WO2001092339A1 - Molecular marker for mitosis - Google Patents

Abstract

The present invention relates to modified peptide sequences that serve as unique molecular markers of cell division. More particularly the present invention is directed to the amino terminus of a histone H3-like protein, CENP-A, and antibodies that bind to this peptide. Only cells that are dividing are phosphorylated at Ser7 of this peptide (see Fig. 2C) and an antibody specific for the phosphorylated peptides can be used to identify such cells.

Description

Molecular Marker for Mitosis

This application claims priority under 35 U.S.C. § 119(e) to provisional patent application no. 60/208,261, filed May 31, 2000, the disclosure of which is incorporated herein by reference in its entirety.

US Government Rights

This invention was made with United States Government support under Grant Nos. GM 49022 and GM 39068, awarded by the National Institutes of Health. The United States Government has certain rights in the invention.

Field of the Invention

The present invention relates to antibodies, binding portions thereof, and probes to a centromere-associated histone H3 variant and uses of these agents for detecting the occurrence of mitosis and meiosis.

Background of the Invention

Eukaryotic cells utilize an elaborate repertoire of evolutionarily conserved intracellular signaling pathways to elicit defined alterations in cellular structure and physiology in response to sudden changes in their external environment. In mammalian cells, a diverse collection of environmental stimuli, including polypeptide growth factors and tumor-promoting phorbol esters, induce a Ras- dependent MAP (mitogen-activated protein) kinase cascade resulting in the transcriptional activation of a subset of immediate-early responsive genes that include the proto-oncogenes c-fos and c-jun (reviewed in Karin et al., "Transcriptional

Control by Protein Phosphorylation: Signal Transmission from the Cell Surface to the Nucleus," Curr. Biol., 5:747-757 (1995); Treisman 1996; Cohen, P. "The Search for Physiological Substrates of MAP and SAP Kinases in Mammalian Cells," Trends in Cell Biol., 7:353-361 (1997)). A central question in the MAPK (MAP kinase) signaling field remains regarding how the activity of MAP kinase pathways controls the transcription process itself. Various transcription factors have been directly implicated in MAPK- controlled gene expression, and, most often, regulation of candidate factors is thought to occur by reversible phosphorylation impacting at multiple levels: translocation to the nucleus; DNA binding; and transcriptional activation. However, the underlying mechanism(s) by which MAPK phosphorylation regulates transcription factor activity and/or ensuing gene expression remains poorly defined.

Eukaryotic genomes are highly compacted in the form of chromatin with histone proteins and the nucleosomal complexes they form being the fundamental structural unit. Each nucleosome particle is comprised of an octameric unit of core histones (2 copies each of histones H2A, H2B, H3, and H4) wrapping a conserved 147 base pairs of DNA. A fifth linker histone (often called HI) associates with the DNA between nucleosomes that are more variable in length. Nucleosomal arrays are then folded into progressively higher-order structures, the precise organization of which has important functional consequences. The four nucleosomal core histones (H2A, H2B, H3 and H4) are among the most conserved proteins known. This high degree of conservation reflects a nearly invariant way in which the DNA template is wrapped by these proteins, a packaging theme adopted by all eukaryotes. The precise organization of DNA in chromatin has important functional consequences. DNA-templated processes such as transcription, replication, repair, recombination and segregation are influenced by the topological complexity of DNA in chromatin, imposed at the most fundamental level by histone proteins.

Protruding from the surface of the chromatin polymer, are histone termini or 'tails' that are decorated by a diverse array of post-translational modifications including: acetylation, phosphorylation, methylation, etc. While detailed molecular mechanisms remain unclear, a rapidly growing body of evidence suggests that tail-directed chromatin remodeling and histone modifications occur as part of a multi-step activation pathway of at least certain eukaryotic genes. For example, post-translational modifications of histone proteins, notably histone acetylation, have long been suspected to contribute to dynamic and local differences in chromatin environments (reviewed in Felsenfeld, G. "Chromatin Unfolds," Cell, 86:13-19 (1996); Grunstein, M. "Histone Acetylation in Chromatin Structure and Transcription," Nature, 389:349:352 (1997)). The architecture of chromatin is likely to impinge on a wide variety of fundamental processes, including cellular transformation (see DePinho, R.A., "Transcriptional Repression: The Cancer- Chromatin Connection," Nature, 391 :533-536 (1998)). Although the regulation of gene expression is probably one of the more intensely studied areas of current molecular biology, fundamental to all proliferating cells, whether they divide by mitosis or meiosis, is the faithful segregation of condensed chromosomes. In 1997, it was reported that histone H3 is uniquely phosphorylated at serine 10 during mitosis and meiosis in a wide range of organisms (Hendzel et al., 1997; Wei and AUis, 1998; Wei et al., 1998). Consistent with the hypothesis that H3 phosphorylation at serine 10 (SerlO) plays an important role with mitotic chromosome condensation in vivo, mutation of the H3 gene in Tetrahymena (S10A) displays abnormal patterns of chromosome segregation leading to extensive chromosome loss during mitosis and meiosis (Wei et al, 1999). Antibodies directed to the phosphorylated H3 at serine 10 (Phos H3) have been used as markers for mitotic cells. However, this modification of H3 also occurs during transcriptional activation of DNA sequences and therefore staining can be detected in interphase cells as well as mitotic cells. Accordingly, there is a need for a marker of mitosis that is not also associated with transcriptional activity. Centromeric DNA itself is packaged with specialized proteins, one of which is a specialized H3 'variant' that is found in organisms ranging from yeast (Cse4p) to humans (CENP-A). In yeast, Cse4p is known to play an essential role in chromosome segregation and/or organizing a specialized chromatin structure at the centromere (Meluh et al., 1998). Both CENP-A and Cse4p differ from general H3 primarily in having unique N-terminal tails, reinforcing the idea that these domains may have a signaling or receptor function that is separate from the DNA- wrapping function.

The present invention is directed to a centromere-associated histone H3 variant, CENP-A, that is uniquely phosphorylated during mitosis. Antibodies generated to phosphorylated CENP-A (hereafter Phos CENP-A) are absolutely specific to the kinetochores of mitotic cells. Thus the Phos CENP-A antibodies of the present invention provide a mitosis and/or proliferation marker that has research and diagnostic potential. Importantly, this antibody does not recognize Phos H3 and there is no interphase staining.

The present invention is directed to compositions comprising an anti- Phos CENP-A antibody and methods of using those compositions to detect dividing cells. The compositions comprise monoclonal or polyclonal antibodies, or binding portions thereof, that specifically bind to Phos CENP-A. These compositions can be used as diagnostics to detect a variety of conditions or molecular biological events. In addition the phosphorylated CENP-A antibodies of the present invention can be coupled with therapeutic agents to provide therapeutic compositions.

Definitions

In describing and claiming the invention, the following terminology will be used in accordance with the definitions set forth below.

As used herein, the term "nucleic acid" encompasses RNA as well as single and double-stranded DNA and cDNA. Furthermore, the terms, "nucleic acid," "DNA," "RNA" and similar terms also include nucleic acid analogs, i.e. analogs having other than a phosphodiester backbone. For example, the so-called "peptide nucleic acids," which are known in the art and have peptide bonds instead of phosphodiester bonds in the backbone, are considered within the scope of the present invention.

The term "peptide" encompasses a sequence of 3 or more amino acids wherein the amino acids are naturally occurring or synthetic (non-naturally occurring) amino acids. Peptide mimetics include peptides having one or more of the following modifications: 1. peptides wherein one or more of the peptidyl ~C(O)NR~ linkages (bonds) have been replaced by a non-peptidyl linkage such as a --CH₂_carbamate linkage (~CH₂OC(O)NR~), a phosphonate linkage, a -CH₂.sulfonamide (-CH ₂-S(O)₂NR~) linkage, a urea (~NHC(O)NH~) linkage, a ~CH₂ -secondary amine linkage, or with an alkylated peptidyl linkage (~C(O)NR~) wherein R is C1.C4 alkyl; 2. peptides wherein the N-terminus is derivatized to a --NRR1 group, to a ~ NRC(O)R group, to a -NRC(O)OR group, to a ~NRS(O)₂R group, to a

~NHC(O)NHR group where R and Rj are hydrogen or C 1..C4 alkyl with the proviso that

R and R are not both hydrogen;

3. peptides wherein the C terminus is derivatized to ~C(O)R₂ where R ₂ is selected from the group consisting of C \ 4 alkoxy, and --NR3R4 where R3 and R4 are independently selected from the group consisting of hydrogen and Cj_C4 alkyl.

Naturally occurring amino acid residues in peptides are abbreviated as recommended by the IUPAC-R7B Biochemical Nomenclature Commission as follows: Phenylalanine is Phe or F; Leucine is Leu or L; Isoleucine is He or I; Methionine is Met or M; Norleucine is Nle; Valine is Vat or V; Serine is Ser or S;

Proline is Pro or P; Threonine is Thr or T; Alanine is Ala or A; Tyrosine is Tyr or Y;

Histidine is His or H; Glutamine is Gin or Q; Asparagine is Asn or N; Lysine is Lys or

K; Aspartic Acid is Asp or D; Glutamic Acid is Glu or E; Cysteine is Cys or C;

Tryptophan is Trp or W; Arginine is Arg or R; Glycine is Gly or G, and X is any amino acid. Other naturally occurring amino acids include, by way of example,

4-hydroxyproline, 5-hydroxylysine, and the like.

As used herein, the term "conservative amino acid substitution" is defined herein as exchanges within one of the following five groups:

I. Small aliphatic, nonpolar or slightly polar residues: Ala, Ser, Thr, Pro, Gly;

II. Polar, negatively charged residues and their amides:

Asp, Asn, Glu, Gin;

III. Polar, positively charged residues:

His, Arg, Lys; IV. Large, aliphatic, nonpolar residues:

Met Leu, He, Val, Cys V. Large, aromatic residues: Phe, Tyr, Trp

As used herein, the term "purified" and like terms relate to the isolation of a molecule or compound in a form that is substantially free of contaminants normally associated with the molecule or compound in a native or natural environment.

"Operably linked" refers to a juxtaposition wherein the components are configured so as to perform their usual function. For example, control sequences or promoters operably linked to a coding sequence are capable of effecting the expression of the coding sequence.

As used herein, the terms "complementary" or "complementarity" are used in reference to polynucleotides (i.e., a sequence of nucleotides) related by the base-pairing rules. For example, for the sequence "A-G-T," is complementary to the sequence "T-C-A."

As used herein, the term "hybridization" is used in reference to the pairing of complementary nucleic acids. Hybridization and the strength of hybridization (i.e., the strength of the association between the nucleic acids) is impacted by such factors as the degree of complementarity between the nucleic acids, stringency of the conditions involved, the length of the formed hybrid, and the G:C ratio within the nucleic acids.

"Therapeutic agent," "pharmaceutical agent" or "drug" refers to any therapeutic or prophylactic agent which may be used in the treatment (including the prevention, diagnosis, alleviation, or cure) of a malady, affliction, disease or injury in a patient.

As used herein, the term "treating" includes alleviating the symptoms associated with a specific disorder or condition and/or preventing or eliminating said symptoms. For example, treating cancer includes preventing or slowing the growth and/or division of cancer cells as well as killing cancer cells. As used herein, the term "pharmaceutically acceptable carrier" encompasses any of the standard pharmaceutical carriers, such as a phosphate buffered saline solution, water and emulsions such as an oil/water or water/oil emulsion, and various types of wetting agents.

As used herein, the term "antigenic fragment of CENP-A" encompasses both natural peptide fragments of CENP-A and synthetic equivalents of those fragments. As used herein, the term "antibody" refers to a polyclonal or monoclonal antibody or a binding fragment thereof such as Fab, F(ab')₂ and Fv fragments.

As used herein, the term "biologically active fragments" of the Phos CENP-A antibody encompasses natural or synthetic portions of the full-length antibody that are capable of specific binding to the peptide of SEQ ID NO: 1.

As used herein, the term "Ser(P)" refers to the phosphorylated form of the amino acid serine.

As used herein, the term "parenteral" includes administration subcutaneously, intravenously or intramuscularly.

Summary of the Invention

The present invention is directed to antibodies that recognize phosphorylated histones and histone variants, compositions comprising such antibodies, and the use of such antibodies for diagnostic and therapeutic applications. More particularly, present invention is directed to antibodies that bind to centromere protein A (CENP-A), a histone H3-like protein found uniquely at centromeres. The amino terminus of CENP-A is phosphorylated during cell division and antibodies that bind to the phosphorylated CENP-A are unique markers of cell mitosis and meiosis.

Brief Description of the Drawings

Figs. 1A & IB. Fig. 1 A shows the amino terminus of the human H3 (SEQ ID NO: 5). Fig. IB shows the amino termini of human CENP-A (SEQ ID NO: 6). Known phosphorylation sites in H3 (see Hendzel et al., 1997; Goto et al., 1999 for details) and the site of phosphorylation in CENP-A (Ser7) are indicated. The phosphorylated (Phos) peptides used to generate the Phos H3 and Phos CENP-A antibodies are underlined and shown in more detail in (Figs. 2A-2D).

Figs.2A - Fig. 2D show the synthetic peptides used to generate antibodies selective for phosphorylated and unmodified H3 and CENP-A amino terminal peptides. Fig. 2A represents the unmodified H3 peptide (Un H3; SEQ ID NO: 7); Fig. 2B represents the phosphorylated (SerlO) H3 peptide (Phos H3; SEQ ID NO: 8); Fig 2C represents the phosphorylated (Ser7) CENP-A peptide (Phos CENP- A; SEQ ID NO: 1) and Fig. 2D represents the unmodified CENP-A peptide (Un CENP-A; SEQ ID NO: 2). The H3 peptides have been aligned with the CENP-A peptides to show conserved motifs between the two regions. The carboxy-terminal cysteine (C* ) is artificial and was used for conjugation purposes. Figs. 3 A - Fig. 3D Specificity of Phos H3 versus Phos CENP-A antibodies with histone peptides. The Phos CENP-A (SEQ ID NO: 1), Un CENP-A (SEQ ID NO: 2) and Phos H3 peptide (SEQ ID NO: 8) were immobilized on separate microtiter dishes and subjected to ELISA analysis. ELISA assays were performed using antisera isolated from four groups of mice: 1. mice immunized with Phos H3 (represented by the symbol O); 2. mice immunized with Phos CENP-A (represented by the symbol 0); 3. mice immunized with Un H3 (represented by the symbol Δ); and 4. mice immunized with Un CENP-A (represented by the symbol D). Fig 3A shows the ELISA results obtained with the immobilized Phos H3 peptide, Fig 3B shows the ELISA results obtained with the immobilized Phos CENP-A, Fig 3C shows the ELISA results obtained with the immobilized Un CENP-A and Fig 4D shows the ELISA results obtained with the immobilized Phos CENP-A peptide when the sera was pre-incubated with 1 mg/ml of Phos CENP-A competitor peptide prior to the ELISA. Each phospho-specific antisera (Phos H3 vs. Phos CENP-A) is specific for the appropriate phosphopeptide and displays essentially no reactivity towards either the other phosphopeptide or the unmodified peptide.

Fig. 4 Specificity of affinity-purified antibodies toward total acid- soluble nuclear protein. Nuclei from randomly growing (R) or nocodozole-arrested mitotic (M; >95% by FACS analyses) HeLa cells were acid-extracted, and soluble protein was precipitated and resolved by 12% SDS PAGE. Samples were examined by Coomassie staining or by immunoblotting using the following antibodies: anti-Un CENP-A, anti-Phos CENP-A or anti-Phos H3. Despite equal histone loading (compare lanes 1 and 2 in the Coomassie stained gel), a single 17 kDa band is detected in the randomly growing sample that is not easily detected in the mitotic samples. In contrast, a slower migrating isoform of CENP-A (>17KDa) is observed in the mitotic samples that is not detected in the randomly growing sample. Both of these CENP-A forms migrate slower than the expected 15 kDa phosphorylated isoform of H3. Fig. 5 Phosphorylation of CENP-A is required for recognition with the Phos CENP-A antibody. Acid-soluble protein from mitotic (M) or randomly growing (R) HeLa cells was incubated in the presence (+) or absence (-) of alkaline phosphatase (AP). Note that incubation with the phosphatase (+AP) removed essentially all of the reactivity of the Phos CENP-A antibody towards the slower migrating phosphorylated (M-phase) isoform of CENP-A (see lanes 5 and 6). In contrast, +AP had no effect on the reactivity of Un CENP-A antibody toward the unmodified CENP-A (see lanes 3 and 4). In some M-phase sample preparations, a doublet of phosphorylated CENP-A has been observed, but detection of the doublet varies considerably from preparation to preparation.

Fig. 6. Quantitation of CENP-A phosphorylation by analysis of fluorescence intensity. 5-6 cells were analyzed for each stage of mitosis (as judged by DAPI staining) with detectable Phos CENP-A signal. No signal was detected in telophase. Mean integrated fluorescence intensity is displayed as a histogram with error bars reflecting the standard error for each stage of the cell cycle. On average, the peak of phosphorylation is in prometaphase, drops 30% in metaphase, and continues to decline until mid-anaphase. Chi-squared analysis demonstrates that these phases of the cell cycle can be distinguished, with P=O for comparisons among the phases.

Detailed Description of the Invention

Histones play a central role in modulating protein assembly on the chromatin fiber. This function of histones is regulated by post-translational modifications that occur on the flexible N-terminal tails of each of the four core histones. The chemical diversity of the modifications, including lysine-N-acetylation, lysine-N-methylation, arginine methylation, ADP-ribosylation, ubiquitination and phosphorylation has led to the hypothesis (the "histone code hypothesis") that each site of modification can serve a distinct function. In particular, modifications (singly or in combination) create changes in the overall charge density of histone tails, which in turn modulate histone interactions with DNA, non-histone proteins and other histones.

The present invention is directed to a unique centromere-associated histone H3 variant, CENP-A. CENP-A is found specifically at centromeres throughout the cell cycle where it is thought to substitute for histone H3 in the nucleosomes of kinetochore-associated chromatin. In addition to containing a conserved H3-like histone fold domain, CENP-A contains an N-terminal tail that shares very little sequence identity with that of histone H3. However, a small region of Histone H3, including several amino acids surrounding the histone H3 SerlO residue are conserved in a corresponding region of CENP-A (see Fig 2A-2D). Since the SerlO residue of Histone H3 has been previously identified as playing a critical role in chromosome condensation (Wei et al, (1998) PNAS 95, 7480-7484 and Wei et al., (1999) Cell 97, 99-109) this region may function similarly in the CENP-A protein to regulate chromosome condensation.

The present invention is directed to the discovery that modification of the amino terminus of the CENP-A protein, and homologous proteins from other species, plays an important role in the regulation of chromosome condensation. More particularly, applicants have discovered that the amino terminus of CENP-A (see Fig. IB) protrudes from the surface of the chromatin and the serine amino acid at the 7th position from the amino terminus (Ser7) is selectively phosphorylated in vivo during mitosis. Therefore, in accordance with one aspect of the present invention methylation of Ser7 of CENP-A serves as a marker of mitosis, and antibodies recognizing this portion of the protein have use as important diagnostic tools. One aspect of the present invention is directed to antigens used to produce antibodies specific to the amino terminus of Phos CENP-A. In one embodiment, a purified antigenic fragment of CENP-A or a synthetic equivalent thereof is provided. The antigen comprises an 11 amino acid long sequence that consists of the motif RZSXXXXAPRZ' (SEQ ID NO: 10/SEQ ID NO: 11) wherein X is any amino acid and Z and Z' are both arginine or both lysine. In other words the motif represents the two sequences: RRSXXXXAPRR (SEQ ID NO: 10) and RKSXXXXAPRK (SEQ ID NO: 11). This motif does not match any other known phosphorylation motif. In one preferred embodiment the antigen consists of the sequence Arg Arg Ser(P) Arg Lys Pro Glu Ala Pro Arg Arg (SEQ ID NO: 9). In an alternative embodiment the antigen comprises an amino acid sequence selected from the group consisting of: Arg Arg Arg Ser(P) Arg Lys Pro Glu Ala Pro Arg Arg Arg Ser (SEQ ID NO: 1); Arg Arg Arg Ser Arg Lys Pro Glu Ala Pro Arg Arg Arg Ser (SEQ ID NO: 2);

Arg Arg Arg Ser(P) Pro Ser Pro Thr Pro Thr Pro Gly Pro Ser Arg Arg (SEQ ID NO:

3);

Arg Arg Arg Ser Pro Ser Pro Thr Pro Thr Pro Gly Pro Ser Arg Arg (SEQ ID NO: 4); and amino acid sequences that differ from SEQ ID NO: 1 , SEQ ID NO: 2, SEQ ID NO: 3 or SEQ ID NO: 4 by one or more conservative amino acid substitutions. In one preferred embodiment, the purified antigenic fragment of CENP-A consists of the sequence Arg Arg Arg Ser(P) Arg Lys Pro Glu Ala Pro Arg Arg Arg Ser (SEQ ID NO: 1); Arg Arg Arg Ser Arg Lys Pro Glu Ala Pro Arg Arg Arg Ser (SEQ ID NO: 2); or an amino acid sequence that differs from SEQ ID NO: 1 or SEQ ID NO: 2 by one or two conservative amino acid substitutions. In one embodiment, the purified antigen for making the anti-Phos CENP-A antibody comprises a phosphorylated polypeptide of the sequence Arg Arg Arg Ser(P) Arg Lys Pro Glu Ala Pro Arg Arg Arg Ser Cys (the "Phos CENP-A peptide"; SEQ ID NO: 1). In another embodiment, the invention is directed to antigens used to produce antibodies specific for the un- phosphorylated amino terminus of the CENP-A protein. The antigen used for this embodiment comprises the sequence Arg Arg Arg Ser Arg Lys Pro Glu Ala Pro Arg Arg Arg Ser Cys (the "Un CENP-A peptide"; SEQ ID NO: 2). In an alternative embodiment, the purified antigen comprises a polypeptide linked to a suitable carrier, such as bovine serum albumin or Keyhole limpet hemocyanin. In one preferred embodiment the Phos CENP-A antigen consists of a peptide having at least eleven consecutive residues of the amino acid sequence: Arg Arg Arg Ser(P) Arg Lys Pro Glu Ala Pro Arg Arg Arg Ser (SEQ ID NO: 1), and derivatives of this amino acid sequence wherein the amino acid sequence contains one or more conservative amino acid substitutions, and a carrier protein linked to the peptide. In another embodiment, the purified antigen used to make anti-Unmodified CENP-A antibody consists of a peptide having at least eleven consecutive residues of the amino acid sequence: Arg Arg Arg Ser Arg Lys Pro Glu Ala Pro Arg Arg Arg Ser, and derivatives of this amino acid sequence wherein the amino acid sequence contains one or more conservative amino acid substitutions, and a carrier protein linked to the peptide. In addition to the antigens described above, the present invention is also directed to antibodies that specifically bind to the amino terminus of CENP-A. In particular, the anti-Phos CENP-A antibody binds to an epitope within the polypeptide portion of the Phos CENP-A amino terminus shown in Fig. IB (SEQ ID NO: 6). Similarly, in another embodiment of the invention anti-Unmodified CENP-A antibody binds to an epitope within the polypeptide portion of the non-phosphorylated CENP-A amino terminus. More particularly, the anti-Phos CENP-A antibody binds to an epitope of the CENP-A protein within a portion of the protein corresponding to amino acids 4 to 17, counting from the N-terminus, wherein amino acid 7 is phosphorylated. Similarly, the anti-Unmodified CENP-A antibody binds to an epitope of a CENP-A protein within the portion of the protein corresponding to amino acids 4 to 17, counting from the N-terminus, wherein amino acid 7 is not phosphorylated.

In one embodiment, the antibody binds specifically to an antigen selected from the group consisting of Arg Arg Arg Ser(P) Arg Lys Pro Glu Ala Pro Arg Arg Arg Ser (SEQ ID NO: 1); Arg Arg Arg Ser Arg Lys Pro Glu Ala Pro Arg Arg Arg Ser (SEQ ID NO: 2); Arg Arg Arg Ser(P) Pro Ser Pro Thr Pro Thr Pro Gly Pro Ser Arg Arg (SEQ ID NO:

3);

Arg Arg Arg Ser Pro Ser Pro Thr Pro Thr Pro Gly Pro Ser Arg Arg (SEQ ID NO: 4); and amino acid sequences that differ from SEQ ID NO: 1 , SEQ ID NO: 2, SEQ ID NO: 3 or SEQ ID NO: 4 by one or more conservative amino acid substitutions. In one preferred embodiment an anti-Phos CENP-A antibody is provided that binds specifically to the sequence Arg Arg Arg Ser(P) Arg Lys Pro Glu Ala Pro Arg Arg Arg Ser (SEQ ID NO: 1), and an anti-Unmodified CENP-A antibody is provided that binds specifically to the sequence Arg Arg Arg Ser Arg Lys Pro Glu Ala Pro Arg Arg Arg Ser (SEQ ED NO: 2).

One method used to generate either the anti-Phos or anti-Unmodified CENP-A antibody involves administration of the respective antigen (Arg Arg Arg Ser(P) Arg Lys Pro Glu Ala Pro Arg Arg Arg Ser (SEQ ID NO: 1) or Arg Arg Arg Ser Arg Lys Pro Glu Ala Pro Arg Arg Arg Ser (SEQ ID NO: 2)) to a laboratory animal, typically a rabbit, to trigger production of antibodies specific for the antigen. The dose and regiment of antigen administration to trigger antibody production as well as the methods for purification of the antibody are well known to those skilled in the art. Typically, such antibodies can be raised by administering the antigen of interest subcutaneously to New Zealand white rabbits which have first been bled to obtain pre- immune serum. The antigens can be injected at a total volume of 100 ul per site at six different sites. Each injected material will contain synthetic surfactant adjuvant pluronic polyols, or pulverized acrylamide gel containing the protein or polypeptide after SDS-polyacrylamide gel electrophoresis.

The rabbits are then bled two weeks after the first injection and periodically boosted with the same antigen three times every six weeks. A sample of serum is then collected 10 days after each boost. Polyclonal antibodies are then recovered from the serum by affinity chromatography using the corresponding antigen to capture the antibody. Ultimately, the rabbits are euthenized with pentobarbital 150 mg/Kg IV. This and other procedures for raising polyclonal antibodies are disclosed in E. Harlow, et. al., editors, Antibodies: A Laboratory Manual (1988), which is hereby incorporated by reference.

The specificity of antibodies may be determined by enzyme-linked immunosorbent assay or immunoblotting, or similar methods known to those skilled in the art. Specific binding of the anti-Phos CENP-A antibody to the Phos CENP-A antigen of SEQ ID NO: 1 is characterized by the antibody failing to significantly cross-react with other antigens, particularly the Unmodified CENP-A antigen (SEQ ID NO: 2). Likewise, specific binding of the anti-Unmodified CENP-A antibody to the Unmodified CENP-A antigen of claim 1 is characterized by the antibody failing to significantly cross-react with other antigens, particularly the Phos CENP-A antigen (SEQ ID NO: 1). As shown in ELISA assays phospho-specific antisera (Phos H3 vs. Phos CENP-A) is specific for the appropriate phosphopeptide and displays essentially no reactivity towards either the other phosphopeptide or the unmodified peptide (See Figs 3A-3D). Furthermore, as shown in Fig. 4 and Fig. 5 the anti-Phos CENP-A antibody is specific for acid soluble nuclear proteins isolated from mitotic cells and this reactivity is removed by phosphotase treatment of the nuclear proteins. These results show the specificity of the anti-Phos CENP-A antibody for its target antigen and its ability to function as a marker for mitotic cells. The present invention also encompasses monoclonal antibodies that specifically bind to either the Phos CENP-A antigen or the Unmodified CENP-A antigen. Monoclonal antibody production may be effected using techniques well- known to those skilled in the art. Basically, the process involves first obtaining immune cells (lymphocytes) from the spleen of a mammal (e.g., mouse) which has been previously immunized with the antigen of interest either in vivo or in vitro. The antibody-secreting lymphocytes are then fused with myeloma cells or transformed cells, which are capable of replicating indefinitely in cell culture, thereby producing an immortal, immunoglobulin-secreting cell line. The resulting fused cells, or hybridomas, are cultured, and the resulting colonies screened for the production of the desired monoclonal antibodies. Colonies producing such antibodies are cloned, and grown either in vivo or in vitro to produce large quantities of antibody. One embodiment of the invention is directed to a hybridoma cell line which produces monoclonal antibodies which bind the Phos CENP-A or Unmodified CENP-A antigens. A description of the theoretical basis and practical methodology of fusing such cells is set forth in Kohler and Milstein, Nature, 256:495 (1975), which is hereby incorporated by reference.

In addition to whole antibodies, fragments of antibodies can retain binding specificity for a particular antigen. Antibody fragments can be generated by several methods, including, but not limited to proteolysis or synthesis using recombinant DNA technology. An example of such an embodiment is selective proteolysis of the anti-Phos CENP-A or anti-Unmodified CENP-A antibody by papain to generate Fab fragments, or by pepsin to generate a F(ab')₂ fragment. These antibody fragments can be made by conventional procedures, as described in J. Goding, Monoclonal Antibodies: Principles and Practice, pp. 98-118 (N.Y. Academic Press 1983), which is hereby incorporated by reference. Other fragments of the anti- Phos CENP-A or anti-Unmodified CENP-A antibodies which retain the specific binding of the whole antibody can be generated by other means known to those skilled in the art. CENP-A is phosphorylated only during cell division and thus the anti-

Phos CENP-A antibody serves as a marker of mitosis and meiosis. Currently, an antibody to the phosphorylated H3 histone is used as a marker of cell division. However, because phosphorylation of H3 occurs during transcriptional activation of DNA sequences as well as during cell division, staining with the Phos H3 antibody can be detected in interphase cells as well as mitotic cells. This short-coming is overcome by the anti-Phos CENP-A antibody which does not recognize Phos H3 or un-phosphorylated CENP-A and does not stain interphase chromosomes.

Furthermore as shown in Fig. 6 the amount of detectable Phos CENP-A present in a cell varies throughout the cell cycle, and thus the anti-Phos CENP-A antibody can also be used to determine the mitotic stage of a cell. Therefore, the anti-Phos CENP- A antibody is a highly specific and extremely reliable marker of cells which are in the process of dividing. The present invention also encompasses a "control" antibody that is specific to the unmodified form of the CENP-A protein, (the "anti-Un CENP-A" antibody). This antibody will bind to non-dividing cells.

The antibodies, or fragments thereof, that recognize Phos CENP-A can be used to detect the occurrence of cell division in a sample. The method comprises the steps of contacting the properly prepared sample with the anti-Phos CENP-A antibody or fragment under conditions that permit the specific binding of the antibody or fragment to its epitope. The presence of bound anti-Phos CENP-A antibody to a cell indicates that the cell is dividing. Advantageously, the anti-Unmodified CENP-A antibody can be used in conjunction with the anti-Phos CENP-A antibodies as a control because the anti-Unmodified CENP-A antibody does not bind to cells which are dividing.

To detect the presence of bound anti-Phos CENP-A in a sample, the anti-Phos CENP-A antibody or fragment may be labeled. Numerous methods for labeling antibodies to assist in their detection are known to those skilled in the art. Labels can include, but are not limited to: an enzyme, a fluorescent material, a chemical luminous material, biotin, avidin, a radiopaque material, a paramagnetic ion and a radioisotope. Alternatively, the bound anti-Phos CENP-A antibody or fragment in a sample may be detected by the use of a second antibody which is anti- immunoglobulin. This anti-immunoglobulin antibody may be labeled to assist in its detection. The use of anti-immunoglobulin antibodies to detect bound primary antibody in a sample is well known to those skilled in the art. In accordance with one embodiment a kit is provided for detecting dividing cells. The kit comprises one or more antibodies directed against an amino acid sequence selected from the group consisting of:

Arg Arg Arg Ser(P) Arg Lys Pro Glu Ala Pro Arg Arg Arg Ser (SEQ ID NO: 1); Arg Arg Arg Ser Arg Lys Pro Glu Ala Pro Arg Arg Arg Ser (SEQ ID NO: 2);

Arg Arg Arg Ser(P) Pro Ser Pro Thr Pro Thr Pro Gly Pro Ser Arg Arg (SEQ ID NO: 3); and

Arg Arg Arg Ser Pro Ser Pro Thr Pro Thr Pro Gly Pro Ser Arg Arg (SEQ ID NO: 4) In one embodiment the kit comprises an antibody that binds specifically to the amino acid sequence Arg Arg Arg Ser(P) Arg Lys Pro Glu Ala Pro Arg Arg Arg Ser (SEQ ID NO: 1) and a second antibody that binds specifically to the amino acid sequence Arg Arg Arg Ser Arg Lys Pro Glu Ala Pro Arg Arg Arg Ser (SEQ ID NO: 2). In one preferred embodiment the antibodies are monoclonal antibodies and in a further embodiment the antibodies are labeled. To this end, the antibodies of the present invention can be packaged in a variety of containers, e.g. , vials, tubes, microtiter well plates, bottles, and the like. Other reagents can be included in separate containers and provided with the kit; e.g., positive control samples, negative control samples, buffers, cell culture media, etc.

The antibodies or fragments of the present invention can be combined with a carrier or diluent to form a composition. In one embodiment, the carrier is a pharmaceutically acceptable carrier. Such carriers and diluents include sterile liquids such as water and oils, with or without the addition of a surfactant and other pharmaceutically and physiologically acceptable carrier, including adjuvants, excipients or stabilizers. Illustrative oils are those of petroleum, animal, vegetable, or synthetic origin, for example, peanut oil, soybean oil, or mineral oil. In general, water, saline, aqueous dextrose, and related sugar solution, and glycols such as, propylene glycol or polyethylene glycol, are preferred liquid carriers, particularly for injectable solutions. The compositions comprising the anti-Phos CENP-A antibody, or a bioactive fragment thereof, and a carrier or diluent can be used in conjunction with the method to detect dividing cells or cancer.

In accordance with one embodiment the antibodies of the present invention are labeled for use in diagnostic imaging. Examples of labels useful for diagnostic imaging in accordance with the present invention are radiolabels such as ^{13 l}I, 11 lln, 123i_s 99m_TC} 32_Pj 125_L 3_H, 14c, and 188Rh, fluorescent labels such as fluorescein and rhodamine, nuclear magnetic resonance active labels, electron dense or radiopaque materials, positron emitting isotopes detectable by a positron emission tomography ("PET") scanner, chemilluminescers such as luciferin, and enzymatic markers such as peroxidase or phosphatase. Short-range radiation emitters, such as isotopes detectable by short-range detector probes, such as a transrectal probe, can also be employed. These isotopes and transrectal detector probes, when used in combination, are especially useful in detecting prostatic fossa recurrences and pelvic nodal disease. The antibodies of the present invention can be labeled with such reagents using techniques known in the art. For example, see Wensel and Meares, Radioimmunoimaging and Radioimmunotherapy, Elsevier, New York (1983), which is hereby incorporated by reference, for techniques relating to the radiolabeling of antibodies. See also, D. Colcher et al., "Use of Monoclonal Antibodies as Radiopharmaceuticals for the Localization of Human Carcinoma Xenografts in

Athymic Mice," Meth. EnzvmQL, 121: 802-816 (1986), which is hereby incorporated by reference.

In the case of a radiolabeled antibody, the antibody is detected or "imaged" in vivo using known techniques such as radionuclear scanning using e.g., a gamma camera or emission tomography. See e.g., A.R. Bradwell et al.,

"Developments in Antibody Imaging," Monoclonal Antibodies for Cancer Detection and Therapv, R. W. Baldwin et al, (eds.), pp. 65-85 (Academic Press 1985), which is hereby incorporated by reference. Alternatively, a positron emission trans axial tomography scanner, such as designated Pet VI located at BrookhavenNational Laboratory, can be used where the radiolabel emits positrons.

In accordance with one preferred embodiment the antibody is labeled with a fluorophore or chromophore using standard moieties known in the art. Since antibodies and other proteins absorb light having wavelengths up to about 310 nm, the fluorescent moieties should be selected to have substantial absorption at wavelengths above 310 nm and preferably above 400 nm. A variety of suitable fluorescers and chromophores are described by Stryer, Science, 162:526 (1968) and Brand, L. et al., Annual Review of Biochemistry , 41 :843.:868 (1972), which are hereby incorporated by reference. The biological agents can be labeled with fluorescent chromophore groups by conventional procedures such as those disclosed in U.S. Patent Nos. 3,940,475, 4,289,747, and 4,376,110, which are hereby incorporated by reference. One group of fluorescers having a number of the desirable properties described above are the xanthene dyes, which include the fluoresceins derived from 3,6-dihydroxy-9- henylxanthhydrol and resamines and rhodamines derived from 3,6-diamino-9- phenylxanthydrol and lissanime rhodamine B. The rhodamine and fluorescein derivatives of 9-o-carboxyphenylxanthhydrol have a 9-o-carboxyphenyl group. Fluorescein compounds having reactive coupling groups, such as amino and isothiocyanate groups, such as fluorescein isothiocyanate and fluorescamine, are readily available.

Another group of fluorescent compounds are the naphthylamines, having an amino group in the α or 13 position. Antibodies can be labeled with fluorochrome or chromophores by the procedures described by Goding. The antibodies can be labeled with an indicating group containing the NMR-active 1 ^F atom, or a plurality of such atoms inasmuch as (i) substantially all of naturally abundant fluorine atoms are the l^F isotope and, thus, substantially all fluorine- containing compounds are NMR-active; (ii) many chemically active polyfluorinated compounds such as trifluoracetic anhydride are commercially available at relatively low cost, and (iii) many fluorinated compounds have been found medically acceptable for use in humans such as the perfluorinated poly ethers utilized to carry oxygen as hemoglobin replacements. After permitting such time for incubation, a whole body NMR determination is carried out using an apparatus such as one of those described by Pykett, Scientific American, 246:78-88 (1982), which is hereby incorporated by reference, to locate and image prostate epithelial cells.

Cancer is characterized by unregulated growth of cells, and thus cancer tissue contains a high prevalence of cells that are undergoing mitosis. Accordingly, the anti-Phos CENP-A antibody can be used to identify tissues that contain a high incidence of mitotic cells and assist in the detection of oncogenesis and cancer. The method of detecting a cancerous cell or tissue involves contacting tissues with a labeled anti-Phos CENP-A antibody or fragment and determining if the antibody or fragment binds to cells in that tissue. Any cell or tissue to which the antibody or fragment binds is identified as dividing and, thus, potentially as cancerous. For example, this procedure can be used to assist the analysis of tissues isolated for a biopsy. Tissues containing an abnormally high number of cells undergoing mitosis are identified as potentially cancerous. In one embodiment a composition comprising a suitable diagnostic imaging label linked to the anti-Phos CENP-A antibody can be administered to a patient to locate or diagnose cancer in vivo. Cancer cells or tissue which may be detected according to this procedure include renal cancerous cells or tissues, urothelial cancerous cells or tissues, colon cancerous cells or tissues, rectal cancerous cells or tissues, lung cancerous cells or tissues, breast cancerous cells or tissues, or cancerous cells or tissues of metastatic adenocarcinoma to the liver.

Methods of administration are well known to those of skill in the art and include, but are not limited to oral and parenteral administration. Dosage forms for parenteral administration, for example intramuscular, intravenous or subcutaneous administration can be formulated in physiological saline using techniques known to those skilled in the art. In accordance with one embodiment the composition is administered intravenously to treated cancer. Administration can be effected continuously or intermittently such that the amount is effective for its intended purpose. The dosage depends on a variety of factors including the age, weight and condition of the patient and the route of administration.

In another embodiment, the antibodies of the current invention are coupled to a bioactive substance or substances and compositions comprising the antigen conjugate are used to treat cancer. The bioactive substance(s) can be selected from a group comprising drugs, toxins, immunomodulators, peptide effectors and isotopes. One specific embodiment is a composition comprised of a pharmaceutically appropriate carrier and the anti-Phos CENP-A antibody (or bioactive fragment thereof) conjugated to an anti-cancer agent such as: adriamycin, mitomycin, cisplatin, vincristine, epirubicin, methotrexate, 5 Fu, aclacinomycin, rincin A and diptheria toxin. Because the anti-Phos CENP-A antibody binds to dividing cells, the anti- cancer agent will be concentrated in tissues containing relatively high levels of dividing cells, such as carcinomas. Therefore, one embodiment of the invention is directed to the use of an anti-Phos CENP-A antibody conjugate for treating cancer. Such treatment involves administering to a person in need of treatment an amount of an anti-Phos CENP-A antibody conjugate composition effective to 1) slow or stop the growth of the cancerous tissue; 2) shrink or prevent an increase in the size of a cancerous tumor; 3) alleviate the symptoms associated with cancer. In one embodiment a composition for treating cancer comprises an anti-Phos CENP-A antibody conjugated to an anti-cancer agent and a pharmaceutically acceptable carrier. In one preferred embodiment the anti-Phos CENP-A antibody is an antibody that binds to the sequence of SEQ ID NO: 1, and more preferably the antibody is a monoclonal antibody. Because the anti-Phos CENP-A antibodies have the potential for use in humans as diagnostic and therapeutic agents, one embodiment of the present invention is humanized versions of the anti-Phos CENP-A and anti-Unmodified CENP-A antibodies. Humanized versions of the antibodies are needed because antibodies from non-human species may be recognized as foreign substances by the human immune system and neutralized such that they are less useful. Humanized antibodies are immunoglobulin molecules comprising a human and non-human portion. More specifically, the antigen combining region (variable region) of a humanized antibody is derived from a non-human source (e.g. murine) and the constant region of the humanized antibody is derived from a human source. The humanized antibody should have the antigen binding specificity of the non-human antibody molecule and the effector function conferred by the human antibody molecule. Typically, creation of a humanized antibody involves the use of recombinant DNA techniques.

In accordance with one embodiment, the mitosis marker antibody of the present invention is modified to remove the majority of the murine antibody sequence to lessen the likelihood of generating human anti-mouse antibody (HAMA) responses in patients administered compositions comprising the antibody. In particular, in one embodiment the humanized antibody comprises a human acceptor framework region (FR) linked to a CDR from a non-human donor immunoglobulin which specifically binds to an antigen of SEQ ID NO: 1. In addition, the native mouse Fv fragment sequences can be modified, using standard techniques known to those skilled in the art, to make the gene more suitable for expression in prokaryotic cells. For example one or more codons of the gene encoding the mouse Fv fragments can be exchanged for codon sequences more commonly used by bacteria. It is anticipated that the removal of infrequent bacterial codons will allow the miniantibody to be expressed in E. coli at higher concentrations.

The antibodies of the present invention can also be linked to an insoluble support to provide a means of isolating mitotically dividing cells from complex mixtures. The support may be in particulate or solid form and could include, but is not limited to: a plate, a test tube, beads, a ball, a filter or a membrane. Methods for fixing antibodies to insoluble supports are known to those skilled in the art. In one embodiment an antibody of the current invention is fixed to an insoluble support that is suitable for use in affinity chromatography. Because the anti-Phos CENP-A antibody binds to cells that are dividing, the anti-Phos CENP-A antibody fixed on an insoluble support can be used to separate dividing cells from a sample. Conversely, the anti-Unmodified CENP-A antibody or fragment fixed on an insoluble support can be used to separate non-dividing cells from a sample. Prior to the present invention, the kinase responsible for bringing about the steady-state balance of H3 and CENP-A phosphorylation during mitosis and meiosis was not known. The Ipllp/aurora kinase has now been identified as the major enzyme system responsible for this activity. Using Saccharamyces cerevisae and Caenarhabditis elegans, it was demonstrate that the mitotic histone H3 kinase is Ipllp/aurora, a previously identified essential serine-threonine kinase required for high-fidelity mitotic and meiotic chromosome segregation in budding yeast. Consistent with genetic evidence that Ipllp kinase acts antagonistically to Glc7p (PPlc phosphatase) in yeast, levels of H3 phosphorylation are restored or elevated in mutants where the activity of Gcl7p is missing or severely compromised. Human homologs of the aurora/Ipl family (referred to as AIK kinases or auroraIpllp like kinases) are amplified in a variety of human cancers and are associated with defects that include missegregation of chromosomes. The present findings shed new light on the enzymology of H3 phosphorylation and serve to reinforce an emerging theme that covalent modification of histones is linked to fundamental processes with far-reaching implications for human biology and disease.

The Ipllp/aurora kinases have been previously described by others (Chan and Botstein, 1993; Glover et al., 1995) with regard to genetic screens leading to missegregation of chromosomes. Similarly, the idea that Glc7p in yeast (PPlc phosphatase in mammals) is the corresponding phosphatase that is also required for high-fidelity chromosome transmission has also been previously described. However, this is the first description of the substrate for these enzymes, and thus provides insights into the mechanisms responsible for defects in mitosis, including the induction of cancer. Furthermore, as shown in Fig.2A-2D there is a common motif (RZSXXXXAPRZ' (SEQ ID NO: 10/SEQ ID NO: 11) wherein X is any amino acid and Z and Z¹ are both arginine or both lysine) shared between the H3 and the CENP-A sequences that flank the phosphorylated serine residue. Therefore, it is likely that the Ipllp/aurora kinase may also be involved in the phosphorylation of CENP-A as well. In accordance with one embodiment of the present invention a method of inhibiting mitosis comprises interfering with Ipllp/aurora kinase's ability to phosphorylate the H3 SerlO and CENP-A Ser7 residues. This inhibitory activity can result from either specific inhibition of the kinase or by blocking the target peptide to prevent its interaction with the kinase. In preferred embodiments the kinase inhibitor selectively inhibits Ipllp/aurora kinases and does not affect other members of the PKA superfamily.

One strategy for selectively inhibiting the aurora/Ipl lp kinase family members derives from the fact that the amino-terminal domain (NTD) of this family of kinases is unique to this branch of the PKA protein kinase family. The NTD domain is 100 amino acids and lacks the catalytic domain of the enzyme. This domain contains three perfect consensus motifs for CDKs ( cyclin-dependent kinases) leading to the intriguing possibility that Ipllp/aurora is one of the key physiological targets of CDKs, and that this is how these CDK cell cycle 'engines' implement downstream cell cycle events, such as chromosome condensation, that is, in turn, mediated by histone H3 phosphorylation. Thus inhibitors of this enzyme system stand as attractive targets for cancer drug therapy.

To detect such inhibitors, a high through-put screening approach can be applied, using the anti-Phos CENP-A antibodies as part of the screening process. In accordance with one embodiment of the invention, a method of screening for potential inhibitors of the kinase responsible for phosphorylation of the N-terminal of CENP-A and analogous proteins is provided. The anti-Phos CENP-A antibody or fragment will be used to quantify the level of phosphorylation of CENP-A in the sample, indicating the relative effectiveness or ineffectiveness of the potential kinase inhibitor.

In accordance with one embodiment of the present invention a method for isolating inhibitors of the kinase that phosphorylates Ser7 of CENP-A ("CENP-A kinase") is provided. The method comprises the steps of providing a sample that comprises the amino acid sequence of SEQ ID NO: 2 and a kinase under conditions suitable for the phosphorylation of the sequence. A potential inhibitor of the kinase is then added to the sample along with the anti-Phos CENP-A antibody and the sample is incubated for a predetermined length of time sufficient to allow phosphorylation of the CENP-A substrate. The sample is then analyzed to quantify the amount of antibody bound to CENP-A substrate. The amount of anti-Phos CENP-A antibody bound to the CENP-A substrate is a direct indication of the level activity of said kinase. By comparing this result to a control reaction (a sample that comprises the amino acid sequence of SEQ ID NO: 2, a kinase and the anti-Phos CENP-A antibody) any decrease in kinase activity observed in the experimental reaction will be due to the potential inhibitor. Thus the level of phosphorylation of CENP-A in the sample indicates the relative effectiveness of the potential kinase inhibitor. Preferably the CENP-A substrate is the peptide Arg Arg Arg Ser Arg Lys Pro Glu Ala Pro Arg Arg Arg Ser (SEQ ID NO: 2) and the kinase is from the Ipllp/auroa kinase family.

Example 1

MATERIALS AND METHODS

Peptide synthesis, conjugation and antibody generation. An unmodified CENP-A peptide was synthesized corresponding to the amino terminus of human CENP-A from amino acids 4-17 (RRRSRKPEAPRRRS; SEQ ID NO: 2, see Fig. 2B for a schematic drawing). A phosphorylated derivative of this peptide was synthesized such that it contained a single phosphorylated serine residue at position 7 (RRRSpRKEAPRRRS; SEQ ID NO: 1). Both peptides contained an artificial, carboxy-terminal cysteine for coupling to Keyhole limpet hemocyanin (Sigma) using standard protocols. Immunization of rabbits, and sera collection were done as previously described (Hendzel et al., (1997), Chromosoma 106,348-360).

Cell culture and induction of mitotic arrest.

HeLa cells were obtained from ATCC and cultured in MEM medium (Gibco/BRL), supplemented with 10% fetal bovine serum, under standard conditions. Routinely, cells were grown to 4 x 10^ cells per 28 mm in fresh MEM medium. Adherent cells were then collected by scraping, washed in PBS, pH 7.5 and then collected by low-speed centrifugation. Typically, these randomly growing populations (R) contained -5% mitotic cells. To induce mitotic arrest (M), cells grown to 4 x 10^ were treated with 15ug/ml nococdozole and incubated for 18hrs followed by agitation to release the loosely-associated mitotic cells. FACS analyses demonstrated that >95% of the cells were arrested in M-phase after this procedure. Cell pellets were resuspended one-half volume ND3250 buffer, collected by centrifugation and stored frozen at -80°C. Nucleus isolation and histone extraction.

Nuclei were isolated essentially according to Hendzel et al. (1997), Chromosoma 106,348-360). Resulting nuclei were immediately resuspended in .4 N sulfuric acid, and acid-soluble proteins, released by incubating nuclei with gentle shaking on ice for 30 min, were generated by pelleting insoluble material by centrifugation at 12,000g for lOmin. The soluble proteins were collected by TCA precipitation on ice for 10 min, pelleted at 12,000g and washed once with acidified acetone (.1 %HCL in acetone) and twice with acetone. Electrophoresis and immunoblotting.

Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-P AGE) was performed as described by Laemmli (1970). Specificity of antibodies used in this report was determined by ELISA assay or by immunoblotting as described previously (Hendzel et al 1997). Balanced protein loads were ensured by staining parallel gels of equivalently loaded samples and/or by staining blots directly with Ponceau Red. All blots were blocked for 1 hour with nonfat dry milk at 5% and incubated with immune sera. All antibody reactions were detected by ECL using horseradish peroxidase-conjugated secondary antibody. Where indicated, the antibody was preincubated with unmodified or phosphorylated CENP-A peptide. In this case, 1ml of antiserum was incubated with 3ug of peptide for 1 h at room temperature prior to applying the antibody-containing solution to the blotted proteins. Immunofluorescence

Asynchronous human CENP-B-GFP U20S cells were plated and grown to confluency on acid-washed glass coverslips (Fisher) for 2 days. Cells were fixed in 1% paraformaldehyde in PBS containing 140 mM NaF to inhibit phosphatases. Coverslips were then washed once with PBS- TX (PBS + 0.1% Triton X -100) and incubated in blocking solution (PBS-TX + 1% BSA) at room temperature for 15 minutes. Primary antibodies were diluted as follows: 1:25 of affinity-purified anti-Un CENP-A or anti-Phos CENP-A, and 1 :500 of rabbit anti-lamin A (a gift from Larry Gerace). Coverslips were incubated with primaries for 30 minutes at 37°C, and then washed two times in PBS-TX for 10 minutes. Secondary antibodies were used at 1:100 dilutions (Jackson Laboratories, West Grove, PA), incubated on coverslips with DAPI for 30 minutes at 37 degrees, washed once in PBS-TX for 10 minutes, washed once in detergent-free PBS for 10 minutes, and finally rinsed once in distilled water before drying. Coverslips were mounted using SloFade Lite (Molecular Probes, Eugene, OR) and visualized on a Delta Vision wide field optical sectioning microscope system based on an Olympus LX70 epifluorescence microscope (Applied Precision, Issaquah, WA). A xl 00 1.35 NA NeofluAr oil immersion lens was used for all images. Images were processed using a constrained iterative deconvolution algorithm. Projection images were prepared from 3 -dimensional images stacks and composite images shown were assembled using Adobe Photoshop 5.5 (Adobe, Mountain View, CA). For quantitation of fluorescence intensities, images were collected at 0.3 um intervals spanning the entire chromatin volume as defined by DAPI staining. An exposure time was selected such that pixel intensities recorded from maximally labeled mitotic cells did not exceed the optimal response range of the CCD camera. This exposure time was used to image 21 cells on a single coverslip in a single data

Collection Session After deconvolution, Softworx analysis software (Applied Precision) was used to integrate Phos CENP-A signal within the volume of each cell using an automated thresholding algorithm to define antibody signals. Visual inspection demonstrated that all labeled centromeres were accurately defined by this procedure. Integrated fluorescence intensity data were imported into a spreadsheet (Excel, Microsoft, Redmond, WA) for analysis and display. Chi-squared analysis demonstrated that independent samples from each stage of the cell cycle were significantly different between the stages (P=O), and error bars report the standard error in the distribution.

Codetection of H3P and CENP-A

For co-detection of phosphorylated histone H3 and CENP-A with rabbit antisera, we utilized a successive labeling method with an additional blocking step to facilitate co-detection without cross-reaction. Optimal results were obtained using the CENP-A antibody first. After washing, the secondary antibody was added (donkey anti-rabbit TRITC, Jackson Laboratories, West Grove, PA) at twice the usual concentration ( 1 :50 instead of 1 :100). After washing, coverslips were blocked for 15 minutes at 37°C with PBS-TX + 1% BSA and 5% normal goat serum. Anti-H3-P (Hendzel et al., 1997) was added at a dilution of 1:3000 in PBS-TX + 1% BSA, incubated for 30 minutes at 37°C, coverslips were washed again, the secondary antibody (donkey anti-rabbit cascade blue, Jackson Laboratories) was used at the usual concentration (1:100), and coverslips were washed again before mounting.

Results

In order to test whether CENP-A is phosphorylated, rabbit antisera was raised against two synthetic phosphorylation-specific peptides (Figs. 2C and 2D). The first peptide, Un CENP-A, contains residues 4-17 of CENP-A. The second peptide, Phos CENP-A, contains the same sequence with a phosphorylated serine 7. To begin evaluating the specificity of these rabbit sera, ELISA assays were performed (Fig. 3A- 3D). The binding of each antibody for its peptide was specific to within 30ng/ml. The binding of the antisera to the opposing peptide was negative to 90 ng/ml peptide. Western blots comparing Nocodazole-blocked HeLa cells with asynchronous cultures illustrate antiserum against peptide Un CENP-A, anti-Un CENP-A, detects a 17 kDa antigen (see Fig. 4). In contrast, antiserum elicited against Phos CENP-A (i.e. anti- Phos CENP-A), detected a slightly slower migrating species that is greatly enriched in mitotically arrested cells. The signal seen with anti-Phos CENP-A was removed when the samples were treated with alkaline phosphatase (Fig. 5), demonstrating that the antibody specifically recognizes a CENP-A phosphoepitope in mitotic HeLa nuclei.

The cellular distribution of phosphorylated CENP-A confirmed the mitotic nature of the modification. Lnmunofluorescence was performed using affinity purified anti-CENP-A antibodies on a human osteosarcoma (U2OS) cell line that constitutively expresses CENP-B-GFP as a marker for centromeres. Interphase cells lacking detectable chromosome condensation (resolved with DAPI staining or anti- H3P) showed no reactivity with anti-Phos CENP-A. Reactivity with anti-Phos CENP- A is first detectable in prophase cells that exhibit visible chromosome condensation. Phosphoepitope staining exhibits characteristic kinetochore double dot morphology. Reactivity with anti-Phos CENP-A is evident at all centromeres in prometaphase cells and persists through metaphase (Fig. 6). Reactivity with anti-Phos CENP-A is lost beginning in anaphase, becoming undetectable in telophase cells. The complementary staining pattern is observed with anti-Un CENP-A antibody, which labels centromeres brightly in all interphase cells, fails to label mitotic cells and reappears in anaphase cells. Anti-Un CENP-A continues to recognize centromeres in cells that exhibit moderate levels of chromosome condensation. In each experiment, some telophase cells were observed that lacked reactivity with either antibody. Weakly reactive telophase cells are also observed with an antiserum against a similar N-terminal unphosphorylated CENP-A peptide. Lack of detectable signal at this time could be due to masking of the epitope by association with other proteins, or additional N- terminal modifications of CENP-A. The long half-life of CENP-A-HA in HeLa cells rules out the idea that CENP-A is degraded at the end of mitosis. Phosphorylation of histone H3 initiates in pericentromeric regions prior to general chromatin phosphorylation. To directly examine the relationship between timing of histone H3 and CENP-A phosphorylation, simultaneous co-detection of the two proteins was performed by immunofluorescence. G2 cells exhibiting the pericentromeric pattern of early histone H3 phosphorylation were negative for Phos CENP-A staining but reacted strongly with anti-Un CENP-A. Similarly, cells with anti-Phos H3 reactivity along chromosome arms reacted strongly with anti-Un CENP- A but cells in a similar stage lacked reactivity with anti-Phos CENP-A. Cells with detectable anti-Phos CENP-A staining exhibited significant chromosome condensation in addition to anti-Phos H3 reactivity along chromosome arms. Little or no signal with anti-Un CENP-A was seen at this stage. These data indicate that CENP-A phosphorylation at Ser7 does not initiate until histone H3 phosphorylation has occurred throughout the chromosome arms.

Examination of the nuclear lamina revealed that CENP-A phosphorylation is detected at all centromeres prior to nuclear envelope breakdown. CENP-A phosphorylation thus constitutes a kinetically distinct phase in phosphorylation of the histone H3 family proteins in preparation of mitosis. Taken together with previous results (Hendzel et al., 1997), three distinct phases of histone H3 -family N-terminal phosphorylation can be defined during G2 and M, prior to nuclear envelope breakdown: pericentromeric H3 phosphorylation, general chromatin H3 phosphorylation, and kinetochore-specific CENP-A phosphorylation. Paired spots of anti-Un CENP-A are first detectable during G2, near the onset of H3 phosphorylation demonstrating that the morphological duplication of kinetochore chromatin is complete in early G2. Reactivity with anti-Phos CENP-A is absent at this stage. Flanking alpha satellite arrays, visualized with CENP-B-GFP (Shelby et al., 1996), undergo morphological changes during the time when histones are being phosphorylated. In interphase cells prior to histone H3 phosphorylation, CENP-B- GFP spots appear as small, spherical dots. In cells in early G2, defined as having fewer than 10 pericentromeric sites of anti-H3-P signal, CENP-B-GFP foci are observed as larger, more irregular spots. These irregular spots are seen associated with paired anti-CENP-A- Ser7P-labeled kinetochores in prophase cells. In prometaphase and metaphase, the CENP-B-GFP-labeled alpha satellite arrays adopt characteristic stretched dumbbell shapes indicative of microtubule attachment, as previously described (Shelby et al., 1996).

Histone H3 phosphorylation is known to begin in G2 and persist through mitosis. The period of CENP-A phosphorylation from prophase to mid- anaphase is estimated to be in the range of 30-60 minutes in cultured human cell lines. However, within this short period, the appearance and disappearance of phosphoepitope reactivity seemed gradual, rather than sudden and complete. Simultaneous and complete staining of every centromere at once might imply that CENP-A phosphorylation could represent a discrete step in kinetochore assembly. A more gradual modulation of phosphorylation might imply involvement in a counting mechanism similar to the spindle checkpoint proteins. Alternatively, gradual modulation could imply involvement in a continuing process, such as chromosome condensation, which is not complete until metaphase. To examine the CENP-A immunofluorescence data quantitatively, the total intensity of anti-Phos CENP-A labeling was measured in 21 cells sampled at 0.3 um intervals through the volume of the nucleus. Cells were staged within mitosis on the basis of chromosome and centromere morphology, and the corresponding fluorescence intensities were plotted as shown in Fig. 6. Cells in prophase exhibit a wide range of fluorescence intensities and variable number of reactive centromeres. The peak of intensity occurs in late prophase or early prometaphase. Phosphorylation or epitope accessibility decreases by about 30% in metaphase and then rapidly declines through anaphase to reach undetectable levels in late anaphase or early telophase.

Claims

Claims

1. A purified antigenic fragment of CENP-A comprising an amino acid sequence selected from the group consisting of:

Arg Arg Arg Ser(P) Arg Lys Pro Glu Ala Pro Arg Arg Arg Ser (SEQ ID NO: 1); Arg Arg Arg Ser Arg Lys Pro Glu Ala Pro Arg Arg Arg Ser (SEQ ID NO: 2);

Arg Arg Arg Ser(P) Pro Ser Pro Thr Pro Thr Pro Gly Pro Ser Arg Arg (SEQ ID NO: 3);

Arg Arg Arg Ser Pro Ser Pro Thr Pro Thr Pro Gly Pro Ser Arg Arg (SEQ ID NO: 4); and amino acid sequences that differ from SEQ ID NO: 1, SEQ ID NO: 2 or SEQ ID

NO: 3 by one or more conservative amino acid substitutions.

2. The purified antigenic fragment of claim 1 wherein the amino acid sequence consists of Arg Arg Arg Ser(P) Arg Lys Pro Glu Ala Pro Arg Arg Arg Ser (SEQ ID NO: 1);

Arg Arg Arg Ser Arg Lys Pro Glu Ala Pro Arg Arg Arg Ser (SEQ ID NO: 2); or an amino acid sequence that differs from SEQ ID NO: 1 or SEQ ID NO: 2 by one conservative amino acid substitution.

3. The purified antigenic fragment of claim 1 wherein the amino acid sequence consists of

Arg Arg Arg Ser(P) Pro Ser Pro Thr Pro Thr Pro Gly Pro Ser Arg Arg (SEQ ID NO: 3);

Arg Arg Arg Ser Pro Ser Pro Thr Pro Thr Pro Gly Pro Ser Arg Arg (SEQ ID NO: 4); or an amino acid sequence that differs from SEQ ID NO: 3 or SEQ ID NO: 4 by one conservative amino acid substitution

4. An antibody that binds specifically to an antigen selected from the group consisting of

Arg Arg Arg Ser(P) Arg Lys Pro Glu Ala Pro Arg Arg Arg Ser (SEQ ID NO: 1); Arg Arg Arg Ser Arg Lys Pro Glu Ala Pro Arg Arg Arg Ser (SEQ ID NO: 2); Arg Arg Arg Ser(P) Pro Ser Pro Thr Pro Thr Pro Gly Pro Ser Arg Arg (SEQ ID O: 3);

Arg Arg Arg Ser Pro Ser Pro Thr Pro Thr Pro Gly Pro Ser Arg Arg (SEQ ID O: 4); and amino acid sequences that differ from SEQ ID NO: 1 , SEQ ID NO: 2, SEQ ID O: 3 or SEQ ID NO: 4 by one or more conservative amino acid substitutions.

5. The antibody of claim 4, wherein the antibody specifically binds to the sequence Arg Arg Arg Ser Arg Lys Pro Glu Ala Pro Arg Arg Arg Ser (SEQ ID NO: 2).

6. The antibody of claim 4, wherein the antibody specifically binds to the sequence Arg Arg Arg Ser(P) Arg Lys Pro Glu Ala Pro Arg Arg Arg Ser (SEQ ID NO: 1).

7. The antibody of claim 6, wherein the antibody is a monoclonal antibody.

8. A fragment of the antibody of claim 4 or 6 which retains binding specificity for the antigenic fragment of claim 1.

9. A composition comprising the antibody of claim 6 and a diluent or pharmaceutically acceptable carrier.

10. The antibody of claim 6 wherein said antibody is coupled to a bioactive substance selected from the group consisting of a drug, a toxin, a immunomodulator, a peptide effector and an isotope. >

11. A pharmaceutical composition comprising a therapeutically effective amount of the antibody of claim 10 and a pharmaceutically acceptable carrier.

12. A method of detecting mitotic cells in a sample, said method comprising the steps of: contacting said sample with the antibody of claim 6; removing unbound and non-specific bond antibody from the sample; and detecting the antibody bound to the sample.

13. The method of claim 12, wherein the antibody is labeled.

14. The method of claim 13, wherein the antibody is labeled with a fluorescent marker.

15. The method of claim 12, wherein the detection step comprises contacting said antibody with a labeled secondary antibody wherein said secondary antibody is an anti-immunoglobulin antibody.

16. An antibody that selectively binds to an epitope contained within the sequence Arg Arg Arg Ser(P) Arg Lys Pro Glu Ala Pro Arg Arg Arg Ser (SEQ ID NO: 1).

17. The antibody of claim 16, wherein the antibody is a monoclonal antibody.

18. A fragment of the antibody of claim 17 which retains binding specificity for the antigenic fragment of claim 1.

19. A kit for detecting mitotic cells, said kit comprising an antibody that specifically binds to the sequence Arg Arg Arg Ser(P) Arg Lys Pro Glu Ala Pro Arg Arg Arg Ser (SEQ ID NO: 1).

20. The kit of claim 19 further comprising an antibody that specifically binds to the sequence Arg Arg Arg Ser Arg Lys Pro Glu Ala Pro Arg Arg Arg Ser (SEQ ID NO: 2).

21. A method of treating cancer in humans, comprising the step of administering to a human in need of such treatment a therapeutically effective amount of the antibody of claim 10.

22. A method of screening for potential inhibitors of a kinase said method comprising the steps of: providing a sample that comprises said kinase and a substrate that is phosphorylated by said kinase; adding a potential inhibitor of said kinase to the sample; contacting the sample with the antibody of claim 4; quantifying said antibody bound to said sample as an indication of the level activity of said kinase.

23. The method of claim 22 wherein the kinase substrate is the peptide Arg Arg Arg Ser Arg Lys Pro Glu Ala Pro Arg Arg Arg Ser (SEQ ID NO: 2).

24. The method of claim 23 wherein said kinase is from the Ipllp/auroa kinase family.