., Curr: body and the bonds between, for example, the biotin and Opin. Biotechnol., 11:408-412, 2000. avidin. The polypeptides attached to the beads can be recoV 0236 Separation of ions according to their m/z ratio can ered and the others washed away. be accomplished with any type of mass analyzer, including US 2016/0033516 A1 Feb. 4, 2016 27 quadrupole mass analyzers (Q), time-of-flight (TOF) mass Surface-enhanced laser desorption-ionization time-of-flight analyzers (for example, linear or reflecting) analyzers, mag (SELDI-TOF) mass spectrometry is used to detect protein netic sector mass analyzers, 3D and linear ion traps (IT), expression, for example by using the PROTEINCHIPTM (Ci Fourier-transform ion cyclotron resonance (FT-ICR) analyz phergen Biosystems, Palo Alto, Calif.). Such methods are ers, Orbitrap analyzers (like LTQ-Orbitrap LC/MS/MS), and well known in the art (for example see U.S. Pat. No. 5,719, combinations thereof (for example, a quadrupole-time-of 060; U.S. Pat. No. 6,897,072; and U.S. Pat. No. 6,881,586). flight analyzer, or Q-TOF analyzer). A triple quadropole SELDI is a solid phase method for desorption in which the instrument can be used such as the Q-trap. analyte is presented to the energy stream on a Surface that 0237. In some embodiments, the mass spectrometric tech enhances analyte capture or desorption. Additional methods nique is tandem mass spectrometry (MS/MS). Typically, in are disclosed in the examples section below. tandem mass spectrometry a protein product, entering the 0240 Briefly, one version of SELDI uses a chromato tandem mass spectrometer is selected and Subjected to colli graphic Surface with a chemistry that selectively captures sion induced dissociation (CID). The spectrum of the result analytes of interest, Such as one or more proteins of interest. ing fragment ion is recorded in the second stage of the mass Chromatographic Surfaces can be composed of hydrophobic, spectrometry, as a so-called CID or ETD spectrum. Because hydrophilic, ion exchange, immobilized metal, or other the CID or ETD process usually causes fragmentation at chemistries. For example, the Surface chemistry can include peptide bonds and different amino acids for the most part binding functionalities based on oxygen-dependent, carbon yield peaks of different masses, a CID or ETD spectrum alone dependent, Sulfur-dependent, and/or nitrogen-dependent often provides enough information to determine the presence means of covalent or noncovalent immobilization of analytes. of a the protein of interest. Suitable mass spectrometer sys The activated surfaces are used to covalently immobilize tems for MS/MS include an ion fragmentor and one, two, or specific “bait' molecules such as antibodies, receptors, or more mass spectrometers, such as those described above. oligonucleotides often used for biomolecular interaction Examples of Suitable ion fragmentors include, but are not studies such as protein-protein and protein-DNA interactions. limited to, collision cells (in which ions are fragmented by 0241 The surface chemistry allows the bound analytes to causing them to collide with neutral gas molecules), photo be retained and unbound materials to be washed away. Sub dissociation cells (in which ions are fragmented by irradiating sequently, analytes bound to the Surface can be desorbed and them with a beam of photons), and Surface dissociation frag analyzed by any of several means, for example using mass mentor (in which ions are fragmented by colliding them with spectrometry. When the analyte is ionized in the process of a solid or a liquid surface). Suitable mass spectrometer sys desorption, such as in laser desorption/ionization mass spec tems can also include ion reflectors or Negative Electron trometry, the detector can be an ion detector. Mass spectrom Transfer Dissociation (NETD) MS, which is run at alkaline eters generally include means for determining the time-of pH and therefore can be used for pHis analysis. flight of desorbed ions. This information is converted to mass. 0238 Prior to mass spectrometry, the sample or fragments However, one need not determine the mass of desorbed ions of the sample, for example made by digestion with the trypsin to resolve and detect them: the fact that ionized analytes strike protease, can be subjected to one or more dimensions of the detector at different times provides detection and resolu chromatographic separation, for example, one or more tion of them. Alternatively, the analyte can be detectably dimensions of liquid or size exclusion chromatography. Rep labeled (for example with a fluorophore or radioactive iso resentative examples of chromatographic separation include tope). In these cases, the detector can be a fluorescence or paper chromatography, thin layer chromatography (TLC), radioactivity detector. liquid chromatography, column chromatography, high per 0242. In an additional example, the method may include formance liquid chromatography (HPLC), fast protein liquid detection of a protein of interest in a sample using an electro chromatography (FPLC), ion exchange chromatography, size chemical immunoassay method. See, e.g., Yu et al., J. Am. exclusion chromatography, affinity chromatography, high Chem. Soc., 128:11199-11205, 2006; Mani et al., ACS Nano, performance liquid chromatography (HPLC), nano-reverse 3:585-594, 2009; Malhotra et al., Anal. Chem., 82:3118 phase liquid chromatography (nano-RPLC), polyacrylamide 3123, 2010. In this method, a primary antibody or antigen gel electrophoresis (PAGE), capillary electrophoresis (CE), binding fragment that specifically binds polypeptides includ reverse phase high performance liquid chromatography (RP ing a histidine phosphorylated at N3 is conjugated to termi HPLC) or other suitable chromatographic techniques. Thus, nally carboxylated single-wall carbon nanotubes (SWNT), in some embodiments, the mass spectrometric technique is multi-wall carbon nanotubes (MWCNT), or gold nanopar directly or indirectly coupled with a one, two or three dimen ticles (AuNP), which are attached to a conductive surface. A sional liquid chromatography technique. Such as column sample (such as a blood, plasma or serum sample) is con chromatography, high performance liquid chromatography tacted with the SWNTs, MWCNTs, or AuNPs, and protein in (HPLC or FPLC), reversed phase, ion exchange chromatog the sample binds to the primary antibody. A second antibody raphy, size exclusion chromatography, affinity chromatogra conjugated directly or indirectly to a redox enzyme (such as phy (such as protein or peptide affinity chromatography, horseradish peroxidase (HRP), cytochrome c, myoglobin, or immunoaffinity chromatography, lectin affinity chromatog glucose oxidase) binds to the primary antibody or to the raphy, etc.), or one, two or three dimensional polyacrylamide protein (for example, in a 'sandwich' assay). In some gel electrophoresis (PAGE), or one or two dimensional cap examples, the second antibody is conjugated to the enzyme. illary electrophoresis (CE) to further resolve the biological In other examples, the second antibody and the enzyme are sample prior to mass spectrometric analysis. both conjugated to a Support (Such as a magnetic bead). 0239. A variety of mass spectrometry methods, including Signals are generated by adding enzyme Substrate (e.g. iTRAQR and MRM, can be used. In some embodiments, hydrogen peroxide if the enzyme is HRP) to the solution quantitative spectroscopic methods, such as SELDI, are used bathing the sensor and measuring the current produced by the to analyze protein expression in a sample. In one example, catalytic reduction. US 2016/0033516 A1 Feb. 4, 2016 28
Methods of Detecting Tumors embodiments, the quantity of one of more polypeptides including a histidine phosphorylated at N3 is determined. In 0243 Cancer is the second leading cause of human death further embodiments, method can include determining the next to coronary disease in the United States. Worldwide, phosphorylation profile of one or more polypeptides includ millions of people die from cancer every year. In the United ing a histidine that can be phosphorylated at N3 present in the States alone, as reported by the American Cancer Society, sample. cancer causes the death of well over a half-million people annually, with over 1.2 million new cases diagnosed per year. 0250 Examples of polypeptides including a histidine While deaths from heart disease have been declining signifi phosphorylated at N3 are presented in Tables 1 and Table 2 cantly, those resulting from cancer generally are on the rise. shown in Example 13. One or more of these polypeptides Cancer is soon predicted to become the leading cause of including a histidine phosphorylated at N3 can be detected. In death. Some embodiments, one or more of these polypeptides 0244 Cancer is an abnormal state in which uncontrolled including a histidine phosphorylated at N3 listed in Table 2 proliferation of one or more cell populations interferes with can be detected. In some embodiments, 1,2,3,4,5,6,7,8,9, normal biological functioning. The proliferative changes are or 10 of the polypeptides listed in Table 1 and/or Table 2 can usually accompanied by other changes in cellular properties, be detected. In other embodiments, 5, 10, 15, 20, 25, 30, 35, including reversion to a less differentiated, more develop 40, 45 or 50 of the polypeptides listed in Table 1 and/or Table mentally primitive state. The in vitro correlate of cancer is 2 can be detected. Any combination of the polypeptides listed called cellular transformation. Transformed cells generally in Table 2 can be used in the present methods. display several or all of the following properties: spherical 0251. In some embodiments, the presence of one or more morphology, expression offetal antigens, growth-factorinde polypeptides that include a histidine phosphorylated at N3, or pendence, lack of contact inhibition, anchorage-indepen the amount of one or more proteins phosphorylated at N3, or dence, and growth to high density. the phosphorylation profile from the sample, is compared to a 0245 Methods are provided herein for evaluating cancer control. The control can be the phosphorylation profile of risk, for example for determining the likelihood that a subject, polypeptides including a histidine phosphorylated at N3, or Such as an otherwise healthy Subject, or a subject Suspected or the quantity of one of more specific polypeptides including a at risk of having a tumor, has a tumor or will likely develop the histidine phosphorylated at N3 that is present in a control tumor in the future, or that a tumor will become malignant or sample. The control sample can be a positive control sample, metastasize. In particular examples, the method can deter Such as a sample from a subject known to have the tumor, or mine if a subject has or will likely develop the tumor in the a negative control sample, such as a sample from a subject future. In further examples, the method can determine the known not to have the tumor. In other embodiments, the likelihood that a pharmaceutical agent (Such as a chemothera control can be a reference standard (Such as an absolute or peutic or biologic) is effective for treating a Subject. relative amount of polypeptides including a histidine phos 0246. In some examples, a biological sample obtained phorylated at N3 expected if the sample is a tumor sample or from the Subject, such as, but not limited to, serum, blood, if the sample is a normal-non-tumor sample). plasma, urine, purified cells (for example, blood cells, such as 0252. In some embodiments, it is determined if a particu white blood cells, B cells, T cells, or mononuclear cells), lar protein of interest includes a histidine phosphorylated at saliva, a biopsy or tissue sample, such as a sample including N3. The amount of a particular polypeptide including a his cells of a tissue sample obtained from the subject are used to tidine phosphorylated at N3 can increase or decrease, for predict the Subject's risk. In specific non-limiting examples, example relative to a control. Exemplary proteins include the sample includes tumor cells. PGAM, and those proteins listed in the Examples section. 0247. In some embodiments, the subject is apparently 0253) Thus, it can be determined if a particular polypep healthy, such as a subject who does not exhibit symptoms of tide including a histidine phosphorylated at N3 is increased or the tumor. In some examples, a healthy Subject is one that if decreased, such as by about 10%, about 20%, about 30%, examined by a medical professional, would be characterized about 40%, about 50%, about 60%, about 70% about 80%, as healthy and free of symptoms of the tumor. The methods about 90%, about 95%, about 96%, about 97%, about 98%, disclosed herein can be used to screen subjects for future about 99% or about 100%, as compared to a control. In other evaluation or treatment for tumors. In other embodiments, the embodiments, detection of an increase or decrease in 1, 2, 3, methods determine the likelihood that a subject will develop 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50 or more the tumor, or whether the tumor will metastasize. The meth polypeptides including a histidine phosphorylated at N3 indi ods disclosed herein can be used to confirm a prior clinical cates that the Subject has the tumor. An increase or decrease in Suspicion of disease. the overall amount of polypeptides phosphorylated at N3 can 0248. The method includes obtaining a sample from a also be detected. Thus, it can be determined if the amount of Subject that includes polypeptides, and determining whether polypeptides phosphorylated at N3 is increased or decreased, polypeptides that include a histidine phosphorylated at N3 are such as by about 10%, about 20%, about 30%, about 40%, present using the antibodies disclosed herein. Any of the about 50%, about 60%, about 70% about 80%, about 90%, methods disclosed above can be used to detect and/or quan about 95%, about 96%, about 97%, about 98%, about 99% or tify polypeptides including a histidine phosphorylated at N3. about 100%, as compared to a control. 0249 Optionally, the identity of the one or more polypep 0254. In more embodiments, the control can be sample tides including a histidine phosphorylated at N3, in order to from a subject known to have the tumor, a sample from a determine the phosphorylation status of one of more specific healthy Subject, or a reference standard. The comparison can polypeptides of interest. Methods for determining the identity allows determination of the presence of the tumor, and/or the of polypeptides including a histidine phosphorylated at N3. likelihood that the tumor will metastasize. The method can including, but not limited to, the use of additional antibodies determine if the tumor is benign or malignant, or it can deter and mass spectrometry are disclosed above. In additional mine if the tumor is aggressive and likely to metastasize. US 2016/0033516 A1 Feb. 4, 2016 29
0255. The tumor can be any tumor of interest, including, time dysplasia is diagnosed. However, treatment can be ini but not limited to, lymphoma, breast cancer, lung cancer and tiated during any stage of the disease, such as but not limited colon cancer. Additional examples are skin, breast, brain, to stage I, stage II, stage III and stage IV cancers. The treat cervical carcinomas, testicular carcinomas, head and neck, ment can be designed to decrease the severity of the Symp lung, mediastinum, gastrointestinal tract, genitourinary sys toms of one of the conditions, or completely removing the tem, gynecological system, breast, endocrine system, skin, symptoms, or reducing metastasis, tumor Volume or number childhood, unknown primary site or metastatic cancer, a sar of tumors. Treatment can also include increasing the immune coma of the Soft tissue and bone, a mesothelioma, a mela response to the tumor, such as by increasing the humoral noma, a neoplasm of the central nervous system, a lym response. In one example, there is an increase in antibodies phoma, a leukemia, a paraneoplastic syndrome, a peritoneal that specifically bind the tumor. In some examples, treatment carcinomastosis. The tumor cells can be from: head and neck is administered to try to prevent a benign tumor converting tumor, comprising tumors of the nasal cavity, paranasal into a malignant or even metastatic lesion. However, in other sinuses, nasopharynx, oral cavity, oropharynx, larynx, examples, treatment is administered to any Subject diagnosed hypopharynx, Salivary glands and paragangliomas, a cancer with cancer. of the lung, comprising non-small cell lung cancer, Small cell 0258. The treatment can be with naturally occurring che lung cancer, a cancer of the mediastinum, a cancer of the motherapeutic agents, non-naturally occurring chemothera gastrointestinal tract, comprising cancer of the esophagus, peutic agent, or combinations thereof. The chemotherapeutic stomach, pancreas, liver, biliary tree, Small intestine, colon, agent can be a cytokine, a chemokine, or a chemical com rectum and anal region, a cancer of the genitourinary system, pound. In one example, for the prevention and treatment of comprising cancer of the kidney, urethra, bladder, prostate, cancer. Such as lung cancer, colon cancer or prostate cancer, urethra, penis and testis, a gynecologic cancer, comprising the treatment can be with a cytokine, including interleukin-2 cancer of the cervix, Vagina, Vulva, uterine body, gestational (IL-2), granulocyte macrophage colony stimulating factor trophoblastic diseases, ovarian, fallopian tube, peritoneal, a (GM-CSF), or interferon, such as interferon (IFN). In another cancer of the breast, a cancer of the endocrine system, com example, this administration is sequential. In other examples, prising a tumor of the thyroid, parathyroid, adrenal cortex, this administration is simultaneous. pancreatic endocrine tumors, carcinoid tumor and carcinoid syndrome, multiple endocrine neoplasias, a sarcoma of the 0259 Examples of additional chemotherapeutic agents Soft tissue and bone, a mesothelioma, a cancer of the skin, a are alkylating agents, antimetabolites, natural products, or melanoma, comprising cutaneous melanomas and intraocular hormones and their antagonists. Examples of alkylating melanomas, a neoplasm of the central nervous system, a agents include nitrogen mustards (such as mechlorethamine, cancer of the childhood, comprising retinoblastoma, Wilms cyclophosphamide, melphalan, uracil mustard or chloram tumor, neurofibromatoses, neuroblastoma, Ewing's sarcoma bucil), alkyl Sulfonates (such as buSulfan), nitrosoureas (Such family of tumors, rhabdomyosarcoma, a lymphoma, com as carmustine, lomustine, Semustine, Streptozocin, or dacar prising non-Hodgkin’s lymphomas, cutaneous T-cell lym bazine). Examples of antimetabolites include folic acid ana phomas, primary central nervous system lymphoma, and logs (such as methotrexate), pyrimidine analogs (such as Hodgkin’s disease, a leukemia, comprising acute leukemias, 5-FU or cytarabine), and purine analogs, such as mercaptopu chronic myelogenous and lymphocytic leukemias, plasma rine or thioguanine. Examples of natural products include cell neoplasms, a cancer of unknown primary site, a perito Vinca alkaloids (such as vinblastine, Vincristine, or Vin neal carcinomastosis, a Kaposi's sarcoma, AIDS-associated desine), epipodophyllotoxins (such as etoposide or tenipo lymphomas, AIDS-associated primary central nervous sys side), antibiotics (such as dactinomycin, daunorubicin, doxo tem lymphoma, AIDS-associated Hodgkin’s disease and rubicin, bleomycin, plicamycin, or mitocycin C), and AIDS-associated anogenital cancers, a metastatic cancer to enzymes (such as L-asparaginase). Examples of miscella the liver, metastatic cancer to the bone, malignant pleural and neous agents include platinum coordination complexes (such pericardial effusions and malignant ascites. In some as cis-diamine-dichloroplatinum II also known as cisplatin), examples, the tumor is a lymphoma, breast cancer, colon Substituted ureas (such as hydroxyurea), methyl hydrazine cancer, prostate cancer or lung cancer. The tumor can be derivatives (such as procarbazine), and adrenocortical Sup benign or malignant. pressants (such as mitotane and aminoglutethimide). Examples of hormones and antagonists include adrenocorti Method of Predicting Responsiveness to a costeroids (such as prednisone), progestins (such as hydrox Therapeutic Agent yprogesterone caproate, medroxyprogesterone acetate, and magestrol acetate), estrogens (such as diethylstilbestrol and 0256 Methods are also provided herein for determining if ethinyl estradiol), antiestrogens (such as tamoxifen), and a cancer in a Subject is responsive to an agent, such as a androgens (such as testerone proprionate and fluoxymester chemotherapeutic agent. The chemotherapeutic agent can be one). Examples of the most commonly used chemotherapy a naturally or non-naturally occurring agent. The chemo drugs include Adriamycin, Alkeran, Ara-C, BiCNU, Busul therapeutic agent can be a biological molecule (e.g., a thera fan, CCNU, Carboplatinum, Cisplatinum, Cytoxan, Dauno peutic antibody), a chemical compound, or a combination rubicin, DTIC, 5-FU, Fludarabine, Hydrea, Idarubicin, Ifos thereof. famide, Methotrexate, Mithramycin, Mitomycin, 0257 Treatment of the conditions described herein are Mitoxantrone, Nitrogen Mustard, Taxol (or other taxanes, generally initiated after the development of a condition such as docetaxel), Velban, Vincristine, VP-16, while some described herein, or after the initiation of a precursor condi more newer drugs include Gemcitabine (Gemzar), Herceptin, tion (such as dysplasia or development of a benign tumor). Irinotecan (Camptosar, CPT-11), Leustatin, Navelbine, Rit Treatment can be initiated at the early stages of cancer, for uxan STI-571, Taxotere, Topotecan (Hycamtin), Xeloda instance, can be initiated before a subject manifests symp (Capecitabine), Zevelin and calcitriol. Non-limiting toms of a condition, such as during a stage I diagnosis or at the examples of immunomodulators that can be used include US 2016/0033516 A1 Feb. 4, 2016 30
AS-101 (Wyeth-Ayerst Labs.), bropirimine (Upjohn), at N3 is increased or decreased, such as by about 10%, about gamma interferon (Genentech), GM-CSF (granulocyte mac 20%, about 30%, about 40%, about 50%, about 60%, about rophage colony stimulating factor; Genetics Institute), IL-2 70% about 80%, about 90%, about 95%, about 96%, about (Cetus or Hoffman-LaRoche), human immune globulin (Cut 97%, about 98%, about 99% or about 100%, as compared to ter Biological), IMREG (from Imreg of New Orleans, La.), a control. In other embodiments, detection of an increase or SK&F 106528, and TNF (tumor necrosis factor; Genentech). decrease in 1,2,3,4,5,6,7,8,9, 10, 15, 20, 25, 30, 35, 40, 45, 0260 The treatment can be with a biologic, such as an 50 or more polypeptides including a histidine phosphorylated antibody (e.g., Cetuximab, Gemtuzumab, Ibritumomab tiuX at N3 indicates that the subject has the tumor. In yet other etan, Nivolumab, Panitumumab, Rituximab, Tositumomab or embodiments, an increase or decrease in the total amount of Trastuzumab) cytokine, chemokine, or other biological mol polypeptides phosphorylated at N3 can also be detected. ecule. In some embodiment, the treatment is a non-naturally Thus, an increase or a decrease in the total amount of polypep occurring monoclonal antibody. tides phosphorylated at N3, such as by about 10%, about 0261 Treatment or treating a tumor includes, but is not 20%, about 30%, about 40%, about 50%, about 60%, about limited to, reduction in tumor growth or tumor burden, 70% about 80%, about 90%, about 95%, about 96%, about enhancement of an anti-tumor immune response, induction of 97%, about 98%, about 99% or about 100%, as compared to apoptosis of tumor cells, inhibition of angiogenesis, enhance a control, indicates that the Subject has the tumor. ment of tumor cell apoptosis, and inhibition of metastases. 0265. The method includes evaluating proteins in a sample Administration of an effective amount of a chemotherapeutic from a Subject, and detecting proteins that included a histidine agent to a subject may be carried out by any means known in phosphorylated at N3 in proteins using the antibodies dis the art including, but not limited to intraperitoneal, intrave closed herein. In some embodiments, the identity of polypep nous, intramuscular, Subcutaneous, transcutaneous, oral, tides including a histidine phosphorylated at N3 can be deter nasopharyngeal or transmucosal absorption. The specific mined. In other embodiments, the quantity of one of more amount or dosage administered in any given case will be polypeptides including a histidine phosphorylated at N3 is adjusted in accordance with the specific cancer being treated, determined. The method can include comparing the phospho the condition, including the age and weight, of the Subject, rylation profile of one or more proteins including a histidine and other relevant medical factors known to those of skill in that can be phosphorylated at N3 present in the sample. the art. 0266. In some embodiments, the presence of a particular 0262. In these embodiments, a sample can be taken from a protein phosphorylated at N3, the amount of one or more subject prior to initiation of therapy. After therapy is initiated, proteins phosphorylated at N3, or the phosphorylation profile an additional sample is taken from the Subject. Any of the from the sample is compared to a reference standard, Such as method disclosed above can be used to determine a change in the phosphorylation profile of polypeptides including a his the amount of the one or more proteins that included a histi tidine phosphorylated at N3, or the quantity of one of more dine phosphorylated at N3 indicates that the therapy is effi specific polypeptides including a histidine phosphorylated at cacious. In addition, the Subject can be monitored over time to N3, or the profile of proteins phosphorylated at N3, in a evaluate the continued effectiveness of the therapeutic proto control sample. The control sample can be a positive control, col. The effect of different dosages can also be evaluated, by Such as a sample from a subject known to respond to the comparing the expression of markers in a sample from the chemotherapeutic agent, or a negative control. Such as a Subject receiving a first dose to the expression of the same sample from a subject known not to respond to the chemo markers in a sample from the Subject receiving a second therapeutic agent. The control can also be a sample from the (different) dose. The methods can be repeated 1, 2, 3, 4, 5, 6, Subject prior to the administration of the therapeutic agent. 7, 8, 9, 10 or more times to determine the lowest dose of a The control also can be a reference standard (Such as an pharmaceutical agent that is effective for treating the Subject, absolute or relative amount of polypeptides having a histidine and/or the shortest duration of administration that is effective phosphorylated at N3 expected if the tumor will respond to for treating the subject. The methods can also be used over the the chemotherapeutic agent or if the tumor will not respond to course of a therapeutic regimen to monitor the efficacy of a the chemotherapeutic agent). pharmaceutical agent for the treatment of the Subject. 0267 In some embodiments, comparison of one of more 0263. Examples of polypeptides including a histidine proteins phosphorylated at N3 in the sample from the subject phosphorylated at N3 are presented in Tables 1 and Table 2 with the tumor to the phosphorylation status of the one or shown in Example 13. One or more of these polypeptides more proteins in samples from Subjects known to be sensitive including a histidine phosphorylated at N3 can be detected. In or resistant to the chemotherapeutic agent, or to the reference Some embodiments, one or more of these polypeptides standard, allows prediction of the responsiveness of the tumor including a histidine phosphorylated at N3 listed in Table 2 to the chemotherapeutic agent. The prediction may indicate can be detected. In some embodiments, 1, 2, 3, 4, 5, 6, 7, 8, 9, that the tumor will respond completely to the chemotherapeu or 10 of the polypeptides listed in Table 1 and/or Table 2 can tic agent, or it may predict that the tumor will be only partially be detected. In other embodiments, 5, 10, 15, 20, 25, 30, 35, responsive or non-responsive (i.e. resistant) to the chemo 40, 45 or 50 of the polypeptides listed in Table 1 and/or Table therapeutic agent. 2 can be detected. Any combination of the polypeptides listed 0268. Once a subject's tumor is predicted to be responsive in Table 2 can be used in the present methods. to a particular chemotherapy, then a treatment plan can be 0264. In some embodiments, it is determined if a particu developed incorporating the chemotherapeutic agent and an lar protein of interest includes a histidine phosphorylated at effective amount of the chemotherapeutic agent(s) can be N3. The amount of a particular polypeptide including a his administered to the subject with the tumor. Those of skill in tidine phosphorylated at N3 can increase or decrease, for the art will appreciate that the methods do not guarantee that example relative to a control. Thus, it can be determined if a the Subjects will be responsive to the chemotherapeutic agent, particular polypeptide including a histidine phosphorylated but the methods will increase the probability that the selected US 2016/0033516 A1 Feb. 4, 2016
treatment will be effective to treat the tumor. Also encom thra, bladder, prostate, urethra, penis and testis, a gynecologic passed is the ability to predict the responsiveness of the tumor cancer, comprising cancer of the cervix, Vagina, Vulva, uter to multiple chemotherapeutic agents and then to develop a ine body, gestational trophoblastic diseases, ovarian, fallo treatment plan using a combination of two or more chemo pian tube, peritoneal, a cancer of the breast, a cancer of the therapeutic agents. endocrine system, comprising a tumor of the thyroid, par athyroid, adrenal cortex, pancreatic endocrine tumors, carci 0269. The disclosed methods can also be used to deter noid tumor and carcinoid syndrome, multiple endocrine neo mine the lowest dose of a chemotherapeutic agent effective to plasias, a sarcoma of the Soft tissue and bone, a treat a Subject. The method includes administering a dose of mesothelioma, a cancer of the skin, a melanoma, comprising the chemotherapeutic agent, and detecting polypeptides that cutaneous melanomas and intraocular melanomas, a neo included a histidine phosphorylated at N3 in proteins using plasm of the central nervous system, a cancer of the child the antibodies disclosed herein. In some embodiments, the hood, comprising retinoblastoma, Wilms tumor, neurofibro identity of polypeptides including a histidine phosphorylated matoses, neuroblastoma, Ewing's sarcoma family of tumors, at N3 can be determined. In other embodiments, the quantity rhabdomyosarcoma, a lymphoma, comprising non of one of more polypeptides including a histidine phospho Hodgkin’s lymphomas, cutaneous T-cell lymphomas, pri rylated at N3 is determined. The method can include compar mary central nervous system lymphoma, and Hodgkin’s dis ing the phosphorylation profile of one or more proteins ease, a leukemia, comprising acute leukemias, chronic including a histidine that can be phosphorylated at N3 present myelogenous and lymphocytic leukemias, plasma cell neo in a sample from the subject prior to treatment with the dose plasms, a cancer of unknown primary site, a peritoneal car of chemotherapeutic agent. cinomastosis, a Kaposi's sarcoma, AIDS-associatedlympho 0270. In some embodiments, it can be determined if a mas, AIDS-associated primary central nervous system particular polypeptide including a histidine phosphorylated lymphoma, AIDS-associated Hodgkin’s disease and AIDS at N3 is increased or decreased, such as by about 10%, about associated anogenital cancers, a metastatic cancer to the liver, 20%, about 30%, about 40%, about 50%, about 60%, about metastatic cancer to the bone, malignant pleural and pericar 70% about 80%, about 90%, about 95%, about 96%, about dial effusions and malignant ascites. In some examples, the 97%, about 98%, about 99% or about 100%, as compared to tumor is a lymphoma, breast cancer, colon cancer, prostate a control. In other embodiments, an increase or decrease in 1, cancer or lung cancer. 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50 or more polypeptides including a histidine phosphorylated at N3 can Method of Identifying Compounds of Use as be detected. An increase or decrease in the total amount of Antibiotics polypeptides phosphorylated at N3 can also be detected. Thus, it can be determined if the total amount of polypeptides 0273. Two-component regulatory systems (TCSs) are phosphorylated at N3 is increased or decreased, such as by found in many bacteria, including gram positive and gram about 10%, about 20%, about 30%, about 40%, about 50%, negative bacteria and are one of the main signal transduction about 60%, about 70% about 80%, about 90%, about 95%, systems. about 96%, about 97%, about 98%, about 99% or about 0274. A typical TCS has two components, a histidine 100%. The increase or decrease is measured as compared to kinase (HK) and a cognate response regulator (RR). HKS the control. Such as a sample from the Subject prior to treat autophosphorylate at conserved histidine residues in ment with the dose of the chemotherapeutic agent. response to environmental or metabolic signals. Phosphoryl 0271 The methods can be repeated 1, 2, 3, 4, 5, 6, 7, 8, 9. groups on the histidine residues of HKs are then transferred to 10 or more times to determine the lowest dosage of a chemo conserved aspartate residues in the receiver domains of cog therapeutic agent that is effective for treating the Subject, nate RRs. Phosphorylation of an RR alters its conformation and/or the shortest duration of administration that is effective and its interactions with other components of the signal trans for treating the subject. The methods can also be used over the duction pathway, which can result in an alteration in the RR to course of a therapeutic regimen to monitor the efficacy of a bind to DNA and influence transcription (see Gilmore et al., J. chemotherapeutic agent for the treatment of the Subject. Bacteriol. 187: 8196-8200, 2005). TCSs have been proposed 0272. The tumor can be any tumor of interest, including, as targets for new antibiotics. The antibodies disclosed herein but not limited to, lymphoma, breast cancer, lung cancer and can be used to evaluate phosphorylation of histidine kinases colon cancer. The tumor can be benign or malignant. Addi in bacteria, and thus can be used to determine if an agent of tional examples are skin, breast, brain, cervical carcinomas, interest increases or decreases phosphorylation. The antibod testicular carcinomas, head and neck, lung, mediastinum, ies disclosed herein can be used to measure phosphorylation gastrointestinal tract, genitourinary system, gynecological of polypeptide that include histidine phosphorylated at N3. system, breast, endocrine system, skin, childhood, unknown primary site or metastatic cancer, a sarcoma of the Soft tissue and bone, a mesothelioma, a melanoma, a neoplasm of the Histidine Kinase and Response Regulator (HKJRR) Pairs that central nervous system, a lymphoma, a leukemia, a paraneo Regulate Virulence Signaling in Pathogenic Bacteria plastic syndrome, a peritoneal carcinomastosis. The tumor cells can be from: head and neck tumor, comprising tumors of Bacteria HK RR the nasal cavity, paranasal sinuses, nasopharynx, oral cavity, Pseudomonas aeruginosa PhoQ PhoP oropharynx, larynx, hypopharynx, Salivary glands and Saimoneia enterica GacS GacA Myobacterium tuberculosis MtrB Mitra paragangliomas, a cancer of the lung, comprising non-Small Staphylococcusatiretts WalK WaR cell lung cancer, Small cell lung cancer, a cancer of the medi Staphylococcusatiretts ArgC ArgA astinum, a cancer of the gastrointestinal tract, comprising Enterococci is faecalis FsrC FSA cancer of the esophagus, stomach, pancreas, liver, biliary tree, Bordeteila pertissis BvgS BvgA Small intestine, colon, rectum and anal region, a cancer of the genitourinary system, comprising cancer of the kidney, ure US 2016/0033516 A1 Feb. 4, 2016 32
0275. In some embodiments, cells expressing the two tries include, but are not limited to: peptoids (e.g., PCT Pub component regulator system, including a histidine kinase and lication No. WO 91/19735), encoded peptides (e.g., PCT a cognate response regulator are contacted with an agent of Publication No. WO93/20242), random bio-oligomers (e.g., interest, also referred to as a test agent (see, for example, PCT Publication No. WO92/00091), benzodiazepines (e.g., Fosteret al., Microbiology 150: 885-896, 2004. The cells can U.S. Pat. No. 5.288,514), diversomers such as hydantoins, be bacterial cells, such as, but not limited to, E. coli. The benzodiazepines and dipeptides (Hobbs et al., Proc. Nat. amount of the histidine kinase including a histidine phospho Acad. Sci. USA 90:6909-6913 (1993)), vinylogous polypep rylated at N3, and/or the amount of the cognate response tides (Hagihara et al., J. Amer: Chem. Soc. 114:6568 (1992)), regulator including a histidine phosphorylated at N3 is mea nonpeptidal peptidomimetics with glucose scaffolding (Hir Sured using any of the assays disclosed herein. schmann et al., J. Amer: Chem. Soc. 114:9217-9218 (1992)), 0276. The amount of histidine kinase including a histidine analogous organic syntheses of Small compound libraries phosphorylated at N3, and/or the amount of the cognate (Chen et al., I Amer: Chem. Soc. 116:2661 (1994)), oligocar response regulator including a histidine phosphorylated at N3 bamates (Cho et al., Science 261: 1303 (1993)), and/or pepti can be compared to a control. Such as a reference value, or the dyl phosphonates (Campbell et al., I Org. Chem. 59:658 amount of histidine kinase including a histidine phosphory (1994)), nucleic acid libraries (see Ausubel, Berger and Sam lated at N3, and/or the amount of the cognate response regu brook, all Supra), peptide nucleic acid libraries (see, e.g., U.S. lator including a histidine phosphorylated at N3 present in a Pat. No. 5,539,083), antibody libraries (see, e.g., Vaughn et cell not contacted with the agent of interest, or contacted with al., Nature Biotechnology, 14(3):309-314 (1996)), carbohy an agent known not to affect a TCS. A decrease in a histidine drate libraries (see, e.g., Liang et al., Science, 274: 1520-1522 kinase including a histidine phosphorylated at N3, and/or a (1996) and U.S. Pat. No. 5,593.853), small organic molecule decrease in the amount of the activated cognate response libraries (see, e.g., benzodiazepines, Baum C&EN, January regulator including a histidine phosphorylated at N3 indicates 18, page 33 (1993); isoprenoids, U.S. Pat. No. 5,569,588; that the agentis of use as an antibacterial. In several examples, thiazolidinones and metathiazanones, U.S. Pat. No. 5,549, a decrease in a histidine kinase including a histidine phos 974; pyrrolidines, U.S. Pat. Nos. 5,525,735 and 5,519,134: phorylated at N3 is assessed. The decrease can be about 10%, morpholino compounds, U.S. Pat. No. 5,506,337; benzodi about 20%, about 30%, about 40%, about 50%, about 60%, azepines, U.S. Pat. No. 5,288,514, and the like). about 70% about 80%, about 90%, about 95%, about 96%, 0280. The compounds tested as modulators of phosphory about 97%, about 98%, about 99% or about 100% as com lation can be any Small organic molecule, or a biological pared to a control. In additional embodiments, a decrease in entity, such as a protein, e.g., an antibody or peptide, a Sugar, pAsp in the cognate response regulator indicates the agent is a nucleic acid, e.g., an antisense oligonucleotide or a of use as an antibacterial. The decrease can be about 10%, ribozyme or siRNA, or a lipid. Typically, test compounds will about 20%, about 30%, about 40%, about 50%, about 60%, be small organic molecules, peptides, circular peptides, about 70% about 80%, about 90%, about 95%, about 96%, siRNA, antisense molecules, ribozymes, and lipids. about 97%, about 98%, about 99% or about 100% as com 0281 Essentially any chemical compound can be used as pared to a control. a potential modulator, although most often compounds can be 0277. In one embodiment, high throughput screening dissolved in aqueous or organic (especially DMSO-based) methods are used that involve providing a combinatorial Solutions. The assays are designed to screen large chemical Small organic molecule or peptide library containing a large libraries by automating the assay steps and providing com number of potential therapeutic compounds (potential modu pounds from any convenient Source to assays, which are lator or ligand compounds). Such "combinatorial chemical typically run in parallel (e.g., in microtiter formats on micro libraries' or “ligand libraries' are then screened in one or titer plates in robotic assays). It will be appreciated that there more assays, as described herein, to identify those library are many Suppliers of chemical compounds, including Sigma members (particular chemical species or subclasses) that dis (St. Louis, Mo.), Aldrich (St. Louis, Mo.), Sigma-Aldrich (St. play a desired characteristic activity. The compounds thus Louis, Mo.), Fluka Chemika-Biochemica Analytika (Buchs identified can serve as conventional “lead compounds' or can Switzerland) and the like. themselves be used as potential or actual therapeutics. 0282. Devices for the preparation of combinatorial librar 0278 A combinatorial chemical library is a collection of ies are commercially available (see, e.g.,357 MPS,390 MPS, diverse chemical compounds generated by either chemical Advanced Chem Tech, Louisville Ky., Symphony, Rainin, synthesis or biological synthesis, by combining a number of Woburn, Mass., 433A Applied Biosystems, Foster City, chemical “building blocks” Such as reagents. For example, a Calif., 9050 Plus, Millipore, Bedford, Mass.). In addition, linear combinatorial chemical library Such as a polypeptide numerous combinatorial libraries are themselves commer library is formed by combining a set of chemical building cially available (see, e.g., ComGenex, Princeton, N.J., blocks (amino acids) in every possible way for a given com Asinex, Moscow, Ru, Tripos, Inc., St. Louis, Mo., ChemStar, pound length (i.e., the number of amino acids in a polypeptide Ltd, Moscow, RU, 3D Pharmaceuticals, Exton, Pa., Martek compound). Millions of chemical compounds can be synthe Biosciences, Columbia, Md., etc.). sized through Such combinatorial mixing of chemical build 0283 Any of the assays disclosed herein can be adapted ing blocks. for high throughput screening. In high throughput assays, 0279 Preparation and screening of combinatorial chemi either soluble or solid state, it is possible to screen up to cal libraries is well known to those of skill in the art. Such several thousand different modulators or ligands in a single combinatorial chemical libraries include, but are not limited day. In particular, each well of a microtiter plate can be used to, peptide libraries (see, e.g., U.S. Pat. No. 5,010, 175, Furka, to run a separate assay against a selected potential modulator, Int. J. Pept. Prot. Res. 37:487-493 (1991) and Houghtonet al., or, if concentration or incubation time effects are to be Nature 354:84-88 (1991)). Other chemistries for generating observed, every 5-10 wells can test a single modulator. Thus, chemical diversity libraries can also be used. Such chemis a single standard microtiter plate can assay about 100 (e.g., US 2016/0033516 A1 Feb. 4, 2016
96) modulators. If 1536 well plates are used, then a single 0288. In one example, the kit further includes one or more plate can easily assay from about 100- about 1500 different chemotherapeutic agents, for example in a container separate compounds. It is possible to assay many plates per day; assay from the antibody. screens for up to about 6,000, 20,000, 50,000, or more than 0289. The following examples are provided to illustrate 100,000 different compounds are possible using integrated particular features of certain embodiments, but the scope of systems. the claims should not be limited to those features exemplified. Kits EXAMPLES 0284 Kits are also provided. The kits will typically 0290 Phospho-specific, monoclonal antibodies (mAbs) include an antibody or antigenbinding fragment that specifi for phosphoester-forming (P. O) amino acids (phospho cally binds a polypeptide including a histidine phosphory serine, phosphothreonine and phosphotyrosine) can be used lated at N3, and/or a conjugate thereof. in the study of protein phosphorylation in cellular signaling. 0285 More than one of the conjugates or antibodies or Histidine (His) phosphorylation is well studied in bacterial antigen binding fragments that specifically bind a polypep signal transduction; however, its role in mammalian signaling tide including a histidine phosphorylated at N3 can be remains largely unexplored due to the lack of pHis mAbs and included in the kit. Thus, the kit can include two or more lability of the phosphoramidate (P N) bond. Both nitrogen antibodies that specifically bind a polypeptide including a atoms (N1 and N3) in histidine's imidazole side chain can be histidine phosphorylated at N3, or a multivalent or bivalent phosphorylated to give rise to one of two pHis isomers; antibody orantigenbinding fragment that specifically binds a 1-phosphohistidine (1-pHis) and 3-phosphohistidine polypeptide including a histidine phosphorylated at N3 and a (3-pHis). conjugate thereof, or a combination thereof, wherein in some 0291 Disclosed herein are mabs that bind specifically to examples each antibody is in a separate container forming the pHis and can distinguish between both pHis isomers. Anti kit. In some embodiments, an antigen binding fragment or bodies were raised in rabbits by immunization with stable conjugate including an antigen binding fragment. Such as an pHis analogues incorporated into degenerate peptide librar FV fragment, is included in the kit. In one example, Such as for ies. Two novel screening assays were developed based on the in vivo uses, the antibody can be a scFv fragment. isomer specific auto-phosphorylation of NME1 (Nm23-H1/ NDPK) and phosphoglycerate mutase (PGAM), which gen 0286 The kit can include a container and a label or pack erate either 1-pHis or 3-pHis respectively. These assays, in age insert on or associated with the container. Suitable con combination with immunoblotting bacterial and mammalian tainers include, for example, bottles, vials, Syringes, etc. The cell lysates and sequencing mAb IgG variable domains, were containers may be formed from a variety of materials such as used to characterize anti-1-pHis and anti-3-pHis antibodies glass or plastic. The container typically holds a composition and select hybridoma clones for establishment of monoclonal including one or more of the disclosed antibodies, antigen cell lines. The sequence independence of these mabs was binding fragments, or conjugates. In several embodiments the determined by peptide dot blot arrays. The pHis mAbs dis container may have a sterile access port (for example the closed herein lack sequence specificity (for antigen binding) container may be an intravenous solution bag or a vial having and do not cross-react with phosphotyrosine or the other pHis a stopper piercable by a hypodermic injection needle). A label isomer. Thus, they can be used for identification and study of or package insert indicates that the composition is used for pHis Substrates in any species using a variety of immunologi treating the particular condition. cal, proteomic and biological assays. 0287. The label or package insert typically will further include instructions for use of a disclosed antibodies or frag Example 1 ments thereof, or conjugates thereof, for example, in a detec tion method. The package insert typically includes instruc Incorporation of Non-Hydrolyzable pHis Analogues tions customarily included in commercial packages of into Degenerate Peptide Libraries diagnostic products that contain information about the usage of the antibodies, such as in particular types of assays. The 0292 Previous attempts to make pHis antibodies using instructional materials may be written, in an electronic form pHis itself as the antigen have been unsuccessful, presumably (such as a computer diskette or compact disk) or may be because the labile phosphoramidate (P N) bond is hydro visual (such as video files). The kits may also include addi lyzed too rapidly after immunization to elicit an immune tional components to facilitate the particular application for response (McAllister et al., Biochemical Society transactions which the kit is designed. Thus, for example, the kit may 41, 1072 (August, 2013)). Until recently, the difficulties in additionally contain means of detecting a label (Such as creating stable pHis peptides have precluded generation of enzyme substrates for enzymatic labels, filter sets to detect pHis-specific monoclonal antibodies (mAbs). The develop fluorescent labels, appropriate secondary labels such as a ment of non-hydrolyzable pHis analogues (Kee et al., Journal secondary antibody, or the like). The kits may additionally of the American Chemical Society, 132, 14327 (October, include buffers and other reagents routinely used for the prac 2010)) has allowed us to develop a novel strategy for genera tice of a particular method. Such kits and appropriate contents tion of both 1-pHis- and 3-pHis-specific mAbs. Phosphonate are well known to those of skill in the art. Kits may include (P C) analogues of both isomers (1-phosphoryltriazolylala recombinant proteins for use as a positive control. For nine 1-pTza and (3-phosphoryltriazolylalanine 3-pTza) example recombinantly expressed and purified PGAM can be can be synthesized by combining the same starting materials included along with 2.3-DPG and instructions for performing (an azidoalanine derivative and an alkyne) in a click-chemis in vitro phosphorylation reactions and analysis by a modified try reaction using different catalysts. Two peptide libraries SDS-PAGE method that has been optimized for the preserva were synthesized consisting of 1-pTza or 3-pTza flanked by tion and detection of a histidine phosphorylated at N3. randomized, neutral, Small side chain amino acids (alanine US 2016/0033516 A1 Feb. 4, 2016 34
A and glycine G)) to serve as immunogens to promote pHis. Immunoblotting with 3-pHis antisera from rabbit 7303 generation of sequence-independent anti-pHis antibodies revealed a heat-sensitive band at the correct size for GST (FIG. 1B). An unphosphorylated version of the peptide librar PGAM (~45 kDa) that was absent when reactions lacked ies (with His in place of the pHis analog (FIG. 1B) was also 2,3-DPG (lane 6). PGAM was subsequently cloned into a synthesized as a negative control. MS analysis of the peptide bacterial expression vector that allowed cleavage of the GST libraries confirmed that incorporation of Ala and Gly for analysis of untagged protein. PGAM was purified from E. occurred randomly and fit with the expected distribution of coli (FIG. 2D) and incubated with or without 2.3-DPG. calculated molecular weights for nine groups of peptides Immunoblotting with anti-3-pHis antisera revealed a heat sharing the same composition of 0-8 Ala and/or Gly residues sensitive band at the correct size for un-tagged PGAM (-25 (FIG. 1C). The N-terminal Cys was used to ligate the pTza kDa, FIG. 2E) phosphorylation at the auto-catalytic residue libraries to the carrier protein Keyhole limpet hemocyanin (H11) was confirmed by mass spectrometry (FIG. 7B). As (KLH) and three rabbits were immunized for each pHis iso was observed for the 1-pHis antibodies, not all antisera that mer to increase the chance of obtaining antibodies with the recognized the pTza analogue could bind actual pHis. In this desired characteristics (FIG. 1D). Rabbits were chosen for case, only antisera from 7303 showed a robust signal to immunization due to recent advances in rabbit hybridoma and 3-pHis on PGAM. At this point, antisera from 7304 did show monoclonal antibody (RabMAb) technology and unique 3-pHis-specific signal, but it was barely detectable above advantages of the rabbit immune system including; strong background. After several subsequent boosts with the 3-pTza immune response to Small epitopes, ability to recognize post peptide library, rabbit 7304 did eventually generate antibod translational modifications with high specificity and pM ies that were on par with 7303. Splenocytes would eventually affinity (Dei Tos et al., Amer: J. Clin. Path. 124, 295 (2005)). be combined from both rabbits to generate hybridomas after an initial attempt with cells from 7303 alone failed to yield Example 2 any mAbs that could bind 3-pHis, though they could bind the 3-pTza analogue. Generation of 3-pHis Antibodies and Development 0294 To confirm that the anti-3-pHis antibodies did not of PGAM In Vitro Screening Assay cross-react with 1-pHis, in vitro phosphorylated NME1 was analyzed along side PGAM (FIG. 2F) and no 1-pHis signal 0293 Bleeds from rabbits immunized with 3-pTza pep tide library (7302, 7303 and 7304) were screened by dot blot was observed. Identical samples blotted with anti-1-pHis using the immunizing, 3-pTza library (FIG. 2A). The 1-pTza antibodies serve as a positive control for phosphorylation of peptide library, the His control library and a pTyr peptide NME1. To determine 3-pHis antibody sensitivity, in vitro were included as controls. Antisera from all three rabbits phosphorylated PGAM was diluted 1:5 (250ng, 50 ng and 10 detected only the 3-pTza immunizing library and did not ng), treated with or without heating and spotted directly on cross-react with the other pHis isomer, the His library or a nitrocellulose (FIG. 2G). A multiclonal/monoclonal 3-pHis pTyr peptide (FIG. 2A). The data as well as (several) crystal Ab was able to detect phospho-PGAM. in a heat-sensitive structures (e.g., PDB ID: 1NSP) show that NME1/2 auto a. phosphorylation generates only 1-pHis; however, it would be Example 3 advantageous to use the same protein for screening antibodies to both pHis isomers. It was thus attempted to allow the Affinity Purification of Polyclonal Anti-1-pHis and generation of both pHis isomers on NME1 through mutagen Anti-3-pHis Antibodies esis of E129. Several mutants were compared with WT 0295) A second version of the 1-pTza and 3-pTza peptide NME1 in an in vitro phosphorylation assay. While each libraries (FIG. 1B) was synthesized with a PEG-linker (poly mutant tested was still able to autophosphorylate, neither ethylene glycol) inserted between the N-terminal Cys residue E129L, E129Q nor E129D (E129K did not express in E. coli) and the Ala/Gly/pTza peptide (FIG. 3A). The PEG-linker elicited any isomer Switching Suggesting other biochemical, libraries were immobilized on agarose beads and used to structural or steric factors also favor 1-pHis over 3-pHis. affinity-purify polyclonal pHis antibodies from rabbit antis Therefore a different enzyme was needed for generation of era. By providing a greater distance between the agarose resin 3-pHis and PGAM was chosen due to solved crystal struc and the pTza analogue, the PEG-linker minimizes steric inter tures of His phosphorylated PGAM and PGAM co-crystal ference to improve binding of pHis antibodies. Fractions lized with its phosphate donor (2,3-diphosphoglycerate 2,3- from the purification were analyzed by SDS-PAGE followed DPG) that show the precise location of the phosphate on H11 by Coomassie staining (FIGS. 3B and 3D) to determine (PDB ID: 2H4Z and 1E58, FIG.2B). PGAM converts 3-phos which fractions contained IgG. Elution fractions (E3 to E11) phoglycerate to 2-phosphoglycerate during glycolysis using were analyzed by immunoblotting of in vitro phosphorylated a unique “ping-pong mechanism in which a catalytic His NME1 and PGAM for anti-1-pHis (rabbit 7305) and anti-3- residue transfers phosphate by forming a 3-pHis phosphoen pHis (rabbit 7303) antibodies respectively (FIGS. 3C and Zyme intermediate. 2.3-DPG directly phosphorylates only 3E). Fractions E6 to E11 (and beyond) contained anti-pHis the N3 nitrogen on PGAM (H11)(Vander Heiden et al., Sci antibodies that could detect as little as 5 ng phospho-NME1 or ence 329, 1492 (Sep. 17, 2010); Davies et al., Acta Crystal phospho-PGAM. Identical membranes were probed with logr 67, 1044 (Sep. 1, 2011); Wang et al., J. Biol. Chem. 281, crude antisera as a positive control. 39642 (Dec. 22, 2006)). To determine if PGAM could be Example 4 phosphorylated in vitro, a GST-PGAM fusion (Novus Bio logical) was incubated with increasing concentrations of 2,3- pTza Peptide Dot Blot Screening and DPG 1 um to 1 mM for 10 min at 30° (FIG. 2C). Reactions Characterization of Anti-1-pHis and Anti-3-pHis were stopped by addition of 5xpH 8.8 sample buffer and Antibodies immediately analyzed by a modified SDS-PAGE method. 0296 Synthetic peptide dot blot arrays were used to fur Identical samples were heated to 95°C. for 10 minto abolish ther demonstrate the pHis isoform specificity of the antibod US 2016/0033516 A1 Feb. 4, 2016 ies and determine if they have any amino acid sequence pTyr peptides (based on sequences of Nck, Eck and Fak specificity. Peptides of defined sequence were chosen based tyrosine kinases) were spotted on nitrocellulose membranes on the best-characterized mammalian pHis proteins: ACLY. with along with their un-phosphorylated counterparts. Anti NME1, PGAM, histone H4, KCa3.1 and GNB1. Peptides pTyrantibodies (4G10) detected only the pTyr peptides (FIG. were synthesized with either His, 1-pTza or 3-pTza flanked 4D) where as none of the peptides were detected by anti-3- by 4 amino acids on either side. Serial dilutions of each pHis (FIG. 4E) or 1-pHis mAbs (FIG. 4F). peptide (500 ng to 160 pg) and the immunizing pTza and control His peptide libraries were spotted onto nitrocellulose Example 6 and blotted with affinity-purified, polyclonal anti-1-pHis or 3-pHis antibodies (FIGS. 3C and 3E). The anti-3-pHis anti Screening of Anti-3-pHis Hybridomas bodies bound only the 3-pTza peptides and the anti-1-pHis antibodies bound only the 1-pTza peptides, regardless of 0298 Anti-3-pHis hybridomas were generated from sple sequence (FIG. 4A). Identical membranes were also probed nocytes harvested from rabbits 7303 and 7304 since antisera with several anti-1-pHis mAbs in order to screen and select from both of these rabbits was able to specifically bind those with the broadest sequence recognition for future pro 3-pHis. Hybridomas were subcloned from four parental teomic studies (FIG. 4B). The anti-1-pHis mAbs all showed multi-clonal cell lines (MC39, MC44, MC56 and MC60) the same binding profiles and displayed some preference for selected from 30 ELISA-positive lines. Up to 12 subclones the NME1/2 H118 peptide. However, with the exception of were obtained from each cell line and cell supernatants were the KCa3.1 peptide, all 1-pTza peptides were detectable initially screened by a 3-pTza peptide ELISA assay and posi down to 20 ng. As observed previously, ability of an antibody tive clones were selected for secondary Screening. Since to bind pTza does not always correlate with ability to bind 3-pHis is involved in bacterial two-component signaling, an genuine pHis, so further validation and characterization of E. coli based screening assay was used (FIG. 6A). these mabs using pHis in proteins is necessary. Since it was 0299 E. coli transformed with a pGEX-PGAM plasmid shown that the anti-3-pHis antibodies do not cross-react with were induced (with IPTG for 3 hr) and crude lysates were either 3-pTza or His peptides, peptide arrays including just loaded on preparative minigels (with a single sample well). 3-pTza peptides were provided to determine the sequence After transfer, PVDF membranes were clamped into a slot specificity of the top 3-pHis mAbs selected from our hybri blotting apparatus to screen up to 40 cell Supernatants simul doma screening efforts (FIG. 4C). A PGAM peptide was taneously (BioRad Miniprotean II Multiscreen Apparatus). included in these arrays since phospho-PGAM was the basis Blotting solutions were normalized to 0.5 ug/ml IgG for of our screening assay. In contrast to the 1-pHis mAbs tested, each cell Supernatant and membranes were probed simulta the 3-His mAbs displayed some variation in binding profiles. neously with a mouse anti-GST antibody to control for pro The 3-pHis mAb 7303-MC-39 was able to detect all 3-pTza tein loading and GST-NME1 expression and also to assess peptides down to about 800 pg, however binding to the detection of auto-phosphorylated GST-PGAM by anti-3- KCa3.1 peptide was relatively poor (~100 ng). 3-pHis mAb pHis mAbs. 30 mM octyl-13-D-glucopyranoside was added 7304-MC-56 showed similar binding characteristics, how to lysis buffer to better solubilize membrane spanning, bac ever it was better at detecting the KCa3.1 peptide (~4 ng) terial histidine kinases. A small-scale Screen was performed while worse at binding the GNB1 peptide. Two or more pHis in parallel using identical E. coli lysates treated with or with mAbs could therefore be combined for use as a multiclonal out heat as a negative control (we had previously shown that antibody (e.g. the anti-pTyr mAb cocktail 4G10R Platinum, phospho-PGAM is sensitive to heat treatment) to determine EMD Millipore) for large-scale proteomic identification of which bands detected by anti-3-pHis mAbs were 3-pHis spe pHis Substrates in order to broaden the sequence coverage cific vs. background or non-specific (FIG. 6B). Identical provided by any one pHis mAb. Surprisingly, a highly membranes were probed with crude anti-3-pHis antisera as a sequence-specific clone was identified that showed high positive control and, as expected, the anti-3-pHis mAbs had affinity for the A/G motif peptide based on the sequence of vastly improved background and non-specific signal levels. ACLY.3-pHis mAb 7304-MC44 was able to detect the ACLY Membranes were probed simultaneously with a mouse anti peptide and the immunizing peptide library down to ~160 pg. GST antibody as a loading control. This indicates that, in this case, the randomized Ala/Gly resi 0300 Since the positive control 3-pHis protein, GST dues in the degenerate peptide library influenced the PGAM, is highly overexpressed in these cells, it is not sur sequence specificity of this mAb. Despite its lack of sequence prising that it represents the strongest signal detected by the independence, it shows very high sensitivity for this sequence anti-3-pHis mAbs, however many other heat-sensitive bands are also detected by the anti-3-pHis mAbs: SC39, SC.56 and context which is present in at least two important pHis Sub SC60 indicating they lack sequence specificity (FIGS.6A and strates, ACLY and SCS. 6B). SC44 only weakly detects PGAM, but strongly detects Example 5 bacterial SCS (FIG. 6B) and mammalian SCS and ACLY (FIGS. 6C and 6D) which both have the sequence motif: pTyr Peptide Dot Blots and Immunoblotting Reveal G-H-A-G-A (FIG. 6E)) similar to the ACLY peptide SC44 No Cross-Reactivity of pHis mAbs showed preference for in the pTza peptide dot blots (FIG. 4C). Cell lysates prepared from a stably transfected HEK293 0297 Since some of the early pTyrantibodies were shown cell line expressing FLAG-Nm23-H1 were blotted with the to cross-react with pHis (Frackelton, et al., Mol. Cell. Biol. 3, anti-3-pHis mAb SC39 which detects 3-pHis on endog 1343 (Aug. 1, 1983, 1983)) and recently reported "pan-pHis’ enously phosphorylated PGAM, but not 1-pHis on FLAG polyclonal antibodies displayed just a 10-fold higher selec NME1 (FIG. 6F). These lysates were treated with or without tivity for pHis over pTyr (Kee et al., Nat. Chem. Biol. 9, 416 heat to demonstrate that the signal detected by the anti-3-pHis (07//print, 2013)), the pHis mAbs were tested for cross-reac mAbs was heat labile, but the same protein band detected by tivity using synthetic pTyr peptides. Serial dilutions of three anti-PGAM antibodies was not. Identical lysates were blotted US 2016/0033516 A1 Feb. 4, 2016 36 with the anti-3-pHis mAb SC44 (FIG. 6G) and while many 60% NaCO/40% NaHCO, (31), 6 Murea, 1 mM Sodium heat-sensitive bands are detected, none of these correspond to ortho-Vanadate (activated), 30 mM octyl-3-D-glucopyrano patterns of bands detected by anti-1-pHis mAbs (FIG. 5B). side (Sigma, Cat. 08001) supplemented with protease inhibi tors (PMSF, pepstatin, leupeptin and aprotinin). Cells were Example 7 sonicated and clarified (10 min. (a) 15,000xg, 4°C.) before diluting lysates 1:5 with Wash/Binding Buffer to decrease Immunoblotting of Cell Lysates Using 1-pHis and urea concentration to 1M prior to binding the affinity column. 3-pHis Antibodies Lysates were passed over the column 2 times and the column 0301 To test for pTyr cross-reactivity of pHis mAbs on was washed with 4x10 ml Wash/Binding Buffer. pHis pro cell lysates, Src-transformed NIH/3T3 fibroblast cell line teins/peptides were eluted with 6x500 ul 100 mM triethy (Psrc 11) were pre-incubated with 1 mM ortho-Vanadate for lamine (TEA), pH 11 (Sigma). Elutions were immediately 30 minto enhance pTyr signals. Non-transformed fibroblasts neutralized with 1M ammonium bicarbonate and 20 ul (pancreatic stellate cells PaSC) were tested in parallel as a samples were saved from each fraction for SDS-PAGE and negative control. western blot analysis. Elution fractions were then lyophilized 0302) An anti-pTyr mAb (4G10) detected an elevated sig in order to remove volatile buffer components, store and nal in the psrc11 cells but not in the PaSC negative control, but preserve pHis modified proteins/peptides until LC-MS/MS neither the 1-pHis nor 3-pHis mAbs detected the elevated could be performed. pTyr signal in Psrc 11 cells. Interestingly, many heat-sensitive 0306 Immunoblotting of fractions from the 1-pHis mAb bands were detected by the anti-pHis mAbs (FIG.5A). These and 3-pHis mAb affinity columns was performed to demon pHis mAbs also do not cross-react with pSer, pThr, the other strate that enrichment of pHis proteins was achieved (FIGS.9 pHis isomer or non-phosphorylated His. We used our anti-1- and 10). For mass spectrometry analysis of proteins eluted pHis mAbs to immunoblot a number of different pancreatic from the pHis mAb columns (Example 12), elution fractions cancer cell lysates and observed common patterns of heat 1-2 and 3-6 were pooled and analyzed together. Immunoblot sensitive bands (FIG. 5B) indicating many proteins in these ting of the elution fractions with anti-pHis mAbs (FIGS. cancer cells are similarly regulated by phosphorylation on 9A-9B and 10 A-B) shows that the pHis was maintained His. Testing of the 3-pHis mAbs by blotting bacterial and throughout the immunoaffinity purification. Immunoblotting mammalian cell lysates (FIG. 5C) demonstrated that many, of elution fractions with protein-specific antibodies includ ing; anti-NME1/2, anti-tubulin and anti-ACLY antibodies heat-sensitive 3-pHis bands were observed. shows that these proteins were bound by the 1-pHis mAb Example 8 column. Subsequent analysis by MS (Example 12) also iden tified these specific proteins in the pHis mAb column elution Immunofluorescence of pHis Proteins Using fractions. Anti-3-pHis mAbs Example 10 0303. In addition to validating the anti-3pHis mAbs for immunoblotting, they were tested in other applications, Substrates including and immunofluorescence (IF). 0304 Primary murine macrophages were stained with our 0307 Primary amino acid sequences of pHis substrates anti-3-pHis mAbs (FIGS. 8A and 8B). A staining pattern were aligned to look for motifs or commonalities in the resi distinct from that obtained with anti-1-pHis mAb staining dues flanking pHis. Protein names, sequence (SEQID NOS. was observed, suggesting that different sets of proteins are 10-34) and amino acid positions of the pHis residues are regulated by 1-pHis and 3-pHis in an isoform-specific man shown. ner. In contrast to the anti-1-pHis staining, punctate structures were observed throughout the cytosol, and especially pro nounced puncta were visible in the nuclei of these cells. This DAYTYTEHARRKTVTAMDVVY Histone H4 His indicates that some unknown, but specific compartments or organelles have increased 3-pHis signals compared with KYTKYSEHDMNKWLDELKGD Annexin I H246 other regions of the cell. As a negative control, slides were boiled for 10 min in acetic acid and this treatment success fully abolished the observed anti-1-pHis staining. MESSFGERHEAEERIMESRSEI Annexin I H293 Example 9 LKKALEHLEEWLAELKTPA Annexin I H103
Immunoaffinity Chromatography Histone H4 H18 0305 Anti-1-pHis and anti-3-pHis affinity resins were generated by crosslinking the mabs to protein-A agarose WITHGKCHYRESEKYSSWAN NSP2 H225 beads using DSS or BS3 (Pierce). mAbs were coupled to beads at 1 mg IgG/ml of settled protein-A beads. After FROSEREKHRK-EREWNSMV KCa3.1 H358 crosslinking, resins were packed in 10 ml columns (BioRad) and equilibrated with Wash/Binding Buffer (50 mM Tris pH KRWISHNSERGIWKHLE PGAM 1 H186 8.0, 30 mMSodium carbonate (prepared by dilution of Lysis Buffer with 1 M Tris pH 7.0). Cell lysates were prepared by rinsing with cold TD buffer and scraping cells into 500ul cold YKEYLERHGESAWNLENRFS PGAM 1 H11 Denaturing Lysis Buffer (Sodium carbonate buffer pH 10.0 US 2016/0033516 A1 Feb. 4, 2016 37
- Continued Example 12 RCIFVCHERMDVVFGKYWL STS1 (UASH3) H391 3-pHis mAb Immunofluorescence Reveals Staining of Centrosomes, Spindle Poles and Midbodies KSVLVVRHGERVDOIFGKA STS2 0309 Macrophages stained with 3-pHis mAbs displayed a SSESOFGHAGACANSETA ACL H760 pattern distinct from 1-pHis staining. Punctate structures were observed throughout the cytosol; however, no co-local ization was observed when antibodies specific for organelle PPGRRMGHAGAIIAGGKGGA SCS H299 markers (e.g., ATP Synthase, LC3, Rab5, C-tubulin and LAMP1, (FIGS. 12A-12B) were tested for co-staining. In MKGVRMGHSAIVE SCS (yeast) contrast to macrophages, staining of HeLa cells with 3-pHis mAbs was primarily nuclear (though curiously absent from WRGQRDNAGAATEEFIK Aldolase C (rat) D319 nucleoli) and distinctive cell cycle-dependent patterns were observed. Cells in prometaphase through telophase displayed QEEMTYSHDNEECGITSVSF GB GNB1 H266 remarkable 3-pHis staining of spindle poles (FIGS. 11 F 11K). Interphase cells displayed staining of centrosomes and cells in prophase were observed with duplicated centrosomes GKCPLNPHSHEGTYGSIFTNA P-Selectin Hff1. (FIG. 11G). An apparent burst of 3-pHis signals was observed in dividing cells and this seemed to last from prometaphase CPNPHSHLTYSWFENAAF P-Selectin H773 through anaphase. To confirm this observation, HeLa cells were co-stained with 3-pHis mAbs and spindle pole markers NAIFRAAHSEESSAGEFGFS Chea. H48 Aurora-A and Y-tubulin (FIGS. 12H-12I). To demonstrate that 3-pHis mAbs stained primarily spindle poles and not HEIFRAAHEKGMSAEMGYT CHEA BACSU H46 spindles, cells were co-stained with C-tubulin (FIG. 12J). 3-pHis mAbs also stained structures devoid of Aurora-A, Y-tubulin and C-tubulin in both HeLa and U2Os cells and OVERNIHGSDSWKSAEKEI Nm23 -H2 H118 these appeared to be the midbody of cells in late telophase. (FIGS. 11H-11K and 12C-12E). A series of negative controls OVERNIHGSDSWESAEKEI Nm23-H1 H118 using the immunizing pTza peptide libraries were performed. Only the 1-pTza peptides could block 1-pHis staining (FIGS. VFIANISHEERTPLNGILGM Sln1 H576 12F-12I, 12P-12Q) while only the 3-pTza peptides could block 3-pHis staining (FIGS. 12K-12N, 12R-12S). Addition TELDNIGHFEKGSSAALGLQ Ypd1 (HPt) H64 ally, boiling slides for 10 min in citrate buffer reduced both 1-pHis and 3-pHis staining (FIGS. 12J and 12O). ANSRSSAHMSSNAIOR Coc10 H314 Example 13 Example 11 Enrichment and Identification of Proteins by pHis Immunofluorescence Staining Reveals Association mAb Immunoaffinity Purification and SILAC of 1-pHis with Outer Membrane of Phagosomes LC-MS/NIS 0308 To test the ability of these mabs to detect pHis 0310 Traditional immunoprecipitation methods are not proteins by immunofluorescence staining, HeLa cells were amenable to pHis preservation and detection. A method for stained with the 1-pHis mAb SC 1-1. A distinct staining pat immunoaffinity purification of pHis substrates using immo tern was observed in which most cells had a large (1-2 um) bilized pHis mAbs was developed. Reusable pHis mAb resins compartment that stained strongly in the Surrounding region were packed in chromatography columns and used to enrich but lacked interior pHis staining (FIG. 11A). It was surmised pHis phosphoproteins from cell lysates prior to analysis by that these might be acidic compartments such as phagosomes or autophagosomes, and this was tested by using primary LC-MS/MS. pNME1 and pPGAM were used to test the pHis murine macrophages to look for specific staining of phago isomer selectivity of the high density mAb columns. NME1 Somes. Macrophages isolated from bone marrow were incu and PGAM were phosphorylated in vitro, denatured (6 M bated with fluorescently-labeled dextrans to track internaliza urea, pH 10), mixed together and incubated with either a tion into phagosomes. Cells were also incubated with 1-pHis or 3-pHis mAb column. Purification fractions were LysoTracker prior to fixation to label acidic compartments. immunoblotted with 1- and 3-pHis mAbs as well as NME1 1-pHis staining was absent in nuclei as well as the interior of and PGAM antibodies and quantification demonstrates that phagosomes in macrophages co-labeled with the internalized pNME1 was enriched in elution fractions from the 1-pHis dextrans and LYSOTRACKER(R), but staining was pro mAb column while pPGAM was enriched in elutions from nounced in the regions surrounding these compartments the 3-pHis mAb column. (FIGS. 11B-11D). Remodeling of the actin cytoskeleton Sup ports the extension of pseudopodia at sites of particle engulf 0311. In order to determine which proteins bind the col ment and F-actin assembles around nascent phagosomes. Co umns in a pHis-dependent manner and which are likely false staining with mAb SC 1-1 and phalloidin-TRITC revealed a positives that bind non-specifically, stable isotope labeling lack of co-localization of 1-pHis with actin filaments (FIG. was used by amino acids in cell culture (SILAC) to metaboli 11E). cally label FLAG-NME1 293 cells. Initially, both the light US 2016/0033516 A1 Feb. 4, 2016 and heavy labeled (Arg'C/N and Lys 'C) cells were proteins were enriched in the untreated, light vs. heavy lysates lysed using identical denaturing and alkaline conditions (6M that had been treated to reduce pHis. urea, pH 10) to preserve pHis. The heavy lysates were then acidified (pH 6) and heated at 65° C. for 30 min to reduce 0312 Significant enrichment was observed for NME1/2 pHis. A dramatic decrease of pHis in the heavy lysates was (55-fold) by the 1-pHis mAb column as well as enrichment of confirmed by immunoblotting. Both lysates were neutralized, PGAM (4-fold) and other known 3-pHis proteins including: diluted (1:5 to 1 Murea, pH 8), mixed together and one half histone H4 (22-fold) and ACLY (11-fold). Proteins corre was passed over the 1-pHis mAb column while the other half sponding to recently identified pHis phosphopeptides (Lapek was passed over the 3-pHis mAb column. LC-MS/MS analy et al., 2015, Naunyn-Schmiedeberg's archives of pharmacol sis was performed on tryptic peptides derived from proteins ogy388, 161-173) including: TUBB, TCP1/CCT1,YWHAB, that eluted off the columns at pH 11. A SILAC ratio (light/ LDHA, RPS3A and GAPDH were also enriched from 5 to heavy) was calculated for each peptide to determine which 11-fold (Tables 1 and 2). TABLE 1. SILAC Ratios Determined by LC-MS/MS Analysis Indicate Enrichment of Known pHis Proteins by pHis mAb Immunoaffinity Purification SEQ ID 1pH 1-pH 3-pH 3-pH Protein Sequence'k NO: Site E1 E2 E1 E2
NME1 OWGRNIIHGSDSWES 35 H.118 15. OS 51.86 118 2.14
NME2 OWGRNIIHGSDSWKS 36 36H118 1559 55.96 3.46
NME4 HISRNWIHASDSWEG 37 371
NMEs DDLRNALHGSNDFAA 38 H127
NME6 TDTRNTTHGSDSVVS 39 H129
NMEf DGIRNAAHIGPDSFAS 4 O H2O6
Histone H4 GKGGAKRHRKWLRDN 41 H18 1914 9.8 22.82 17.9
KCa31 FROVRLKHRKLREQV 42 H358
TRPVs LRONTLGHLNLGLNL 43 H711
GNB1 OELMTYSHDNIICGI 44 H226 1.21
PGAM YKLWLIRHGESAWNL 45 H11 4.25 4. O1 2. 44
ACLY SSEWOFGHAGACANQ 46 H760 5.29 7.2 11. 62 2.99
SCS PPGRRMGHAGAIIAG 47 H299 1.68
P-Selectin GKCPLNPHSHLGTYG 48 H771
TUBB GNNWAKGHYTEGAEL 49 H105 3.55 4. 49 4.33 2. O9
TCP1 EETERSLHDAIMIWR SO H34 6 6.10 5.99 5.98 3.2O
YWHAB KTALCFRHLMKOLLN 51 H2O2 2. O4 7.43 1.63
LDHA GEMMDLOHGSLFLRT 52 H67 4.18 6.50 6. 6 O 3. O8
RPS3A LGKLMELHGEGSSSG 53 H232 7.21 9. 10 1.O. 41 4. 98
GAPDH TMEKAGAHLQGGAKR 54 H111 3.4 O 4. 45 4.76 3.32
Annotated pHis sites in protein sequences are highlighted in bold. SILAC ratio (light/heavy) indicates fold enrichment of proteins by pHis mAb affinity purification. Values from two sequential elution fractions (elution 1 E1 and elution 2 E2 are reported as the median value of all quantified peptides that Correspond to the listed protein. 2 not expressed in Cell line tested; b related protein family members or isoform(s) were enriched; sequences from pHis phosphopeptides detected by MS (Lapek et al., 2015, Naunyn-Schmiedeberg's archives of pharmacology 388, 161-173); - indicates not detected. See also Table 2.
US 2016/0033516 A1 Feb. 4, 2016 49
TABLE 2-continued
28O 156 786 350 630 SO6 1-pHis 3-pHis -pHis 1-pHis 1-pHis 3-pHis only only or 3-pHis and 3-pHis TOMM22 TOMM7OA TOP2A TOP2B TOR1AIP1 TRAP1
UBA2 UBE2D2 UBE2D3 UBE2G2 UBE2O UCHLS UFM1 UHRF1 UPF1 UQCR10 UQCRC1 UQCRC2 USO1 WAT1 WDAC2 WDAC3 WTA1 VTN WARS WDR36 WDR5 WDR61 WDR77 WDR82 XPO1 XPOS XRCCS XRCC6 YARS YWHAB YWHAE YWHAG YWHAH YWHAQ YWHAZ ZNF 638
0314 Examples of polypeptides including a histidine detecting a tumor or determining the effectiveness of a che phosphorylated at N3 are presented in above. One or more of motherapeutic agent. In some embodiments, 1, 2, 3, 4, 5, 6, 7, these polypeptides including a histidine phosphorylated at N3 8, 9, or 10 of the listed polypeptides can be detected. In other can be detected in the methods disclosed herein, such as for embodiments, 5, 10, 15, 20, 25, 30, 35, 40, 45 or 50 of these US 2016/0033516 A1 Feb. 4, 2016 50 polypeptides can be detected. Any combination of the diluted to 1 mg/ml and stored in 1.5 ml aliquots at -20°C. For polypeptides listed in Tables 1 and/or Table 2 can be used in the primary immunization, Freund's Complete Adjuvant (0.5 the presently disclosed methods. In some embodiments, the ml per rabbit) was emulsified with antigen (0.5 ml per rabbit) methods utilize any combination of the polypeptides listed in using 5 ml syringes. Antigens were administered via intrad ermal injection of 50 ul at 20 sites on the back. Every three Table 2. In some examples a combination of any of the listed weeks, Subsequent boosts were administered intradermally in “pHis3 only' phosphorylated polypeptides is utilized. Incomplete Freund's Adjuvant. Bleeds were collected in 10 ml tubes from the central ear artery ten days after each boost. Example 14 Rabbit antisera was collected after spinning down blood (2,400xg for 10 min at 4°C.) that was allowed to clot for Materials and Methods 24-48 hr. Antisera was frozen at -20°C. for longterm storage. 0315 Materials and Chemicals: 0319 Synthetic Peptide Synthesis: 0316 Reagents and their sources were as follows: FLAG 0320 Sequences of synthetic peptides and pTyrphospho NME1/Nm23-H1 mammalian expression vector was from peptides used in this study are as follows; Nck pY105 Addgene (Cat. 25000), GST-PGAM1 (Cat. H00005223-P01) (CGERLpYDLNMPAYVK (SEQ ID NO: 55), Nck Y105 was from Novus Biological, pCEX-6P-1 GST-fusion vector (CGERLYDLNMPAYVK (SEQID NO. 56)), Eck (Eph.A2) (Cat. 28-9546-48) and PreScission Protease (27-0843-01) pY588 (CLKPLKTpYVD (SEQID NO:57)), Eck (EphA2) were from GE Life Sciences, RosettaTM 2 (DE3) competent Y588 (CLKPLKTYVD (SEQ ID NO:58)) and FAK pY397 cells (Cat. 71397), the pTyr mAb clone 4G10 (Cat. 05-321) (AVSVSETDDpYAEIIDEEDTYT (SEQ ID NO. 59)). Pep was from EMD Millipore, 2,3-diphospho-D-glyceric acid tides were synthesized using Fmoc Solid phase synthesis. pentasodium salt (Cat. SC-2 13964) and NME1 mAb (Cat. 0321) Peptide Dot Blot Screening of Rabbit Antisera: SC-136141) were from Santa Cruz, Alexa Fluor R 680 goat 0322 Peptide dot blots were used initially to screen rabbit anti-rabbit IgG secondary antibody (Cat. A-21109), GST antiseratiter. The 1-pTza and 3-pTza peptide libraries. His mAb (Cat. 13-6700), Oregon Green-Dextran?R488 and control library and a pTyr peptide (Nck pY105) were dis LysoTracker Red DND-99 (Cat. L-7528) were from Life Solved in water at a stock concentration of 1 mg/ml. 1:5 serial Technologies, goat anti-mouse IgG (H+L) secondary anti dilutions (500, 100, 20, 5, 1 and 0.2 ng/ul) were prepared for body, DyLight 800 conjugate (Cat. 35521) was from Pierce each peptide and 1 ul of each dilution was spotted on nitro and Casein Blocking Buffer was from BioRad. Amicon cellulose membrane and allowed to dry for 1-2 hr at RT. Ultrafree 0.5-5K MWCO centrifugal filters (Cat. Membranes were blocked for 1 hr at RT in Casein Blocking UFC500396), Immobilon-FL PVDF membranes (Cat. Buffer (0.1% casein, 0.2xPBS-/-) and incubated with rabbit IPFL00010), the Mini-PROTEAN II Multiscreen Apparatus antisera or pre-immune serum (diluted 1:1,000 in Blocking (Cat. 170-4017) and Casein blocking solution (Cat. 161 Buffer with 0.1% Tween-20) for 1 hr at RT or overnight at 4° 0783) was from BioRad. Glutathione resin (Cat. L00206) was C. All subsequent steps were as described for “immunoblot from Genscript, Ampicillin, chloramphenicol, Adenosine ting with anti-pHis antibodies’. 5'-triphosphate disodium salt (Cat. A2383), SDS, Trizma 0323 Protein Expression and Purification: base, glycine, Isopropyl B-D-1-thiogalactopyranoside (Cat. 0324 NME1, NME2 and PGAM were subcloned into the 16758), Anti-FLAG M2 mAb (Cat. F1804), Anti-C-tubulin pGEX-6P-1 GST-fusion vector. The following primers were (Cat. T5168), Anti-y-tubulin (T6557), Freund's Complete Adjuvant (Cat. F5881), Freund's Incomplete Adjuvant (Cat. used for PCR amplification and insertion of BamHI and F5506), iodoacetamide and octyl-f-D-glucopyranosideside EcoRI restriction sites; (Cat. 08001) were from Sigma-Aldrich. Anti-Aurora A mAb was from Abcam (Ab13824). The SulfoLink Coupling Resin NME1-Fw, (Cat.44999), the chemical crosslinkers DSS (Cat. 21555)and (SEQ ID NO: 6O) BS3 (Cat. 21580) and SILAC reagents (Cat. 89983 and 5'- GATCGGATCCATGGCCAACTGTGAGCGTAC-3' 89990) were from Pierce/Thermo Scientific. All protein elec NME1-Rev, trophoresis equipment including; Four Gel Caster (Cat. (SEQ ID NO : 61) SE275), Mighty Small II Mini Deluxe Vertical Unit (Cat. 5'-GATCGAATTCTCATTCATAGATCCAGTTCTC-3', , SE260-10A-0.75), Mighty Small Mini Transfer Tank (Cat. NME2-Fw, TE22) were from Hoefer, RPMI 1640 and 1% Glutamax (Cat. (SEQ ID NO: 62) 35050-061) were from Gibco/Life technologies (Cat. 11875 5'- GATCGGATC-CATGGCCAACCTGGAGCGCAC-3' 119), Rabbit Hybridoma Supplement A was from Epitomics/ AbCam (Cat. EP-401), cell culture grade 55 mM 2-Mercap NME2-Rev, (SEQ ID NO: 63) toethanol was from Invitrogen (Cat. 2198-023), IS-MAB-CD 5'- GATCGAATTCTTATTCATAGAC-CCAGTCATG-3' Serum-free medium was from Irvine Scientific (Cat. 91104) and and 1% antibiotic/antimycotic solution was from Cellgro (Cat. 30-0004-CI). Tris(2-carboxyethyl)phosphine hydro PGAM-Fw, chloride (TCEP) was from Roche Applied Science. (SEQ ID NO: 64) Paraformaldehyde (PFA) was from Electron Microscopy Sci 5'- GATCGGATCCATGGCCGCCTACAAACTGGTG-3' ences (Cat. 15710). PGAM-Rew (SEO ID NO : 65) 0317. Immunization of Rabbits s" - GATCGAATTCTCACTTCTTGGCCTTGCCCTG-3' 0318 New Zealand White rabbits (three per antigen) were immunized using a standard prime-boost regimen and pre ROSETTATM2 (DE3) competent cells were transformed with immune serum was collected from each rabbit prior to immu pGEX-NME1, pGEX-NME2 or pGEX-PGAM and starter nization. The pTza peptide libraries were coupled to KLH, cultures from single colonies were grown at 37°C. for 16 hr US 2016/0033516 A1 Feb. 4, 2016 in LB broth supplemented with 100 ug/ml ampicillin and 34 or 12.5% resolving gels. Electrophoresis buffer recipes were ug/ml chloramphenicol with shaking at 225 RPM. Expres as follows: Running Buffer: (1x20 L, pH 8.5) 20g SDS, 60 g sion cultures were diluted from starter cultures with the same Trizma Base, 288 g glycine, dH0 to 20 L, Transfer Buffer: medium to an A600 of 0.2. Protein expression was induced (1x4 L. pH 8.5) 56.7 g glycine, 4g SDS, 12 g Trizma Base, with 1 mMIPTG at an A600 of 0.6 for 3 hr at 30° C. Bacteria 800 ml MeOH, dH0 to 4 L. All electrophoresis steps were were pelleted (5,000xg for 10 min at 4°C.) and resuspended performed at 4°C. and samples were resolved at 90-100V for in 1 ml GST Lysis/Wash Buffer (PBS, pH 8.0, 1% Triton 2-3 hr. Proteins were transferred to Immunoblon-FL PVDF X-100, 5% glycerol, 1 mM DTT)/50 ml culture. Lysates were membranes at 30V for 12-18 hr at 4° C. and immediately sonicated on ice and clarified by centrifugation (14,000xg for incubated for 45-60 min at RT or >2 hr at 4° C. in Casein 30 min at 4°C.). Glutathione resin was equilibrated with GST Blocking Buffer (0.1% casein, 0.2xPBS -/-). Primary anti lysis/wash buffer and 1 ml washed resin/200 ml culture was bodies were diluted in blocking buffer with 0.1% Tween-20, incubated with clarified bacterial lysates for 2 hr at 4° C. incubated with membranes for 1 hr at RT, or 3-18 hr at 4°C. Resin was then pelleted and the Supernatant was removed Membranes were washed at least three times for 10 min each before washing at least 3 times with 10 ml wash buffer. with 0.1% TBST before incubation with secondary antibod Washed resin was resuspended in 2 ml PreScission Protease ies for 45-50 min at room temperature. Rabbit anti-pHis Buffer (20 mM Tris pH=7.0, 150 mM NaCl, 1 mM DTT, 0.5 antisera was stored at -20°C. was used at 1:1,000 for dot blots mM EDTA) and cleavage of GST-tag was performed over and western blots. Affinity purified, polyclonal anti-pHis night at 4°C. using 2 ul PreScission Protease (5U/200 ml antibodies were stored at 4° C. and used at 1:200. After culture). Cleaved resin was pelleted (1000xg for 5 min at 4 incubation with secondary antibodies, membranes were C.) and supernatants were transferred to fresh tubes. Buffer washed least four times for 10 min each with 0.1% TBST. exchange into Storage Buffer B (50 mM Tris-HCl, pH 8.0, Immunoblots and Coomassie stained gels were imaged on a 100 mM NaCl, 1 mM DTT) was performed using centrifugal LI-COR Odyssey Infrared Imaging System. Duplexing of filters and proteins were concentrated to ~1 ug/ul. Purified primary antibodies was performed using both channels of the proteins were supplemented with 10% glycerol and stored at Odyssey by co-incubating membranes with rabbit primary -80° C. Quantification of purified proteins was performed by antibodies with mouse; anti-FLAG-M2, anti-GST or anti densitometry of Coomassie stained gels using a BSA stan NME1. For rabbit antibodies, Alexa Fluor R 680 Goat Anti dard curve. Rabbit IgG secondary antibodies were diluted 1:20,000 in 0325 NME and PGAM In Vitro Phosphorylation Assays: blocking buffer supplemented with 0.1% tween-20 and 0326 Invitro autophosphorylation of purified NME1 and 0.01% SDS. For mouse primary antibodies, Goat Anti-Mouse NME2 (10-30 ngful) was performed in TMD buffer (20 mM IgG secondary antibody (DyLight 800 conjugate) were Tris-HCl, pH 8.0.5 mMMgCl, 1 mMDTT) at RT. Fresh ATP diluted 1:20,000 in blocking buffer supplemented with 0.1% was added to initiate reactions which were allowed to proceed tween-20 and 0.01% SDS and incubated alone or co-incu at RT for 10 min. Reactions were stopped by addition of 5xpH bated with Alexa Fluor R 680 Goat Anti-Rabbit secondary 8.8 sample buffer and analyzed immediately by SDS-PAGE antibodies for duplexed primary antibodies. (see immunoblotting with anti-pHis antibodies). Reactions 0329 Slot Blot Screening of Hybridoma Cell Superna lacking ATP or treated briefly with heat or acid served as tants: negative controls. In vitro autophosphorylation of PGAM 0330. A slot blotting apparatus was used to screen up to 40 was performed as described for NME1 except 2,3-diphospho anti-pHis hybridoma cell Supernatants simultaneously. Pre glycerate (2,3-DPG) was used as the phosphate donor instead parative slab gels were cast using custom Teflon combs to of ATP and incubations were carried out at 30° C. Reactions create stacking gels that contained one large sample well and lacking 2.3-DPG or treated briefly with heat served as nega a single lane for loading protein molecular weight standards. tive controls. Heat treatment was performed after addition of SDS-PAGE was performed as described above for immuno 5xpH 8.8 sample buffer for 10-15 min at 95°C. Acid treat blotting with anti-pHis antibodies. Briefly, PVDF membranes ment was performed by adding 25 ul 1N HCl to a 100 ul were clamped into the BioRad Miniprotean II Multiscreen reaction and incubating at 37°C. for 15 min. Reactions were Apparatus and blotting was performed as instructed by the neutralized with 25ul 1 NNaOH before addition of 5xpH 8.8 manufacturer. IgG concentrations of hybridoma cell-super sample buffer. natants (obtained from IgG ELISA assays performed by 0327 Immunoblotting with Anti-pHis Antibodies: Epitomics) were normalized to 0.5 ug/ml for screening of 0328. In general, immunoblotting with anti-pHis antibod anti-pHis mAbs (dilution factors ranged from 1:5 to 1:500) by ies was performed with modifications to standard procedures dilution with casein blocking buffer supplemented with 0.1% to help preserve pHis for detection. Buffers were adjusted to Tween-20. 600ul of each diluted mAb was pipetted into each pH 8-9 to stabilize pHis and methods were modified to avoid chamber and incubated on top of the membrane for at 4°C. for heating samples. Protein samples were prepared in pH 8.8 3 hr. After three 10 min washes of the membranes in the sample buffer (5x=10% SDS, 250 mM Tris-HCl, pH 8.8, apparatus with 0.1% TBST, the membranes were transferred 0.02% Bromophenol blue, 50% glycerol, 50 mM EDTA, 500 to blotting containers and washed again using larger Volumes mMDTT) for electrophoresis. Mammalian whole cell lysates of 0.1% TBST. Incubation with secondary antibodies imag were prepared by rinsing 70-100% confluent 10 cm dishes ing was performed as described above. twice with 5 ml cold TD buffer (TBS -/-, pH 8). Cells were 0331 Affinity Purification of Polyclonal pHis Antibodies: scraped directly into 2xpH 8.8 sample buffer, incubated on 0332 Affinity columns for purification of polyclonal anti ice and a cup horn Sonicator was used (3-5x5 sec bursts) to bodies from rabbitantisera were prepared by covalently cou disrupt cells and shear DNA. Lysates were clarified by cen pling 2 mg of either PEG-1-pTza or PEG-3-pTza peptide trifugation (14,000xg for 5-15 min at 4°C.) and analyzed libraries to 2 ml SulfoLink agarose resin according to manu immediately using freshly prepared Bis-Tris polyacrylamide facturers instructions. 5 ml of the corresponding anti-1-pTza minigels with a modified, pH 8.8 stacking gel and either 10% oranti-3-pTza antisera was thawed on ice and diluted 1:2 with US 2016/0033516 A1 Feb. 4, 2016 52
PBS (pH 7.4). Diluted antiserum was clarified by centrifuga by centrifugation at 1,100 RPM for 5 min. 20-30 ug RNA was tion (8,000xg for 20 min at 4°C.) and a sample was taken for isolated from each hybridoma using the Qiagen RNA Easy analysis (“Input'). The columns were equilibrated with 15 ml Mini Kit according to the manufacturer's instructions. RT PBS and the clarified antiserum was passed over columns PCR was performed to using the Superscript III First-Strand three times. The flow through (FT) was collected and the Synthesis System (Life Technologies Cat. 18080-051) to syn column was then washed twice with PBS (15 ml="Wash 1”, thesize cDNA from RNA primed with oligo(dT) primers. 1 ml="Wash 2'). The antibodies were eluted by addition of PCR primers used to amplify and sequence IgG V and V. 0.1 M glycine (pH 2.5) buffer and 15x1 ml and 4x2 ml elution regions are listed. (SEQ ID NOS. 66-88) fractions were collected and immediately neutralized to pH 7 with sodium phosphate. A final wash step was performed with TABLE 3 PBS (15 ml="Wash 3, 1 ml="Wash 4). Samples from each elution and wash fraction and a 10 ul sample of column Rabbit V, V, primers material (“col”) were saved for SDS-PAGE analysis followed Light Chain by Coomassie staining (FIG. 3D) to monitor binding and elution of IgG. Elution fractions were also tested for anti-3- Vk1 A 5 GTGATGACCCAGACTCCA 3 pHis antibodies by immunoblotting in vitro phosphorylated Vk1 C s' GTGCTGACCCAGACTCCA 3" PGAM (FIG.3E). 0333 pTza Peptide Dot Blot Arrays: Vk2 A 5 GATATGACCCAGACTCCA 3 0334 1-pTza, 3-pTza or His was incorporated into syn thetic peptides of defined sequences from mammalian pro Vk2 C s' GATCTGACCCAGACTCCA 3" teins with mapped pHis sites. The peptides used were as wk3 s' TTTGATTTCCACATTGGTGCC 3 follows: ACLY-like H760 (AGAG-X-AGAG, SEQ ID NO: 89), PGAM H11 (VLIR-X-GESA, SEQID NO:90), NME1 wk4 s' TAGGATCTCCAGCTCGGTCCC 3' H118 (RNII-X-GSDS, SEQ ID NO: 91), Histone H4 H18 (GAKR-X-RKVL, SEQID NO:92), KCa3.1 H358 (VRLK vk5 C s' TTTGACCACCACCTCGGTCCC 3 X-RKLR, SEQ ID NO: 93) and GNB1 H266 (MTYS-X- vk5 G s' TTTGACGACCACCTCGGTCCC 3 DNII, SEQ ID NO: 94) where X=His, 1-pTza or 3-pTza. Peptides were dissolved in water at a stock concentration of 1 W.1 s' GTGCTGACT CAGTCGCCCTC 3' mg/ml. 1:5 serial dilutions (500, 100, 20, 5, 1 and 0.2 ng/ul) were prepared for each peptide and 1 ul of each dilution was v2 s' GCCTGTGACGGTCAGCTGGGTCCC 3' spotted on nitrocellulose membrane and allowed to dry for 1-2 hr at RT. The pTza peptide stock solutions had a pH of 4-5 Heavy Chain so these were neutralized by addition of 25 ul of 1M Tris VH1 A 5 AGTCGGTGGAGGAGTCCAGG 3." buffer pH 8.0. Immunoblotting was performed as described above. VH1 G 5 AGTCGGTGGAGGAGTCCGGG 3." 0335 Cell Culture and Stable Cell Line Generation: 0336 Human embryonic kidney cells (HEK 293), HeLa, WH2 5 AGTCGGTGAAGGAGTCCGAG 3." Psrc 11 and pancreatic stellate cells (PaSCs) were cultured in VH3 C 5 AGTCGCTGGAGGAGTCCGGG 3." a 37°C., 5.0% CO, incubator. HEK 293 and HeLa cells were grown in DMEM (4.5 g/liter glucose, L-glutamine, and VH3 T 5 AGTCGTTGGAGGAGTCCGGG 3." sodium pyruvate) supplemented with 10% FBS without anti biotics. Prsc1 1 were grown in DMEM supplemented with 4% VH4 CA 5 AGCAGCAGCTGATGGAGTCCGG 3." FCS and Pen/Strep. VH4 GA 5 AGGAGCAGCTGATGGAGTCCGG 3." 0337 For generation of stably transfected FLAG-NME1 cell lines, HEK 293 cells were transiently transfected with 15 VH4 CG 5 AGCAGCAGCTGGTGGAGTCCGG 3." ug FLAG-NME1 mammalian expression vector in a 10 cm dish using the calcium phosphate method. 48 hr post-trans VH4 GG 5 AGGAGCAGCTGGTGGAGTCCGG 3." fection cells were split and plated in 96-well plates and stable wh5 AC 5 AGAGACGGTGACCAGGGTGCC 3' transfectants were selected with G418. After 10 days in selec tion medium, Surviving clones were trypsinized and wh5 GC 5 GGAGACGGTGACCAGGGTGCC 3' expended in 6-well plates. Single colonies were selected, expanded and cell lysates were analyzed by immunoblotting wh5 AT 5 AGAGATGGTGACCAGGGTGCC 3' with anti-NME1 and anti-FLAG antibodies to confirm stable wh5 GT 5 GGAGATGGTGACCAGGGTGCC 3' integration. 0338 Rabbit Hybridoma Cell Culture: 0339 pHis hybridoma cell lines were maintained with 0342 cDNA from RT-PCR reactions was analyzed by gel Growth Medium (1xHAT 240E medium:500 ml RPMI 1640, electrophoresis and reactions yielding products of the correct 40 ml Rabbit Hybridoma Supplement A (Epitomics), 55uM size (300-350 bp) were sequenced with both forward and 2-Mercaptoethanol and 10% FBS) in a 37° C., 5% CO, incu reverse primers. bator. Briefly, cultures were seeded at 1x10 cells/ml and split (0343 pHis mAb Production and Purification: at 70-80% confluency by aspirating media and replacing with 0344 pHis hybridomas were expanded from 10 cm2 fresh medium. Cell lines were stored in liquid N in freezing dishes to T175 flasks in 60 ml Growth Medium. Once con media (90% FBS, 10% DMSO). fluent, cells were collected by centrifugation at 1,100 RPM 0340 Sequencing pHis Antibody IgGV, and V. Regions: for 5 min in 2x50 mL tubes. 22.5 ml supernatant was removed 0341 Anti-1-pHis and Anti-3-pHis hybridomas were cul from each tube and cells were resuspended in the remaining tured as described above and -750,000 cells were collected 2x7.5 ml medium. Cells were transferred back into the same US 2016/0033516 A1 Feb. 4, 2016
T175 flask and 45 ml fresh Serum-Free Medium (SFM; IS original database was reversed (Penget al., JProteome Res. 2: MABCD chemically defined medium (Irvine Scientific), 1% 43-50 (2003)) supplemented with UniProt sequences for antibiotic/antimycotic Supplement and 1% Glutamax) was either human NM23 or human PGAM1. The ProLuCID added. Cells were acclimated to this low-serum (2.5%) con search was performed using full enzyme specificity, static dition for 3 days. Cells were spun, as before, into 2x50 ml modification of cysteine due to carboxyamidomethylation tubes and all media was aspirated from pellets. Cells were (57.02146) and differential modification of histidine, serine, resuspended in 2x7.5 ml SFM and transferred back into their threonine and tyrosine due to phosphorylation (79.9663). The respective T175 flasks with 45 ml (60 ml total) SFM. Cells data was searched using a precursor mass tolerance of 50 ppm were grown in SFM until cell viability was approximately and a fragment ion mass tolerance of 10 ppm. ProLuCID 50% (-7-10 days). To harvestantibodies, cells were collected search results were assembled and filtered using the DTASe by centrifugation. Cell Supernatants were spun again in fresh lect (version 2.0) algorithm (Tabb et al., J Proteomics Res. tubes at 3,000 RPM for an additional 15 min. For antibody 1:21-26, (2002)). All peptide-spectra matches had less than purification, 1 ml Protein-A-agarose beads were incubated 10 ppm mass error. Phosphorylation site assignment was overnight at 4°C. with 50 ml SFM hybridoma cell superna confirmed by manual annotation of spectra. tant. The Protein-A-agarose beads were pelleted at 4,000xg 0350 Stable Isotope Labeling (SILAC) and pHis mAb for 5 min at 4° C. and washed with 3x with 10 ml PBS (pH Immunoaffinity Purification of Proteins for LC-MS/MS: 7.4). Anti-pHis IgG was eluted with two sequential additions of 1 ml Elution Buffer (200mMGlycine, pH 2.8), which were 0351 1-pHis mAb SC 1-1 and 3-pHis mAb SC39-4 affin immediately neutralized with 1.0 M Tris-HCl (pH 8.3). Anti ity resins were generated by crosslinking purified mAbs to pHis mAb concentrations were measured by IgG A280 and protein-A agarose beads using DSS or BS3. mAbs were stored at 4°C. Purified mAbs were used at a concentration of coupled to beads at 1 mg IgG/ml of protein-A beads. After 0.5 ug/ml (1:2000) and validated by immunoblotting cell crosslinking, pHis mAb resins (-750 ul each) were packed in lysates and dot blotting in vitro phosphorylated NME1 10 ml chromatography columns (BioRad) and stored at 4°C. (1-pHis) or PGAM (3-pHis). in or equilibrated with Wash/Binding Buffer (50 mM Tris, 30 mM sodium carbonate pH 8 (prepared by dilution of 100 mM 0345 Mass Spectrometry-Detection of pHis Sites on sodium carbonate buffer pH 10 with HO and 1 M Tris pH 7)). NME1 and PGAM: Stable isotope labeling by amino acids in cell culture (SI (0346. In vitro phosphorylated NME1 and PGAM samples LAC) was performed on FLAG-NME1 293 cells which were were first denatured in 8 Murea and then reduced and alky metabolically labeled with Arg (C/N) and Lys ('C). lated with 10 mMTCEP and 55 mM iodoacetamide respec These “heavy” labeled cells were used as an internal negative tively. The samples were diluted to 2 Murea with 100 mM control for pHis mAb binding. Non-labeled, “light FLAG Tris pH 8.5 and then digested with trypsin Promega at room NME1 293 cells were cultured in parallel. For cell lysis, temperature for 4 hours. precautions were taken to preserve pHis and avoid salts and 0347 Each protein digest was pressure-loaded onto 250 detergents that can interfere with downstream MS analysis. micron i.d. fused silica capillary Polymicro Technologies Both “heavy” and “light” cells were lysed under identical columns with a Kasil frit packed with 3 cm of 5 micron C18 denaturing conditions to inhibit phosphatase activity and resin Phenomenex. After desalting, each column was con alkaline pH was used to stabilize pHis. All cell lysates (10x10 nected to a 100 micron i.d. fused silica capillary Polymicro cm plates) were prepared by rinsing cells (80-90% conflu Technologies analytical column with a 5 micron pulled-tip, ent) with cold TD buffer and scraping cells into 500 ul cold packed with 10 cm of 5 micron C18 resin Phenomenex). Denaturing Lysis Buffer (100 mM sodium carbonate pH 10 0348. Each column was placed inline with an Easy 60% NaCO/40% NaHCO), 6 Murea, 30 mMoctyl-3-D- NanoLC II pump Thermo Scientific and the eluted peptides glucopyranoside Supplemented with protease inhibitors were electrosprayed directly into a Q Exactive mass spec (PMSF, pepstatin, leupeptin and aprotinin)). All “light' trometer Thermo Scientific. The buffer solutions used were lysates were pooled together and all “heavy lysates were 10 mM ammonium bicarbonate pH 5 (buffer A) and 100% pooled prior to Sonication and clarification (10 min (a) methanol (buffer B). The 90 minute elution gradient had the 15,000xg, 4°C.). Light lysates were set aside on ice and kept following profile: 10% buffer Bat 5 minutes, to 55% buffer B at pH 10 to preserve pHis while the heavy lysates were treated at 50 minutes, to 99% buffer B at 65 minutes and continuing to reduce or abolish pHis by acidification (pH 6) and moder to 75 minutes. A cycle consisted of one full scan mass spec ate heating (65° C. for 30 min). The combination of decreased trum (400-1600 m/z) at 70 K resolution followed by up to 10 pH and moderate heat treatment was found to be important for data-dependent MS/MS (fixed first mass, 100 m/z) at 17.5 K significant reduction of pHis, since neither treatment alone resolution using a normalized collision energy (NCE) of 25 was sufficient. Extreme heating (e.g. 95-100°C.) was avoided with 20% stepped NCE. Charge state exclusion was selected to decrease carbamylation of proteins in the presence of urea. Such that only +2 and +3 ions were selected for fragmenta Both “heavy” and “light” lysates were then diluted 1:5 with tion. Dynamic exclusion was set at 10 seconds. Application of Wash/Binding Buffer to decrease urea concentration to 1 M mass spectrometer scan functions and HPLC solvent gradi and neutralized pH to 8. The “heavy” and “light” lysates were ents were controlled by the Xcalibur data system Thermo pooled and passed over 1-pHis and 3-pHis mAb columns 2 Scientific. times. The column was washed four times with 10 ml Wash/ (0349 MS/MS spectra were extracted using RawXtract Binding Buffer. pHis proteins were eluted in three fractions (version 1.9.9) (McDonald et al., Rapid Commun Mass Spec (E1 to E3) with 6x600 ul 100 mM triethylamine (TEA), pH trom. 18:2162-21682004 (2004)). MS/MS spectra were 11. Samples were saved from each fraction for analysis by searched with the ProLuCID algorithm (Eng et al., JAm Soc SDS-PAGE and immunoblotting (FIG. 11). Elution fractions Mass Spectrom. 5: 976-989, (1994)) againsta Saccharomyces were frozen on dry ice and lyophilized overnight to remove cerevisiae and Escherichia coli database concatenated to a volatile buffer components. Elution fractions were stored at decoy database in which the sequence for each entry in the -80° C. until LC-MS/MS was performed. A full list of pro US 2016/0033516 A1 Feb. 4, 2016 54 teins identified and the SILAC ratio for each peptide quanti (6.020129). The data was searched using a precursor mass fied using this method is shown in Example 13 and peptides tolerance of 50 ppm and a fragmention mass tolerance of 600 from known pHis proteins quantified using this method are ppm. Both “light' and “heavy’ ProLuCID search results were listed in Table 1. assembled and filtered using the DTASelect (version 2.0) 0352. The 1-pHis and 3-pHis mAb columns were tested algorithm (Tabb et al., 2002). A minimum of one peptide was for isomer selectivity by mixing pNME1 (1-pHis positive required for each protein identification and peptides were control) and pPGAM (3-pHis positive control) together. In required to be fully tryptic. All peptide-spectra matches had vitro phosphorylation reactions using 80 ug recombinant less than 10 ppm mass error. The protein false positive rate NME1 or PGAM were performed separately in 800 ulTMD was below one percent for all experiments. Quantification buffer with 1 mM ATP at RT or 2.3-DPG at 30° C. respec was performed using the Census algorithm (Parket al., 2008). tively for 10 min.2 ml Sodium carbonate buffer pH 10 with 10 The proline conversion correction option was selected. Pep Murea (6 M final concentration) was then added to each tide SILAC ratios were normalized based on the analysis of reaction to denature proteins, stabilize pHis and replicate the pre-column mixed lysates (data not shown). A determinant alkaline and denaturing lysis conditions used to prepare cell factor of 0.8 and a singleton profile score of 0.9 were used to lysates for the pHis mAb immunoaffinity purification filter quantified peptides. The composite score was selected described above. The two reactions were then pooled together for reporting SILAC ratios. For each quantified protein, three (2.8 ml pNME1+2.8 ml pPGAM) and diluted 1:5 with 28 ml pieces of information are reported: the number of quantified Wash/Binding Buffer to reduce urea to 1 M and pH to 8. peptides, the median peptide SILAC ratio and all peptide 1-pHis and 3-pHis mAb columns were equilibrated with SILAC ratios. SILAC ratios reported are the untreated Wash/Binding Buffer and the half of the diluted pNME1 and (“light') sample divided by the acid/heat-treated (“heavy”) pPGAM reaction mixtures were passed over each pHis mAb sample. A SILAC ratio greater than two was considered to be column two times. The columns were washed with 4x10 ml indicative of specific binding to the pHis columns. Wash/Binding Buffer and 3x600 ul elution fractions were 0356 Immunofluorescence: collected using 100 mM TEA pH 11. Samples of each elution 0357 Primary murine macrophages were differentiated were mixed with 5xpH 8.8 sample buffer and stored at -80° from bone marrow progenitors (Zhang et al., 2008) plated on C. for analysis by SDS-PAGE and immunoblotting with pHis, cover slips and incubated O/N in fresh medium. Cells were NME1 and PGAM antibodies. incubated with 10 ug/ml Oregon Green-Dextran R488 and/or 0353 Mass Spectrometry: LysoTracker (50 nM) for 1-2 hrprior to fixation with 4% PFA 0354 Lyophilized elution samples were first denatured in for 10 min. Negative controls were performed by boiling 8 Murea and then reduced and alkylated with 10 mMTCEP slides for 5-10 min in 0.01 M citrate buffer or by pre-incuba and 55 mM iodoacetamide respectively. The samples were tion of pHis mAbs with pTza blocking peptides 5 g/ml. diluted to 2 M urea with 100 mM Tris pH 8.5 and then Cells were permeabilized in blocking buffer (PBS, 5% serum digested with trypsin Promega overnight at 37° C. Each (2' Ab species), 2% BSA, 0.1% Tween) with 0.1% Triton protein digest was pressure-loaded onto 250 micron i.d. fused X100 for 1 hr at 4°C. Primary antibodies were diluted to 1 silica capillary Polymicro Technologies columns with a ug/ml in blocking buffer and incubated with slides for 2 hr at Kasil frit packed with 3 cm of 5 micron C18 resin Phenom 4° C. Slides were washed 5x with cold PBS--0.1% Tween and enex. After desalting, each column was connected to a 100 incubated with 2". Abdiluted 1:400 in blocking buffer for 1 micron i.d. fused silica capillary Polymicro Technologies hr at 4°C. Slides were mounted on cover slips after washing analytical column with a 5 micron pulled-tip, packed with 10 5x with cold PBS+0.1% Tween. See also Extended Experi cm of 5 micron C18 resin Phenomenex. Each column was mental Procedures for immunostaining of HeLa cells. placed inline with a 1200 quaternary HPLC pump Agilent 0358 1-pHis and 3-pHis Immunofluorescence Staining of and the eluted peptides were electrosprayed directly into a HeLa Cells: LTQ Orbitrap Velos mass spectrometer Thermo Scientific. 0359. HeLa cells were plated on cover slips in 6-well The buffer solutions used were 5% acetonitrile/0.1% formic plates and grown until 30-50% confluent. Cells were washed acid (buffer A) and 80% acetonitrile/0.1% formic acid (buffer with sterile filtered PBS (pH 7.4) and fixed for 20 min at RT B). The 120 minute elution gradient had the following profile: in 4% PFA (16% PFA diluted 1:4 in PBS). After fixation, cells 10% buffer Bat 10 minutes, to 45% buffer Bat 90 minutes, to were washed 2x with PBS and then permeabilized with PBS 100% buffer Bat 100 minutes and continuing to 110 minutes. (pH 9.0)+0.1% Triton X-100 at RT for 15 min. Cells were A cycle consisted of one full scan mass spectrum (300-1600 then washed 3x with PBS (pH 9.0) before blocking in sterile m/z) at 60 K resolution followed by up to 20 data-dependent filtered, 0.1% TBST with 4% BSA at RT for 30 min. Cover collision induced dissociation (CID) MS/MS spectra. Charge slips were transferred to parafilm, incubated with primary state exclusion was selected Such that only +2 and +3 ions antibodies (1-pHis mAb SC 1-1, diluted 1:100 FIG. 10A) at were selected for fragmentation. Dynamic exclusion was set RT for 90 min and washed 3x with 0.1% TBST for 5 min. at 120 seconds. Application of mass spectrometer scan func Secondary antibodies (anti-rabbit 488 nm and anti-mouse tions and HPLC solvent gradients were controlled by the 568 nm) were diluted 1:2,000 in TBST plus 1% BSA and Xcalibur data system Thermo Scientific. incubated with coverslips at RT for 60 min in the dark. Cells 0355 MS/MS spectra were extracted using RawXtract were then washed 3 times with TBST at RT for 5 min. To stain (version 1.9.9) (McDonaldet al., 2004). MS/MS spectra were nuclei, coverslips were incubated with PBS plus DAPI at searched with the ProLuCID algorithm (Eng et al., 1994) 1:4,000 for 2 min and washed 3 times with PBS. Alternative against a human Uniprot database concatenated to a decoy methods for fixation were used for co-staining experiments. database in which the sequence for each entry in the original Methanol fixation (Aurora A and C-tubulin) was performed database was reversed (Peng et al., 2003). For protein identi by incubating cells at -20°C. for 15 min in methanol. Pre fications, the “light ProLuCID search was performed using permeabilization (Y-tubulin and O-tubulin) was performed by no enzyme specificity and static modification of cysteine due incubation of cells 45 sec in 0.5% Triton X-100 followed by to carboxyamidomethylation (57.02146). A second “heavy 4% PFA for 15 min at pH 9.0. Coverslips were mounted and ProLuCID search was performed and additionally considered stored in the dark prior to Scanning on a confocal microscope static modification of arginine (10.008269) and lysine under 60x magnification. US 2016/0033516 A1 Feb. 4, 2016 55
0360. In view of the many possible embodiments to which the scope of the invention. Rather, the scope of the disclosure the principles of the disclosure may be applied, it should be is defined by the following claims. We therefore claim as our recognized that the illustrated embodiments are only invention all that comes within the scope and spirit of these examples of the disclosure and should not be taken as limiting claims.
SEQUENCE LISTING
<16 Os NUMBER OF SEO ID NOS: 94
<21 Os SEQ ID NO 1 &211s LENGTH: 2O3 212s. TYPE: PRT <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Synthetic polypeptide
<4 OOs SEQUENCE: 1 Glu Ser Gly Gly Arg Lieu Val Thr Pro Gly Gly Ser Lieu. Thir Lieu. Thr 1. 5 1O 15 Cys Thr Val Ser Gly Phe Ser Leu Ser Arg Tyr Asn Met Gly Trp Val 2O 25 3 O Arg Glin Ala Pro Gly Lys Gly Lieu. Glu Trp Ile Gly Trp Ile Pro Phe 35 4 O 45 Arg Gly Ser Leu Lys Tyr Ala Thr Trp Ala Thr Gly Arg Phe Thr Ile SO 55 60 Ser Arg Thr Ser Thr Thr Val Asp Lieu. Arg Met Thr Gly Lieu. Thir Ala 65 70 7s 8O Ala Asp Thr Ala Thr Tyr Phe Cys Val Arg Ser Ser Asp Gly Phe Asp 85 90 95 Leu Trp Gly Pro Gly Thr Lieu Val Thr Val Ser Ser Gly Glin Pro Llys 1OO 105 110 Ala Pro Ser Val Phe Pro Leu Ala Pro Cys Cys Gly Asp Thr Pro Ser 115 12O 125 Ser Thr Val Thr Lieu. Gly Cys Lieu Val Lys Gly Tyr Lieu Pro Glu Pro 13 O 135 14 O Val Thr Val Thir Trp Asn Ser Gly Thr Lieu. Thir Asn Gly Val Arg Thr 145 15 O 155 16 O Phe Pro Ser Val Arg Glin Ser Ser Gly Lieu. Tyr Ser Leu Ser Ser Val 1.65 17 O 17s Val Ser Val Thr Ser Ser Ser Gln Pro Val Thr Cys Asn Val Ala His 18O 185 190 Pro Ala Thr Asn Thr Llys Val Asp Llys Thr Val 195 2 OO
<21 Os SEQ ID NO 2 &211s LENGTH: 2O7 212s. TYPE: PRT <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Synthetic polypeptide
<4 OOs SEQUENCE: 2 Glu Ser Gly Arg Gly Lieu Val Glin Pro Gly Gly Ser Lieu. Thir Lieu. Thr 1. 5 1O 15
Cys Thr Ala Ser Gly Phe Ser Ile Asp Ser Tyr Gly Phe Ser Trp Val 2O 25 3 O Arg Glin Ala Pro Gly Lys Gly Lieu. Glu. His Ile Gly Tyr Lieu. Thir Ala 35 4 O 45 US 2016/0033516 A1 Feb. 4, 2016 56
- Continued Gly Gly Arg Ala Phe Tyr Ala Ser Trp Ala Lys Ser Arg Ser Thir Ile SO 55 6 O Thr Arg Asn Thr Asn Glu Asn Thr Val Thr Lieu Lys Met Thr Ser Lieu. 65 70 7s 8O Thir Ala Ala Asp Thr Ala Thr Tyr Phe Cys Ala Lys Lieu. Gly Ser Gly 85 90 95 Asn Pro Val Ala Ile Trp Gly Pro Gly Thr Lieu Val Thr Val Ser Ser 1OO 105 11 O Gly Glin Pro Lys Ala Pro Ser Val Phe Pro Leu Ala Pro Cys Cys Gly 115 12 O 125 Asp Thr Pro Ser Ser Thr Val Thr Lieu. Gly Cys Lieu Val Lys Gly Tyr 13 O 135 14 O Lieu Pro Glu Pro Val Thr Val Thir Trp Asin Ser Gly Thr Lieu. Thir Asn 145 150 155 160 Gly Val Arg Thr Phe Pro Ala Val Arg Glu Ser Ser Gly Lieu. Tyr Ser 1.65 17O 17s Lieu. Asn Ser Val Gly Lys Val Thr Ser Ser Ser Gln Pro Val Thr Cys 18O 185 19 O Asn Val Ala His Pro Ala Thr Asn. Thir Lys Val Asp Llys Thr Val 195 2OO 2O5
<210s, SEQ ID NO 3 &211s LENGTH: 210 212. TYPE PRT <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Synthetic polypeptide
<4 OOs, SEQUENCE: 3 Ser Val Lys Glu Ser Glu Gly Gly Lieu. Ile Llys Pro Gly Gly Ile Lieu. 1. 5 1O 15 Thr Lieu. Thr Cys Thr Ala Ser Gly Phe Ser Leu Ser Ser Tyr Gly Phe 2O 25 3O Ser Trp Val Arg Glin Ala Pro Gly Lys Gly Lieu. Glu. His Ile Gly Tyr 35 4 O 45 Lieu. His Ala Asn Gly Arg Ala Tyr Tyr Ala Thir Trp Ala Lys Ser Arg SO 55 6 O Ser Thir Ile Thr Arg Asn Thr Asn Lieu. Asn Thr Val Thr Lieu Gln Leu 65 70 7s 8O Thir Ser Lieu. Thir Ala Ala Asp Thr Ala Thr Tyr Phe Cys Ala Lys Ile 85 90 95 Gly Ser Val Ser Asp Val Ala Ile Trp Gly Pro Gly Thr Lieu Val Thr 1OO 105 11 O Val Ser Ser Gly Glin Pro Lys Ala Pro Ser Val Phe Pro Leu Ala Pro 115 12 O 125
Cys Cys Gly Asp Thr Pro Ser Ser Thr Val Thr Lieu. Gly Cys Lieu Val 13 O 135 14 O
Lys Gly Tyr Lieu Pro Glu Pro Val Thr Val Thir Trp Asn Ser Gly Thr 145 150 155 160
Lieu. Thir Asn Gly Val Arg Thr Phe Pro Ser Val Arg Glin Ser Ser Gly 1.65 17O 17s
Lieu. Tyr Ser Leu Ser Ser Val Val Ser Val Thr Ser Ser Ser Glin Pro 18O 185 19 O US 2016/0033516 A1 Feb. 4, 2016 57
- Continued Val Thr Cys Asn Val Ala His Pro Ala Thr Asn. Thir Lys Val Asp Llys 195 2OO 2O5
Thir Wall 21 O
<210s, SEQ ID NO 4 &211s LENGTH: 210 212. TYPE: PRT <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Synthetic polypeptide
<4 OOs, SEQUENCE: 4 Ser Val Lys Glu Ser Glu Gly Gly Lieu Phe Llys Pro Thr Asp Thr Lieu. 1. 5 1O 15 Thr Lieu. Thr Cys Thr Val Ser Gly Phe Ser Lieu. Thir Thr Tyr Gly Phe 2O 25 3O Ser Trp Val Arg Glin Ala Pro Gly Lys Gly Lieu. Glu Trp Ile Gly Tyr 35 4 O 45 Val Arg Ser Asp Gly Arg Ile Tyr Tyr Thir Ser Trp Ala Lys Ser Arg SO 55 6 O Ser Thr Lieu. Thr Arg Asn Thr Asn Lieu. Asn Thr Val Thr Lieu. Ile Met 65 70 7s 8O Thir Ser Lieu. Thr Val Ala Asp Thr Ala Thr Tyr Phe Cys Ala Lys Ile 85 90 95 Gly Ser Gly Thr Gly Val Ala Ile Trp Gly Pro Gly Thr Lieu Val Thr 1OO 105 11 O Val Ser Ser Gly Glin Pro Lys Ala Pro Ser Val Phe Pro Leu Ala Pro 115 12 O 125 Cys Cys Gly Asp Thr Pro Ser Ser Thr Val Thr Lieu. Gly Cys Lieu Val 13 O 135 14 O Lys Gly Tyr Lieu Pro Glu Pro Val Thr Val Thir Trp Asn Ser Gly Thr 145 150 155 160 Lieu. Thir Asn Gly Val Arg Thr Phe Pro Ser Val Arg Glin Ser Ser Gly 1.65 17O 17s Lieu. Tyr Ser Leu Ser Ser Val Val Ser Val Thr Ser Ser Ser Glin Pro 18O 185 19 O Val Thr Cys Asn Val Ala His Pro Ala Thr Asn. Thir Lys Val Asp Llys 195 2OO 2O5
Thir Wall 21 O
<210s, SEQ ID NO 5 &211s LENGTH: 216 212. TYPE: PRT <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Synthetic polypeptide
<4 OOs, SEQUENCE: 5
Ala Glin Phe Wal Met Thr Glin. Thir Pro Ala Ser Wall Glu Ala Wal Wall 1. 5 1O 15
Gly Gly. Thr Val Thir Ile Llys Cys Glin Ala Ser Arg Asp Thr Gly Asp 2O 25 3O
Gly Lieu. Ile Trp Tyr Glin Gln Llys Pro Gly Glin Pro Pro Lys Arg Lieu. 35 4 O 45 US 2016/0033516 A1 Feb. 4, 2016 58
- Continued
Ile Tyr Lys Ala Ser Thr Val Ala Ser Gly Val Pro Ser Arg Phe SO 55 6 O
Gly Arg Gly Ser Gly Thr Asp Phe Thr Lieu. Thir Ile Ser Asp Luell Glu 65 70 7s 8O
Ala Asp Ala Ala Thr Tyr Tyr Cys His Ser Asn Phe Asn Arg 85 90 95
Trp Thr Tyr Gly Asn Ala Phe Gly Gly Gly Thr Glu Wall Wall Wall 1OO 105 11 O
Gly Asp Pro Val Ala Pro Thr Val Lieu. Ile Phe Pro Pro Ala Ala 115 12 O 125
Glin Val Ala Thr Gly Thr Val Thir Ile Val Cys Val Ala Asn 13 O 135 14 O
Phe Pro Asp Val Thr Val Thir Trp Glu Val Asp Gly Thir Thir Glin Thir 145 150 155 160
Thir Gly Ile Glu Asn Ser Lys Thr Pro Glin Asn Ser Ala Asp Cys Thir 1.65 17O 17s
Asn Lieu. Ser Ser Thir Lieu. Thir Lieu. Thir Ser Thr Glin Tyr Asn Ser 18O 185 19 O
His Lys Glu Tyr Thr Cys Llys Val Thr Glin Gly Thr Thir Ser Wall Wall 195 2OO 2O5
Glin Ser Phe Asn Arg Gly Asp Cys 21 O 215
<210s, SEQ ID NO 6 &211s LENGTH: 217 212. TYPE: PRT <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Synthetic polypeptide
<4 OOs, SEQUENCE: 6
Asp Pro Wal Met Thr Pro Thr Pro Ser Phe Thir Ser Ala Ala Wall Gly 1. 5 1O 15
Gly Thr Val Thir Ile Asn Cys Glin Ser Ser Glin Ser Wall Trp Arg Asn 2O 25
Lys Asn Lieu Ala Trp Tyr Glin Glin Llys Pro Gly Glin Pro Pro Arg 35 4 O 45
Lell Ile Tyr Ala Ile Ala Thr Lieu. Asp Ser Gly Val Pro Ser Arg Phe SO 55 6 O
Ser Gly Ser Gly Ser Gly Thr Glin Phe Thr Lieu. Thr Ile Ser Asp Wall 65 70 7s
Glin Cys Asp Asp Ala Ala Thr Tyr Tyr Cys Val Gly His Gly Ser 85 90 95
Glu Asn Asp Ala Tyr Tyr Ala Phe Gly Gly Gly Thr Glu Wall Wall Wall 1OO 105 11 O
Gly Asp Pro Val Ala Pro Thr Val Lieu. Ile Phe Pro Pro Ser Ala 115 12 O 125
Asp Leu Val Ala Thr Gly Thr Val Thir Ile Val Cys Wall Ala Asn 13 O 135 14 O
Tyr Phe Pro Asp Val Thr Val Thir Trp Glu Val Asp Gly Thir Thir Glin 145 150 155 160
Thir Thr Gly Ile Glu Asn Ser Lys Thr Pro Glin Asn Ser Ala Asp 1.65 17O 17s US 2016/0033516 A1 Feb. 4, 2016 59
- Continued
Thr Tyr Asn Lieu Ser Ser Thr Lieu. Thir Lieu. Thir Ser Thr Glin Tyr Asn 18O 185 19 O Ser His Lys Glu Tyr Thr Cys Llys Val Thr Glin Gly Thr Thr Ser Val 195 2OO 2O5 Val Glin Ser Phe Asn Arg Gly Asp Cys 21 O 215
<210s, SEQ ID NO 7 &211s LENGTH: 223 212. TYPE: PRT <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Synthetic polypeptide
<4 OO > SEQUENCE: 7 Asp Pro Val Met Thr Glin Thr Pro Ser Ser Thr Ser Ala Ala Val Gly 1. 5 1O 15 Gly Thr Val Thir Ile Asn Cys Glin Ser Ser Glu Ser Ile Tyr Asn Asn 2O 25 3O Lys Asn Lieu Ala Trp Tyr Glin Glin Llys Pro Gly Glin Ser Pro Arg Arg 35 4 O 45 Lieu. Ile Tyr Ser Ile Ser Thr Lieu Ala Ser Gly Val Ser Ser Arg Phe SO 55 6 O Lys Gly Ser Gly Ser Gly Thr Glin Phe Thr Lieu. Thir Ile Ser Asp Val 65 70 75 8O Gln Cys Asp Asp Ala Ala Thr Tyr Tyr Cys Val Gly Tyr Tyr Tyr Ser 85 90 95 Gly Gly Tyr Tyr Tyr Ser Gly Ser Ala Ala Tyr Tyr Ala Phe Gly Gly 1OO 105 11 O Gly Thr Glu Val Val Val Lys Gly Asp Pro Val Ala Pro Thr Val Lieu. 115 12 O 125 Ile Phe Pro Pro Ser Ala Asp Leu Val Ala Thr Gly Thr Val Thir Ile 13 O 135 14 O Val Cys Val Ala Asn Lys Tyr Phe Pro Asp Val Thr Val Thir Trp Glu 145 150 155 160 Val Asp Gly. Thir Thr Glin Thir Thr Gly Ile Glu Asn Ser Lys Thr Pro 1.65 17O 17s Gln Asn Ser Ala Asp Cys Thr Tyr Asn Lieu. Ser Ser Thr Lieu. Thir Lieu. 18O 185 19 O Thir Ser Thr Glin Tyr Asn Ser His Lys Glu Tyr Thr Cys Llys Val Thr 195 2OO 2O5 Gln Gly. Thir Thr Ser Val Val Glin Ser Phe Asn Arg Gly Asp Cys 21 O 215 22O
<210s, SEQ ID NO 8 &211s LENGTH: 216 212. TYPE: PRT <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Synthetic polypeptide
<4 OOs, SEQUENCE: 8 Asp Gly Val Met Thr Pro Thr Pro Ala Ser Ala Ser Ala Gly Val Gly 1. 5 1O 15
Gly Thr Val Thir Ile Asn Cys Glin Ser Ser Glin Ser Ile Tyr Lys Lys US 2016/0033516 A1 Feb. 4, 2016 60
- Continued
2O 25 3O Tyr Ile Ala Trp Tyr Glin Gln Llys Pro Gly Glin Pro Pro Lys Arg Lieu. 35 4 O 45 Ile Tyr Ser Thr Ser Thr Lieu Ala Ser Gly Val Ser Ser Arg Phe Lys SO 55 6 O Gly Ser Gly Ser Gly Thr Glin Phe Thr Lieu. Thir Ile Ser Asp Val Glin 65 70 7s 8O Cys Asp Asp Val Ala Thr Tyr Tyr Cys Val Gly Tyr Tyr Ile Ile Thr 85 90 95 Asn Asp Ala Tyr Tyr Ser Phe Gly Gly Gly Thr Glu Val Val Val Lys 1OO 105 11 O Gly Asp Pro Val Ala Pro Thr Val Lieu. Ile Phe Pro Pro Ser Ala Asp 115 12 O 125 Lieu Val Ala Thr Gly Thr Val Thir Ile Val Cys Val Ala Asn Llys Tyr 13 O 135 14 O Phe Pro Asp Val Thr Val Thir Trp Glu Val Asp Gly Thr Thr Glin Thr 145 150 155 160 Thr Gly Ile Glu Asn Ser Lys Thr Pro Glin Asn Ser Ala Asp Cys Thr 1.65 17O 17s Tyr Asn Lieu Ser Ser Thr Lieu. Thir Lieu. Thir Ser Thr Glin Tyr Asn Ser 18O 185 19 O His Lys Glu Tyr Thr Cys Llys Val Thr Glin Gly Thr Thr Ser Val Val 195 2OO 2O5 Glin Ser Phe Asin Arg Gly Asp Cys 21 O 215
<210s, SEQ ID NO 9 &211s LENGTH: 10 212. TYPE: PRT <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Synthetic peptide 22 Os. FEATURE: 221 NAMEAKEY:A MOD RES <222s. LOCATION: (1) . . (1) 223 OTHER INFORMATION: ACETYLATION 22 Os. FEATURE: <221s NAME/KEY: MISC FEATURE <222s. LOCATION: (2) ... (5) <223> OTHER INFORMATION: Xaa = Gly or Ala 22 Os. FEATURE: <221s NAME/KEY: MISC FEATURE <222s. LOCATION: (6) . . (6) <223> OTHER INFORMATION: Xaa = His, 1-pTza or 3-pTza 22 Os. FEATURE: <221s NAME/KEY: MISC FEATURE <222s. LOCATION: (7) . . (10) <223> OTHER INFORMATION: Xaa = Gly or Ala 22 Os. FEATURE: <221s NAME/KEY: MOD RES <222s. LOCATION: (10) ... (10) 223 OTHER INFORMATION: AMIDATION
<4 OOs, SEQUENCE: 9
Cys Xaa Xala Xala Xaa Xaa Xaa Xala Xala Xala 1. 5 1O
<210s, SEQ ID NO 10 &211s LENGTH: 21 212. TYPE: PRT <213> ORGANISM: Artificial Sequence US 2016/0033516 A1 Feb. 4, 2016 61
- Continued
22 Os. FEATURE: <223> OTHER INFORMATION: Synthetic peptide 22 Os. FEATURE: <221s NAME/KEY: MOD RES <222s. LOCATION: (8) ... (8) 223 OTHER INFORMATION: PHOSPHORYLATION
<4 OOs, SEQUENCE: 10 Asp Ala Val Thr Tyr Thr Glu. His Ala Lys Arg Llys Thr Val Thir Ala 1. 5 1O 15 Met Asp Val Val Tyr 2O
<210s, SEQ ID NO 11 &211s LENGTH: 21 212. TYPE: PRT <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Synthetic peptide 22 Os. FEATURE: <221s NAME/KEY: MOD RES <222s. LOCATION: (8) ... (8) 223 OTHER INFORMATION: PHOSPHORYLATION
<4 OOs, SEQUENCE: 11 Llys Tyr Thr Lys Tyr Ser Llys His Asp Met Asn Llys Val Lieu. Asp Lieu. 1. 5 1O 15 Glu Lieu Lys Gly Asp 2O
<210s, SEQ ID NO 12 &211s LENGTH: 21 212. TYPE: PRT <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Synthetic peptide 22 Os. FEATURE: <221s NAME/KEY: MOD RES <222s. LOCATION: (8) ... (8) 223 OTHER INFORMATION: PHOSPHORYLATION
<4 OOs, SEQUENCE: 12 Met Lys Gly Val Gly Thr Arg His Lys Ala Lieu. Ile Arg Ile Met Val 1. 5 1O 15 Ser Arg Ser Glu Ile 2O
<210s, SEQ ID NO 13 &211s LENGTH: 21 212. TYPE: PRT <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Synthetic peptide 22 Os. FEATURE: <221s NAME/KEY: MOD RES <222s. LOCATION: (8) ... (8) 223 OTHER INFORMATION: PHOSPHORYLATION
<4 OOs, SEQUENCE: 13 Lieu Lys Lys Ala Lieu. Thr Gly His Lieu. Glu Glu Val Val Lieu Ala Lieu. 1. 5 1O 15
Lieu Lys Thr Pro Ala 2O US 2016/0033516 A1 Feb. 4, 2016 62
- Continued
<210s, SEQ ID NO 14 &211s LENGTH: 19 212. TYPE: PRT <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Synthetic peptide 22 Os. FEATURE: <221s NAME/KEY: MOD RES <222s. LOCATION: (8) ... (8) 223 OTHER INFORMATION: PHOSPHORYLATION
<4 OOs, SEQUENCE: 14 Gly Lys Gly Gly Ala Lys Arg His Arg Llys Val Lieu. Arg Asp Asn. Ile 1. 5 1O 15 Gln Gly Ile
<210s, SEQ ID NO 15 &211s LENGTH: 19 212. TYPE: PRT <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Synthetic peptide 22 Os. FEATURE: <221s NAME/KEY: MOD RES <222s. LOCATION: (8) ... (8) 223 OTHER INFORMATION: PHOSPHORYLATION
<4 OOs, SEQUENCE: 15 Val Ile Thr His Gly Lys Gly His Tyr Arg Ile Val Lys Tyr Ser Ser 1. 5 1O 15
Wall Ala Asn
<210s, SEQ ID NO 16 &211s LENGTH: 19 212. TYPE: PRT <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Synthetic peptide 22 Os. FEATURE: <221s NAME/KEY: MOD RES <222s. LOCATION: (8) ... (8) 223 OTHER INFORMATION: PHOSPHORYLATION
<4 OOs, SEQUENCE: 16 Phe Arg Glin Val Arg Lieu Lys His Arg Llys Lieu. Arg Glu Glin Val Asn 1. 5 1O 15
Ser Met Wall
<210s, SEQ ID NO 17 &211s LENGTH: 2O 212. TYPE: PRT <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Synthetic peptide 22 Os. FEATURE: <221s NAME/KEY: MOD RES <222s. LOCATION: (8) ... (8) 223 OTHER INFORMATION: PHOSPHORYLATION
<4 OOs, SEQUENCE: 17 Lys Arg Val Lieu. Ile Ala Ala His Gly Asn. Ser Lieu. Arg Gly Ile Val 1. 5 1O 15 Llys His Lieu. Glu 2O US 2016/0033516 A1 Feb. 4, 2016 63
- Continued
<210s, SEQ ID NO 18 &211s LENGTH: 2O 212. TYPE: PRT <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Synthetic peptide 22 Os. FEATURE: <221s NAME/KEY: MOD RES <222s. LOCATION: (8) ... (8) 223 OTHER INFORMATION: PHOSPHORYLATION
<4 OOs, SEQUENCE: 18 Tyr Lys Lieu Val Lieu. Ile Arg His Gly Glu Ser Ala Trp Asn Lieu. Glu 1. 5 1O 15 Asn Arg Phe Ser 2O
<210s, SEQ ID NO 19 &211s LENGTH: 21 212. TYPE: PRT <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Synthetic peptide 22 Os. FEATURE: <221s NAME/KEY: MOD RES <222s. LOCATION: (8) ... (8) 223 OTHER INFORMATION: PHOSPHORYLATION
<4 OOs, SEQUENCE: 19 Arg Cys Lieu. Phe Val Cys Arg His Gly Glu Arg Met Asp Val Val Phe 1. 5 1O 15 Gly Lys Tyr Trp Lieu. 2O
<210s, SEQ ID NO 2 O &211s LENGTH: 19 212. TYPE: PRT <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Synthetic peptide 22 Os. FEATURE: <221s NAME/KEY: MOD RES <222s. LOCATION: (8) ... (8) 223 OTHER INFORMATION: PHOSPHORYLATION
<4 OOs, SEQUENCE: 2O Llys Ser Val Lieu Val Val Arg His Gly Glu Arg Val Asp Glin Ile Phe 1. 5 1O 15 Gly Lys Ala
<210s, SEQ ID NO 21 &211s LENGTH: 2O 212. TYPE: PRT <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Synthetic peptide 22 Os. FEATURE: <221s NAME/KEY: MOD RES <222s. LOCATION: (8) ... (8) 223 OTHER INFORMATION: PHOSPHORYLATION
<4 OOs, SEQUENCE: 21 Ser Ser Glu Val Glin Phe Gly His Ala Gly Ala Cys Ala Asn Glin Ala 1. 5 1O 15 US 2016/0033516 A1 Feb. 4, 2016 64
- Continued
Ser Glu Thir Ala 2O
<210s, SEQ ID NO 22 &211s LENGTH: 2O 212. TYPE: PRT <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Synthetic peptide 22 Os. FEATURE: <221s NAME/KEY: MOD RES <222s. LOCATION: (8) ... (8) 223 OTHER INFORMATION: PHOSPHORYLATION
<4 OOs, SEQUENCE: 22 Pro Pro Gly Arg Arg Met Gly His Ala Gly Ala Ile Ile Ala Gly Gly 1. 5 1O 15 Lys Gly Gly Ala 2O
<210s, SEQ ID NO 23 &211s LENGTH: 14 212. TYPE: PRT <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Synthetic peptide
<4 OOs, SEQUENCE: 23 Met Lys Gly Val Arg Met Gly His Ser Gly Ala Ile Val Glu 1. 5 1O
<210s, SEQ ID NO 24 &211s LENGTH: 17 212. TYPE: PRT <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Synthetic peptide
<4 OOs, SEQUENCE: 24 Trp Arg Gly Glin Arg Asp Asn Ala Gly Ala Ala Thr Glu Glu Phe Ile 1. 5 1O 15 Lys
<210s, SEQ ID NO 25 &211s LENGTH: 2O 212. TYPE: PRT <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Synthetic peptide 22 Os. FEATURE: <221s NAME/KEY: MOD RES <222s. LOCATION: (8) ... (8) 223 OTHER INFORMATION: PHOSPHORYLATION
<4 OOs, SEQUENCE: 25 Gln Glu Lieu Met Thr Tyr Ser His Asp Asn Ile Ile Cys Gly Ile Thr 1. 5 1O 15
Ser Wal Ser Phe 2O
<210s, SEQ ID NO 26 &211s LENGTH: 2O 212. TYPE: PRT <213> ORGANISM: Artificial Sequence US 2016/0033516 A1 Feb. 4, 2016 65
- Continued
22 Os. FEATURE: <223> OTHER INFORMATION: Synthetic peptide 22 Os. FEATURE: <221s NAME/KEY: MOD RES <222s. LOCATION: (8) ... (8) 223 OTHER INFORMATION: PHOSPHORYLATION 22 Os. FEATURE: <221s NAME/KEY: MOD RES <222s. LOCATION: (10) ... (10) 223 OTHER INFORMATION: PHOSPHORYLATION
<4 OOs, SEQUENCE: 26 Gly Lys Cys Pro Leu. Asn Pro His Ser His Leu Gly Thr Tyr Gly Val 1. 5 1O 15
Phe Thir Asn Ala 2O
<210s, SEQ ID NO 27 &211s LENGTH: 2O 212. TYPE: PRT <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Synthetic peptide 22 Os. FEATURE: <221s NAME/KEY: MOD RES <222s. LOCATION: (6) . . (6) 223 OTHER INFORMATION: PHOSPHORYLATION 22 Os. FEATURE: <221s NAME/KEY: MOD RES <222s. LOCATION: (8) ... (8) 223 OTHER INFORMATION PHOSPHORYLATION
<4 OOs, SEQUENCE: 27 Cys Pro Leu. Asn Pro His Ser His Leu Gly Thr Tyr Gly Val Phe Thr 1. 5 1O 15
Asn Ala Ala Phe 2O
<210s, SEQ ID NO 28 &211s LENGTH: 2O 212. TYPE: PRT <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Synthetic peptide 22 Os. FEATURE: <221s NAME/KEY: MOD RES <222s. LOCATION: (8) ... (8) 223 OTHER INFORMATION: PHOSPHORYLATION
<4 OOs, SEQUENCE: 28 Asn Ala Ile Phe Arg Ala Ala His Ser Ile Lys Gly Gly Ala Gly Thr 1. 5 1O 15 Phe Gly Phe Ser 2O
<210s, SEQ ID NO 29 &211s LENGTH: 2O 212. TYPE: PRT <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Synthetic peptide 22 Os. FEATURE: <221s NAME/KEY: MOD RES <222s. LOCATION: (8) ... (8) 223 OTHER INFORMATION: PHOSPHORYLATION
<4 OOs, SEQUENCE: 29 US 2016/0033516 A1 Feb. 4, 2016 66
- Continued
His Asp Ile Phe Arg Ala Ala His Thr Lieu Lys Gly Met Ser Ala Thr 1. 5 1O 15 Met Gly Tyr Thr 2O
<210s, SEQ ID NO 3 O &211s LENGTH: 2O 212. TYPE: PRT <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Synthetic peptide 22 Os. FEATURE: <221s NAME/KEY: MOD RES <222s. LOCATION: (8) ... (8) 223 OTHER INFORMATION: PHOSPHORYLATION
<4 OOs, SEQUENCE: 30 Glin Val Gly Arg Asn. Ile Ile His Gly Ser Asp Ser Wall Lys Ser Ala 1. 5 1O 15 Glu Lys Glu Ile 2O
<210s, SEQ ID NO 31 &211s LENGTH: 2O 212. TYPE: PRT <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Synthetic peptide 22 Os. FEATURE: <221s NAME/KEY: MOD RES <222s. LOCATION: (8) ... (8) 223 OTHER INFORMATION: PHOSPHORYLATION
<4 OOs, SEQUENCE: 31 Glin Val Gly Arg Asn. Ile Ile His Gly Ser Asp Ser Val Glu Ser Ala 1. 5 1O 15 Glu Lys Glu Ile 2O
<210s, SEQ ID NO 32 &211s LENGTH: 2O 212. TYPE: PRT <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Synthetic peptide 22 Os. FEATURE: <221s NAME/KEY: MOD RES <222s. LOCATION: (8) ... (8) 223 OTHER INFORMATION: PHOSPHORYLATION
<4 OOs, SEQUENCE: 32 Val Phe Ile Ala Asn. Ile Ser His Glu Lieu. Arg Thr Pro Lieu. Asn Gly 1. 5 1O 15
Ile Leu Gly Met 2O
<210s, SEQ ID NO 33 &211s LENGTH: 2O 212. TYPE: PRT <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Synthetic peptide 22 Os. FEATURE: <221s NAME/KEY: MOD RES US 2016/0033516 A1 Feb. 4, 2016 67
- Continued
<222s. LOCATION: (8) ... (8) 223 OTHER INFORMATION: PHOSPHORYLATION
<4 OOs, SEQUENCE: 33 Thr Glu Lieu. Asp Asn Lieu. Gly His Phe Lieu Lys Gly Ser Ser Ala Ala 1. 5 1O 15 Lieu. Gly Lieu. Glin 2O
<210s, SEQ ID NO 34 &211s LENGTH: 16 212. TYPE: PRT <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Synthetic peptide 22 Os. FEATURE: <221s NAME/KEY: MOD RES <222s. LOCATION: (8) ... (8) 223 OTHER INFORMATION: PHOSPHORYLATION
<4 OOs, SEQUENCE: 34 Ala Asn. Ser Arg Ser Ser Ala His Met Ser Ser Asn Ala Ile Glin Arg 1. 5 1O 15
<210s, SEQ ID NO 35 &211s LENGTH: 15 212. TYPE: PRT <213> ORGANISM: Artificial Sequence & 22 O FEATURE; <223> OTHER INFORMATION: Synthetic peptide 22 Os. FEATURE: <221s NAME/KEY: MOD RES <222s. LOCATION: (8) ... (8) 223 OTHER INFORMATION: PHOSPHORYLATION
<4 OOs, SEQUENCE: 35 Glin Val Gly Arg Asn. Ile Ile His Gly Ser Asp Ser Val Glu Ser 1. 5 1O 15
<210s, SEQ ID NO 36 &211s LENGTH: 15 212. TYPE: PRT <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Synthetic peptide 22 Os. FEATURE: <221s NAME/KEY: MOD RES <222s. LOCATION: (8) ... (8) 223 OTHER INFORMATION: PHOSPHORYLATION
<4 OOs, SEQUENCE: 36 Glin Val Gly Arg Asn. Ile Ile His Gly Ser Asp Ser Wall Lys Ser 1. 5 1O 15
<210s, SEQ ID NO 37 &211s LENGTH: 15 212. TYPE: PRT <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Synthetic peptide 22 Os. FEATURE: <221s NAME/KEY: MOD RES <222s. LOCATION: (8) ... (8) 223 OTHER INFORMATION: PHOSPHORYLATION
<4 OO > SEQUENCE: 37 US 2016/0033516 A1 Feb. 4, 2016 68
- Continued His Ile Ser Arg Asn Val Ile His Ala Ser Asp Ser Val Glu Gly 1. 5 1O 15
<210s, SEQ ID NO 38 &211s LENGTH: 15 212. TYPE: PRT <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Synthetic peptide 22 Os. FEATURE: <221s NAME/KEY: MOD RES <222s. LOCATION: (8) ... (8) 223 OTHER INFORMATION: PHOSPHORYLATION
<4 OOs, SEQUENCE: 38 Asp Asp Lieu. Arg Asn Ala Lieu. His Gly Ser Asn Asp Phe Ala Ala 1. 5 1O 15
<210s, SEQ ID NO 39 &211s LENGTH: 15 212. TYPE: PRT <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Synthetic peptide 22 Os. FEATURE: <221s NAME/KEY: MOD RES <222s. LOCATION: (8) ... (8) 223 OTHER INFORMATION: PHOSPHORYLATION
<4 OOs, SEQUENCE: 39 Thr Asp Thr Arg Asn. Thir Thr His Gly Ser Asp Ser Val Val Ser 1. 5 1O 15
<210s, SEQ ID NO 4 O &211s LENGTH: 15 212. TYPE: PRT <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Synthetic peptide 22 Os. FEATURE: <221s NAME/KEY: MOD RES <222s. LOCATION: (8) ... (8) 223 OTHER INFORMATION: PHOSPHORYLATION
<4 OOs, SEQUENCE: 4 O Asp Gly Ile Arg Asn Ala Ala His Gly Pro Asp Ser Phe Ala Ser 1. 5 1O 15
<210s, SEQ ID NO 41 &211s LENGTH: 15 212. TYPE: PRT <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Synthetic peptide 22 Os. FEATURE: <221s NAME/KEY: MOD RES <222s. LOCATION: (8) ... (8) 223 OTHER INFORMATION: PHOSPHORYLATION
<4 OOs, SEQUENCE: 41 Gly Lys Gly Gly Ala Lys Arg His Arg Llys Val Lieu. Arg Asp Asn 1. 5 1O 15
<210s, SEQ ID NO 42 &211s LENGTH: 15 212. TYPE: PRT <213> ORGANISM: Artificial Sequence US 2016/0033516 A1 Feb. 4, 2016 69
- Continued
22 Os. FEATURE: <223> OTHER INFORMATION: Synthetic peptide 22 Os. FEATURE: <221s NAME/KEY: MOD RES <222s. LOCATION: (8) ... (8) 223 OTHER INFORMATION: PHOSPHORYLATION
<4 OOs, SEQUENCE: 42 Phe Arg Glin Val Arg Lieu Lys His Arg Llys Lieu. Arg Glu Glin Val 1. 5 1O 15
<210s, SEQ ID NO 43 &211s LENGTH: 15 212. TYPE: PRT <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Synthetic peptide 22 Os. FEATURE: <221s NAME/KEY: MOD RES <222s. LOCATION: (8) ... (8) 223 OTHER INFORMATION: PHOSPHORYLATION
<4 OOs, SEQUENCE: 43 Lieu. Arg Glin Asn. Thir Lieu. Gly His Lieu. Asn Lieu. Gly Lieu. Asn Lieu. 1. 5 1O 15
<210s, SEQ ID NO 44 &211s LENGTH: 15 212. TYPE: PRT <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Synthetic peptide 22 Os. FEATURE: <221s NAME/KEY: MOD RES <222s. LOCATION: (8) ... (8) 223 OTHER INFORMATION: PHOSPHORYLATION
<4 OOs, SEQUENCE: 44 Gln Glu Lieu Met Thr Tyr Ser His Asp Asn Ile Ile Cys Gly Ile 1. 5 1O 15
<210s, SEQ ID NO 45 &211s LENGTH: 15 212. TYPE: PRT <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Synthetic peptide 22 Os. FEATURE: <221s NAME/KEY: MOD RES <222s. LOCATION: (8) ... (8) 223 OTHER INFORMATION: PHOSPHORYLATION
<4 OOs, SEQUENCE: 45 Tyr Lys Lieu Val Lieu. Ile Arg His Gly Glu Ser Ala Trp Asn Lieu. 1. 5 1O 15
<210s, SEQ ID NO 46 &211s LENGTH: 15 212. TYPE: PRT <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Synthetic peptide 22 Os. FEATURE: <221s NAME/KEY: MOD RES <222s. LOCATION: (8) ... (8) 223 OTHER INFORMATION: PHOSPHORYLATION
<4 OOs, SEQUENCE: 46 US 2016/0033516 A1 Feb. 4, 2016 70
- Continued
Ser Ser Glu Val Glin Phe Gly His Ala Gly Ala Cys Ala Asn Glin 1. 5 1O 15
<210s, SEQ ID NO 47 &211s LENGTH: 15 212. TYPE: PRT <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Synthetic peptide 22 Os. FEATURE: <221s NAME/KEY: MOD RES <222s. LOCATION: (8) ... (8) 223 OTHER INFORMATION: PHOSPHORYLATION
<4 OOs, SEQUENCE: 47 Pro Pro Gly Arg Arg Met Gly His Ala Gly Ala Ile Ile Ala Gly 1. 5 1O 15
<210s, SEQ ID NO 48 &211s LENGTH: 15 212. TYPE: PRT <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Synthetic peptide 22 Os. FEATURE: <221s NAME/KEY: MOD RES <222s. LOCATION: (8) ... (8) 223 OTHER INFORMATION: PHOSPHORYLATION
< 4 OO SEQUENCE: 48 Gly Lys Cys Pro Leu. Asn Pro His Ser His Leu Gly Thr Tyr Gly 1. 5 1O 15
<210s, SEQ ID NO 49 &211s LENGTH: 15 212. TYPE: PRT <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Synthetic peptide 22 Os. FEATURE: <221s NAME/KEY: MOD RES <222s. LOCATION: (8) ... (8) 223 OTHER INFORMATION: PHOSPHORYLATION
<4 OOs, SEQUENCE: 49 Gly Asn. Asn Trp Ala Lys Gly His Tyr Thr Glu Gly Ala Glu Lieu. 1. 5 1O 15
<210s, SEQ ID NO 50 &211s LENGTH: 15 212. TYPE: PRT <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Synthetic peptide 22 Os. FEATURE: <221s NAME/KEY: MOD RES <222s. LOCATION: (8) ... (8) 223 OTHER INFORMATION: PHOSPHORYLATION
<4 OOs, SEQUENCE: 50 Glu Glu Thr Glu Arg Ser Lieu. His Asp Ala Ile Met Ile Val Arg 1. 5 1O 15
<210s, SEQ ID NO 51 &211s LENGTH: 15 212. TYPE: PRT US 2016/0033516 A1 Feb. 4, 2016 71
- Continued <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Synthetic peptide 22 Os. FEATURE: <221s NAME/KEY: MOD RES <222s. LOCATION: (8) ... (8) 223 OTHER INFORMATION: PHOSPHORYLATION
<4 OOs, SEQUENCE: 51 Llys Thr Ala Lieu. Cys Phe Arg His Lieu Met Lys Glin Lieu. Lieu. Asn 1. 5 1O 15
<210s, SEQ ID NO 52 &211s LENGTH: 15 212. TYPE: PRT <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Synthetic peptide 22 Os. FEATURE: <221s NAME/KEY: MOD RES <222s. LOCATION: (8) ... (8) 223 OTHER INFORMATION: PHOSPHORYLATION
<4 OOs, SEQUENCE: 52 Gly Glu Met Met Asp Leu Gln His Gly Ser Leu Phe Lieu. Arg Thr 1. 5 1O 15
<210s, SEQ ID NO 53 &211s LENGTH: 15 212. TYPE PRT <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Synthetic peptide 22 Os. FEATURE: <221s NAME/KEY: MOD RES <222s. LOCATION: (8) ... (8) 223 OTHER INFORMATION: PHOSPHORYLATION
<4 OOs, SEQUENCE: 53 Lieu. Gly Llys Lieu Met Glu Lieu. His Gly Glu Gly Ser Ser Ser Gly 1. 5 1O 15
<210s, SEQ ID NO 54 &211s LENGTH: 15 212. TYPE: PRT <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Synthetic peptide 22 Os. FEATURE: <221s NAME/KEY: MOD RES <222s. LOCATION: (8) ... (8) 223 OTHER INFORMATION: PHOSPHORYLATION
<4 OOs, SEQUENCE: 54 Thir Met Glu Lys Ala Gly Ala His Lieu. Glin Gly Gly Ala Lys Arg 1. 5 1O 15
<210s, SEQ ID NO 55 &211s LENGTH: 15 212. TYPE: PRT <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Synthetic peptide 22 Os. FEATURE: <221s NAME/KEY: MOD RES <222s. LOCATION: (6) . . (6) 223 OTHER INFORMATION: PHOSPHORYLATION US 2016/0033516 A1 Feb. 4, 2016 72
- Continued
<4 OO > SEQUENCE: 55 Cys Gly Glu Arg Lieu. Tyr Asp Lieu. Asn Met Pro Ala Tyr Val Lys 1. 5 1O 15
<210s, SEQ ID NO 56 &211s LENGTH: 15 212. TYPE: PRT <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Synthetic peptide
<4 OOs, SEQUENCE: 56 Cys Gly Glu Arg Lieu. Tyr Asp Lieu. Asn Met Pro Ala Tyr Val Lys 1. 5 1O 15
<210s, SEQ ID NO 57 &211s LENGTH: 10 212. TYPE: PRT <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Synthetic peptide 22 Os. FEATURE: <221s NAME/KEY: MOD RES <222s. LOCATION: (8) ... (8) 223 OTHER INFORMATION: PHOSPHORYLATION
<4 OO > SEQUENCE: 57 Cys Lieu Lys Pro Lieu Lys Thr Tyr Val Asp 1. 5 1O
<210s, SEQ ID NO 58 &211s LENGTH: 10 212. TYPE: PRT <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Synthetic peptide
<4 OOs, SEQUENCE: 58 Cys Lieu Lys Pro Lieu Lys Thr Tyr Val Asp 1. 5 1O
<210s, SEQ ID NO 59 &211s LENGTH: 21 212. TYPE: PRT <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Synthetic peptide 22 Os. FEATURE: <221s NAME/KEY: MOD RES <222s. LOCATION: (10) ... (10) 223 OTHER INFORMATION: PHOSPHORYLATION
<4 OO > SEQUENCE: 59 Ala Val Ser Val Ser Glu Thir Asp Asp Tyr Ala Glu Ile Ile Asp Glu 1. 5 1O 15
Glu Asp Thr Tyr Thr 2O
<210s, SEQ ID NO 60 &211s LENGTH: 30 &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Synthetic polynucleotide US 2016/0033516 A1 Feb. 4, 2016 73
- Continued
<4 OOs, SEQUENCE: 60 gatcggat.cc atggccaact gtgagcgtac 3 O
<210s, SEQ ID NO 61 &211s LENGTH: 31 &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Synthetic polynucleotide
<4 OOs, SEQUENCE: 61 gatcgaattic ticatt catag atccagttct c 31
<210s, SEQ ID NO 62 &211s LENGTH: 30 &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Synthetic polynucleotide
<4 OOs, SEQUENCE: 62 gatcggat.cc atggccalacc tigagcgcac 3 O
<210s, SEQ ID NO 63 &211s LENGTH: 31 &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence & 22 O FEATURE; <223> OTHER INFORMATION: Synthetic polynucleotide
<4 OOs, SEQUENCE: 63 gatcgaattic titatt catag acc cagt cat g 31
<210s, SEQ ID NO 64 &211s LENGTH: 31 &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Synthetic polynucleotide
<4 OOs, SEQUENCE: 64 gatcggat.cc atggcc.gc.ct acaaactggit g 31
<210s, SEQ ID NO 65 &211s LENGTH: 31 &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Synthetic polynucleotide
<4 OOs, SEQUENCE: 65 gatcgaattic ticacttcttg gcc ttgcc ct g 31
<210s, SEQ ID NO 66 &211s LENGTH: 18 &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Synthetic polynucleotide
<4 OOs, SEQUENCE: 66 gtgatgaccc agacticca 18 US 2016/0033516 A1 Feb. 4, 2016 74
- Continued
<210s, SEQ ID NO 67 &211s LENGTH: 18 &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Synthetic polynucleotide
<4 OO > SEQUENCE: 67 gtgctgaccc agacticca 18
<210s, SEQ ID NO 68 &211s LENGTH: 18 &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Synthetic polynucleotide
<4 OOs, SEQUENCE: 68 gatatgaccc agacticca 18
<210s, SEQ ID NO 69 &211s LENGTH: 18 &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Synthetic polynucleotide
<4 OOs, SEQUENCE: 69 gatctgaccc agacticca 18
<210s, SEQ ID NO 70 &211s LENGTH: 21 &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Synthetic polynucleotide
<4 OO > SEQUENCE: 7 O tittgatttico acattggtgc c 21
<210s, SEQ ID NO 71 &211s LENGTH: 21 &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Synthetic polynucleotide
<4 OOs, SEQUENCE: 71 taggat ct co agctcggit co c 21
<210s, SEQ ID NO 72 &211s LENGTH: 21 &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Synthetic polynucleotide
<4 OOs, SEQUENCE: 72 tittgaccacc acct cqgtcc c 21
<210s, SEQ ID NO 73 &211s LENGTH: 21 &212s. TYPE: DNA US 2016/0033516 A1 Feb. 4, 2016 75
- Continued <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Synthetic polynucleotide
<4 OO > SEQUENCE: 73 tittgacgacc acct cqgtcc c 21
<210s, SEQ ID NO 74 &211s LENGTH: 2O &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Synthetic polynucleotide
<4 OOs, SEQUENCE: 74 gtgctgactic agt cqCCCtc 2O
<210s, SEQ ID NO 75 &211s LENGTH: 24 &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Synthetic polynucleotide
<4 OO > SEQUENCE: 75 gcctgtgacg gtcagotggg tocc 24
<210 SEQ ID NO 76 &211s LENGTH: 2O &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Synthetic polynucleotide
<4 OO > SEQUENCE: 76 agtcggtgga gagtcCagg 2O
<210s, SEQ ID NO 77 &211s LENGTH: 2O &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Synthetic polynucleotide
<4 OO > SEQUENCE: 77 agtcggtgga gagtc.cggg 2O
<210s, SEQ ID NO 78 &211s LENGTH: 2O &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Synthetic polynucleotide
<4 OO > SEQUENCE: 78 agtcggtgaa gagtc.cgag 2O
<210s, SEQ ID NO 79 &211s LENGTH: 2O &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Synthetic polynucleotide US 2016/0033516 A1 Feb. 4, 2016 76
- Continued
<4 OO > SEQUENCE: 79 agtc.gctgga ggagtc.cggg
<210s, SEQ ID NO 8O &211s LENGTH: 2O &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Synthetic polynucleotide
<4 OOs, SEQUENCE: 80 agtcgttgga ggagtc.cggg
<210s, SEQ ID NO 81 &211s LENGTH: 22 &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Synthetic polynucleotide
<4 OOs, SEQUENCE: 81 agcagcagct gatggagt cc gg 22
<210s, SEQ ID NO 82 &211s LENGTH: 22 &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence & 22 O FEATURE; <223> OTHER INFORMATION: Synthetic polynucleotide
<4 OOs, SEQUENCE: 82 aggagcagct gatggagtCc gig 22
<210s, SEQ ID NO 83 &211s LENGTH: 22 &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Synthetic polynucleotide
<4 OOs, SEQUENCE: 83 agcagcagct getggagtCc gig 22
<210s, SEQ ID NO 84 &211s LENGTH: 22 &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Synthetic polynucleotide
<4 OOs, SEQUENCE: 84 aggagcagct ggtggagtCc 99 22
<210s, SEQ ID NO 85 &211s LENGTH: 21 &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Synthetic polynucleotide
<4 OOs, SEQUENCE: 85 agagacggtg accagggtgc C 21 US 2016/0033516 A1 Feb. 4, 2016 77
- Continued
<210s, SEQ ID NO 86 &211s LENGTH: 21 &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Synthetic polynucleotide
<4 OOs, SEQUENCE: 86 ggaga.cggtg acCagggtgc C 21
<210s, SEQ ID NO 87 &211s LENGTH: 21 &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Synthetic polynucleotide
<4 OO > SEQUENCE: 87 agagatggtg accagggtgc C 21
<210s, SEQ ID NO 88 &211s LENGTH: 21 &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Synthetic polynucleotide
<4 OOs, SEQUENCE: 88 ggagatggtg acCagggtgc C 21
<210s, SEQ ID NO 89 &211s LENGTH: 9 212. TYPE: PRT <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Synthetic peptide 22 Os. FEATURE: <221s NAME/KEY: MISC FEATURE <222s. LOCATION: (5) . . (5) <223> OTHER INFORMATION: Xaa = His, 1-pTza or 3-pTza
<4 OOs, SEQUENCE: 89 Ala Gly Ala Gly Xaa Ala Gly Ala Gly 1. 5
<210s, SEQ ID NO 90 &211s LENGTH: 9 212. TYPE: PRT <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Synthetic peptide 22 Os. FEATURE: <221s NAME/KEY: MISC FEATURE <222s. LOCATION: (5) . . (5) <223> OTHER INFORMATION: Xaa = His, 1-pTza or 3-pTza
<4 OOs, SEQUENCE: 90 Val Lieu. Ile Arg Xaa Gly Glu Ser Ala 1. 5
<210s, SEQ ID NO 91 &211s LENGTH: 9 212. TYPE: PRT <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: US 2016/0033516 A1 Feb. 4, 2016 78
- Continued <223> OTHER INFORMATION: Synthetic peptide 22 Os. FEATURE: <221s NAME/KEY: MISC FEATURE <222s. LOCATION: (5) . . (5) <223> OTHER INFORMATION: Xaa = His, 1-pTza or 3-pTza <4 OOs, SEQUENCE: 91 Arg Asn. Ile Ile Xaa Gly Ser Asp Ser 1. 5
<210s, SEQ ID NO 92 &211s LENGTH: 9 212. TYPE: PRT <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Synthetic peptide 22 Os. FEATURE: <221s NAME/KEY: MISC FEATURE <222s. LOCATION: (5) . . (5) <223> OTHER INFORMATION: Xaa = His, 1-pTza or 3-pTza
<4 OOs, SEQUENCE: 92 Gly Ala Lys Arg Xaa Arg Llys Val Lieu. 1. 5
SEO ID NO 93 LENGTH: 9 TYPE PRT ORGANISM: Artificial Sequence FEATURE; OTHER INFORMATION: Synthetic peptide FEATURE: NAME/KEY: MISC FEATURE LOCATION: (5) . . (5) OTHER INFORMATION: Xaa = His 1-pTza or 3-pTza
<4 OOs, SEQUENCE: 93 Val Arg Lieu Lys Xaa Arg Llys Lieu. Arg 1. 5
SEQ ID NO 94 LENGTH: 9 TYPE PRT ORGANISM: Artificial Sequence FEATURE: OTHER INFORMATION: Synthetic peptide FEATURE: NAME/KEY: MISC FEATURE LOCATION: (5) . . (5) OTHER INFORMATION: Xaa = His 1-pTza or 3-pTza
<4 OOs, SEQUENCE: 94 Met Thr Tyr Ser Xaa Asp Asn Ile Ile 1. 5
We claim: prises a H-CDR1, a H-CDR2, and a H-CDR3, wherein 1. A method for detecting the presence of a tumor in a the antibody or antigen binding fragment comprises: Subject, comprising: a) the H-CDR1, the H-CDR2, and the H-CDR3 of the contacting a sample comprising polypeptides from the Sub heavy chain variable region of the amino acid ject with a monoclonal antibody orantigenbinding frag sequence set forth as SEQID NO: 1; ment thereof under conditions sufficient to form an b) the H-CDR1, the H-CDR2, and the H-CDR3 of the immune complex with a polypeptide comprising a his heavy chain variable region of the amino acid tidine phosphorylated at N3 (3-pHis) if present in the sequence set forth as SEQID NO: 2; sample, wherein the monoclonal antibody comprises a c) the H-CDR1, the H-CDR2, and the H-CDR3 of the heavy chain variable region and a light chain variable heavy chain variable region of the amino acid region, wherein the heavy chain variable region com sequence set forth as SEQID NO: 3; or US 2016/0033516 A1 Feb. 4, 2016 79
d) the H-CDR1, the H-CDR2, and the H-CDR3 of the c) the heavy chain variable region of the monoclonal anti heavy chain variable region of the amino acid body comprises amino acid 1-115 of SEQID NO:3 and sequence set forth as SEQID NO: 4, the light chain variable region of the monoclonal anti and wherein the monoclonal antibody specifically binds body comprises amino acids 1-120 of SEQID NO: 7, or a polypeptide comprising a histidine phosphorylated d) the heavy chain variable region of the monoclonal anti at N3 (3-pHis); body comprises amino acids 1-115 of SEQID NO: 4 and detecting the presence of one or more polypeptides that are the light chain variable region of the monoclonal anti phosphorylated at N3; and body comprises amino acids 1-113 of SEQID NO: 8. comparing the amount of the one or more polypeptides that 5. The method of claim 1, wherein the control represents an are phosphorylated at N3 to a control, amount of the one or more polypeptides that are phosphory wherein a change in the amount of the one or more lated at N3 in a sample from a healthy subject or a standard polypeptides that are phosphorylated at N3 as compared value. to the control indicates the presence of the tumor in the 6. A method of determining if a subject with a tumor is Subject. responsive to a chemotherapeutic agent, comprising 2. The method of claim 1, wherein the light chain variable contacting a tumor sample comprising polypeptides from region comprises a L-CDR1, a L-CDR2, and a L-CDR3, the Subject with a monoclonal antibody or antigenbind wherein the antibody orantigenbinding fragment comprises: ing fragment thereofunder conditions sufficient to form a) the L-CDR1, the L-CDR2, and the L-CDR3 of the light an immune complex with a polypeptide comprising a chain variable region of the amino acid sequence set histidine phosphorylated at N3 (3-pHis) if present in the forth as SEQID NO: 5; sample, wherein the monoclonal antibody comprises a b) the L-CDR1, the L-CDR2, and the L-CDR3 of the light heavy chain variable region and a light chain variable chain variable region of the amino acid sequence set region, wherein the heavy chain variable region com forth as SEQID NO: 6; prises a H-CDR1, a H-CDR2, and a H-CDR3, wherein c) the L-CDR1, the L-CDR2, and the L-CDR3 of the light the antibody or antigen binding fragment comprises: chain variable region of the amino acid sequence set a) the H-CDR1, the H-CDR2, and the H-CDR3 of the forth as SEQID NO: 7; or heavy chain variable region of the amino acid sequence d) the L-CDR1, the L-CDR2, and the L-CDR3 of the light set forth as SEQID NO: 1; chain variable region of the amino acid sequence set b) the H-CDR1, the H-CDR2, and the H-CDR3 of the forth as SEQID NO: 8. heavy chain variable region of the amino acid sequence 3. The method of claim 1, wherein set forth as SEQID NO: 2: a) the HCDR1, HCDR2, and HCDR3 of the monoclonal c) the H-CDR1, the H-CDR2, and the H-CDR3 of the antibody comprise amino acids 28, 45-52, and 88-97 of heavy chain variable region of the amino acid sequence SEQ ID NO: 1, respectively, and the LCDR1, LCDR2, set forth as SEQID NO:3: or and LCDR3 of the monoclonal antibody comprise d) the H-CDR1, the H-CDR2, and the H-CDR3 of the amino acids 28-22, 51-53, and 90-102 of SEQID NO: 5 heavy chain variable region of the amino acid sequence respectively; set forth as SEQID NO: 4, b) the HCDR1, HCDR2, and HCDR3 of the monoclonal and wherein the monoclonal antibody specifically binds antibody comprise amino acids 21-28, 46-52, and a polypeptide comprising a histidine phosphorylated 91-101 of SEQID NO: 2, respectively, and the LCDR1, at N3 (3-pHis); LCDR2, and LCDR3 of the monoclonal antibody com detecting the presence of one or more polypeptides phos prise amino acids 27-34, 52-54,91-103 of SEQID NO: 6, respectively; phorylated at N3; and comparing the amount of the one or more polypeptides c) the HCDR1, HCDR2, and HCDR3 of the monoclonal antibody comprise amino acids 24-31, 49-55,94-104 of phosphorylated at N3 to a control, SEQ ID NO:3, respectively, and the LCDR1, LCDR2, wherein a change in the amount of the one or more and LCDR3 of the monoclonal antibody comprise polypeptides phosphorylated at N3 as compared to a amino acids 27-34, 52-54, and 91-109 of SEQID NO: 7, control indicates that the chemotherapeutic agent is of respectively; or use for treating the Subject. d) the HCDR1, HCDR2, and HCDR3 of the monoclonal 7. The method of claim 6, wherein the light chain variable antibody comprise amino acids 24-31, 49-55,94-104 of region comprises a L-CDR1, a L-CDR2, and a L-CDR3, SEQ ID NO: 4, respectively, and the LCDR1, LCDR2, wherein the antibody or antigenbinding fragment comprises: and LCDR3 of the monoclonal antibody comprise a) the L-CDR1, the L-CDR2, and the L-CDR3 of the light amino acids amino acids 27-33, 51-53 and 90-102 of chain variable region of the amino acid sequence set SEQ ID NO: 8, respectively. forth as SEQID NO: 5; 4. The method of claim 3, wherein b) the L-CDR1, the L-CDR2, and the L-CDR3 of the light a) the heavy chain variable region of the monoclonal anti chain variable region of the amino acid sequence set body comprises amino acids 1-108 of SEQID NO: 1 and forth as SEQID NO: 6; the light chain variable region of the monoclonal anti c) the L-CDR1, the L-CDR2, and the L-CDR3 of the light body comprises amino acids 1-113 of SEQID NO: 5; chain variable region of the amino acid sequence set b) the heavy chain variable region of the monoclonal anti forth as SEQID NO: 7; or body comprises amino acids 1-112 of SEQID NO: 2 and d) the L-CDR1, the L-CDR2, and the L-CDR3 of the light the light chain variable region of the monoclonal anti chain variable region of the amino acid sequence set body comprises amino acids 1-114 of SEQID NO: 6: forth as SEQID NO: 8. US 2016/0033516 A1 Feb. 4, 2016 80
8. The method of claim 6, wherein b) the H-CDR1, the H-CDR2, and the H-CDR3 of the a) the HCDR1, HCDR2, and HCDR3 of the monoclonal heavy chain variable region of the amino acid sequence antibody comprise amino acids 28, 45-52, and 88-97 of set forth as SEQID NO: 2: SEQ ID NO: 1, respectively, and the LCDR1, LCDR2, c) the H-CDR1, the H-CDR2, and the H-CDR3 of the and LCDR3 of the monoclonal antibody comprise heavy chain variable region of the amino acid sequence amino acids 28-22, 51-53, and 90-102 of SEQID NO: 5 set forth as SEQID NO:3: or respectively; d) the H-CDR1, the H-CDR2, and the H-CDR3 of the b) the HCDR1, HCDR2, and HCDR3 of the monoclonal heavy chain variable region of the amino acid sequence antibody comprise amino acids 21-28, 46-52, and set forth as SEQID NO: 4, and wherein the monoclonal 91-101 of SEQID NO: 2, respectively, and the LCDR1, antibody specifically binds a polypeptide comprising a LCDR2, and LCDR3 of the monoclonal antibody com histidine phosphorylated at N3 (3-pHis); and prise amino acids 27-34, 52-54,91-103 of SEQID NO: detecting the presence of histidine kinase and/or cognate 6, respectively; response regulator phosphorylated at N3; c) the HCDR1, HCDR2, and HCDR3 of the monoclonal wherein a decrease in the amount of histidine kinase and a antibody comprise amino acids 24-31, 49-55,94-104 of cognate response regulator phosphorylated at N3 as SEQ ID NO:3, respectively, and the LCDR1, LCDR2, compared to a control indicates the agent of interest is an and LCDR3 of the monoclonal antibody comprise antibiotic. amino acids 27-34, 52-54, and 91-109 of SEQID NO: 7, 12. The method of claim 11, wherein the light chain vari respectively; or able region comprises a L-CDR1, a L-CDR2, and a L-CDR3, d) the HCDR1, HCDR2, and HCDR3 of the monoclonal wherein the antibody or antigenbinding fragment comprises: antibody comprise amino acids 24-31, 49-55,94-104 of a) the L-CDR1, the L-CDR2, and the L-CDR3 of the light SEQ ID NO: 4, respectively, and the LCDR1, LCDR2, chain variable region of the amino acid sequence set and LCDR3 of the monoclonal antibody comprise forth as SEQID NO: 5; amino acids amino acids 27-33, 51-53 and 90-102 of b) the L-CDR1, the L-CDR2, and the L-CDR3 of the light SEQ ID NO: 8, respectively. chain variable region of the amino acid sequence set 9. The method of claim 6, wherein forth as SEQID NO: 6; a) the heavy chain variable region of the monoclonal anti c) the L-CDR1, the L-CDR2, and the L-CDR3 of the light body comprises amino acids 1-108 of SEQID NO: 1 and chain variable region of the amino acid sequence set the light chain variable region of the monoclonal anti forth as SEQID NO: 7; or body comprises amino acids 1-113 of SEQID NO: 5; d) the L-CDR1, the L-CDR2, and the L-CDR3 of the light b) the heavy chain variable region of the monoclonal anti chain variable region of the amino acid sequence set body comprises amino acids 1-112 of SEQID NO: 2 and forth as SEQID NO: 8. the light chain variable region of the monoclonal anti 13. The method of claim 11, wherein a) the HCDR1, HCDR2, and HCDR3 of the monoclonal body comprises amino acids 1-114 of SEQID NO: 6: antibody comprise amino acids 28, 45-52, and 88-97 of c) the heavy chain variable region of the monoclonal anti SEQ ID NO: 1, respectively, and the LCDR1, LCDR2, body comprises amino acid 1-115 of SEQID NO:3 and and LCDR3 of the monoclonal antibody comprise the light chain variable region of the monoclonal anti amino acids 28-22, 51-53, and 90-102 of SEQID NO: 5 body comprises amino acids 1-120 of SEQID NO: 7, or respectively; d) the heavy chain variable region of the monoclonal anti b) the HCDR1, HCDR2, and HCDR3 of the monoclonal body comprises amino acids 1-115 of SEQID NO: 4 and antibody comprise amino acids 21-28, 46-52, and the light chain variable region of the monoclonal anti 91-101 of SEQID NO: 2, respectively, and the LCDR1, body comprises amino acids 1-113 of SEQID NO: 8. LCDR2, and LCDR3 of the monoclonal antibody com 10. The method of claim 6, wherein the control is an prise amino acids 27-34, 52-54,91-103 of SEQID NO: amount of the one or more polypeptides that are phosphory 6, respectively; lated at N3 in a sample from the subject prior to treatment c) the HCDR1, HCDR2, and HCDR3 of the monoclonal with the chemotherapeutic agent or a standard value. antibody comprise amino acids 24-31, 49-55,94-104 of 11. A method of identifying an antibiotic, comprising SEQ ID NO:3, respectively, and the LCDR1, LCDR2, contacting a bacterial cell expressing a histidine kinase and and LCDR3 of the monoclonal antibody comprise a cognate response regulator with an agent of interest, amino acids 27-34, 52-54, and 91-109 of SEQID NO: 7, measuring an amount of phosphorylated histidine kinase respectively; or and/or cognate response regulator bound by a mono d) the HCDR1, HCDR2, and HCDR3 of the monoclonal clonal antibody or antigen binding fragment thereof antibody comprise amino acids 24-31, 49-55,94-104 of under conditions sufficient to form an immune complex SEQ ID NO: 4, respectively, and the LCDR1, LCDR2, with a polypeptide comprising a histidine phosphory and LCDR3 of the monoclonal antibody comprise lated at N3 (3-pHis), wherein the monoclonal antibody amino acids amino acids 27-33, 51-53 and 90-102 of comprises a heavy chain variable region and a light SEQ ID NO: 8, respectively. chain variable region, wherein the heavy chain variable 14. The method of claim 11, wherein region comprises a H-CDR1, a H-CDR2, and a a) the heavy chain variable region of the monoclonal anti H-CDR3, wherein the antibody or antigen binding frag body comprises amino acids 1-108 of SEQID NO: 1 and ment comprises: the light chain variable region of the monoclonal anti a) the H-CDR1, the H-CDR2, and the H-CDR3 of the body comprises amino acids 1-113 of SEQID NO: 5; heavy chain variable region of the amino acid sequence b) the heavy chain variable region of the monoclonal anti set forth as SEQID NO: 1; body comprises amino acids 1-112 of SEQID NO: 2 and US 2016/0033516 A1 Feb. 4, 2016
the light chain variable region of the monoclonal anti c) the L-CDR1, the L-CDR2, and the L-CDR3 of the light body comprises amino acids 1-114 of SEQID NO: 6: chain variable region of the amino acid sequence set c) the heavy chain variable region of the monoclonal anti forth as SEQID NO: 7; or body comprises amino acid 1-115 of SEQID NO:3 and d) the L-CDR1, the L-CDR2, and the L-CDR3 of the light the light chain variable region of the monoclonal anti chain variable region of the amino acid sequence set body comprises amino acids 1-120 of SEQID NO: 7, or forth as SEQID NO: 8. d) the heavy chain variable region of the monoclonal anti 19. The method of claim 17, wherein body comprises amino acids 1-115 of SEQID NO: 4 and a) the HCDR1, HCDR2, and HCDR3 of the monoclonal the light chain variable region of the monoclonal anti antibody comprise amino acids 28, 45-52, and 88-97 of body comprises amino acids 1-113 of SEQID NO: 8. SEQ ID NO: 1, respectively, and the LCDR1, LCDR2, 15. The method of claim 11, wherein the control is an and LCDR3 of the monoclonal antibody comprise amount of histidine kinase and a cognate response regulator amino acids 28-22, 51-53, and 90-102 of SEQID NO: 5 in a cell not contacted with the agent of interest or a standard respectively; value. b) the HCDR1, HCDR2, and HCDR3 of the monoclonal 16. The method of claim 11, wherein the bacterial cell is a antibody comprise amino acids 21-28, 46-52, and gram negative or gram positive bacterial cell. 91-101 of SEQID NO: 2, respectively, and the LCDR1, 17. A method of identifying specific polypeptides that LCDR2, and LCDR3 of the monoclonal antibody com comprise a histidine phosphorylated at N3 in a sample from a prise amino acids 27-34, 52-54,91-103 of SEQID NO: Subject, comprising 6, respectively; contacting a sample comprising polypeptides from the Sub c) the HCDR1, HCDR2, and HCDR3 of the monoclonal ject with a solid Substrate comprising a monoclonal anti antibody comprise amino acids 24-31, 49-55,94-104 of body or antigen binding fragment thereof under condi SEQ ID NO:3, respectively, and the LCDR1, LCDR2, tions sufficient to form an immune complex with a and LCDR3 of the monoclonal antibody comprise polypeptide comprising a histidine phosphorylated at amino acids 27-34, 52-54, and 91-109 of SEQID NO: 7, N3 (3-pHis), wherein the monoclonal antibody com respectively; or prises a heavy chain variable region and a light chain d) the HCDR1, HCDR2, and HCDR3 of the monoclonal variable region, wherein the heavy chain variable region antibody comprise amino acids 24-31, 49-55,94-104 of comprises a H-CDR1, a H-CDR2, and a H-CDR3, SEQ ID NO: 4, respectively, and the LCDR1, LCDR2, wherein the antibody or antigen binding fragment com and LCDR3 of the monoclonal antibody comprise prises: amino acids amino acids 27-33, 51-53 and 90-102 of a) the H-CDR1, the H-CDR2, and the H-CDR3 of the SEQ ID NO: 8, respectively. heavy chain variable region of the amino acid sequence 20. The method of claim 17, wherein set forth as SEQID NO: 1; a) the heavy chain variable region of the monoclonal anti b) the H-CDR1, the H-CDR2, and the H-CDR3 of the body comprises amino acids 1-108 of SEQID NO: 1 and heavy chain variable region of the amino acid sequence the light chain variable region of the monoclonal anti set forth as SEQID NO: 2: body comprises amino acids 1-113 of SEQID NO: 5; c) the H-CDR1, the H-CDR2, and the H-CDR3 of the b) the heavy chain variable region of the monoclonal anti heavy chain variable region of the amino acid sequence body comprises amino acids 1-112 of SEQID NO: 2 and set forth as SEQID NO:3: or the light chain variable region of the monoclonal anti d) the H-CDR1, the H-CDR2, and the H-CDR3 of the body comprises amino acids 1-114 of SEQID NO: 6: heavy chain variable region of the amino acid sequence c) the heavy chain variable region of the monoclonal anti set forth as SEQID NO: 4, body comprises amino acid 1-115 of SEQID NO:3 and and wherein the monoclonal antibody specifically binds the light chain variable region of the monoclonal anti a polypeptide comprising a histidine phosphorylated body comprises amino acids 1-120 of SEQID NO: 7, or at N3 (3-pHis); d) the heavy chain variable region of the monoclonal anti eluting the polypeptides from the Solid Substrate; and body comprises amino acids 1-115 of SEQID NO: 4 and performing mass spectrometry or an immunoassay to the light chain variable region of the monoclonal anti detect the presence of one or more specific proteins; body comprises amino acids 1-113 of SEQID NO: 8. thereby identifying specific polypeptides including a his 21. The method of claim 17, wherein the sample is from a tidine phosphorylated at N3 in the sample. subject with a tumor. 18. The method of claim 17, wherein the light chain vari 22. The method of claim 21, wherein the antigen binding able region comprises a L-CDR1, a L-CDR2, and a L-CDR3, fragment is a Fv, Fab, F(ab'), sclV or a schV fragment. wherein the antibody orantigenbinding fragment comprises: 23. The method of claim 21, wherein the monoclonal anti a) the L-CDR1, the L-CDR2, and the L-CDR3 of the light body or antigen binding fragment is conjugated to a detect chain variable region of the amino acid sequence set able label. forth as SEQID NO: 5; 24. The method of claim 23, wherein the detectable marker b) the L-CDR1, the L-CDR2, and the L-CDR3 of the light is a fluorescent, enzymatic, heavy metal or radioactive chain variable region of the amino acid sequence set marker. forth as SEQID NO: 6;