WO2008153636A1 - Phospho-specific antibodies to p13k regulatory subunit and uses thereof - Google Patents

Abstract

The invention discloses ten newly discovered PI3K regulatory subunit phosphorylation sites, tyrosines 467, 452, 463, and 470 in PI3KR1 (PI3Kp85 alpha), tyrosines 464, 460, and 467 in PI3KR2 (PI3Kp85 beta), and tyrosines 199, 184, and 202 in PI3KR3 (PI3Kp55 gamma), and provides reagents, including polyclonal and monoclonal antibodies, that selectively bind to PI3K when phosphorylated at one of the disclosed sites. Also provided are assays utilizing this reagent, including methods for determining the phosphorylation of PI3K in a biological sample, selecting a patient suitable for PI3K inhibitor therapy, profiling PI3K activation in a test tissue, and identifying a compound that modulates phosphorylation of PI3K in a test tissue, by using a detectable reagent, such as the disclosed antibody, that binds to PI3K only when phosphorylated at a disclosed site. The sample or test tissue may be taken from a subject suspected of having cancer, such as lymphoma, glioma, and colon cancer, involving altered PI3K signaling.

Description

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PHOSPHO-SPECIFIC ANTIBODIES TO PI3K REGULATORY

SUBUNIT AND USES THEREOF

RELATED APPLICATIONS

This application claims priority to and the benefit of USSN 11/503,335, filed August 11 , 2006, presently pending, which itself claims priority to PCT/US04/26199, filed August 12, 2004, now abandoned, and USSN 60/833,752, filed July 27, 2006, presently pending, and PCT/US06/00979, filed January 12, 2006, presently pending, which itself claims priority to USSN 60/651,583, filed February 10, 2005, now abandoned, and PCT/US04/42940, filed December 21 , 2004, presently pending, and PCT/US06/10868, filed March 24, 2006, presently pending, which itself claims priority to USSN 60/670,447, filed April 12, 2005, now abandoned, and USSN 60/833,752, filed July 27, 2006, presently pending, USSN 60/830,550, filed July 13, 2006, presently pending, USSN 11/744,753, filed on May 4, 2007, now pending, PCT/US07/73540, filed on July 13, 2007, now pending, and PCT/US07/ 16889, filed July 27, 2007, now pending, the disclosures of which are hereby incorporated herein in their entirety by reference.

FIELD OF THE INVENTION

The invention relates generally to antibodies, and more particularly to activation state-specific antibodies to receptor tyrosine kinases and their uses.

BACKGROUND OF THE INVENTION

Many diseases are characterized by disruptions in cellular signaling pathways that lead to pathologies including uncontrolled growth and proliferation of cancerous cells, as well as aberrant inflammation processes. Such defects include changes in the activity of lipid kinases, a class of enzymes that catalyze the transfer of phosphate groups to lipids. These phosphorylated lipids, in turn, recruit important downstream proteins that propagate the signals originating from upstream signaling mediators, such as receptor tyrosine kinase and antigen receptors. For example, the protein kinase Akt is recruited by phospholipids to the plasma membrane where it is activated. Once activated, Akt plays a pivotal role in survival both of normal and cancerous tissues.

Phosphoinositide 3 -kinases (PDKs) are a family of lipid kinases that play pivotal roles in signaling pathways downstream from multiple cell surface receptors, controlling growth, proliferation, and cell survival. Active PDKs consist of two subunits: a regulatory subunit with a molecular weight of either 85 or 55 kD (p85 or p55), and a catalytic subunit of molecular weight 110 kD

(pi 10). While it is clear that the regulatory subunits are critical to the function of PDK, they also transmit signals independently of PI 3-kinase (Ueki et al, J Biol Chem. Nov 28;278(48): 48453-66 (2003)). It has recently been demonstrated that p85-alpha can induce apoptosis via the inducible transcription factor NFAT3 independent of the PDK signaling pathway (Song et al, MoI. Cell. Biol. 27: 2713-2731 (2007)).

Generation of phosphoinositides by hormones binding to receptors is the most common means of transducing signals across the cell membrane. In unstimulated cells, the phosphoinositide 3 -kinases (PIK3) that convert phosphatidylinositol(4,5) bisphosphate to phosphatidylinositol(3,4,5) trisphosphate are found as inactive heterodimers in the cytosol, consisting of one catalytic and one regulatory protein. Upon stimulation of membrane receptors, the regulatory domain is recruited to binding proteins at the membrane, and the catalytic domain is released to act on its lipid substrate (J Endocrinol. 2007 Aug;194(2):243-56). The liberated regulatory domain further influences signalling by stimulating the activity of the lipid phosphatase PTEN(Proc Natl Acad Sci U S A. 2006 Aug 8; 103(32): 12093-7), although the molecular details of its action are unknown. Phosphorylations of the three members of the regulatory subunit family are described in this patent. Consistent with their essential role in initiating and controlling signalling, the PIK3s have been implicated in cancer (Cancer Res. 2001 Aug 15;61(16):5985-91. Cancer Cell. 2008 Mar;13(3):235-48), and metabolic diseases(Proc Natl Acad Sci U S A. 2006 Aug 8; 103(32): 12093-7). Influenza A virus binds to PIK3R2 in order to facilitate its own replication (Proc Natl Acad Sci U S A. 2006 Sep 19;103(38):14194-9). Understanding of the mechanisms by which the PIK3 regulatory proteins are controlled is important for the treatment and diagnosis of these diseases.

We have observed that the paralogue proteins PIK3R1, PIK3R2, and PIK3R3 are phosphorylated on multiple tyrosine residues. Phosphorylation is a well-known method by which signalling is controlled. We describe 12 phosphorylation sites on PIK3R1 (Y59, Y73, Y76, Y426, Y452, Y463, Y467, Y470, Y504, Y556, Y657, and Y679), 11 sites on PIK3R2 (Y74, Y365, Y423, Y449, Y453, Y460, Y464, Y467, Y577, Y605, Y671), and 7 sites on PIK3R3 (Yl 84, Yl 88, Y195, Yl 99, Y202, Y373, and Y407). Several of these sites reside in the first SH3 domain, a domain for interaction with other proteins (PIK3R1 Y59, Y73, Y76; PIK3R2 Y74). Others reside within a phosphotyrosine binding pocket, another domain for binding signalling molecules (eg. PIK3R1 Y679; PIK3R2 Y365, Y671 ; PIK3R3 Y407). Molecular probes such as antibodies to these sites would provide essential tools for studying the function of the PI3K regulatory proteins as well as their roles in cancer, diabetes, and influenza (PhosphoSite®, Cell Signaling Technology, Danvers, MA. Human PSD™, Biobase Corporation, Beverly, MA).

Three closely related regulatory subunits have been described: p85-alpha (PI3KR1), p85-beta (PI3KR2), and p55-gamma (PI3KR3) (also referred to as PIK3R1-R3). A limited number of phosphorylation sites have previously been reported on PIK3R1 and PIK3R3. The published sites on PIK3R1 are serine 83 (Cosentino et al, Oncogene Oct 02 (2006)), tyrosines 368, 580 and 607 (Hayashi et al, J Biol Chem Apr; 268(10): 7107-17 (1993)), tyrosine 508 (Kavanaugh et al, Biochemistry Sep 13; 33(36): 1 1046-50 (1994)), tyrosines 528 and 556 (Kwon et al, Endocrinology Mar; 147(3): 1458-65 (2006)), tyrosine 608 - A -

(Dhand et al, EMBO J Feb; 13(3): 522-33 (1994)), and tyrosine 688 (von Willebrand et al, J. Biol. Chem. Feb; 273(7): 3994-4000 (1998)). The only previously published phosphorylation site on PIK3R3 is tyrosine 341 (Pons et al, MoI Cell Biol. Aug;15(8): 4453-65 (1995)). To date, no PI3KR2 phosphorylation sites have been described.

The PI3K pathway is implicated in various human diseases including diabetes, heart failure, and many cancers (see e.g., Kim et al, Curr Opin. Investig. Drugs. Dec;6(12): 1250-8 (2005)) including colorectal cancer, acute myeloid leukemia, breast cancer, gliomas, and ovarian cancer. Inhibitors of PI3K are being studied as potential therapeutics in a variety of diseases including cancer, heart failure and autoimmune/inflammatory disorders. For example, the PI3K inhibitor SFl 126 is being investigated clinically for the treatment of cancer including multiple myelomas by Semafore Pharmaceuticals, Inc.

Although a limited number of PI3K phosphorylation sites are known, and a few antibodies for their study available, there remains a need for the identification of additional phosphorylation sites relevant to activity of this kinase. Accordingly, new and improved reagents for the detection of PI3K activity would be desirable, including development of reagents against newly identified sites of PI3K phosphorylation. Since phosphorylation-dependent over- activation of PI3K is associated with diseases such as lymphoma, glioma, and colon cancer, reagents enabling the specific detection of PI3K activation would be useful tools for research and clinical applications.

SUMMARY OF THE INVENTION

The invention discloses ten novel Phosphatidylinositol 3 Kinase (PI3K) regulatory subunit phosphorylation sites, and provides antibodies, both polyclonal and monoclonal, which selectively bind to PI3KR1-R3 only when phosphorylated at one of these novel sites. The novel sites are tyrosines 467, 452, 463, and 470 in PI3KR1 (PI3Kp85 alpha), tyrosines 464, 460, and 467 in PI3KR2 (PI3Kp85 beta), and tyrosines 199, 184, and 202 in PI3KR3 (PI3Kp55 gamma), occurring on the three paralogs of human POK regulatory subunit. Several of the sites (e.g. Tyr 467 in PI3KR1, Tyr 464 in PI3KR2, and Tyr 199 in PI3KR3) are highly homologous across the three paralogs). Also provided are methods of determining the phosphorylation of PI3K in a biological sample, identifying a patient suitable for PI3K inhibitor therapy, profiling PI3K activation in a test tissue, and identifying a compound that modulates phosphorylation of PI3K in a test tissue, by using a detectable reagent, such as the disclosed antibodies, that binds to PI3K when phosphorylated at one of the disclosed sites. In preferred embodiments, the sample or test tissue is taken from a subject suspected of having cancer, such as lymphoma, glioma, and colon cancer, characterized by or involving PI3K activity.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 - is a multiple sequence alignment showing the amino acid sequences (1- letter code) of human PI3K regulatory subunit paralogs PI3KR1, PI3KR3, and PI3KR2 (SEQ ID NOs: 1-3). Tyrosines 59, 73, 76, 426, 452, 463, 467, 470, 504, 556, 657, and 679 in PI3KR1 (PI3Kp85 alpha), tyrosines 74, 365, 423, 449, 453, 460, 464, 467, 577, 605, and 671 in PI3KR2 (PI3Kp85 beta), and tyrosines 184, 188, 195, 199, 202, 373, and 407 in PI3KR3 (PI3Kp55 gamma) are shown. Tyrosines presently disclosed are shown in bold. Asterisks indicate amino acid identity among all three paralogs. The amino acid sequences of these paralogs of PI3K are publicly available at NCBI REFPEPT database (Accession Nos. NP_852664.1(PIK3R1), NP_005018.1(PIK3R2), NP_003620.2(PIK3R3)).

FIG. 2 - Western blot analysis of extracts from NIH/3T3-Src cells, untreated or treated with lambda phosphatase and from C2C12 cells, untreated or treated with H2O2, using a phospho-PI3K p85 (Tyr464)/p55 (Tyr 199) Antibody (top panel). The same blot was probed with Akt Antibody showing equal loading (bottom panel). DETAILED DESCRIPTION OF THE INVENTION

In accordance with the present invention, ten novel phosphorylation sites in human PI3K regulatory subunit have now been identified. The novel sites are tyrosines 467, 452, 463, and 470 in PI3KR1 (PI3Kp85 alpha), tyrosines 464, 460, and 467 in PI3KR2 (PI3Kp85 beta), and tyrosines 199, 184, and 202 in PI3KR3 (PI3Kp55 gamma), are most are highly homologous phosphorylation sites occurring across these three human PI3K regulatory subunit paralogs {see Figure 1). The sites each occur in the coiled coil domain of their respective paralog. Although a handful of PI3K regulatory subunit phosphorylation sites have previously been described {see Cosentino et al, supra.; Hayashi et al, supra.; Kavanaugh et al, supra; Kwon et al, supra.; Dhand et al, supra.; von Willebrand et al, supra.; Pons et al, supra.), the ten tyrosine phosphorylation sites disclosed herein are novel.

The newly identified PI3K regulatory subunit phosphorylation sites were first described by the present inventors in United States Patent Application

20080038752 (Moritz et al.), PCT/US06/00979 (Goss et al.), USSN 60/651,583 (Guo et al), PCT/US04/42940 (Guo et al), PCT/US06/ 10868 (Guo et al), USSN 60/833,752 (Guo et al), USSN 60/830,550 (Hornbeck et al), and were discovered by globally phospho-profiling cellular models of human cancers, including leukemia and carcinoma, using the PhosphoScan® technique described in U.S. Patent Nos. 7, 198,896, and 7,300,753, Rush et al, as further described in Example 1 herein. The phospho-profiling identified a total of over 1700 novel tyrosine phosphorylation sites in a multitude of different signaling proteins, including the phosphorylation sites at tyrosines 467, 452, 463, and 470 in PI3KR1 (PI3Kp85 alpha), tyrosines 464, 460, and 467 in PI3KR2 (PI3Kp85 beta), and tyrosines 199, 184, and 202 in PI3KR3 (PI3Kp55 gamma) presently described. As a result of this discovery, peptide antigens may now be designed to raise phospho-specific antibodies that bind a PI3K regulatory subunit (paralogs R1-R3) only when phosphorylated at one (or more) of the disclosed phosphorylation sites. These new reagents enable previously unavailable assays for the detection of PI3K phosphorylation at these sites.

The invention provides, in part, phospho-specific antibodies that bind to POK regulatory subunit only when phosphorylated at a tyrosine phosphorylation site selected from the group consisting of tyrosines 467, 452, 463, and 470 in PI3KR1 (PI3Kp85 alpha) (SEQ ID NO: 1), tyrosines 464, 460, and 467 in PI3KR2 (PI3Kp85 beta) (SEQ ID NO: 3), and tyrosines 199, 184, and 202 in PI3KR3 (PI3Kp55 gamma) (SEQ ID NO: 2), respectively. Also provided are methods of using a detectable reagent that binds to a disclosed phosphorylated PI3K protein to detect PI3K phosphorylation and activation in a biological sample or test tissue suspected of containing phosphorylated PI3K or having altered PI3K activity, as further described below. In a preferred embodiment, the detectable reagent is a PI3K antibody of the invention. All references cited herein are hereby incorporated herein by reference.

A. Antibodies and Cell Lines

PI3K phosphospecific antibodies of the present invention bind to PI3K regulatory subunit only when phosphorylated at a tyrosine phosphorylation site selected from the group consisting of tyrosines 467, 452, 463, and 470 in PI3KR1 (PI3Kp85 alpha) (SEQ ID NO: 1), tyrosines 464, 460, and 467 in PI3KR2 (PI3Kp85 beta) (SEQ ID NO: 3), and tyrosines 199, 184, and 202 in PI3KR3 (PI3Kp55 gamma) (SEQ ID NO: 2), respectively, but do not substantially bind to PI3K when not phosphorylated at these respective sites, nor to PI3K when phosphorylated at other tyrosine residues. The PI3K antibodies of the invention include (a) monoclonal antibody which binds phospho-PI3K sites described above, (b) polyclonal antibodies which bind to phospho-PI3K sites described above, (c) antibodies (monoclonal or polyclonal) which specifically bind to the phospho-antigen (or more preferably the epitope) bound by the exemplary PI3K phospho-specific antibodies disclosed in the Examples herein, and (d) fragments of (a), (b), or (c) above which bind to the antigen (or more preferably the epitope) bound by the exemplary antibodies disclosed herein. Such antibodies and antibody fragments may be produced by a variety of techniques well known in the art, as discussed below. Antibodies that bind to the phosphorylated epitope (i.e., the specific binding site) bound by the exemplary PI3K antibodies of the Examples herein can be identified in accordance with known techniques, such as their ability to compete with labeled PD K antibodies in a competitive binding assay. The preferred epitopic site of the PI3K antibodies of the invention is a peptide fragment consisting essentially of about 11 to 17 amino acids comprising a phosphorylated tyrosine site described herein (tyrosines 467, 452, 463, and 470 in PI3KR1 (PI3Kp85 alpha) (SEQ ID NO: 1), tyrosines 464, 460, and 467 in PI3KR2 (PI3Kp85 beta) (SEQ ID NO: 3), and tyrosines 199, 184, and 202 in PI3KR3 (PI3Kp55 gamma) (SEQ ID NO: 2), respectively), wherein about 5 to 8 amino acids are positioned on each side of the tyrosine phosphorylation site (for example, residues 194-203 of SEQ ID NO: 2).

The invention is not limited to PI3K antibodies, but includes equivalent molecules, such as protein binding domains or nucleic acid aptamers, which bind, in a phospho-specific manner, to essentially the same phosphorylated epitope to which the PI3K antibodies of the invention bind. See, e.g., Neuberger et al, Nature 312: 604 (1984). Such equivalent non-antibody reagents may be suitably employed in the methods of the invention further described below.

The term "antibody" or "antibodies" as used herein refers to all types of immunoglobulins, including IgG, IgM, IgA, IgD, and IgE, and any sub-isotype, including IgGl, IgG2a, IgG2b, IgG3 and IgG4, IgEl, IgE2 etc, and may include including F_ab or antigen-recognition fragments thereof. The antibodies may be monoclonal or polyclonal and may be of any species of origin, including (for example) mouse, rat, rabbit, horse, or human, or may be chimeric antibodies. See, e.g., M. Walker et al. , Molec. Immunol. 26: 403- 11 (1989); Morrision et al. , Proc. Nat 'I. Acad. ScL 81: 6851 (1984); Neuberger et al, Nature 312: 604 (1984)). The antibodies may be recombinant monoclonal antibodies produced according to the methods disclosed in U.S. Pat. No. 4,474,893 (Reading) or U.S. Pat. No. 4,816,567 (Cabilly et al.). The antibodies may also be chemically constructed by specific antibodies made according to the method disclosed in U.S. Pat. No. 4,676,980 (Segel et al).

The chimeric antibody is an antibody having portions derived from different antibodies. For example, a chimeric antibody may have a variable region and a constant region derived from two different antibodies. The donor antibodies may be from different species. In certain embodiments, the variable region of a chimeric antibody is non-human, e.g., murine, and the constant region is human.

"Genetically altered antibodies" refer to antibodies wherein the amino acid sequence has been varied from that of a native antibody. Because of the relevance of recombinant DNA techniques to this application, one need not be confined to the sequences of amino acids found in natural antibodies; antibodies can be redesigned to obtain desired characteristics. The possible variations are many and range from the changing of just one or a few amino acids to the complete redesign of, for example, the variable or constant region. Changes in the constant region will, in general, be made in order to improve or alter characteristics, such as complement fixation, interaction with membranes and other effector functions. Changes in the variable region will be made in order to improve the antigen binding characteristics.

The term "PI3K antibodies" means phospho-specific antibodies that selectively PI3K regulatory subunit only when phosphorylated at a tyrosine phosphorylation site selected from the group consisting of tyrosines 467, 452, 463, and 470 in PI3KR1 (PI3Kp85 alpha) (SEQ ID NO: 1), tyrosines 464, 460, and 467 in PI3KR2 (PI3Kp85 beta) (SEQ ID NO: 3), and tyrosines 199, 184, and 202 in PI3KR3 (PI3Kp55 gamma) (SEQ ID NO: 2), respectively, both monoclonal and polyclonal, as disclosed herein. The term "does not bind" with respect to such antibodies means does not substantially react with as compared to binding to phospho-PI3K. The antibodies may bind the regulatory subunit alone or when complexed with the catalytic subunit to form the complete PI 3 K holoenyzme. The term "does not bind", when appeared in context of an antibody's binding to one phospho-form (e.g., phosphorylated form) of a sequence, means that the antibody does not substantially react with the other phospho-form (e.g., non-phosphorylated form) of the same sequence. One of skill in the art will appreciate that the expression may be applicable in those instances when (1) a phospho-specific antibody either does not apparently bind to the non-phospho form of the antigen as ascertained in commonly used experimental detection systems (Western blotting, IHC, Immunofluorescence, etc.); (2) where there is some reactivity with the surrounding amino acid sequence, but that the phosphorylated residue is an immunodominant feature of the reaction. In cases such as these, there is an apparent difference in affinities for the two sequences. Dilutional analyses of such antibodies indicates that the antibodies apparent affinity for the phosphorylated form is at least 10-100 fold higher than for the non- phosphorylated form; or where (3) the phospho-specific antibody reacts no more than an appropriate control antibody would react under identical experimental conditions. A control antibody preparation might be, for instance, purified immunoglobulin from a pre-immune animal of the same species, an isotype- and species-matched monoclonal antibody. Tests using control antibodies to demonstrate specificity are recognized by one of skill in the art as appropriate and definitive. The term "detectable reagent" means a molecule, including an antibody, peptide fragment, binding protein domain, etc., the binding of which to a desired target is detectable or traceable. Suitable means of detection are described below. In particular embodiments, the antibodies of the present application are attached to labeling moieties, such as a detectable marker. One or more detectable labels can be attached to the antibodies. Exemplary labeling moieties include radiopaque dyes, radiocontrast agents, fluorescent molecules, spin-labeled molecules, enzymes, or other labeling moieties of diagnostic value, particularly in radiologic or magnetic resonance imaging techniques.

Polyclonal antibodies of the invention may be produced according to standard techniques by immunizing a suitable animal (e.g., rabbit, goat, etc.) with an antigen encompassing a PI3K phosphorylation site described herein, collecting immune serum from the animal, and separating the polyclonal antibodies from the immune serum, in accordance with known procedures. In a preferred embodiment, the antigen is a phospho-peptide antigen comprising the site sequence surrounding and including the respective phosphorylated tyrosine residue described herein, the antigen being selected and constructed in accordance with well-known techniques. See, e.g., ANTIBODIES: A LABORATORY MANUAL, Chapter 5, p. 75-76, Harlow & Lane Eds., Cold Spring Harbor Laboratory (1988); Czernik, Methods In Enzymology, 201: 264-283 ( 1991 ); Merrifield, J. Am. Chem. Soc. 85: 21 -49 ( 1962)). An exemplary peptide antigen, CSKEYDRLyEEYTRT (where y = phosphotyrosine) (SEQ ID NO: 4) for PI3K p55(Tyrl99) is described in the Examples, below. It will be appreciated by those of skill in the art that longer or shorter phosphopeptide antigens may be employed. As used herein, the term "epitope" refers to the smallest portion of a protein capable of selectively binding to the antigen binding site of an antibody. It is well accepted by those skilled in the art that the minimal size of a protein epitope capable of selectively binding to the antigen binding site of an antibody is about five or six to seven amino acids.See Id. Polyclonal PI3K antibodies produced as described herein may be screened as further described below. Monoclonal antibodies of the invention may be produced in a hybridoma cell line according to the well-known technique of Kohler and Milstein. Nature 265: 495-97 (1975); Kohler and Milstein, Eur. J. Immunol. 6: 51 1 (1976); see also, CURRENT PROTOCOLS IN MOLECULAR BIOLOGY, Ausubel et al. Eds. (1989). Monoclonal antibodies so produced are highly specific, and improve the selectivity and specificity of diagnostic assay methods provided by the invention. For example, a solution containing the appropriate antigen may be injected into a mouse or other species and, after a sufficient time (in keeping with conventional techniques), the animal is sacrificed and spleen cells obtained. The spleen cells are then immortalized by fusing them with myeloma cells, typically in the presence of polyethylene glycol, to produce hybridoma cells. Rabbit fusion hybridomas, for example, may be produced as described in U. S Patent No. 5,675,063, C. Knight, Issued October 7, 1997. The hybridoma cells are then grown in a suitable selection media, such as hypoxanthine-aminopterin- thymidine (HAT), and the supernatant screened for monoclonal antibodies having the desired specificity, as described below. The secreted antibody may be recovered from tissue culture supernatant by conventional methods such as precipitation, ion exchange or affinity chromatography, or the like.

Monoclonal Fab fragments may also be produced in Escherichia coli by recombinant techniques known to those skilled in the art. See, e.g., W. Huse, Science 246: 1275-81 ( 1989); Mullinax et al. , Proc. Nat 7 Acad. Sci. 87: 8095 (1990). If monoclonal antibodies of one isotype are preferred for a particular application, particular isotypes can be prepared directly, by selecting from the initial fusion, or prepared secondarily, from a parental hybridoma secreting a monoclonal antibody of different isotype by using the sib selection technique to isolate class-switch variants (Steplewski, et al., Proc. Nat 'I. Acad. ScL, 82: 8653 (1985); Spira et al, J. Immunol. Methods, 74: 307 (1984)).

Monoclonal antibodies of the invention may be produced recombinantly by expressing the encoding nucleic acids in a suitable host cell under suitable conditions. Accordingly, the invention further provides host cells comprising the nucleic acids and vectors described herein.

Other antibodies specifically contemplated are oligoclonal antibodies. As used herein, the phrase "oligoclonal antibodies" refers to a predetermined mixture of distinct monoclonal antibodies. See, e.g., PCT publication WO 95/20401 ; U.S.

Patent Nos. 5,789,208 and 6,335,163. In one embodiment, oligoclonal antibodies consisting of a predetermined mixture of antibodies against one or more epitopes are generated in a single cell. In other embodiments, oligoclonal antibodies comprise a plurality of heavy chains capable of pairing with a common light chain to generate antibodies with multiple specificities (e.g., PCT publication WO 04/009618). Oligoclonal antibodies are particularly useful when it is desired to target multiple epitopes on a single target molecule. In view of the assays and epitopes disclosed herein, those skilled in the art can generate or select antibodies or mixtures of antibodies that are applicable for an intended purpose and desired need.

Recombinant antibodies against the phosphorylation sites identified in the invention are also included in the present application. These recombinant antibodies have the same amino acid sequence as the natural antibodies or have altered amino acid sequences of the natural antibodies in the present application. They can be made in any expression systems including both prokaryotic and eukaryotic expression systems or using phage display methods (see, e.g., Dower et al., WO91/17271 and McCafferty et al., WO92/01047; U.S. Pat. No. 5,969,108, which are herein incorporated by reference in their entirety).

Antibodies can be engineered in numerous ways. They can be made as single-chain antibodies (including small modular immunopharmaceuticals or SMIPs™), Fab and F(ab')₂ fragments, etc. Antibodies can be humanized, chimerized, deimmunized, or fully human. Numerous publications set forth the many types of antibodies and the methods of engineering such antibodies. For example, see U.S. Patent Nos. 6,355,245; 6,180,370; 5,693,762; 6,407,213; 6,548,640; 5,565,332; 5,225,539; 6,103,889; and 5,260,203.

The genetically altered antibodies should be functionally equivalent to the above-mentioned natural antibodies. In certain embodiments, modified antibodies provide improved stability or/and therapeutic efficacy. Examples of modified antibodies include those with conservative substitutions of amino acid residues, and one or more deletions or additions of amino acids that do not significantly deleteriously alter the antigen binding utility. Substitutions can range from changing or modifying one or more amino acid residues to complete redesign of a region as long as the therapeutic utility is maintained. Antibodies of this application can be modified post-translationally (e.g., acetylation, and/or phosphorylation) or can be modified synthetically (e.g., the attachment of a labeling group). The genetically altered antibodies used in the invention include CDR grafted humanized antibodies. In one embodiment, the humanized antibody comprises heavy and/or light chain CDRs of a non-human donor immunoglobulin and heavy chain and light chain frameworks and constant regions of a human acceptor immunoglobulin. The method of making humanized antibody is disclosed in U.S. Pat. Nos: 5,530,101; 5,585,089; 5,693,761; 5,693,762; and 6,180,370 each of which is incorporated herein by reference in its entirety.

Antibodies with engineered or variant constant or Fc regions can be useful in modulating effector functions, such as, for example, antigen-dependent cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC). Such antibodies with engineered or variant constant or Fc regions may be useful in instances where a parent singling protein (Table 1 ) is expressed in normal tissue; variant antibodies without effector function in these instances may elicit the desired therapeutic response while not damaging normal tissue. Accordingly, certain aspects and methods of the present disclosure relate to antibodies with altered effector functions that comprise one or more amino acid substitutions, insertions, and/or deletions.

Antigen-binding fragments of the antibodies of the invention, which retain the binding specificity of the intact antibody, are also included in the invention. Examples of these antigen-binding fragments include, but are not limited to, partial or full heavy chains or light chains, variable regions, or CDR regions of any phosphorylation site-specific antibodies described herein.

In some instances the antibody fragments are truncated chains (truncated at the carboxyl end). In certain embodiments, these truncated chains possess one or more immunoglobulin activities (e.g., complement fixation activity). Examples of truncated chains include, but are not limited to, Fab fragments (consisting of the VL, VH, CL and CHl domains); Fd fragments (consisting of the VH and CHl domains); Fv fragments (consisting of VL and VH domains of a single chain of an antibody); dAb fragments (consisting of a VH domain); isolated CDR regions; (Fab')₂ fragments, bivalent fragments (comprising two Fab fragments linked by a disulphide bridge at the hinge region). The truncated chains can be produced by conventional biochemical techniques, such as enzyme cleavage, or recombinant DNA techniques, each of which is known in the art. These polypeptide fragments may be produced by proteolytic cleavage of intact antibodies by methods well known in the art, or by inserting stop codons at the desired locations in the vectors using site-directed mutagenesis, such as after CHl to produce Fab fragments or after the hinge region to produce (Fab')₂ fragments. Single chain antibodies may be produced by joining VL- and VH- coding regions with a DNA that encodes a peptide linker connecting the VL and VH protein fragments. Papain digestion of antibodies produces two identical antigen-binding fragments, called "Fab" fragments, each with a single antigen-binding site, and a residual "Fc" fragment, whose name reflects its ability to crystallize readily. Pepsin treatment of an antibody yields an F(ab')₂ fragment that has two antigen- combining sites and is still capable of cross-linking antigen. "Fv" usually refers to the minimum antibody fragment that contains a complete antigen-recognition and -binding site. This region consists of a dimer of one heavy- and one light-chain variable domain in tight, non-covalent association. It is in this configuration that the three CDRs of each variable domain interact to define an antigen-binding site on the surface of the V_H-V_L dimer. Collectively, the CDRs confer antigen-binding specificity to the antibody. However, even a single variable domain (or half of an Fv comprising three CDRs specific for an antigen) has the ability to recognize and bind antigen, although likely at a lower affinity than the entire binding site.

The Fab fragment also contains the constant domain of the light chain and the first constant domain (CHl) of the heavy chain. Fab' fragments differ from Fab fragments by the addition of a few residues at the carboxy terminus of the heavy chain CHl domain including one or more cysteines from the antibody hinge region. Fab' -SH is the designation herein for Fab' in which the cysteine residue(s) of the constant domains bear a free thiol group. F(ab')₂ antibody fragments originally were produced as pairs of Fab' fragments that have hinge cysteines between them. Other chemical couplings of antibody fragments are also known.

"Single-chain Fv" or "scFv" antibody fragments comprise the V_H and V_L domains of an antibody, wherein these domains are present in a single polypeptide chain. In certain embodiments, the Fv polypeptide further comprises a polypeptide linker between the V_H and V_L domains that enables the scFv to form the desired structure for antigen binding. For a review of scFv see Pluckthun in The Pharmacology of Monoclonal Antibodies, vol. 113, Rosenburg and Moore, eds. (Springer- Verlag: New York, 1994), pp. 269-315. Bispecific antibodies may be monoclonal, human or humanized antibodies that have binding specificities for at least two different antigens. In the present case, one of the binding specificities is for the phosphorylation site, the other one is for any other antigen, such as for example, a cell-surface protein or receptor or receptor subunit. Alternatively, a therapeutic agent may be placed on one arm. The therapeutic agent can be a drug, toxin, enzyme, DNA, radionuclide, etc.

In some instances, the antigen-binding fragment can be a diabody. The term "diabody" refers to small antibody fragments with two antigen-binding sites, which fragments comprise a heavy-chain variable domain (V_H) connected to a light-chain variable domain (V_L) in the same polypeptide chain (V_H-V_L). By using a linker that is too short to allow pairing between the two domains on the same chain, the domains are forced to pair with the complementary domains of another chain and create two antigen-binding sites. Diabodies are described more fully in, for example, EP 404,097; WO 93/11161; and Hollinger et al., Proc. Natl. Acad. Sci. USA, 90: 6444-6448 ( 1993). Camelid antibodies refer to a unique type of antibodies that are devoid of light chain, initially discovered from animals of the camelid family. The heavy chains of these so-called heavy-chain antibodies bind their antigen by one single domain, the variable domain of the heavy immunoglobulin chain, referred to as VHH. VHHs show homology with the variable domain of heavy chains of the human VHIII family. The VHHs obtained from an immunized camel, dromedary, or llama have a number of advantages, such as effective production in microorganisms such as Saccharomyces cerevisiae.

In certain embodiments, single chain antibodies, and chimeric, humanized or primatized (CDR-grafted) antibodies, as well as chimeric or CDR-grafted single chain antibodies, comprising portions derived from different species, are also encompassed by the present disclosure as antigen-binding fragments of an antibody. The various portions of these antibodies can be joined together chemically by conventional techniques, or can be prepared as a contiguous protein using genetic engineering techniques. For example, nucleic acids encoding a chimeric or humanized chain can be expressed to produce a contiguous protein. See, e.g., U.S. Pat. Nos. 4,816,567 and 6,331,415; U.S. Pat. No. 4,816,397; European Patent No. 0,120,694; WO 86/01533; European Patent No. 0,194,276 Bl; U.S. Pat. No. 5,225,539; and European Patent No. 0,239,400 Bl. See also, Newman et al., BioTechnology, 10: 1455-1460 (1992), regarding primatized antibody. See, e.g., Ladner et al., U.S. Pat. No. 4,946,778; and Bird et al., Science, 242: 423-426 (1988)), regarding single chain antibodies.

In addition, functional fragments of antibodies, including fragments of chimeric, humanized, primatized or single chain antibodies, can also be produced. Functional fragments of the subject antibodies retain at least one binding function and/or modulation function of the full-length antibody from which they are derived.

Also contemplated are other equivalent non-antibody molecules, such as protein binding domains or aptamers, which bind, in a phospho-specific manner, to an amino acid sequence comprising a novel phosphorylation site of the invention. See, e.g., Neuberger et al, Nature 312: 604 (1984). Aptamers are oligonucleic acid or peptide molecules that bind a specific target molecule. DNA or RNA aptamers are typically short oligonucleotides, engineered through repeated rounds of selection to bind to a molecular target. Peptide aptamers typically consist of a variable peptide loop attached at both ends to a protein scaffold. This double structural constraint generally increases the binding affinity of the peptide aptamer to levels comparable to an antibody (nanomolar range).

The invention also provides hybridoma clones, constructed as described above, that produce PI3K monoclonal antibodies of the invention. Similarly, the invention includes recombinant cells producing a PI3K antibody as disclosed herein, which cells may be constructed by well known techniques; for example the antigen combining site of the monoclonal antibody can be cloned by PCR and single-chain antibodies produced as phage-displayed recombinant antibodies or soluble antibodies in E. coli {see, e.g., ANTIBODY ENGINEERING PROTOCOLS, 1995 , Humana Press, Sudhir Paul editor.)

PDK antibodies of the invention, whether polyclonal or monoclonal, may be screened for epitope and phospho-specificity according to standard techniques. See, e.g. Czernik et al, Methods in Enzymology, 201: 264-283 (1991). For example, the antibodies may be screened against the phospho and non-phospho peptide library by ELISA to ensure specificity for both the desired antigen (i.e. that epitope including a tyrosine phosphorylation site disclosed herein) and for reactivity only with the phosphorylated form of the antigen. Peptide competition assays may be carried out to confirm lack of reactivity with other PI3K phospho- epitopes. The antibodies may also be tested by Western blotting against cell preparations containing PI3K, e.g. cell lines over-expressing PI3K, to confirm reactivity with the desired phosphorylated target.

Specificity against the desired phosphorylated epitopes may also be examined by construction PI3K mutants lacking phosphorylatable residues at positions outside the desired epitope known to be phosphorylated, or by mutating the desired phospho-epitope and confirming lack of reactivity . PI3K antibodies of the invention may exhibit some cross-reactivity with non-PI3K epitopes. This is not unexpected as most antibodies exhibit some degree of cross-reactivity, and anti-peptide antibodies will often cross-react with epitopes having high homology to the immunizing peptide. See, e.g., Czernik, supra. Cross-reactivity with non- PDK proteins is readily characterized by Western blotting alongside markers of known molecular weight. Amino acid sequences of cross-reacting proteins may be examined to identify sites highly homologous to a PDK sequence surrounding any of the phosphory lated tyrosines disclosed herein.

A "phosphorylatable" amino acid refers to an amino acid that is capable of being modified by addition of a phosphate group (any includes both phosphorylated form and unphosphorylated form). Alternatively, the tyrosine may be deleted. Residues other than the tyrosine may also be modified (e.g., delete or mutated) if such modification inhibits the phosphorylation of the tyrosine residue. For example, residues flanking the tyrosine may be deleted or mutated, so that a kinase can not recognize/phosphorylate the mutated protein or the peptide. Standard mutagenesis and molecular cloning techniques can be used to create amino acid substitutions or deletions.

In certain cases, polyclonal antisera may be exhibit some undesirable general cross-reactivity to phosphotyrosine, which may be removed by further purification of antisera, e.g. over a phosphotyramine column. PDK phospho- specific antibodies raised against one of the disclosed subunit paralog phosphorylation sites may also cross-react with one or more of the nearly identical sites in the other paralogs, as expected. For example, a phospho- specific antibody raised against the PDKR2 (Tyr464) site may cross-react with the nearly-identical PDKR3 (Tyrl99) site, which differ by only two amino acids.

PDK antibodies may be further characterized via immunohistochemical (IHC) staining using normal and diseased tissues to examine PDK phosphorylation and activation status in diseased tissue. IHC may be carried out according to well-known techniques. See, e.g., ANTIBODIES: A LABORATORY MANUAL, Chapter 10, Harlow & Lane Eds., Cold Spring Harbor Laboratory (1988). Briefly, paraffin-embedded tissue (e.g. tumor tissue) is prepared for immunohistochemical staining by deparaffinizing tissue sections with xylene followed by ethanol; hydrating in water then PBS; unmasking antigen by heating slide in sodium citrate buffer; incubating sections in hydrogen peroxide; blocking in blocking solution; incubating slide in primary antibody and secondary antibody; and finally detecting using ABC avidin/biotin method according to manufacturer's instructions.

B. Detection & Profiling Methods

The methods disclosed herein may be employed with any biological sample suspected of containing phosphorylated POK, and in particular, PDK regulatory subunit phosphorylated at a tyrosine phosphorylation site selected from the group consisting of tyrosines 467, 452, 463, and 470 in PI3KR1 (PI3Kp85 alpha) (SEQ ID NO: 1), tyrosines 464, 460, and 467 in PI3KR2 (PI3Kp85 beta) (SEQ ID NO: 3), and tyrosines 199, 184, and 202 in PI3KR3 (PI3Kp55 gamma) (SEQ ID NO: 2). Biological samples taken from human subjects for use in the methods disclosed herein are generally biological fluids such as serum, blood plasma, fine needle aspirate, ductal lavage, bone marrow sample or ascites fluid. In the alternative, the sample taken from the subject can be a tissue sample (e.g., a biopsy tissue), such as tumor tissue. The term "subject" refers to a vertebrate, such as for example, a mammal, or a human. Although present application are primarily concerned with the treatment of human subjects, the disclosed methods may also be used for the treatment of other mammalian subjects such as dogs and cats for veterinary purposes. In one embodiment, the invention provides a method for detecting phosphorylated PI3K in a biological sample by (a) contacting (binding) a biological sample suspected of containing phosphorylated PI3K with at least one antibody that binds to a Phosphatidylinositol 3 Kinase (PI3K) regulatory subunit only when phosphorylated at a tyrosine phosphorylation site selected from the group consisting of tyrosines 467, 452, 463, and 470 in PI3KR1 (PI3Kp85 alpha) (SEQ ID NO: 1), tyrosines 464, 460, and 467 in PI3KR2 (PI3Kp85 beta) (SEQ ID NO: 3), and tyrosines 199, 184, and 202 in PI3KR3 (PI3Kp55 gamma) (SEQ ID NO: 2) under conditions suitable for formation of a reagent-PI3K complex, and (b) detecting the presence of the complex in the sample, wherein the presence of the complex indicates the presence of phosphorylated PI3K in the sample. Biological samples may be obtained from subjects suspected of having a disease involving altered PI3K expression or activity (e.g., lymphoma, glioma, colon cancer, lung cancer, and ovarian cancer). Samples may be analyzed to monitor subjects who have been previously diagnosed as having cancer, to screen subjects who have not been previously diagnosed as carrying cancer, or to monitor the desirability or efficacy of therapeutics targeted at PI3K. Subjects may be either children or adults. In the case of colon cancer, for example, the subjects will most frequently be adult males. In another embodiment, the invention provides a method for profiling

PI3K activation in a test tissue suspected of involving altered PI3K activity, by (a) contacting the test tissue with at least one antibody that binds to a PI3K regulatory subunit only when phosphorylated at a tyrosine phosphorylation site selected from the group consisting of tyrosines 467, 452, 463, and 470 in PI3KR1 (PI3Kp85 alpha) (SEQ ID NO: 1), tyrosines 464, 460, and 467 in PI3KR2 (PI3Kp85 beta) (SEQ ID NO: 3), and tyrosines 199, 184, and 202 in PI3KR3 (PI3Kp55 gamma) (SEQ ID NO: 2) under conditions suitable for formation of a reagent-PI3K complex, (b) detecting the presence of the complex in the test tissue, wherein the presence of the complex indicates the presence of phosphorylated PI3K in the test tissue, and (c) comparing the presence of phosphorylated PI3K detected in step(b) with the presence of phosphorylated PI3K in a control tissue, wherein a difference in PI3K phosphorylation profiles between the test and control tissues indicates altered PI3K activation in the test tissue. In a preferred embodiment, the reagent is a PI3K antibody of the invention. In other preferred embodiments, the test tissue is a cancer tissue, such as lymphoma, glioma, and colon cancer tissue, suspected of involving altered PI3K phosphorylation.

The methods described above are applicable to examining tissues or samples from PI3K related cancers, particularly colorectal cancer, acute myeloid leukemia, breast cancer, gliomas, and ovarian cancer, in which phosphorylation of PI3K at any of the novel sites disclosed herein has predictive value as to the outcome of the disease or the response of the disease to therapy. It is anticipated that the PI3K antibodies will have diagnostic utility in a disease characterized by, or involving, altered PI3K activity or altered PI3K phosphorylation. The methods are applicable, for example, where samples are taken from a subject has not been previously diagnosed as having lymphoma, glioma, and colon cancer, nor has yet undergone treatment for lymphoma, glioma, and colon cancer, and the method is employed to help diagnose the disease, monitor the possible progression of the cancer, or assess risk of the subject developing such cancer involving PI3K phosphorylation. Such diagnostic assay may be carried out prior to preliminary blood evaluation or surgical surveillance procedures.

Such a diagnostic assay may be employed to identify patients with activated PI3K who would be most likely to respond to cancer therapeutics targeted at inhibiting PI3K activity. Such a selection of patients would be useful in the clinical evaluation of efficacy of existing or future PI3K inhibitors, as well as in the future prescription of such drugs to patients. Accordingly, in another embodiment, the invention provides a method for selecting a patient suitable for PI3K inhibitor therapy, said method comprising the steps of (a) obtaining at least one biological sample from a patient that is a candidate for PI3K inhibitor therapy,

(b) contacting the biological sample with at least one PI3K phospho-specific antibody described herein under conditions suitable for formation of a reagent- PI3K complex, and (c) detecting the presence of the complex in the biological sample, wherein the presence of said complex indicates the presence of phosphorylated PI3K in said test tissue, thereby identifying the patient as potentially suitable for PI3K inhibitor therapy.

Alternatively, the methods are applicable where a subject has been previously diagnosed as having, e.g. lymphoma, glioma, and colon cancer, and possibly has already undergone treatment for the disease, and the method is employed to monitor the progression of such cancer involving PI3K phosphorylation, or the treatment thereof.

In another embodiment, the invention provides a method for identifying a compound which modulates phosphorylation of PI3K in a test tissue, by (a) contacting the test tissue with the compound, (b) detecting the level of phosphorylated PI3K in said the test tissue of step (a) using at least one PI3K phospho-specific antibody described herein under conditions suitable for formation of an antiboy-PI3K complex, and (c) comparing the level of phosphorylated PI3K detected in step(b) with the presence of phosphorylated PI3K in a control tissue not contacted with the compound, wherein a difference in PI3K phosphorylation levels between the test and control tissues identifies the compound as a modulator of PI3K phosphorylation. In some preferred embodiments, the test tissue is a taken from a subject suspected of having cancer and the compound is a PI 3 K inhibitor. The compound may modulate PI 3 K activity either positively or negatively, for example by increasing or decreasing phosphorylation or expression of PI3K. PI3K phosphorylation and activity may be monitored, for example, to determine the efficacy of an anti-PI3K therapeutic, e.g. a PI3K inhibitor.

Conditions suitable for the formation of antibody-antigen complexes or reagent-PI3K complexes are well known in the art (see part (d) below and references cited therein). It will be understood that more than one PI3K antibody may be used in the practice of the above-described methods. For example, PI3KR1 (PI3Kp85 alpha) (Tyr467) phospho-specific antibody and a PI3KR2 (PI3Kp85 beta) (Tyr460) phospho-specific antibody may be simultaneously employed to detect phosphorylation of both tyrosines in these two subunit paralogs in one step. Alternatively, multiple antibodies may be simultaneously employed to detect phosphorylation of multiple tyrosines on a single subunit paralog in one step.

C. Immunoassay Formats & Diagnostic Kits

Assays carried out in accordance with methods of the present invention may be homogeneous assays or heterogeneous assays. In a homogeneous assay the immunological reaction usually involves a PI3K-specific reagent (e.g. a PI3K antibody of the invention), a labeled analyte, and the sample of interest. The signal arising from the label is modified, directly or indirectly, upon the binding of the antibody to the labeled analyte. Both the immunological reaction and detection of the extent thereof are carried out in a homogeneous solution. Immunochemical labels that may be employed include free radicals, radioisotopes, fluorescent dyes, enzymes, bacteriophages, coenzymes, and so forth.

In a heterogeneous assay approach, the reagents are usually the specimen, a PI3K-specific reagent (e.g., the PI3K antibody of the invention), and suitable means for producing a detectable signal. Similar specimens as described above may be used. The antibody is generally immobilized on a support, such as a bead, plate or slide, and contacted with the specimen suspected of containing the antigen in a liquid phase. The support is then separated from the liquid phase and either the support phase or the liquid phase is examined for a detectable signal employing means for producing such signal. The signal is related to the presence of the analyte in the specimen. Means for producing a detectable signal include the use of radioactive labels, fluorescent labels, enzyme labels, and so forth. For example, if the antigen to be detected contains a second binding site, an antibody which binds to that site can be conjugated to a detectable group and added to the liquid phase reaction solution before the separation step. The presence of the detectable group on the solid support indicates the presence of the antigen in the test sample. Examples of suitable immunoassays are the radioimmunoassay, immunofluorescence methods, enzyme-linked immunoassays, and the like.

Immunoassay formats and variations thereof that may be useful for carrying out the methods disclosed herein are well known in the art. See generally E. Maggio, Enzyme-Immunoassay, (1980) (CRC Press, Inc., Boca Raton, FIa.); see also, e.g., U.S. Pat. No. 4,727,022 (Skold et al, "Methods for Modulating Ligand-Receptor Interactions and their Application"); U.S. Pat. No. 4,659,678 (Forrest et al, "Immunoassay of Antigens"); U.S. Pat. No. 4,376,1 10 (David et al, "Irnmunometric Assays Using Monoclonal Antibodies"). Conditions suitable for the formation of reagent-antibody complexes are well described. See id. Monoclonal antibodies of the invention may be used in a "two-site" or "sandwich" assay, with a single cell line serving as a source for both the labeled monoclonal antibody and the bound monoclonal antibody. Such assays are described in U.S. Pat. No. 4,376,110. The concentration of detectable reagent should be sufficient such that the binding of phosphorylated PI3K is detectable compared to background.

PI3K antibodies disclosed herein may be conjugated to a solid support suitable for a diagnostic assay {e.g., beads, plates, slides or wells formed from materials such as latex or polystyrene) in accordance with known techniques, such as precipitation. Antibodies of the invention, or other PI3K binding reagents, may likewise be conjugated to detectable groups such as radiolabels {e.g.,³⁵S,¹²⁵I,¹³¹I), enzyme labels {e.g., horseradish peroxidase, alkaline phosphatase), and fluorescent labels {e.g., fluorescein) in accordance with known techniques. PI3K antibodies of the invention may also be optimized for use in a flow cytometry assay to determine the activation status of PI 3 K in patients before, during, and after treatment with a drug targeted at inhibiting PI3K phosphorylation at a tyrosine site disclosed herein. For example, bone marrow cells or peripheral blood cells from patients may be analyzed by flow cytometry for PI3K phosphorylation, as well as for markers identifying various hematopoietic cell types. In this manner, PI3K activation status of the malignant cells may be specifically characterized.

Flow cytometry may be carried out according to standard methods. See, e.g. Chow et al, Cytometry (Communications in Clinical Cytometry) 46: 72-78 (2001). Briefly and by way of example, the following protocol for cytometric analysis may be employed: fixation of the cells with 1% paraformaldehyde for 10 minutes at 37⁰C followed by permeabilization in 90% methanol for 30 minutes on ice. Cells may then be stained with the primary PI3K antibody, washed and labeled with a fluorescent-labeled secondary antibody. Alternatively, the cells may be stained with a fluorescent-labeled primary antibody. The cells would then be analyzed on a flow cytometer {e.g. a Beckman Coulter EPICS-XL) according to the specific protocols of the instrument used. Such an analysis would identify the presence of activated PI3K in the malignant cells and reveal the drug response on the targeted PI3K protein. Alternatively, PI3K antibodies of the invention may be optimized for use in other clinically-suitable applications, for example bead-based multiplex-type assays, such as IGEN, Luminex™ and/or Bioplex™ assay formats, or otherwise optimized for antibody arrays formats.

The use of the antibodies in a RIA assay are additionally contemplated. The radioimmunoassay (RIA) is an analytical technique which depends on the competition (affinity) of an antigen for antigen-binding sites on antibody molecules. Standard curves are constructed from data gathered from a series of samples each containing the same known concentration of labeled antigen, and various, but known, concentrations of unlabeled antigen. Antigens are labeled with a radioactive isotope tracer. The mixture is incubated in contact with an antibody. Then the free antigen is separated from the antibody and the antigen bound thereto. Then, by use of a suitable detector, such as a gamma or beta radiation detector, the percent of either the bound or free labeled antigen or both is determined. This procedure is repeated for a number of samples containing various known concentrations of unlabeled antigens and the results are plotted as a standard graph. The percent of bound tracer antigens is plotted as a function of the antigen concentration. Typically, as the total antigen concentration increases the relative amount of the tracer antigen bound to the antibody decreases. After the standard graph is prepared, it is thereafter used to determine the concentration of antigen in samples undergoing analysis.

In an analysis, the sample in which the concentration of antigen is to be determined is mixed with a known amount of tracer antigen. Tracer antigen is the same antigen known to be in the sample but which has been labeled with a suitable radioactive isotope. The sample with tracer is then incubated in contact with the antibody. Then it can be counted in a suitable detector which counts the free antigen remaining in the sample. The antigen bound to the antibody or immunoadsorbent may also be similarly counted. Then, from the standard curve, the concentration of antigen in the original sample is determined.

Diagnostic kits for carrying out the methods disclosed above are also provided by the invention. Such kits comprise at least one detectable reagent that binds to PI3K when phosphorylated at a novel tyrosine phosphorylation site disclosed herein (a phosphorylation site selected from the group consisting of tyrosines 467, 452, 463, and 470 in PI3KR1 (PI3Kp85 alpha) (SEQ ID NO: 1), tyrosines 464, 460, and 467 in PI3KR2 (PI3Kp85 beta) (SEQ ID NO: 3), and tyrosines 199, 184, and 202 in PI3KR3 (PI3Kp55 gamma)). In a preferred embodiment, the reagent is a PI3K antibody of the invention. In one embodiment, the diagnostic kit comprises (a) a PI3K antibody of the invention conjugated to a solid support and (b) a second antibody conjugated to a detectable group. The reagents may also include ancillary agents such as buffering agents and protein stabilizing agents, e.g., polysaccharides and the like. The diagnostic kit may further include, where necessary, other members of the signal -producing system of which system the detectable group is a member (e.g., enzyme substrates), agents for reducing background interference in a test, control reagents, apparatus for conducting a test, and the like. In another embodiment a kit (e.g. a kit for the selection of a patient suitable for PI3K inhibitor therapy) comprises (a) a PI3K antibody as described herein, and (b) a specific binding partner (i.e. secondary antibody) conjugated to a detectable group.

The primary (phospho-PDK) detection antibody may itself be directly labeled with a detectable group, or alternatively, a secondary antibody, itself labeled with a detectable group, that binds to the primary antibody may be employed. Labels (including dyes and the like) suitable as detectable agents are well known in the art. Ancillary agents as described above may likewise be included. The test kit may be packaged in any suitable manner, typically with all elements in a single container along with a sheet of printed instructions for carrying out the test.

D. Therapeutic Uses

The invention provides methods and compositions for therapeutic uses of the peptides or proteins comprising a phosphorylation site of the invention, and phosphorylation site-specific antibodies of the invention. The invention provides for a method of treating or preventing a disease such as, for example, carcinoma in a subject, wherein the carcinoma is associated with the phosphorylation state of a novel phosphorylation site, whether phosphorylated or dephosphorylated, comprising: administering to a subject in need thereof a therapeutically effective amount of a peptide comprising a novel phosphorylation site and/or an antibody or antigen-binding fragment thereof that specifically bind a novel phosphorylation site of the invention. The antibodies maybe full-length antibodies, genetically engineered antibodies, antibody fragments, and antibody conjugates of the invention.

In one aspect, the disclosure provides a method of treating carcinoma, for example, in which a peptide or an antibody that reduces at least one biological activity of a targeted signaling protein is administered to a subject. For example, the peptide or the antibody administered may disrupt or modulate the interaction of the target signaling protein with its ligand. Alternatively, the peptide or the antibody may interfere with, thereby reducing, the down-stream signal transduction of the parent signaling protein. An antibody that specifically binds the novel tyrosine phosphorylation site only when the tyrosine is phosphorylated, and that does not substantially bind to the same sequence when the tyrosine is not phosphorylated, thereby prevents downstream signal transduction triggered by a phospho-tyrosine. Alternatively, an antibody that specifically binds the unphosphorylated target phosphorylation site reduces the phosphorylation at that site and thus reduces activation of the protein mediated by phosphorylation of that site. Similarly, an unphosphorylated peptide may compete with an endogenous phosphorylation site for same kinases, thereby preventing or reducing the phosphorylation of the endogenous target protein. Alternatively, a peptide comprising a phosphorylated novel tyrosine site of the invention but lacking the ability to trigger signal transduction may competitively inhibit interaction of the endogenous protein with the same down-stream ligand(s).

The antibodies of the invention may also be used to target cancer cells for effector-mediated cell death. The antibody disclosed herein may be administered as a fusion molecule that includes a phosphorylation site-targeting portion joined to a cytotoxic moiety to directly kill cancer cells. Alternatively, the antibody may directly kill the cancer cells through complement-mediated or antibody- dependent cellular cytotoxicity. Accordingly in one embodiment, the antibodies of the present disclosure may be used to deliver a variety of cytotoxic compounds. Any cytotoxic compound can be fused to the present antibodies. The fusion can be achieved chemically or genetically (e.g., via expression as a single, fused molecule). The cytotoxic compound can be a biological, such as a polypeptide, or a small molecule. As those skilled in the art will appreciate, for small molecules, chemical fusion is used, while for biological compounds, either chemical or genetic fusion can be used.

Non-limiting examples of cytotoxic compounds include therapeutic drugs, radiotherapeutic agents, ribosome-inactivating proteins (RIPs), chemotherapeutic agents, toxic peptides, toxic proteins, and mixtures thereof. The cytotoxic drugs can be intracellularly acting cytotoxic drugs, such as short-range radiation emitters, including, for example, short-range, high-energy α-emitters. Enzymatically active toxins and fragments thereof, including ribosome- inactivating proteins, are exemplified by saporin, luffin, momordins, ricin, trichosanthin, gelonin, abrin, etc. Procedures for preparing enzymatically active polypeptides of the immunotoxins are described in WO84/03508 and WO85/03508, which are hereby incorporated by reference. Certain cytotoxic moieties are derived from adriamycin, chlorambucil, daunomycin, methotrexate, neocarzinostatin, and platinum, for example. Exemplary chemotherapeutic agents that may be attached to an antibody or antigen-binding fragment thereof include taxol, doxorubicin, verapamil, podophyllotoxin, procarbazine, mechlorethamine, cyclophosphamide, camptothecin, ifosfamide, melphalan, chlorambucil, bisulfan, nitrosurea, dactinomycin, daunorubicin, doxorubicin, bleomycin, plicomycin, mitomycin, etoposide (VPl 6), tamoxifen, transplatinum, 5-fluorouracil, vincristin, vinblastin, or methotrexate.

Procedures for conjugating the antibodies with the cytotoxic agents have been previously described and are within the purview of one skilled in the art. Alternatively, the antibody can be coupled to high energy radiation emitters, for example, a radioisotope, such as¹³¹I, a γ-emitter, which, when localized at the tumor site, results in a killing of several cell diameters. See, e.g., S. E. Order, "Analysis, Results, and Future Prospective of the Therapeutic Use of Radiolabeled Antibody in Cancer Therapy", Monoclonal Antibodies for Cancer Detection and Therapy, Baldwin et al. (eds.), pp. 303-316 (Academic Press 1985), which is hereby incorporated by reference. Other suitable radioisotopes include α-emitters, such as²¹²Bi,²¹³Bi, and^{21 1}At, and β-emitters, such as¹⁸⁶Re and⁹⁰Y.

Because many of the signaling proteins in which novel tyrosine phosphorylation sites of the invention occur also are expressed in normal cells and tissues, it may also be advantageous to administer a phosphorylation site- specific antibody with a constant region modified to reduce or eliminate ADCC or CDC to limit damage to normal cells. For example, effector function of an antibodies may be reduced or eliminated by utilizing an IgGl constant domain instead of an IgG2/4 fusion domain. Other ways of eliminating effector function can be envisioned such as, e.g., mutation of the sites known to interact with FcR or insertion of a peptide in the hinge region, thereby eliminating critical sites required for FcR interaction. Variant antibodies with reduced or no effector function also include variants as described previously herein.

The peptides and antibodies of the invention may be used in combination with other therapies or with other agents. Other agents include but are not limited to polypeptides, small molecules, chemicals, metals, organometallic compounds, inorganic compounds, nucleic acid molecules, oligonucleotides, aptamers, spiegelmers, antisense nucleic acids, locked nucleic acid (LNA) inhibitors, peptide nucleic acid (PNA) inhibitors, immunomodulatory agents, antigen-binding fragments, prodrugs, and peptidomimetic compounds. In certain embodiments, the antibodies and peptides of the invention may be used in combination with cancer therapies known to one of skill in the art.

In certain aspects, the present disclosure relates to combination treatments comprising a phosphorylation site-specific antibody described herein and immunomodulatory compounds, vaccines or chemotherapy. Illustrative examples of suitable immunomodulatory agents that may be used in such combination therapies include agents that block negative regulation of T cells or antigen presenting cells (e.g., anti-CTLA4 antibodies, anti-PD-Ll antibodies, anti-PDL-2 antibodies, anti-PD-1 antibodies and the like) or agents that enhance positive co- stimulation of T cells (e.g., anti-CD40 antibodies or anti 4- IBB antibodies) or agents that increase NK cell number or T-cell activity (e.g., inhibitors such as IMiDs, thalidomide, or thalidomide analogs). Furthermore, immunomodulatory therapy could include cancer vaccines such as dendritic cells loaded with tumor cells, proteins, peptides, RNA, or DNA derived from such cells, patient derived heat-shock proteins (hsp's) or general adjuvants stimulating the immune system at various levels such as CpG, Luivac®, Biostim®, Rlbomunyl®, Imudon®, Broncho vaxom® or any other compound or other adjuvant activating receptors of the innate immune system (e.g., toll like receptor agonist, anti-CTLA-4 antibodies, etc.). Also, immunomodulatory therapy could include treatment with cytokines such as IL-2, GM-CSF and IFN-gamma.

Furthermore, combination of antibody therapy with chemotherapeutics could be particularly useful to reduce overall tumor burden, to limit angiogenesis, to enhance tumor accessibility, to enhance susceptibility to ADCC, to result in increased immune function by providing more tumor antigen, or to increase the expression of the T cell attractant LIGHT.

Pharmaceutical compounds that may be used for combinatory anti-tumor therapy include, merely to illustrate: aminoglutethimide, amsacrine, anastrozole, asparaginase, beg, bicalutamide, bleomycin, buserelin, busulfan, camptothecin, capecitabine, carboplatin, carmustine, chlorambucil, cisplatin, cladribine, clodronate, colchicine, cyclophosphamide, cyproterone, cytarabine, dacarbazine, dactinomycin, daunorubicin, dienestrol, diethylstilbestrol, docetaxel, doxorubicin, epirubicin, estradiol, estramustine, etoposide, exemestane, filgrastim, fludarabine, fludrocortisone, fluorouracil, fluoxymesterone, flutamide, gemcitabine, genistein, goserelin, hydroxyurea, idarubicin, ifosfamide, imatinib, interferon, irinotecan, letrozole, leucovorin, leuprolide, levamisole, lomustine, mechlorethamine, medroxyprogesterone, megestrol, melphalan, mercaptopurine, mesna, methotrexate, mitomycin, mitotane, mitoxantrone, nilutamide, nocodazole, octreotide, oxaliplatin, paclitaxel, pamidronate, pentostatin, plicamycin, porfimer, procarbazine, raltitrexed, rituximab, streptozocin, suramin, tamoxifen, temozolomide, teniposide, testosterone, thioguanine, thiotepa, titanocene dichloride, topotecan, trastuzumab, tretinoin, vinblastine, vincristine, vindesine, and vinorelbine.

These chemotherapeutic anti-tumor compounds may be categorized by their mechanism of action into groups, including, for example, the following classes of agents: anti-metabolites/anti-cancer agents, such as pyrimidine analogs (5-fluorouracil, floxuridine, capecitabine, gemcitabine and cytarabine) and purine analogs, folate inhibitors and related inhibitors (mercaptopurine, thioguanine, pentostatin and 2-chlorodeoxyadenosine (cladribine)); antiproliferative/antimitotic agents including natural products such as vinca alkaloids (vinblastine, vincristine, and vinorelbine), microtubule disruptors such as taxane (paclitaxel, docetaxel), vincristine, vinblastine, nocodazole, epothilones and navelbine, epidipodophyllotoxins (etoposide, teniposide), DNA damaging agents (actinomycin, amsacrine, anthracyclines, bleomycin, busulfan, camptothecin, carboplatin, chlorambucil, cisplatin, cyclophosphamide, Cytoxan, dactinomycin, daunorubicin, doxorubicin, epirubicin, hexamethylmelamineoxaliplatin, iphosphamide, melphalan, mechlorethamine, mitomycin, mitoxantrone, nitrosourea, plicamycin, procarbazine, taxol, taxotere, teniposide, triethylenethiophosphoramide and etoposide (VP 16)); antibiotics such as dactinomycin (actinomycin D), daunorubicin, doxorubicin (adriamycin), idarubicin, anthracyclines, mitoxantrone, bleomycins, plicamycin (mithramycin) and mitomycin; enzymes (L-asparaginase which systemically metabolizes L- asparagine and deprives cells which do not have the capacity to synthesize their own asparagine); antiplatelet agents; antiproliferative/antimitotic alkylating agents such as nitrogen mustards (mechlorethamine, cyclophosphamide and analogs, melphalan, chlorambucil), ethylenimines and methylmelamines

(hexamethylmelamine and thiotepa), alkyl sulfonates-busulfan, nitrosoureas (carmustine (BCNU) and analogs, streptozocin), trazenes - dacarbazinine (DTIC); antiproliferative/antimitotic antimetabolites such as folic acid analogs (methotrexate); platinum coordination complexes (cisplatin, carboplatin), procarbazine, hydroxyurea, mitotane, aminoglutethimide; hormones, hormone analogs (estrogen, tamoxifen, goserelin, bicalutamide, nilutamide) and aromatase inhibitors (letrozole, anastrozole); anticoagulants (heparin, synthetic heparin salts and other inhibitors of thrombin); fibrinolytic agents (such as tissue plasminogen activator, streptokinase and urokinase), aspirin, dipyridamole, ticlopidine, clopidogrel, abciximab; antimigratory agents; antisecretory agents (breveldin); immunosuppressives (cyclosporine, tacrolimus (FK-506), sirolimus (rapamycin), azathioprine, mycophenolate mofetil); immunomodulatory agents (thalidomide and analogs thereof such as lenalidomide (Revlimid, CC-5013) and CC-4047 (Actimid)), cyclophosphamide; anti-angiogenic compounds (TNP-470, genistein) and growth factor inhibitors (vascular endothelial growth factor (VEGF) inhibitors, fibroblast growth factor (FGF) inhibitors); angiotensin receptor blocker; nitric oxide donors; anti-sense oligonucleotides; antibodies (trastuzumab); cell cycle inhibitors and differentiation inducers (tretinoin); mTOR inhibitors, topoisomerase inhibitors (doxorubicin (adriamycin), amsacrine, camptothecin, daunorubicin, dactinomycin, eniposide, epirubicin, etoposide, idarubicin and mitoxantrone, topotecan, irinotecan), corticosteroids (cortisone, dexamethasone, hydrocortisone, methylprednisolone, prednisone, and prenisolone); growth factor signal transduction kinase inhibitors; mitochondrial dysfunction inducers and caspase activators; and chromatin disruptors.

In certain embodiments, pharmaceutical compounds that may be used for combinatory anti-angiogenesis therapy include: (1) inhibitors of release of "angiogenic molecules," such as bFGF (basic fibroblast growth factor); (2) neutralizers of angiogenic molecules, such as anti-βbFGF antibodies; and (3) inhibitors of endothelial cell response to angiogenic stimuli, including collagenase inhibitor, basement membrane turnover inhibitors, angiostatic steroids, fungal-derived angiogenesis inhibitors, platelet factor 4, thrombospondin, arthritis drugs such as D-penicillamine and gold thiomalate, vitamin D₃ analogs, alpha-interferon, and the like. For additional proposed inhibitors of angiogenesis, see Blood et al., Biochim. Biophys. Acta, 1032:89-1 18 (1990), Moses et al., Science, 248:1408-1410 (1990), Ingber et al., Lab. Invest., 59:44-51 (1988), and U.S. Pat. Nos. 5,092,885, 5,112,946, 5,192,744, 5,202,352, and 6,573,256. In addition, there are a wide variety of compounds that can be used to inhibit angiogenesis, for example, peptides or agents that block the VEGF-mediated angiogenesis pathway, endostatin protein or derivatives, lysine binding fragments of angiostatin, melanin or melanin-promoting compounds, plasminogen fragments (e.g., Kringles 1-3 of plasminogen), troponin subunits, inhibitors of vitronectin α_vβ3, peptides derived from Saposin B, antibiotics or analogs (e.g., tetracycline or neomycin), dienogest-containing compositions, compounds comprising a MetAP-2 inhibitory core coupled to a peptide, the compound EM- 138, chalcone and its analogs, and naaladase inhibitors. See, for example, U.S. Pat. Nos. 6,395,718, 6,462,075, 6,465,431, 6,475,784, 6,482,802, 6,482,810, 6,500,431, 6,500,924, 6,518,298, 6,521,439, 6,525,019, 6,538,103, 6,544,758, 6,544,947, 6,548,477, 6,559,126, and 6,569,845.

E. Pharmaceutical Formulations and Methods of Administration

Methods of administration of therapeutic agents, particularly peptide and antibody therapeutics, are well-known to those of skill in the art.

Peptides of the invention can be administered in the same manner as conventional peptide type pharmaceuticals. Preferably, peptides are administered parenterally, for example, intravenously, intramuscularly, intraperitoneally, or subcutaneously. When administered orally, peptides may be proteolytically hydrolyzed. Therefore, oral application may not be usually effective. However, peptides can be administered orally as a formulation wherein peptides are not easily hydrolyzed in a digestive tract, such as liposome-microcapsules. Peptides may be also administered in suppositories, sublingual tablets, or intranasal spray. If administered parenterally, a preferred pharmaceutical composition is an aqueous solution that, in addition to a peptide of the invention as an active ingredient, may contain for example, buffers such as phosphate, acetate, etc., osmotic pressure-adjusting agents such as sodium chloride, sucrose, and sorbitol, etc., antioxidative or antioxy genie agents, such as ascorbic acid or tocopherol and preservatives, such as antibiotics. The parenterally administered composition also may be a solution readily usable or in a lyophilized form which is dissolved in sterile water before administration.

The pharmaceutical formulations, dosage forms, and uses described below generally apply to antibody-based therapeutic agents, but are also useful and can be modified, where necessary, for making and using therapeutic agents of the disclosure that are not antibodies. To achieve the desired therapeutic effect, the phosphorylation site- specific antibodies or antigen-binding fragments thereof can be administered in a variety of unit dosage forms. The dose will vary according to the particular antibody. For example, different antibodies may have different masses and/or affinities, and thus require different dosage levels. Antibodies prepared as Fab or other fragments will also require differing dosages than the equivalent intact immunoglobulins, as they are of considerably smaller mass than intact immunoglobulins, and thus require lower dosages to reach the same molar levels in the patient's blood. The dose will also vary depending on the manner of administration, the particular symptoms of the patient being treated, the overall health, condition, size, and age of the patient, and the judgment of the prescribing physician. Dosage levels of the antibodies for human subjects are generally between about 1 mg per kg and about 100 mg per kg per patient per treatment, such as for example, between about 5 mg per kg and about 50 mg per kg per patient per treatment. In terms of plasma concentrations, the antibody concentrations may be in the range from about 25 μg/mL to about 500 μg/mL. However, greater amounts may be required for extreme cases and smaller amounts may be sufficient for milder cases.

Administration of an antibody will generally be performed by a parenteral route, typically via injection such as intra-articular or intravascular injection (e.g., intravenous infusion) or intramuscular injection. Other routes of administration, e.g., oral (p.o.), may be used if desired and practicable for the particular antibody to be administered. An antibody can also be administered in a variety of unit dosage forms and their dosages will also vary with the size, potency, and in vivo half-life of the particular antibody being administered. Doses of a phosphorylation site-specific antibody will also vary depending on the manner of administration, the particular symptoms of the patient being treated, the overall health, condition, size, and age of the patient, and the judgment of the prescribing physician. The frequency of administration may also be adjusted according to various parameters. These include the clinical response, the plasma half-life of the antibody, and the levels of the antibody in a body fluid, such as, blood, plasma, serum, or synovial fluid. To guide adjustment of the frequency of administration, levels of the antibody in the body fluid may be monitored during the course of treatment. Formulations particularly useful for antibody-based therapeutic agents are also described in U.S. Patent App. Publication Nos. 20030202972, 20040091490 and 20050158316. In certain embodiments, the liquid formulations of the application are substantially free of surfactant and/or inorganic salts. In another specific embodiment, the liquid formulations have a pH ranging from about 5.0 to about 7.0. In yet another specific embodiment, the liquid formulations comprise histidine at a concentration ranging from about 1 mM to about 100 mM. In still another specific embodiment, the liquid formulations comprise histidine at a concentration ranging from 1 mM to 100 mM. It is also contemplated that the liquid formulations may further comprise one or more excipients such as a saccharide, an amino acid (e.g., arginine, lysine, and methionine) and a polyol. Additional descriptions and methods of preparing and analyzing liquid formulations can be found, for example, in PCT publications WO 03/106644, WO 04/066957, and WO 04/091658.

Wetting agents, emulsifiers and lubricants, such as sodium lauryl sulfate and magnesium stearate, as well as coloring agents, release agents, coating agents, sweetening, flavoring and perfuming agents, preservatives and antioxidants can also be present in the pharmaceutical compositions of the application.

In certain embodiments, formulations of the subject antibodies are pyrogen-free formulations which are substantially free of endotoxins and/or related pyrogenic substances. Endotoxins include toxins that are confined inside microorganisms and are released when the microorganisms are broken down or die. Pyrogenic substances also include fever-inducing, thermostable substances (glycoproteins) from the outer membrane of bacteria and other microorganisms. Both of these substances can cause fever, hypotension and shock if administered to humans. Due to the potential harmful effects, it is advantageous to remove even low amounts of endotoxins from intravenously administered pharmaceutical drug solutions. The Food & Drug Administration ("FDA") has set an upper limit of 5 endotoxin units (EU) per dose per kilogram body weight in a single one hour period for intravenous drug applications (The United States Pharmacopeial Convention, Pharmacopeial Forum 26 (1):223 (2000)). When therapeutic proteins are administered in amounts of several hundred or thousand milligrams per kilogram body weight, as can be the case with monoclonal antibodies, it is advantageous to remove even trace amounts of endotoxin.

The amount of the formulation which will be therapeutically effective can be determined by standard clinical techniques. In addition, in vitro assays may optionally be used to help identify optimal dosage ranges. The precise dose to be used in the formulation will also depend on the route of administration, and the seriousness of the disease or disorder, and should be decided according to the judgment of the practitioner and each patient's circumstances. Effective doses may be extrapolated from dose-response curves derived from in vitro or animal model test systems. The dosage of the compositions to be administered can be determined by the skilled artisan without undue experimentation in conjunction with standard dose-response studies. Relevant circumstances to be considered in making those determinations include the condition or conditions to be treated, the choice of composition to be administered, the age, weight, and response of the individual patient, and the severity of the patient's symptoms. For example, the actual patient body weight may be used to calculate the dose of the formulations in milliliters (mL) to be administered. There may be no downward adjustment to "ideal" weight. In such a situation, an appropriate dose may be calculated by the following formula:

Dose (mL) = [patient weight (kg) x dose level (mg/kg)/ drug concentration (mg/mL)]

For the purpose of treatment of disease, the appropriate dosage of the compounds (for example, antibodies) will depend on the severity and course of disease, the patient's clinical history and response, the toxicity of the antibodies, and the discretion of the attending physician. The initial candidate dosage may be administered to a patient. The proper dosage and treatment regimen can be established by monitoring the progress of therapy using conventional techniques known to those of skill in the art.

The formulations of the application can be distributed as articles of manufacture comprising packaging material and a pharmaceutical agent which comprises, e.g., the antibody and a pharmaceutically acceptable carrier as appropriate to the mode of administration. The packaging material will include a label which indicates that the formulation is for use in the treatment of prostate cancer.

The following Examples are provided only to further illustrate the invention, and are not intended to limit its scope, except as provided in the claims appended hereto. The present invention encompasses modifications and variations of the methods taught herein which would be obvious to one of ordinary skill in the art.

EXAMPLE 1

Identification of Novel PI3K Regulatory Subunit Phosphorylation Sites by

Global Phospho-Profiling

In order to discover previously unknown signal transduction protein phosphorylation sites, PhosphoScan® peptide isolation and characterization techniques (as described in U.S. Patent No. 7,198,896, Rush et al.) were employed to identify phosphotyrosine- containing peptides in cell extracts from several dozen human cancer lines, including leukemia and carcinoma cell lines. This work was first described by the present inventors in USSN 11/503,335 (Moritz et al), PCT/US06/00979 (Goss et al), USSN 60/651,583 (Guo et al), PCT/US04/42940 (Guo et al), PCT/US06/10868 (Guo et al), USSN 60/833,752 (Guo et al), USSN 60/830,550 (Hornbeck et al), the disclosures of which are incorporated herein by reference in their entirety. Briefly, tryptic phosphotyrosine-containing peptides were purified and analyzed from extracts of each of cancer cell lines as follows. Cells were cultured in DMEM medium or RPMI 1640 medium supplemented with 10% fetal bovine serum and penicillin/streptomycin. Cells were harvested by low speed centrifugation. After complete aspiration of medium, cells were resuspended in 1 mL lysis buffer per 1.25 x 10⁸ cells (20 mM HEPES pH 8.0, 9 M urea, 1 mM sodium vanadate, supplemented or not with 2.5 mM sodium pyro-phosphate, 1 mM β-glycerol-phosphate) and sonicated.

Sonicated cell lysates were cleared by centrifugation at 20,000 x g, and proteins were reduced with DTT at a final concentration of 4.1 mM and alkylated with iodoacetamide at 8.3 mM. For digestion with trypsin, protein extracts were diluted in 20 mM HEPES pH 8.0 to a final concentration of 2 M urea and soluble TLCK-trypsin (Worthington) was added at 10-20 μg/mL. Digestion was performed for 1-2 days at room temperature. Trifluoroacetic acid (TFA) was added to protein digests to a final concentration of 1%, precipitate was removed by centrifugation, and digests were loaded onto Sep-Pak Ci₈ columns (Waters) equilibrated with 0.1% TFA. A column volume of 0.7-1.0 ml was used per 2 x 10 cells. Columns were washed with 15 volumes of 0.1% TFA, followed by 4 volumes of 5% acetonitrile (MeCN) in 0.1% TFA. Peptide fraction I was obtained by eluting columns with 2 volumes each of 8, 12, and 15% MeCN in 0.1% TFA and combining the eluates. Fractions II and III were a combination of eluates after eluting columns with 18, 22, 25% MeCN in 0.1% TFA and with 30, 35, 40% MeCN in 0.1% TFA, respectively. All peptide fractions were lyophilized. Peptides from each fraction corresponding to 2 x 10⁸ cells were dissolved in 1 ml of IAP buffer (20 mM Tris/HCl or 50 mM MOPS pH 7.2, 10 mM sodium phosphate, 50 mM NaCl) and insoluble matter (mainly in peptide fractions III) was removed by centrifugation. IAP was performed on each peptide fraction separately. The phosphotyrosine monoclonal antibody P-Tyr-100 (Cell Signaling Technology, Inc., catalog number 9411) was coupled at 4 mg/ml beads to protein G or protein A agarose (Roche), respectively. Immobilized antibody (15 μl, 60 μg) was added as 1 : 1 slurry in IAP buffer to 1 ml of each peptide fraction, and the mixture was incubated overnight at 4° C with gentle rotation. The immobilized antibody beads were washed three times with 1 ml IAP buffer and twice with 1 ml water, all at 4° C. Peptides were eluted from beads by incubation with 75 μl of 0.1% TFA at room temperature for 10 minutes.

Alternatively, one single peptide fraction was obtained from Sep-Pak Cl 8 columns by elution with 2 volumes each of 10%, 15%, 20 %, 25 %, 30 %, 35 % and 40 % acetonitirile in 0.1% TFA and combination of all eluates. IAP on this peptide fraction was performed as follows: After lyophilization, peptide was dissolved in 1.4 ml IAP buffer (MOPS pH 7.2, 10 mM sodium phosphate, 50 mM NaCl) and insoluble matter was removed by centrifugation. Immobilized antibody (40 μl, 160 μg) was added as 1 : 1 slurry in IAP buffer, and the mixture was incubated overnight at 4° C with gentle shaking. The immobilized antibody beads were washed three times with 1 ml IAP buffer and twice with 1 ml water, all at 4° C. Peptides were eluted from beads by incubation with 55 μl of 0.15% TFA at room temperature for 10 min (eluate 1), followed by a wash of the beads (eluate 2) with 45 μl of 0.15% TFA. Both eluates were combined.

Analysis by LC-MS/MS Mass Spectrometry.

40 μl or more of IAP eluate were purified by 0.2 μl StageTips or ZipTips. Peptides were eluted from the microcolumns with 1 μl of 40% MeCN, 0.1% TFA (fractions I and H) or 1 μl of 60% MeCN, 0.1% TFA (fraction III) into 7.6 μl of 0.4% acetic acid/0.005% heptafluorobutyric acid. This sample was loaded onto a 10 cm x 75 μm PicoFrit capillary column (New Objective) packed with Magic Cl 8 AQ reversed-phase resin (Michrom Bioresources) using a Famos autosampler with an inert sample injection valve (Dionex). The column was then developed with a 45-min linear gradient of acetonitrile delivered at 200 nl/min (Ultimate, Dionex), and tandem mass spectra were collected in a data-dependent manner with an LCQ Deca XP Plus ion trap mass spectrometer essentially as described by Gygi et al., supra.

Database Analysis & Assignments.

MS/MS spectra were evaluated using TurboSequest in the Sequest Browser package (v. 27, rev. 12) supplied as part of Bio Works 3.0 (ThermoFinnigan). Individual MS/MS spectra were extracted from the raw data file using the Sequest Browser program CreateDta, with the following settings: bottom MW, 700; top MW, 4,500; minimum number of ions, 20; minimum TIC, 4 x 10⁵; and precursor charge state, unspecified. Spectra were extracted from the beginning of the raw data file before sample injection to the end of the eluting gradient. The IonQuest and VuDta programs were not used to further select MS/MS spectra for Sequest analysis. MS/MS spectra were evaluated with the following TurboSequest parameters: peptide mass tolerance, 2.5; fragment ion tolerance, 0.0; maximum number of differential amino acids per modification, 4; mass type parent, average; mass type fragment, average; maximum number of internal cleavage sites, 10; neutral losses of water and ammonia from b and y ions were considered in the correlation analysis. Proteolytic enzyme was specified except for spectra collected from elastase digests.

Searches were performed against the NCBI human protein database (either as released on April 29, 2003 and containing 37,490 protein sequences or as released on February 23, 2004 and containing 27,175 protein sequences). Cysteine carboxamidomethylation was specified as a static modification, and phosphorylation was allowed as a variable modification on serine, threonine, and tyrosine residues or on tyrosine residues alone. It was determined that restricting phosphorylation to tyrosine residues had little effect on the number of phosphorylation sites assigned.

In proteomics research, it is desirable to validate protein identifications based solely on the observation of a single peptide in one experimental result, in order to indicate that the protein is, in fact, present in a sample. This has led to the development of statistical methods for validating peptide assignments, which are not yet universally accepted, and guidelines for the publication of protein and peptide identification results {see Carr et al, MoI. Cell Proteomics 3: 531-533 (2004)), which were followed in this Example. However, because the immunoaffinity strategy separates phosphorylated peptides from unphosphorylated peptides, observing just one phosphopeptide from a protein is a common result, since many phosphorylated proteins have only one tyrosine- phosphorylated site. For this reason, it is appropriate to use additional criteria to validate phosphopeptide assignments. Assignments are likely to be correct if any of these additional criteria are met: (i) the same sequence is assigned to co- eluting ions with different charge states, since the MS/MS spectrum changes markedly with charge state; (ii) the site is found in more than one peptide sequence context due to sequence overlaps from incomplete proteolysis or use of proteases other than trypsin; (iii) the site is found in more than one peptide sequence context due to homologous but not identical protein isoforms; (iv) the site is found in more than one peptide sequence context due to homologous but not identical proteins among species; and (v) sites validated by MS/MS analysis of synthetic phosphopeptides corresponding to assigned sequences, since the ion trap mass spectrometer produces highly reproducible MS/MS spectra. The last criterion is routinely employed to confirm novel site assignments of particular interest.

All spectra and all sequence assignments made by Sequest were imported into a relational database. Assigned sequences were accepted or rejected following a conservative, two-step process. In the first step, a subset of high- scoring sequence assignments was selected by filtering for XCorr values of at least 1.5 for a charge state of +1, 2.2 for +2, and 3.3 for +3, allowing a maximum RSp value of 10. Assignments in this subset were rejected if any of the following criteria were satisfied: (i) the spectrum contained at least one major peak (at least 10% as intense as the most intense ion in the spectrum) that could not be mapped to the assigned sequence as an a, b, ory ion, as an ion arising from neutral-loss of water or ammonia from a b ory ion, or as a multiply protonated ion; (ii) the spectrum did not contain a series of b or y ions equivalent to at least six uninterrupted residues; or (iii) the sequence was not observed at least five times in all the studies we have conducted (except for overlapping sequences due to incomplete proteolysis or use of proteases other than trypsin). In the second step, assignments with below-threshold scores were accepted if the low-scoring spectrum showed a high degree of similarity to a high-scoring spectrum collected in another study, which simulates a true reference library-searching strategy. All spectra supporting the final list of 424 assigned sequences identified (data not shown) were reviewed by at least three people to establish their credibility.

The phospho-profiling of the examined cell lines identified a total of over

1700 novel tyrosine phosphorylation sites in a multitude of different signaling proteins, including the phosphorylation sites at tyrosines 467, 452, 463, and 470 in PI3KR1 (PI3Kp85 alpha), tyrosines 464, 460, and 467 in PI3KR2 (PI3Kp85 beta), and tyrosines 199, 184, and 202 in PI3KR3 (PI3Kp55 gamma) presently described.

EXAMPLE 2

Development of the Phospho-PI3K p85 (Tyr458)/p55 (Tyrl99) Polyclonal

Antibody

A 15 amino acid phospho-peptide antigen, CSKEYDRLyEEYTRT (where y = phosphotyrosine) (SEQ ID NO: 4), corresponding to residues 192-205 of human PI3K p55 encompassing the tyrosine 199 plus cysteine on the N- terminus for coupling, was constructed according to standard synthesis techniques using a Rainin/Protein Technologies, Inc., Symphony peptide synthesizer. See ANTIBODIES: A LABORATORY MANUAL, supra.; Merrifield, supra.

These peptides were coupled to KLH, and rabbits are then injected intradermally (ID) on the back with antigen in complete Freunds adjuvant (500 μg antigen per rabbit). The rabbits were boosted with the same antigen in incomplete Freund adjuvant (250 μg antigen per rabbit) every three weeks. After the fifth boost, the bleeds were collected. The sera were purified by Protein A- affinity chromatography as previously described {see ANTIBODIES: A LABORATORY MANUAL, Cold Spring Harbor, supra.). The eluted immunoglobulins are then loaded onto a resin-CSKEYDRLyEEYTRT Knotes column. After washing the column extensively, the phospho-PI3K p85 (Tyr458)/p55 (Tyrl99) antibodies were eluted and kept in antibody storage buffer.

The antibody was further tested for phospho-specificity by Western blot analysis. NIH/3T3 and C2C12 cells may be obtained from ATCC in Manassas, VA. NIH/3T3 cells were transfected with src and stable clones were selected using puromycin. NIH/3T3-src cells are cultured in DMEM supplemented with 10% CS and 1.5μg/ml puromycin. NIH/3T3-src cells were treated with λ protein phosphatase (0 units/ml vs. 4000units /ml) for Ih at 37C, washed with PBS and lysed. C2C 12 cells are cultured DMEM supplemented with 10% FBS. C2C 12 cells were stimulated with H₂O₂ (OμM vs. 50 μM) for 20 minutes at 37C, washed with PBS and directly lysed in cell lysis buffer. Loading buffer was added to all cell lysates and the mixture was boiled for 5 minutes. 20 μl (~20 μg protein) of sample was loaded onto an 8% SDS-PAGE gel. A standard Western blot was performed according to the Immunoblotting

Protocol set out in the Cell Signaling Technology 2005-06 Catalogue and Technical Reference, p. 415. The phospho-PI3K p85 (Tyr458)/p55 (Tyrl99) polyclonal antibody is used at dilution 1 : 1000 (for further details see product #4228 at www.cellsignal.com). The results of the Western blot - see Figure 2 - show that the antibody, only recognizes a -85 kDa phospho-protein (phospho- PI3K p85 (Tyr458)) and a -55 kDa phospho-protein (phospho-PI3K p55 (Tyrl99)) activated by Src or H₂O₂. The antibody does not recognize the non- tyrosine phosphorylated PI3K p85 (Tyr458)/p55 (Tyrl99) in λ protein phosphatase treated NIH/3T3-src or non-stimulated C2C12 cells. EXAMPLE 3

Production of a Phospho-PI3K p55 (Tyrl99) Phosphospecific Monoclonal

Antibody

A PI3K p55 (Tyrl99) phosphospecific rabbit monoclonal antibody, may be produced from spleen cells of the immunized rabbit described in Example 2, above, following standard procedures (Harlow and Lane, 1988). The rabbit splenocytes are fused to proprietary fusion partner cells according to a standard protocol (see generally Loyola School of Medicine protocol (Helga Spieker- Polet) at http://www.meddean.luc.edu/ lumen/DeptWebs/microbio/KNIGHT/PROTOC/Hybridom.htm.)

Colonies originating from the fusion may be screened by ELISA for reactivity to the phospho-peptide and non-phospho-peptide and by Western blot analysis. Colonies found to be positive by ELISA to the phospho-peptide while negative to the non-phospho-peptide are further characterized by Western blot analysis. Colonies found to be positive by Western blot analysis are then subcloned by limited dilution. Rabbit ascites are produced from the single clone obtained from subcloning.

Specificity may be determined by Western Blot as described in Example 2 above, using non-tyrosine phosphorylated PI3K p85 (Tyr458)/p55 (Tyrl99) in λ protein phosphatase treated NIH/3T3-src or non-stimulated C2C12 cells for a negative control. Rabbit monoclonal antibody raised to PI3K p55(Tyrl99) is expected to cross-react with the nearly-identical PI3K p85 (Tyr458) site, as described above in Example 2 for the polyclonal antibody.

EXAMPLE 4 Detection of PI3K Phosphorylation In Cytometric Assay

The PI3K phosphospecific antibodies described in Examples 2 or 3 may be used in flow cytometry to detect phospho-PI3K in a biological sample. A sample of cells may be taken to be analyzed by Western blot analysis. The remaining cells are fixed with 1% paraformaldehyde for 10 minutes at 37⁰C, followed by cell permeabilization 90% with methanol for 30 minutes on ice. The fixed cells are then stained with the phospho-PI3K primary antibody for 60 minutes at room temperature. The cells are then washed and stained with an Alexa 488-labeled secondary antibody for 30 minutes at room temperature. The cells may then be analyzed on a Beckman Coulter EPICS-XL flow cytometer.

The cytometric results are expected to match the Western results described above, further demonstrating the specificity of the PI3K antibody for the activated/phosphorylated PI3K protein.

EXAMPLE 5

Detection of Constitutively Active PI3K in Cells using Flow Cytometry

PI3K phosphospecific antibody described in Examples 2 or 3 above may also be used in flow cytometry to detect phospho-PI3K in a biological sample. Serum-starved cells may be incubated with or without a PI3K inhibitor SFl 126 for 4 hours at 37⁰C. The cells are then fixed with 2% paraformaldehyde for 10 minutes at 37⁰C followed by cell permeabilization 90% with methanol for 30 minutes on ice. The fixed cells are stained with the Alexa 488-conjugated PI3K primary antibody for 1 hour at room temperature. The cells may then be analyzed on a Beckman Coulter EPICS-XL flow cytometer.

The cytometric results are again expected to demonstrate the specificity of the PI3K antibody for the activated PI3K protein and the assay's ability to detect the activity and efficacy of a PI3K inhibitor. In the presence of the drug, a population of the cells will show less staining with the antibody, indicating that the drug is active against PI3K.

Claims

WHAT IS CLAIMED IS:

1. An isolated antibody that binds to a Phosphatidylinositol 3 Kinase (PI3K) regulatory subunit only when phosphorylated at a tyrosine phosphorylation site selected from the group consisting of tyrosines 467, 452, 463, and 470 in PI3KR1 (PI3Kp85 alpha) (SEQ ID NO: 1), tyrosines 464, 460, and 467 in PI3KR2 (PI3Kp85 beta) (SEQ ID NO: 3), and tyrosines 199, 184, and 202 in PI3KR3 (PI3Kp55 gamma) (SEQ ID NO: 2).

2. The antibody of claim 1, wherein said antibody is polyclonal.

3. The antibody of claim 1, wherein said antibody is monoclonal.

4. A hybridoma cell line producing the antibody of claim 3.

5. The hybridoma cell line of claim 4, wherein said cell line is a rabbit hybridoma or a mouse hybridoma.

6. A monoclonal antibody produced by the hybridoma cell line of claim 5.

7. A method for detecting phosphorylated PI3K in a biological sample, said method comprising the steps of:

(a) contacting a biological sample suspected of containing phosphorylated PI3K with at least one antibody of claim 1 under conditions suitable for formation of an antibody-PI3K complex; and

(b) detecting the presence of said complex in said sample, wherein the presence of said complex indicates the presence of phosphorylated

PI 3 K in said sample.

8. The method of claim 7, wherein said biological sample is taken from a subject suspected of having cancer.

9. A method of identifying a compound that modulates phosphorylation of PDK in a test tissue, said method comprising the steps of:

(a) contacting said test tissue with said compound;

(b) detecting the level of phosphorylated PI3K in said test tissue of step (a) using at least one antibody of claim 1 under conditions suitable for formation of a antibody-PI3K complex;

(c) comparing the level of phosphorylated PI3K detected in step(b) with the presence of phosphorylated PI3K in a control tissue not contacted with said compound, wherein a difference in PI3K phosphorylation levels between said test tissue and said control tissue identifies said compound as a modulator of PI3K phosphorylation.

10. The method of claim 9, wherein said test tissue is taken from a subject suspected of having cancer.

11. The method of claim 9, wherein said compound is a PI3K inhibitor.

12. A kit for the detection of phosphorylated PI3K in a biological sample, said kit comprising (a) at least one detectable antibody of claim 1 , and (b) at least one secondary reagent.