AU2005242130A1 - Compositions and methods for detecting stress-inducible proteins - Google Patents

Description

06-12-'05 17:23 FROM- T-751 P007/078 F-502 J'IOO/oI Ru0011ion 3.2 Re9ulation 3.2

AUSTRALIA

Patents Act 1990 COMPLETE SPECIFICATION STANDARD PATENT

(ORIGINAL)

Name of Applicant(s): Actual Inventor(s): StressGen Biotechnologies Corp., of #120-4243 Glanford Avenue, Victoria, British Columbia VSZ 4B9, CANADA Heather A. BOUX Geraldine S. WONG Henry RODRIGUEZ Address for Service: DAVIES COLLISON CAVE, Patent Trademark Attorneys, of 1 Nicholson Street, Melbourne, 3000, Victoria, Australia Ph: 03 9254 2777 Fax: 03 9254 2770 Attorney Code: DM "Compositions and methods for detecting stress-inducible proteins" Invention Title: The following statement is a full description of this invention, including the best method of performing it known to us:- COMS ID No: SBMI-02132445 Received by IP Australia: Time 17:45 Date 2005-12-06 06-12-'05 17:23 FROM- T-751 P008/078 F-502 ci \COMPOSITIONS AND METHODS FOR DETECTING STRESS-INQUCIBLE

PROTEJNS

SThe present invention features compositions and methods for detecting the stressinducible protein Hsp70B'. More specifically, the invention features antibodies that specifically bind Hsp70B' or fragments, antigenically equivalent portions, or epitopes thereof.

o BACKGROUND OF THE INVENTION Cells within most organisms have evolved a mechanism known as the "cellular stress S. response" to cope with adverse changes in Lheir environment. The response is a universal cellular defense mechanism that results in increased expression of a class of proteins referred to as "heat shock" or "stress" proteins. The conditions that trigger the response include. a rise in temperature, hypoxia, irradiation, nutritional deficiencies, acute exercise, infection, or exposure to a metabolic insult such as a proinflammatory cyxokine, a heavy metal, an amino acid analogue.

or a metabolic poison (Kelly er at., Appl. Physiol. 81:2379-2385. 1996: Minowada and Welch.

C *i Chn Invesr 95:3-12. 1995).

Stress proteins are also essential for normal cellular funcion d many are.consiutively S To d y r constitutivel i. expressed. They are believed to help regulate the cell cycle and cellular differentiation and to mainmtain the cell at critical stages of organ development (Bimbaum, Springer Semin.

20 Immunopathol. 17:107-118, 1995). Some stress protcins are molecular chaperones that facilitate the correct folding or conformation of nascent polypeptides, direct intracellular trafficking of proteins, protect proteins against denaturation, and assist in the renaturation of unfolded proteins (Macario, Int. J. Clin. Lab Res. 25:59-70, 1995). Stress proteins also participate in antigen presentation and nuclear receptor binding and act as anti-apoptotic agents.

The Hsp70 family of stress proleins includes at least I I different genes that encode highly related protein isoforms ranging in size from 66 kDa to 78 kDa (Tavaria e ol., Cell Stress Chaperones 1:23-28, 1996). Members of this family help regulate protein synthesis and translocation, protein-protein interactions, thermotolcrance, and protein degradation (Mangunen el al., Cell Stress Chaperones 2:168-174, 1 997).

COMS ID No: SBMI-02132445 Received by IP Australia: Time 17:45 Date 2005-12-06 06-12-'05 17:23 FROM- T-751 P009/078 F-502 WO 01/42423 PCT/Usw9afl3e1 o Members of the humran hsplo gene family also display considerable structural and O ~~scqluence zhnilarity; the greatest sequence divergence is in ch nrnlae ein and extreme Cl C-termijnaj coding regions (Leung L7, Genomcs 2:74-79, 1992). Individual fsp7O family members differ in their levels of basal expression and are induced uinder different conditions s(Leung ei al-, Genomics 12:74-79, 1992). The majority offllsp7O protein isoforros are snthesized constitutively, but their expression may be up-reguiled foiloigepsr o 0 ~cflviJOnflntal insult, These proteins bind AT? through an AlP-binding cassette at their Nterminus and have a large C-termninus peptide-bindjng domain (Main et a L, Guidebook to Vulecular Chapenes and Protein-Foldsng Catalyst, Sambrookc Tooze Publiciaton, Oxford University Press, 1997). This peptdde binding function allows H-sp7O proteins to play a .4.significaint role in the protection. and folding of nascent proteini after synthesis. in the Cl translocation of proteins throughi membranes, and in the protection arid repair of stress-induced protein damnage (Mirtowads and Wech, J Clin. Invest. 95:J -12, 1995).

Cl Members of the human l-lsplO protein family associate with distinct cellular is compartmernts. Prominent family members include; i) the constitutive HseM (or cognate) pirotein, which is present within the cntosol and nucleus. ii) the highly stress-inducible I-Isp7OA prolci,. which is present within the cytosol, nucleus, and nucelus (this protein is present at basal lewis in unstressed hwnan cells). iii) the strictly stress-iiducibtc 1-sp7Ofl' protein and its closely retated isoform l-sp7OB3, iv) the constitutive glucose regulated 79 k~ln protein (or DiP).

which is present within the lumen of the endoplasmic reticulum, and v) the glucose regulated k~la Pr-otein (Grp7S or nuHsp 75), which is present within mniochondria (Tava-ia er al.. Cell Stress Chaperones 1;23-28. 1996).

Antibodies have been raised against fisp70 family members that arc expressed at basal levels and whose expression can be induced to high levels (iLe, Grp7S, and l-sp7OA) and to The constitutive 1-k*7O family mcmbars Hsc7O, DiP). However, there are no ztntibodies that specifically bind the strictly inducible lisp705' protein or its homologue, isp700., Thus, Immwtnologacal based assays (such simznunobloning, EtA, and iminunohislochemisr)y have been practiced with antibodies that ar noat strictly stress inducible. The results obtained with these assays are amibiguous because the Hsp7O family of proteins is so comnplex- While there is 3D some indication that J-sp7OA and Qrp7o am uipregulated under conditions of stress, the significant bsal level of the inducible H-spJOA protein in normal tissue, neoplassic tissue, and 2 COMS ID No: SBMI-02132445 Received by IP Australia: Time 17:45 Date 2005-12-06 06-12-'05 17:23 FROM- T-751 P010/078 F-502 WO OI/42423 PCT/USOo/3341 Scell lines (Bachelet e al., Cell Stress Chaperones 3;168-176. 1998: Bratton et al,. In, J o Hyperhermia 13:157-168, 1997; Sztankay e al., Journal of Autoimnzniry 7:219-230. 1994), and an extreme variation in baseline levels in unstressed cells (Pockley et al., Immunol Invest CL) 27:367-77. 1998), confounds interpretation limits the utility of previous studies. The dual function of the sp70 family is also problematic. Hsp70 sress proteins function both I constitutively (by performing cellular "housekeeping" functions) and inductively (by responding to adverse changes to the cellular environment). The assays developed to date assess incremental increases in an already expressed protein (Hsp70A) but, because preexisting basal Slevels fluctuate so much, the results are dificult to interpret. Thus, there is a need for antibodies that specifically bind the srictly stress inducible Hsp70B' protein. The novel compositions of the present invention fulfill this need.

o SUMMARY OF THE INVENTION The present invention is based, in part. on the identificalion of immunogenic peptide sequences from the human Hsp70B' protein. Antibodies that specifically bind this protein can be used to distinguish between the expression of HSC70/HSP70 proteins, which occurs while a cell is functioning normally and when.it is responding to stress, and the Hsp70B' protein, which is only produced in responsc to stress substances or events that are detrimental to the heahh of the cell or organism). Hsp70B- is unique among IIsp70 family proteins because neither mRNA nor Hsp70B' protein has been detected in unstressed cells. Accordingly. the present invention features compositions and methods for determining whether a cell (or a population of cells, such as those in cell culure or within a tissue) expresses Hsp70B', A positive reaction to an Hsp70B' antibody not only provides evidence of stress in a particular cell.

but also provides an indication of the general state of the health of the organism in which that cell resides (or from which it was obtained). As described below, the compositions of the invention antibodies that specifically bind an Hsp70B' protein, an antigenic fragment, or an epitope thereof) can be used to determine whether a cell a human cell), an organ (e.g the skin or the liver), or an entire organism (e.g a human) has been exposed to a stressor.

The invention also features peptides that correspond to, or are antigenically equivalent to, regions of the Hsp70B' protein. The peptide can consist of five or more 5, 6, 7 8. 10. or 12) consecutive amino acids from Hsp70B' protein (beginning at the second, fourth, sixth, or 0* COMS ID No: SBMI-02132445 Received by IP Australia: Time 17:45 Date 2005-12-06 06-12-'05 17:24 FROM- T-751 P011/078 F-502 -4eighth residue), such as the following amino acid sequences; VO VPGGSSCGTQARQGDPSTGPI (SEQ IDNO:1) (e.g CGTQARQGDPSTGPI (SEQ ID NO:2 and CGTQARQGDPST (SEQ ID RDKIPEEDRRKMQDKC (SEQ ID NO:4) RDKIPEEDRRKMQ (SEQ ID NO:5; when these peptides are linked to keyhole O 5 limpet hemocyanin (KLH), they can include cysteine residues); AHVFHVKGSLQEESLRDKIPEEDRRKMQ (SEQ ID NO:6) F^ AHVFHVKGSLQEES (SEQ ID NO:7); MQAPRELAVGID (SEQ ID NO:8), which is Cl located in the N-terminal of Hsp70B' and, when linked to KLH includes a cysteine residue MQAPRELAVGID(C) (SEQ ID GSLQEESLRDKIPEE (SEQ ID 10 The peplides of the invention can contain at least one amino acid substitution 1, 2, or 3 of the residues in the peptides of the invention can be replaced with another amino acid residue; altematively, up to about 50% 10%, 25%, 30%, 40% or 50%) of the residues in the .peptides can be substituted). The substitution can constitute a conservative amino acid substitution. Conservative substitutions include interchanges of alanine and valine. valine and 1s isoleucine, leucine and isoleucine, aspartic acid and glutamic acid. threonine and serine. and S: others of a similar nature (for example, any in which the neutral, positive or negative charge of the original amino acid residue is maintained). Conservative amino acid substitutions are well known to those of ordinary skill in he art. Preferably, peptides containing substitutions will be Santigenically equivalent to the naturally occurring peptide sequence a peptide containing a S 20 substitution will have a relative tire index that is no less than half as great as the relative titre index of the naturally occurring peptide). The peptides of the invention can also be attached to a carrier KLH or ovalbumin) that enhances their immunogenicity or circulating half-life.

Unless otherwise noted, a "protein" is a full-length protein a full length Hsp70B' protein) and a "peptide" is a portion of a full-length protein (e.g five or more consecutive amino acid residues present within the Hsp 7 08' protein). A "polypeptide" may be either a protein or peptide.

In related aspects, the invention features antibodies that specifically bind Hsp70B' or one of the Hsp70B' peptides disclosed herein and methods of obtaining those antibodies. The antibodies can be polyclonal or monoclonal antibodies, and can be produced by methods well known to those of ordinary skill in the an. These methods typically include immunizing an COMS ID No: SBMI-02132445 Received by IP Australia: Time 17:45 Date 2005-12-06 06-12-'05 17:24 FROM- T-751 P012/078 F-502 yWO 01/42423 S1i2PCT/lSO/33341 Sanimal with Hsp70B' or an Hsp 7 08' peptide, but can be carried out instead by immunizing an animal with a nucleic acid molecule that encodes Hsp70B' or an Hsp70B' peptide.

The antibodies of the invention may be used to specifically bind, and thereby detect.

Hsp70B' in virtually any immunoassay an assay carried out by.binding Hsp70B' proteins S 5 or peptides that are immobilized (e.g on a membrane or column) or present in a cell (by. e.g., immunohistochemistry)). Accordingly, the invention features kits that include antibodies that Sspecifically bind an Hsp70B' protein or peptide. The kits can also optionally include an n Hsp70B' protein or peptide (as a positive control), an irrelevant protein one to which the c supplied antibody does not bind; as a negative control), secondary antibodies. other reagents.

C 1 0 buffers, or solutions, and instructions.

SThe details of one or more embodiments of the invention are set forth in the accompa- O nying drawings and the description below. Other features, objects, and advantages of the invention will be apparent from the description and drawings, and from the claims.

BRJEF DESCRIPTION OF THE DRAWINGS Fig. I is a seres of plots representing the displacement curves for Hsp70B' antibodies in the presence of Hsp70B' standard and HSP70 homologues. is the CB2 displacement curve, is the CD displacement curve, and is the 70B' WP displacement curve.

Fig. 2 is a representation ofthe human Hsp70B' amino acid sequence (SEQ ID NO: 11).

20 DETAILED DESCRIPTION The invention features immunogenic peptides whose sequence is present in the protein or whose sequence varies from the sequence of the Hsp70B' protein in such a limited o .way as to remain an antigenic equivalent of the naturally occurring peptide. For example, an 25 Hsp70B' protein or peptide that contains one or more amino acid substitutions one or more conservative amino acid substitutions) can be antigenically equivalent to the naturally occurring protein or peptide fragments thereof. Proteins and peptides that, upon administration to an animal, elicit the production of antibodies that specifically bind to Hsp7OB protein include the following: VPOGSSCGTQARQGDPSTOPI (SEQ ID N: I) (e.g.

CGTQARQGDPSTGPI (SEQ ID NO:2 and CGTQARQGDPST (SEQ ID (2) RDKIPEEDRRKMQDKC (SEQ ID NO:4) RDKIPEEDRRKMQ (SEQ ID COMS ID No: SBMI-02132445 Received by IP Australia: Time 17:45 Date 2005-12-06 06-12-'05 17:24 FROM- T-751 P013/078 F-502 POPEErTOouP07n.1 neodo o C'l -6when these peptides are linked to keyhole limpet hemocyanin (KLH), they can include (N- O terminal cysteine residues); AHVFHVKGSLQEESLRDKIPEEDRRKMQ (SEQ ID NO:6) AHVFHVKGSLQEES (SEQ ID NO:7); MQAPRELAVGID (SEQ ID NO:8), which is located in the N-terminal of Hsp70B' and, when linked to KLH includes a cysteine residue 0 5 (i MQAPRELAVGID(C) (SEQ ID GSLQEESLRDKIPEE (SEQ ID NO: l and the Hsp70B' protein (SEQ ID NO:11 I).

C, Portions of any.of these peptides can also be used to generate antibodies. More specifically, five or more consecutive amino acid residues (i amino acid O residues linked to one another by peptide bonds in the same sequential order as they appear in o1 the naturally occurring sequence) can be used. The starting point can be anywhere within the sequence of Hsp70B' or the Hsp70B' peptides disclosed herein, up to the fifth-to-last amino acid S* residue. Peptides based on the sequences disclosed herein can contain at least 5, 6, 7, 8. 9. I 1I. 12, 13. 14, 15. 18. 20, 25. or 28 consecutive amino acid residues. Moreover, they can begin at, for example, the second, fifth, ninth, tenth, or welth residue in any of the peptides disclosed is herein.

The Hsp70B' protein or any of the Hsp70B' peptides can be attached to a carrier that enhances their immunogenicity. The carrier is any substance that, when attached to the protein or peptide, results in the production ofmore antibodies than when it is omitted from the protein Por peptide. The carrier can be attached to the protein or peptide cpvalently or noncovalently so 20 long as the two entities remain attached to one another when administered to an animal. More specifically, the carrier can be an amino acid-based substance such as keyhole limpet hemocyanin (KLH). Regardless of the means of attachment, one or more groups chemically reactive groups or one or more amino acid residues) can be used to facilitate bonding between the protein or peptide and the carrier. For example, a cysteine residue can be added to either end of Hsp70B' or to any of the Hsp70B' peptides described herein to facilitate coupling with a carrier. If desired, more than one carrier can be used, and a spacer one or more amino acid residues) can be added between the protein or peptide and the carrier.

Antibodies may generally be prepared by any of a variety.of techniques known to those of ordinary skill in the an (see. Harlow and Lane, Antibodies: A Laboratory Manual. Cold Spring Harbor Laboratory, 1988). For example, an immunogen that includes a Hsp70B' peptide is initially injected into suitable animals mice, rats. rabbits, sheep and goats) according to a COMS ID No: SBMI-02132445 Received by IP Australia: Time 17:45 Date 2005-12-06 06-12-'05 17:25 FROM- T-751 P014/78 F-502 WO 01/42423 PCT/USOD33341 Spredetermined schedule with one or more booster immunizations, and blood samples are O obtained from the animals periodically. Polyclonal antibodies specific for the Hsp708 peptide can then be purified from the antisera by, for example, affinity chromatography in which the same peptide sequence administered is coupled to a suitable solid support.

S To obtain Hsp7OB'-specific antibodies, an animal can be immunized with Hsp70B' or O one of the Hsp70B' peptides disclosed herein. The term "immuni2ed" refers to at least a first administration of antigen, and optionally includes subsequent administration a second or Sthird administration) and additional periodic boosting. Immunization typically includes Sadministration or the antigen an Hsp70B' protein or a fragment or epitope thereof that evokes an immune response) and an adjuvant. The Hsp70B' protein or peptide administered can be purified from a natural source. chemically synthesized, or rccomibinantly produced.

l. Regardless of the length of the amino acid sequence used to immunize an animal. sera (or ancigen-specific B cells or other antibody containing fluids) are collected, and the antibody response is evaluated, typically by imminoassay. High-titre preparations are generally pooled and the protein-specific or peptide-specific antibody is purified on an immunoaflinity column to which the protein or peptide is immobilized. Where full length Hsp70B' protein (or any portion of the protein that is longer than the peptides disclosed herein) is used to generate antibodies by immunization or otherwisc,;an Hsp70B' peptide (such as those disclosed herein) is typically used to purify the antibodies. The discovery ofamino acid sequences within Hsp70B' that can yield antibodies that distinguish Hsp70B' from other Hsp70 family members permits antibodies to be obtained in many ways known to those of ordinary skill in the art.

In addition to polyclonal antibodies, the antibodies generated can be monoclonal antibodies, fragments of polyclonal or monoclonal antibodies such as F(ab') 2 and Fab fragments, as well as any naturally occurring or recombinantly produced binding partners (i.e.

molecules that specifically bind Hsp70B'). In addition to the Hsp70B' protein or peptide (which functions as an antigen), the composition administered can include a carrier vehicle and immunostimulatory substances that enhance immunogenicity adjuvants). The carrier vehicle can include aluminum salts, water-in-oil emulsions, biodegradable oil vehicles, oil-inwater emulsions, biodegradable microcapsules, and liposomes. The immunoslimulatory substances can include include N-acetytmuramyl-L-alanine-)Disoglutamine (MDP). lipopolysaccharides (LPS), glucan, IL-12, GM-CSF, gamma interferon. and t COMS ID No: SBMI-02132445 Received by IP Australia: Time 17:45 Date 2005-12-06 06-12-'5 17:25 FROM- T-751 P015/078 F-502 WO 01142423 PCTfUSO/33341 O Monoclonal antibodies specific for Hsp70B' peptides can be prepared, for example, using the technique of Kohler and Milstein (Eur Immunol 6:511-519, 1976), and improvcments thereto. Briefly, these methods involve the preparation of immortal cell lines that produce antibodies having the desired specificity reactivity wilh the Hsp70B' peptide of interest).

Such cell lines may be produced, for example, from spleen cells obtained from an immunized O animal, The spleen cells are then immortalized by. for example, fusion with a myeloma cell fusion partner, preferably one that is syngeneic with the immunized animal. For example, the o spleen cells and myeloma cells may be combined with an agent that promotes membrane fusion polyethylene glycol or a nonionic detergent), and then plated at low density on a selective 10 medium that supports the growth of hybrid cells, but not myeloma cells. The selection technique can be HAT (hypoxanthine, aminopterin, thymidine) selection. After a sufficient time (typically t I to 2 weeks), colonies of hybrids are observed. Single colonies are selected and tested for binding activity against the polypeptide. Hybridomas having high reactivity and specificity are C( preferred. Monoclonal antibodies may be isolated from the supernatants of growing hybridoma colonies. Accordingly, such hybridomas and the monoclonal antibodies they produce e..

monoclonal antibodies that specifically bind to Hsp70B' or Hsp70B' peptides) are specifically encompassed by the present invention.

Techniques that enhance the yield of antibodies are known in the art and can be used in the context of the present invention. For example, the hybridoma cell line can be injected into the peritoneal cavity of a suitable vertebrate host, such as a mouse, and monoclonal antibodies may then be harvested from the ascites fluid or the blnod of that host. Contaminants can be removed by conventional techniques, such as chromatography, gel filtration, precipitation, and extraction. For example, anti-Hsp70B' antibodies can be purified by chromatography on immobilized Protein G or Protein A using standard techniques.

Instead of administering Hsp70B' or Hsp70B' peptides, animals can be indirectly immunized by administering nucleic acid molecules encoding Hsp70B or a Hsp70B' peptide.

Accordingly, nucleic acid molecules that encode the Hsp70B' peptides disclosed herein, alone or in the context of an expression, and cells that contain those molecules are within the scope of the invention. These nucleic acid molecules can be delivered with recombinant viral vectors retroviruses (see WO 90/07936, WO 91/02805, WO 93/25234. WO 93/25698, and WO 94/03622), adenovirus (see Berkner, Biorechniques 6;616-627, 1988; Li et Hum Gene 8 COMS ID No: SBMI-02132445 Received by IP Australia: Time 17:45 Date 2005-12-06 06-12-'05 17:25 FROM- T-751 P016/78 F-502 WO01/423 PCTAUS0oo0n3 O Thr. 4:403-409, 1993; Vincent e al., Nat. lene. 1:130-134, 1993: and Kolls et al.. Proc. Nail SAcad Sci, USA 91:215-219, 1994), pox virus (see U.S. Patent Nos. 4 7 69,330 and 5.017,487; and WO 89/01973)), naked DNA (see WO 90/11092), nucleic acid molecule complexed to a polycationic molecule (see WO 93/03709), and nucleic acid associated with liposomcs (see s Wang t al., Proc. Nad. Acad. Sei. USA 84:7851, 1987). The DNA can be linked to killed or Sinactivated adenovirus (see Curiel at a, Hum. Gene Ther. 1:147-154, 1992; Cotton er al., Proc.

Nadl Aad. Sci. USA 89:6094, 1992). Other suitable compositions include DNA-ligand (see Wu er al., J Biol Chem. 264:16985-16987, 1989) and lipid-DNA combinations (see Felgner et at., Proc. Nadl. Acad. Sci USA B4:7413.7417. 1989). In addition, the efficiency with which naked so DNA is taken up by cells can be increased by coating the DNA onto biodegradable beads.

In some cases, antigen-binding fragments of antibodies are preferred. These fragments ci include Fab fragments, which may be prepared using standard techniques by digestion with papain to yield Fab and Fe fragments). The Fab and Fc fragments can be separated by affinity Ci chromatography on immobilized protein A columns), using standard techniques, See. e.g., Weir, Handbook ofExperimental lmmunology, 1986. Blackwell Scientific, Boston.

Multifunctional fusion proteins having specific binding affinities for pre-selected antigens by virtue of immunoglobuiin V-region domains encoded by DNA sequences linked inframe to sequences encoding various effector proteins are known in the art, for example, as disclosed in EP-B 1-0318554 and U.S. Patent Nos. 5,132.405 and 5,091,513, and 5,476.786.

to Such cffector proteins include polypeptide domains that can be used to detect binding of the fusion protein by any of a variety of routinely practiced techniques, including but not limited to a biotiin mimetic sequence (see. Luo et JBioechnol. 6f:225, 1998 and references cited therein), direct covalent modification with a detectable labeling moiety, non-covalent binding to a specific labeled reporter molecule, enzymatic modification of a detectable substrate or immobilization (covalent or non-covalent) on a solid-phase support.

Single chain antibodies that can be used in the methods described herein can also be generated and selected by a method such as phage display (see. U.S. Patent No. 5,223,409, Schlebusch et al., Hybridoma 16:47,1997, and references cited therein). Briefly, in this method.

DNA sequences are inserted into the gene III or gene VIII gene of a filamentous phage, such as so M13. Several vectors with multicloning sites have been developed for insertion (McLafferty et Gene 128;29-36, 1993; Scott and Smith, Sciene 249:386-390, 1990; Smith and Scott.

9 COMS ID No: SBMI-02132445 Received by IP Australia: Time 17:45 Date 2005-12-06 06-12-'05 17:25 FROM- T-751 F017/078 F-502

I

WO01/4242 PCT/US00/33341 SMethods Enrymol 217:228-257, 1993). The inserted DNA sequences can be randomly 0 generated or can be variants of a known binding domain for binding to Hsp70B' peptides.

o Single chain antibodies can be readily generated using this method. Generally, the inserts encode from 5 to 20 amino acid residues. The peptide encoded by the inserted sequence is S displayed on the surface of the bacteriophage. Bacteriophage expressing a binding domain for a O Hsp70B' peptide are selected by binding rb an immobilized Hsp70B' peptide, for example a recombinant polypeptide prepared using methods well known in the art and nucleic acid coding Ssequences as disclosed by Chang e al. (Proc. Nt. Acad Sci. USA 93:136, 1996) or by Kojima ePral. Biol. Chem. 270:21984. 1995). Unbound phage is removed by a wash. typically C 1to comaning 10 mM Tris, I mM EDTA. and without salt or with a low salt concentration. Bound phage is eluted with a salt containing buffer, for example. The NaCI concentration is increased m in a step-wise fashion until all the phage is eluted. Typically, phage binding with higher affinity will be ree c ased by higher salt concentrations. Eluted phage is propagated in the bacteria host.

N Punther rounds of slection may be performed to select for a few phage binding with high iS affinity. The DNA sequence of the insert in the binding phage is then determined, Once the predicted amino acid sequence of the binding peptide is known, sufficient peptide for use as an antibody specific for a human Hsp70B' protein or peptide can be made either by recombinant means or synthetically. Recombinant means are used when the antibody is produced as a fusion protein. The peptide can also be generated as a tandem array of two or more similar or dissimilar peptidcs, in order to maximize affinity or binding.

Antibodies that specifically bind an I1sp70B protein a murine, porcine, bovine, equine, or human Hsp70B' protein) can be used in vitro or in vivo to evaluate. diagnose, or form a prognosis regarding a specific cell or a disease slate. These antibodies are molecular markers of exposure to a stressful environment an cnvironment where the temperature is increased beyond physiological norms, there is a shortage of oxygen, or an infectious organism) or substance a toxin, a proinflammatory cytokinc. a heavy metal, an amino acid analogue, or a metabolic poison), Notably, the antibodies can serve as markers of an adverse sub-lethal effect of stress. To detect an antigenic determinant reactive with an antibody specific for a human peptide, the detection reagent is typically an antibody. which may be prepared as o3 described herein. The variety of assay formats known to those of ordinary skill in the art include, but are not limited to, enzyme immunoassay (ESA), enzyme linked immunosorbent COMS ID No: SBMI-02132445 Received by IP Australia: Time 17:45 Date 2005-12-06 06-12-'05 17:26 FROM- T-751 F018/078 F-502 WO 01/42423 PCT/USo33341 f assay (ELISA), radioimmunoassay (RIA), immunofluorimetry, immunoprecipitation, Sequilibrium dialysis, immunodiffusion and other techniques. See, Harlow and Lane, o Antibodies: A Laboratory Manual. Cold Spring Harbor Laboratory, 1998: Weir, D,M..

i) AHandbook of Experimental Immunology, 1986, Blackwell Scientific. Boston. For example, the Sassay may be performed in a Western blot formal in which a protein preparation from the Sbiological sample is subjected to gel electrophoresis, transferred to a suitable membrane, and allowed to react with the antibody. The presence of the antibody can then be detected using a suitable detection reagent, as is well known in the art and described below.

The assay can also involve antibodies immobilized on a solid support a test well in a microtiter plate, a nitrocellulose filter or other suitable membrane, a bead or disc, such as glass, fiberglass, latex or a plastic such as polystyrene or polyvinylchloride). The immobilized i antibody can bind to the target Hsp70B' peptide or protein and thereby separate it from substantially all of the rest of the sample. The bound polypeptide can then be detected with a C] second antibody reactive with a distinct polypeptide antigenic determinant, for example. a reagent that contains a detectable reporter moiety. For example, the immobilized antibody and the secondary antibody which each recognize distinct antigenic determinants may be two monocional antibodies. Alternatively, a competitive assay may be utilized, in which a polypeptide is labeled with a detectable reporter moiety and allowed to bind to the irmobilized polypeptide specific antibody after incubation of the immobilized antibody with the sample. The o0 extent to which components of lhe sample inhibit the binding of the labeled polypeptide to the antibody is indicative of the reactivity of the sample with the immobili2ed antibody, and as a result, indicative of the level ofHsp70B' polypeptide in the sample.

In some cases, the assay used to detect Hsp70B' polypeptides in a sample is a twoantibody sandwich assay. This assay can be performed by first contacting a Hsp70B' antibody that has been immobilized on a solid support (commonly the well of a microtiter plate) with the biological sample. Soluble molecules that naturally occur in the sample and have an antigenic determinant that is reactive with the antibody will bind to the immobilized antibody and thereby form an antigen-antibody complex or an immune complex. A 30-minute incubation at room temperature is generally sufficient for complex formation. Unbound constituents of the sample 3D are then removed from the immobilized immune complexes and a second antibody specific for a polypeptidr is added. The antigen-combining site of the second antibody does not

II

COMS ID No: SBMI-02132445 Received by IP Australia: Time 17:45 Date 2005-12-06 06-12-'05 17:26 FROM- T-751 P019/078 F-502 WO 0t/42423 PCT/USOO93334 competitively inhibit binding of the antigen-combining site of the immobilized first antibody.

The second antibody may be detectably labeled as provided herein, such that it may be directly detected. Alternatively, the second antibody may be indirectly detected with a labeled secondary (or -second stage") anti-antibody, or by using a specific detection reagent as provided herein.

Notably. the methods of the invention need not be limited to any particular detection procedure.

O Those familiar with immunoassays understand that there are numerous reagents and configurations for immunologically dctecting a particular antigen in a two-antibody sandwich Simmunoassay.

i When a two-antibody sandwich assay is used, the first, immobilized antibody specific for o a Hsp70B' polypeptide and the second antibody specific for a Hsp7OB' polypcptidc can both be polyclonal antibodies. Alternatively, the first, immobilized antibody specific for polypeptide can be a monoclonal antibody and the second antibody specific for a Spolypeptide can be a polyclonal antibody and vice-versa. It can be preferable, however, to carr N out the assay with the first, immobilized antibody and the second anti-Hsp7OB' antibody being monoclonal antibodies. In yet other configurations, the first, immobilized antibody and/or the second antibody may be any ofthe kinds of antibodies known in the art (for example, Fab fragments. fragments, immunoglobulin V-region fusion proteins or single chain antibodies). Those ofordinary skill in the art will appreciate that the present invention can be practiced with other antibody forms. fragments, derivatives, and the like.

The second antibody may contain a detectable reporter moiety or label such as an enzyme, dye, radionuclide, lumincscent group, fluorescent group or biotin, or the like. The amount of the second antibody that remains bound to the solid support i then determined using a method appropriate for the specific detectable reporter moiety or label. For radioactive groups, scintillation counting or autoradiographic methods are generally appropriate. Antibody-enzyme conjugates may be prepared using a variety of coupling techniques (for review see, Scouten.

Methods in Enrymology 135:30-65. 19B7). Spectroscopic methods may be used to detect dyes (including, for example, colorimetric products of enzyme reactions), luminrscent groups and fluorescent groups. Biotin may be detected using avidin or sireptavidin, coupled to a different reporter group (commonly a radioactive or fluorescent group or an enzyme). Enzyme reporter groups may generally be detected by the addition of substrate (generally for a specific period of time). followed by spectroscopic. Spectrophotometric or other analysis of the reaction 12 COMS ID No: SBMI-02132445 Received by IP Australia: Time 17:45 Date 2005-12-06 06-12-'05 17:27 FROM- T-751 P020/078 F-502 WO 1/42423 FCTUrlso033341 0 products. Stadards and standard additions may be used to determine the level of polypeptidc in a sample, using well known techniques.

o As noted above, the ability to generate anti-Hsp70B Bantibodies to defined epiropes prmilt a variety of in vitro and in vive uses. For example, anti-Hsp70B antibodies may be used to monitor the protein levels ofa specific. sensitive, native, biornarker (Hsp70B') in in vitro 0 bioassays using human cell lines to evaluate the toxicity of chemical compounds. Using antibodies to defined epitopes on the Hsp70B' and Hsp70B proteins allows the specific monitoring of these proteins. Other uses include the evaluation, diagnosis, prognosis and coninued monitoring ot specific disease conditions such as: hypertension, oncology, organ iransplantation, ischaemia and trauma, infection inflammation and fever, heart disease.

autoimmune disorders, neurodegenerative diseases, monitoring spinal cord injuries, neurof) psychology evaluations, and chronic disease states.

O An advantage of the present approach is that one can monitor perturbations in CN homeostasis by monitoring the levels ofHsp70B' in vivo. The Hsp70B' protein can also be used s1 as a biomarker in the fields oforgan transplantation and cytoprocction. This would allow one to identify physiological perturbations even when an individual has not received a definitive diagnosis with respect to a specific condition or to monitor patients exhibiting symptoms of known or unknown cause chronic fatigue syndrome). Such monitoring would help healthcare professionals gauge the severity of a condition, .follow the progress of the patient, and decide when intervention may he needed. Evaluating a stress response (made manifest by expression) may also be useful in the care of patients who are in remission from an autoimnune. chronic condition or neoplasia: to evaluate amniotic fluids or samples of the placenta; and to assess newborn infants who are at risk (due, for example, to premature birth) Monitoring Hsp70B' is also useful in evaluating the fitness of healthy individuals it can be used to assess training programs for high performance athletes). In yet other applications, the compositions and methods of the invention can be used to evaluate the ability of various therapeutic compounds to oppose the stress condition, and in conjunction with in vivo diagnostic imaging to evaluate wounding, inflammation or pathology in patients).

The following examples illustrate, not limit, the invention.

13 COMS ID No: SBMI-02132445 Received by IP Australia: Time 17:45 Date 2005-12-06 06-12-'05 17:27 FROM- 06-1-' 0 1727 FOM-T-751 P021/078 F-502 WO Ot/42423 PCTUSOO333431 o

EXAMPLES

0 following mnaeials were obtained fo h onrjlsplesidrac:aisl (Cat. No. A4405, Sigma Chemical Co., herein "Sigma", St, Louis, MO); 2 21 -azino-di-(3.ethy.

C.)benzithiazoljnc-sujfanic acid) (ARTS) (Car. No. A6499. Molecular Probes Eugene, O)R); s activated Malcimide Keyhole Limpet Cyanin (Cat. No. 77106. Pierce Chemical Co. Rockf'ord, NO IL); Biotin (Cat. No. B32643, Sigma); boric acid (Cat. No. B0252, Sigma); Sephiarosea 413 (Cat.

No. 17-0120-01, LKB/Pharrnacia, Uppsala. Sweden); bovine serum albumn (Cat. No.

100 350, Boebringer Mannuheim, Indianapolis, IN); cyanogen broi de (Cat. No. C6388, Sigma, St. Louis, MO); dialysis tubing SpectraiPor"-t membrane MWCO: 6-8.000 (Cat. No. 132 665, Spectrum Industries Inc., Lakguna Hills, CA); dimethyl fornarnide (DMF) (Cai. No. 22705-6.

Aldrich Chemical Company, Milwaukee, WI); DIG (Cat. No. BP 592-500, Fisher); ethanieichiol (Cat. No. 39,802-0, Aldrich Chemicals, Milwaukece, WI); ether (Cat. No. TX 1275-3, Sciences); ethylenediantincertpcemtic acid (EDTA)(Ca: No. U3P 120-1, Fisher Scientific, Springfield, NJ); l-ethyl- 3 3 'dimethylaminopropyI)..carbodiimide 'HCL, (EDC) (Cat No. 341 006, Calbioehcm. Sam Diego, CA); freund's adjuvant, complete (Cat. No. M-0638-SOB, Lee Laboratoties Grayson, GA); firund's adjuvant. incomplete (Cat. No. M0639-SOR, Let Laboratories), frinted chromatlograhy clumn~s (Column part No. 12131011; Flit: Part No. 12131029, 'Varian Sample Preparatlion Products, Harbor City. CA); gclaxin from bovine skin (CaL No, 09382. Sigma); glycine (Cat. No. 13P38J-5, Fisher); goat anti-rabbit 8GO. biotinylated (Cat No. A 04 18, Sigma); HOBt (Cat. No. 01 .62-0008, Calbiochem-Novabiocheni); horseradish peFoxidrIS (Cat. No. 814 393, Boebringer Mannhieim); H-RP-Streptavidin (Cat. No.

5512. Sigmia); hydrochloric acid (Cat No. 71445-500, Fisher); hydrogen peroxide 10% %w/61 (Cat. No. H 1009, Sigma); methanol (Cat. No. A412-20, Fisher);- mic-rocitre plates. 96 well (Cat.

No. 2595. Coraing-Cosr Pleasanon, CA); N-o-Frpoc protected amnino acids (Calbiochem..

Novabioc'em, San Diego, CA; see 1997-1998 catalog pages 1-45); N-a-Fmoc protectred amino acids attached to Wang Rosin (Uobiocbemn-Novabiochem., see 1997-1998 catalog pages 161.

164); NMAP (Cat. No. GAS 872-50-4, Burdick and Jackson, Muskegon, MI); peptilc (Synthiesizedl by Research Genetics, Inc.,see below); piporidine (Cat. No. 80640. Fluka, available through Sigma); sodium bicarbonate (Cat. No. BP328-l, Fisher); sodium borate (Cat.

No. 09876. Sigma); sodium carbonate (Cat. No. SF3 57. 1, Fisher); sodiumn chloride (Call. No. BP 358-10. Fisher);, sodium hydroxide (Cat. No,.SS255- 1, Fishcr); streptavidin (Cat. No. 1 520.

14 COMS ID No: SBMI-02132445 Received by IP Australia: Time (H:rn) 17:45 Date 2005-12-06 06-12-'05 17:27 FROM- T-751 P022/0a78 F-502 WO 01142423 PCTUSOD/3341 Boehringer Mannheim); thioanisole (Cat. No. T-2765, Sigma); trifluoroacetic acid (Cat. No. TX 1275-3, EM Sciences); Tween-20 (Cat. No. BP 337-500, Fisher); and Webox-(Rubbcrmnnaid o Rectangular Servin' Saver"' Part No. 3862 Wooster, OH).

The following general solutions were prepared: BBS Borate Buffered Saline with s EDTA dissolved in distilled water (pH 8.2 to 8.4 with HCI or NaOH)*. which comains 25 mM S Sodium borate (Borax) 100 mM Boric Acid. 75 mM NaCI. and 5 mM EDTA; 0.1 N HCI in saline, which contains concentrated HCI (8.3 mlU0.917 L distilled water) and 0.154 M NaCI; Glycine (pH 2.0 and pH 3.0) dissolved in distilled water and adjusted to the desired pH, which comtains 0.1 M glycine and 0.154 M NaCI:(4) SX Borate IX Sodium Chloride dissolved ic in distilled water, which contains 0.11 M NaCl, 60 mM sodium borate, and 250 mM boric acid; and substrate buffer in distilled water adjusted to pH 4.0 with sodium hydroxide. which f contains 50 to 100 mM Citric Acid.

The following peptide synthesis solutions were prepared: AA solution, in which C-i ROBt is dissolved in NMP (8.8 gramns HOBt to 1 liter NMP) and Frnoc-N-a-amino is added at a concentration at,.53 M; DIC solution, which is 1 part DIC to 3 parts NMP; a deprotecting solution, which is I panrt piperidine and 3 parts DMF; and Reagent R. which is 2 parts anisole, 3 parts ethanedithiol, 5 parts thioanisol, and 90 parts irifluoroacetic acid.

The following equipment was employed; an MRX plate reader (Dynaech Inc..

Chantilly, VA): a Hamilton Eclipse (Hamilton lnstrumnents. Reno. NV): a Beckman T3-6 centrifuge, refrigerated (Model No. TJ-6 Beckman Instruments. Fullerton, CA); a Chart Recorder (Recorder 1 Pan No. 18-100140, Pharmacia LKB Biotechnology); a UV monitor (Uvicord SII Part No. 18-1004-50, Pharmacia LKB Biotechnology); an Amicon Stirrcd Cell Concentrator (Model 8400, Amicon Inc.. Beverly, MA); 30 kDa MW cut-off filters (Cat. No.

Membranes Cal. No. 13742, Amicon Inc., Beverly. MA); a multi-channel automated pipettor (Cat. No. 4880, Coming Costar Inc.. Cambridge, MA): a pH meter (Coing 240; Comrning Science Products, Corning Glassworks. Corning, NY); an ACT396 peptide synthesizer (Advanced Chem'ech, Louisville, KY); a vacuum dryer (Box is from Labconco, Kansas City, MO: Pump is from Aleatel, Laurel MD); a lyophilizer (Unitop 600sl in tandemrn with Freezemobile 12, both from Virtis, Gardiner,

NY).

0so Metheds: Hsp7OB' Antibodies were produced as follows. Hsp70B' antibodies were produced in rabbits, goaLs and mice with either synthetic peptides or recombinant Is COMS ID No: SBMI-02132445 Received by IP Australia: Time 17:45 Date 2005-12-06 06-12-'05 17:28 FROM- T-751 P023/078 F-502 S-16-

O

protein as immunogen. Eight peptides were chosen from the human Hsp 7 0B' amino acid o sequence. One of the Hsp 70 B' peptides, the NT peptide MQAPRELAVGID(C) (SEQ ID NO:9) corresponded to an N-terminal fragment. The other seven fragments were derived from the C- \O terminal half of the Hsp70B' prolein and included the CC peptide (AHIVFHVKGSLQEES; SEQ ID o 5 NO:7), the CA peptide (DKIPEEDRRKMQ; SEQ ID NO:5), the CD peplide (RDKIPEEDRRKMQDKC; SEQ ID NO:4); the CB peptide (CGTQARQGDPSTGPI; SEQ ID o NO:2), the ECB peptide (VPGGSSCGTQARQGDPSTGPI; SEQ [D NO; the TCB peptide (CGTQARQGDPST; SEQ ID NO:3), and the CE pcptide (GSLQEESLRDKIPEE; SEQ ID c, NO:10). The CB peptide was also resynthesized on a separate occasion and designated CB2.

All peptides were chemically coupled to KLH and animals were immunized with the peptide conjugates. Recombinant human Hsp70B' protein was purified to -90% homogeneity and was O also used as an immunogen Primary immunizations were administered in Freund's complete (N adjuvant and subsequent boosts were made in Freund's incomplete adjuvant. Animals were immunized and boosted on a monthly basis. Sera were collected at various time points and the id antibody response to the immunizing protein or peptide was evaluated in an indirect enzyme immnunoassay (EIA). Titrcs were established as the dilution factor at which the absorbance in the test sample was equal to 0.2 optical density units. In some instances, high-titre antisera from S: each set of animals were pooled and the antigen-specific antibody purified on peptide inmunoaffinity columns.

:26 Peptide was synthesized as follows. The event procedures included an incubation step S. (which allowed resin to be immersed in an appropriate solution; all incubation steps occurred with mixing, a wash (addition of 2 mis of DMF. incubation for 5 minutes and removal of the wash solution), and a wash cycle (consisting of 5 washes). For machine synthesis, sequences of peptide were added to the peptide synthesizer. The C-terminal residue was determined and the O, appropriate Wang Resin was attached to the reaction vessel. The peptides were synthesized from the C-terminus to the N-terminus by adding one amino acid at a time during the synthesis cycles.

The amino acid residue selected for addition to the peptide is controlled by sequence of the peptide that was entered into the database of the synthesizer.

The synthesis per se included a first step in which resin swelled (2 ml of DMF was added for 30 minutes and then removed), a second step in which the peptide was deprotected (I ml deprotecting solution is added to the reaction vessel and incubated for 20 minutes). washed. and COMS ID No: SBMI-02132445 Received by IP Australia: Time 17:45 Date 2005-12-06 06-12-'05 17:28 FROM- T-751 P024/078 F-502 WO 01/42423 PCT/Uai50/33341 Scoupled (750 mi of amino acid solution and 250 ml of DIC solution are added to the reaction Svessel for 30 minutes and then washed out. The coupling step is repeated once before another O wash cycle. The second step is repeated over the length of the peptide, with the amino acid solution changing as the sequence listed in the peptide synthesizer's database dictates. In a third step, final deprotection occurs (the deprotection and washing that occurs during the synthesis cycle are performed one last (ime). Resins are deswelled in methanol by two rinses with S ml methanol, a 5 minute incubation in 5 ml methanol, and a rinse in 5 mL methanol, and then vacuum dried.

SPeptide was removed from the resin by incubation for 2 hours in reagent R and then precipitated into ether. The peptide was then washed in ether, vacuum dried. resolubilized in diH 2 0, frozen, and lyophilized overnight. At this point, hce peptide can be conjugated to KLH as IC follows. The peptide (6 rng) is dissolved in PBS (6 ml) and mixed with 6 mg ofmaleiimide O activated KLH carrier in 6 ml orPBS for a total volume of 12 mL. The emire solution was 0 mixed for two hows, dialyzed in 1 liter of PBS, and lyophilized.

Animals were immunized with pepdde conjugates as follows. Three New Zealand White rabbits were injected in three to four subcutaneous dorsal sites with 250 pg of pepiide-KLH conjugate in Freund's complete adjuvant. Booster shots (100 jg) were administered in Freund's incomplete adjuvant. The total volume of each injection was 1 ml.

The rabbit immunization schedule was as follows: at Day 0 a "pre-immune bleed" was performed and the primary immunization was given; at week 2 a first booster was given; at week 4 a blood sample was obtained; at week 6 a second booster was given; at week 8 a second blood sample was obtained and a third booster was given; at week 10 a third blood sample was obtained; at week 12 a fourth booster was given; and at week 14 a fourth and final blood sample was obtained.

Goats were injected with the same dose of peptide conjugate in Freund's adjuvant as the rabbits received. The immunization schedule was also the same except the booster at week 12 was not given and no blood sample was taken at week 14.

BALB/c mice were immunized intraperitoneally with 50g peptide conjugate in Freund's complete adjuvant on day 0, and in Freund's incomplete adjuvant at weeks 2. 5 and 8. Mouse test bleeds were collected on week 7 and 17 COMS ID No: SBMI-02132445 Received by IP Australia: Time 17:45 Date 2005-12-06 06-12-'05 17:28 FROM- T-751 P025/078 F-502 WO 01/4Z425 PCT/USOW33341 o Rabbits were also immunized with recombinant human Hsp708' protein. More Sspecifically, three New Zealand White rabbits were immunized on day 0 with purified recombinant human Hsp70B' protein in Freund's complete adjuvant Booster shots (100 pg) were given at weeks 3, 4, 6, and 8, and blood samples were collected at weeks 5. 7, 9 and O 5 The blood samples were processed for serum, and the antibody obtained was designated

WP.

To collect rabbit serum, the rabbits were bled (30 to 50 ml) from dhe auricular artery.

o The blood was allowed to clot at room temperature for 15 minutes and the serum was separated en from the clot using an IEC DPR-6000 centrifuge at 5000 x g. Cell-free scrum was decanted gently into a clean test tube and stored at -20*C for affinity puification.

Anti-pcptide titre was determined as follows. All solutions were added by a liquid handling dispenser(the Hamilton Eclipse), with the exception of the wash solution. The anti- Spcptide titres in the rabbits, goats, and mice were determined in an ELISA with peptide on the solid phase. Flexible high binding ELISA plates were passively coated with peptide diluted in BBS (100 pL, 1 pg/well) and the plate was incubated at 4 0 C in a wetbox overnight (air-tight container with moistened cotton balls). The plates were emptied and then washed three times with BBS containing 0.1% Tween-20 (BBS-TW) by repeated filling and emptying using a semiautomMaed plate washer. The plates were blocked by completely filling each well with BBS-TW containing 1% BSA and 0.1% gelatin (BBS-TW-BG) and incubating for 2 hours at room temperature. The plates were emptied and sera of both pre- and post-immune serum were added to wells. The first well contained scra at 1:50 in BBS. The sera were then serially titrated eleven more times across the plate at a ratio of 1:1 for a final (twelfth) dilution of 1:204,800. The plates were incubated overnight at 4"C. The plates were emptied and washed three times as described.

Biotinylated secondary antibodies (100 pl) were added to each microtirre plate test well and incubated for four hours at room temperature. The plates were emptied and washed three times. Horseradish peroxidase-conjugated Sireptavidin (100 pL diluted 1:10,000 in BBS-TW- BG) was added to each well and incubated for two hours at room temperature. The llates were emptied and washed three times. The ABTS was prepared fresh from stock by combining 10 mL of citrate buffer (0.1 M at pH 0.2 mL of the stock solution (15 mg/mL in water) and 10 pL of 30% H 3 0 2 The ABTS solution (100pL) was added to each well and incubated at room temperature. The plates were read at 414 X, 20 minutes following the addition of substrate.

I8 COMS ID No: SBMI-02132445 Received by IP Australia: Time 17:45 Date 2005-12-06 06-12-'05 17:29 FROM- T-751 P026/078 F-502 WO 0 o4423 PCTI/US/33341 S"Titres were established as the reciprocal dilution factor at which the test sample was equal to 0 0.2 absorbance units.

The Anti-Hsp70B' protein titre was determined by indirect ELSA with Hsp 7 0B' on the solid phase. Nunc Maxisorp ELISA plates were passively coated with purified recombinant N *5 human Hsp70B' diluted in PBS (100 pL. 100 ng/well) at 49C overnight. Plares were washed six O times with PBS containing 005% tween-20 (Bio-Rad) and then blocked at room temperature with 200 pl/well blotto non-fat milk (Caration). 0.05% tween-20, 0.02% thimerosal (Fisher Scientific) in PBS). Plates were washed and the antiserum, diluted 1:1000 in blotto, was added n to the wells. The diluted antiserum was serially titrated 5 times at a ratio of 1:3 in blotto for a final dilution of 1:243000. Plates were incubated at room temperature for I hour, followed by washing as described. Peroxidase conjugated anti-rabbit IgG (100 pi/well ofSAB-300.

I Stressen Biotechnologies), diluted 1:25000 in blotto, was added to the wells and the plates Swere incubated for I hour at room temperature. Plates were washed as described and then C developed with teranethylbenzadine (TMB; BioFx) for 5 10 minutes at room temperature.

Color development was stopped by the addition ofacid stop solution (BioFx). Absorbance of each well was measured at 450 run in an EL808 microplate reader (BioTek), interfaced with KC3 software. Tires were established as the reciprocal dilution factor at which the test sample was equal to 0.2 absorbance units.

The peptide affinity purification column was prepared by conjugating 5 mg of peptide to 10 ml of cyanogen bromide-activated Sepharose 4D. and 5 mg of peptide to hydrazine-Scpharose 4B. Briefly, 100 uL of DMF was added to peptide (5 mg) and the mixture was vortexed until the contents were completely wetted. Water was then added (900 pL) and the contems were vorrexed until the peptide dissolved. Half of the dissolved peptide (500 L) was added to separate tubes containing 10 mL of cyanoger-bromide activated Sepharose 4B in 0.1 mL of borate buffered saline at pH 5.4 (BBS), and 10 mL of hydrazine-Sepharose 4B in 0,1 M carbonate buffer adjusted to p-l 4.5 using excess EDC in citrate buffer pH 6.0. The conjugation reactions were allowed to proceed overnight at room temperature. The conjugated Sepharose was pooled and loaded onto fritted columns, washed with 10 mL of BBS, blocked with 10 mL of 1 M glycinc, and washed with 10 mL 0.1 M glycine adjusted to pH 2.5 with HCI and rew3 neutralized in BBS. The column was washed with enough volume for the optical density at 280A to reach baseline.

19 COMS ID No: SBMI-02132445 Received by IP Australia: Time 17:45 Date 2005-12-06 06-12-'05 17:29 FROM- T-751 P027/78 F-502 I I WO 1/42423 PCT/U033341 O To affinity purify antibodies, the peptide affinity column was attached to a UV monitor Sand chart recorder. The titred rabbit antiserum was thawed and pooled. The serum was diluted with one volume of BBS and allowed to flow through the columns at 10 mL per minute. The non-peptide immunoglobulins and other proteins were washed from the column with excess BBS until the optical density at 280 reached baseline. The columns were disconnected and the o affinity purified column was eluted using a stepwise pH gradient from pH 7.0 to pH 1.0. The elution was monitored at 280 nM, and fractions containing antibody (pH 3.0 to pH 1.0) were Scollected directly into excess 0.5 M BBS. Excess buffer (0.5 M BBS) in the collection tubes served to neutralize the antibodies collected in the acidic fractions of the pH gradient.

C 0o The entire procedure was repeated with "depleted: serum to ensure maximal recovery of antibodies. The eluted material was concentrated using a stirred cell apparatus and a membrane \I with a molecular weight cutoffof 30 kD. The concentration of the final preparation was Sdetermined using an optical density reading at 280 nM. The concentralion was determined using C the following formula: mg/mL OD 2 901l.4, Antibody titres were determined by indirect EIA, essentially as already described for the anti-peptide and anli-Hsp70B' antibodies. Briefly. Nunc Maxisorp ELISA plates were passively coated with 100 ng/well of recombinant human Hsp70B'. recombinant human (SPP-755. StressGen Biotechnologies), recombinant bovine Hsc70 (SPP-75 I. StressGen Biolechnologies), recombinant hamster Grp78 (SPP-765. StressOen Biotechnologies). E. coli DnaK (SPP-630, StressGen Biotechnologies) and recombinant M. tuberculosis Hsp71 (StressGen Biotechnologies) diluted in PBS. Similarly, unconjugated CB2, ECD, TCB. and CE peptides were diluted in PBS and coated at 0.5 pg/well. Plates were blocked with 200 ll/well of casein blocking buffer in PBS (Pierce) for 2 hours at room temperature. Test and negative control antibodies were diluted to a starting concentration/dilution of I pg/mi for affinity purified antibodies and 1:1000 for serum antibodies in Stabilzyme Select (SurModics). Diluted antibodies were added to the plate wells and were further diluted by 5 serial titrations at a ratio of 1:3 to a final concentration/dilution of4.1 ng/ml (for purified antibodies) and 1:243000 (for serum antibodies). Plates were incubated for 1 hour at room temperature with diluted primary antibody, followed by another 1 hour room temperature incubation with peroxidase conjugated anti-rabbit IgG (SAB-300, StressGen Biotechnologies), diluted 1:25000 in Stabiltyme Select.

Plates were developed with TMB (BioFx) for 5 10 minutes at room temperature and the COMS ID No: SBMI-02132445 Received by IP Australia: Time 17:45 Date 2005-12-06 06-12-'05 17:29 FROM- T-751 P028/78 F-502

WOO

M

1u123 PCT/USOO/3341 O reaction was stopped with acid stop solution (BioFx). Absorbance of each well was measured at C'l 450 nm in an EL808 microplate reader (BioTek), interfaced with KC3 software. The ritre of the o antibody was represented as the concentration or reciprocal dilution of the antibody that resulted in an absorbance reading of 0.2.

s A relative titre index was established to compare the ttre of the test antibody against a Snegative control antibody. For affinity purified antibodies, the index vnlue was calculated by dividing the litre of the negative control antibody by the titre of the test antibody. For serum o antibodies, the index value was calculated by dividing the titre of the test antibody by the titre of n the negative control antibody.

C 10 CompetWion EIA. Nune Maxisorp ELISA plates (primary plates) were passively coated overnight at 4 0 C with 100ng/well of purified recombinant human Hsp70B' diluted in PBS. After n washing six times with PBS containing 0.05% twccn-20, primary plates were incubated with 0200 pl/wll of Superblock blocking buffer in PBS (Piercc) at room temperature.

Cl While the primary plates were blocking, the free antigen:antibody mixtures were prepared in 96 well Nunc polypropylene plates (secondary plates). First, Hsp70B' standard and Hsp 70 protein homologs cross reactants; recombinant human Hsp70A, recombinant bovine recombinant hamster Grp78. E coli DnaK, and M. luberculosis Hsp71) were diluted to a starting concentration of 10pg/ml and 500pg/nl respectively in BSA diluent (0.12% BSA (Sigma), 0.05% Iween-20 (BioRad). 1:1000 ProClin 200 (Supelco) in PBS). The diluted proteins were added to the secondary plates and then serially titrated four limes at a ratio of 1:5 in BSA diluent to generate a Hsp70B concentration range of 0.016-10 pg/ml and a homolog concentration range of 0.8-500 pgfml in a final volume of 100 l/well. BSA diluent alone served as the 0 g/ml point.

The rabbit CB2 aflinity purified antibody, CE and 70B' WP serum antibodies were diluted to 0.06 pg/ml, 1:10.000 and 1:16,000 respectively in BSA diluent. An equal volume (100 piAvell) of diluted antibody was added to the secondary plate wells with BSA diluent and varying concentrations of Hsp7OB' and homolog. The secondary plate was incubated at room temperature for 1.75 hours, after which 100 il of the free antigen:ntibody mixture was transferred to the blocked and washed primary plates. Primary plates were incubated at room temperature for I hout at room temperature, followed by a 1 hour room temperature incubation with peroxidise conjugated anti-rabbit IgG secondary antibody (SAB-300. StressGen 2t COMS ID No: SBMI-02132445 Received by IP Australia: Time 17:45 Date 2005-12-06 06-12-'05 17:29 FROM- T-751 P029/078 F-502 W oi00 2423 PCT/US0033341 SBiotechnologies) diluted in BSA diluen. The primary plate was developed with TMB (BioFx) O and stopped with acid slop solution (BioFx) after 10 minutes. The absorbance of each well was d) read with a BioTek ELB08 microplate reader set at 450 run. The maximum absorbance (i.e Amax; maximum amount of antibody binding at 0 pg/ml free Hsp70B' or homolog) was o 5 determined and used to calculate the at each Hsp70B' or homolog competition concentration. Antibody displacement curves were generated by plotting the A/Am against the concentration of free Hsp70B' or homolog.

Cloning and Expression of Recombinant Human Hsp708 Human Hsp70B was cloned from heat shocked HeLa cells and expressed recombinantly in E coli. Briefly, 2 X 10' HeLa cells were heat shocked for 2 hours at 44 5 C and then immediately harvested. Poly

RNA

was isolated from the heat shocked HeLa cells with a mRNA isolation kit (Boehringer o Marnheim) and used to synthesize human Hsp70B cDNA by RT-PCR. The 51 pl RT-PCR reaction mixture consisted of I ml of 10 mM dNTP (Perkin Elmer). 2.5 Ml of 100 mM DTT (Bochringer Mannheim). 0.25 pl of 40 units/jl RNase inhibitor (Boehringer Mannheim). 10 pl 15 of 5X RT-PCR buffer containing 7.5 mM MgCI 2 and DMSO (Bochringer Mannheim), 1 pd of enzyme mix (Boehringer Mannheim) containing Expand High Fidelity enzyme mix and AMV S reverse transcriptase, 0.87 1g of poly RNA from heat shocked HeLa cells, and I pg each Sof: primer 1 5'-GAAGCTTCACATATGCAGGCCCCACGGGAGCTC.3' (SEQ IDNO:12) and primer 2: 5'-GAAGCTCGAGTCAATCAACCTCCTCAATGA-.3 (SEQ ID NO: 13).

20 The primer sequences were derived from the human Hsp70B nucleotide sequence (Leung et al, 1990) and designed to introduce a Ndel restriction site upstream of the start codon and a Xhol restriction site downstream of the stop codon. The cDNA was synthesized by incubating the reaction mixture for 30 minutes at 50'C and amplified by PCR in a Perkin Elmer Gene Amp PCR System 2400 which was programmed for 2 minutes at 94 0 C, followed by 10 cycles of 30 seconds at 94'C, 30 seconds at 601C, 2 minutes at 68'C, and 15 cycles of seconds at 94oC, 30 seconds at 60*C, and 2.5 minutes at 68"C; the last extension step was 7 minutes at 68"C. The reaction product was analyzed by agarose gel electrophoresis and ethidium bromide staining. The human Hsp70B' cDNA was digested with Ndei (New England BioLabs) and Xhol (New England BioLabs) and ligated into a similarly digested pET24a 3o (Novagen) expression vector. The resulting expression plasmid carrying human Hsp70B' was 22 COMS ID No: SBMI-02132445 Received by IP Australia: Time 17:45 Date 2005-12-06 06-12-' 05 17:30 FROM- T-751 P030078 F-502 WO 01/42423 PCT/USa/3341 0 transformed into E. coli BLR (DE) and BL21 (DE3) cells. For preparation of bacterial extracts, cells were grown at 370C in LB medium (Difco) containing 30 gglml kanamnycin (Sigma).

When the cclls reached an OD5gs of 0.5, I mM isopropyl I-D.thiogalactopyranosidc (Amersham) was added to the medium and the culture was incubated at 37 0 C for an additional O 5 2.5 hours. Cell pellets were resuspended with lysis buffer (50 mM Tris pH 7.5 (BDH), 150 mM NaC) (BDH), 0.1 mM phenylmethylsulfonyl fluoride (Sigma), pg/mi leupeptin (Sigma) and 1 pg/m) aprotinin (Sigma)) and disrupted by sonication. The Bradford assay (Bio-Rad) was used to determine the protein concentration of the extract. The extract was diluted in SDS-PAGE sample buffer (40 mM Tris-HCI pH 6.8 (BDH), 1% SDS (Bio-Rad), 50 mM DTT (ICN). io glycerol jAnachemia], and 0.003% bromophenol blue (Sigma)) and boiled for 5 minutes in o preparation for SDS-PAGE and immunoblotting.

Cloning ond Expression ofRecombinant Hiso-Humran Hsp70B (Frogmen). The 74 1bp fragment that encodes a portion of the amino terminus region of the human Hsp7OB was obtained from SPD.925, a human Hsp70B stress gene probe (StressGcn Biotechnologies).

SPD-

s 925 is supplied as a plasmid containing 3.15 kb of the 5' non-transcribed Hsp70B gene sequence Sthe 19 bp RNA leader region and the 741 bp protein coding region. Although the protein coding region can be excised from SPD-925 by digestion with Hindll, restriction site.

S modifications were introduced by PCR. The 50 pl PCR reaction mikxture consisted of 8 pl of 1.25 mM dNTP (New England BioLabs), 5 p) of 1OX Expand High Fidelity PCR buffer (Boehringer Mannheim), 0.5 pl of 3.5 units/pl Expand High Fidelity DNA polymnerase (Boehringer Mannheim). 0.05 pg o SPD-925. and I jg each of primer 1: 5 -GAAGCTTCACATATGCAGCCCCACiGGAGCTCG-3 (SEQ ID NO:12) and primer2 5 .TGACAAGCTTAGAATTCTTCCATGAAGTGGT-3 (SEQ ID NO:14)..

SThe primer sequences were derived from the human Hsp70B nucleotide sequence is (Voclmy et atl., 1985). Primer I was designed to introduce an Ndel restriction site upstream of the start codon whereas primer 2 was designed to introduce a stop codon and a HindlI restriction site. The PCR reaction was performed in a Perkin Elmer Gene Amp PCR System 2400 which was programmed for 2 minutes at 94*C, followed by O10 cycles of 30 seconds at 941C, seconds at 60 0 C, I minute at 72 0 C. and 15 cycles of 30 seconds at 94 0 C, 30 seconds at and 1.25 minutes at 72*C; the last extension step was 7 minutes at 72 9 C. The reaction product 23 COMS ID No: SBMI-02132445 Received by IP Australia: Time 17:45 Date 2005-12-06 06-12-' 05 17:30 FROMI- T71 P3/7 T-751 P031/078 F-502 WO 0142413 PCTIUSOO01flS41 was analyzedi by agarose gel eleetrophoresis and ethidium bromide staining, The human E4spYO$3 c-i PCR product was digested with Ndel (New England BioLubs) and HinduI (New England BinL-abs) and ligated into a similarly digested pET2Sa (Novagen) expression vector. The resulting expression plasmid carring hiso-humam Hsp7UB was transformed into E. coi J3LR (DE3) cells. For preparation of bacterial extracts, cells were gmown at 37*C in LB medium O (Difco) containing 30 pg/mt kanaraycin (Sigma). When the cells reached an 00595 of 0.5, I rm isopropyl I 3 -D-rhiogalactopyrnie~ (Amnersham) was added to the medium and the culture wa.3 O incubated at 37 0 C for an addiiional 2.3 hours. Cell pellets were resuspended with SDS-PAGE sample buffer, sonicated, and botled for 5 minutes in preparation for SDS-PAGE and to imrmunoblotting.

Purification qf Recombinant Humnan HspO' Protein. coi BL12 1 (DE3) cells o barb~oring the expression plasinid for full length human Hsp7OB' Were grown in LB media with pg/mI kanamnycij) (Sigma) at 37 t C with. shaking at 250 rpm. All an OD9R of 0.5-0A6 Cl expression of recombinant protein was induced with I mnM isopropyl P-D-zhiogalaciopyranoside (IPTG; Calbiochem) and cells were harvested by centrifugation after 3 hours. Bacterial pellets were resuspended with lysis buffer (25 mM Tris-HCI pH 7.5 (13D3-1), 5 mm EDTA (Sigma), 0,3 mng/mi lysozyme (Sigma), 5 Mlip-atnirtobenzamidine (Sigma), 15 mM 0-mcrcaprocth 'anol (BME; Sigmna). 1 mM phenylmethylsulfonyl fluoride (PMSF, Sigma), I pg/mI aprotiniat (Sigma), I pg/mI leupeptin (Sigma)) and disrupted by sonication- The bacterial suspension was centrifuged and the supemnatarit was loaded onto a Q sepharosc (Amersham Pharmacia) ion exchange coltum. Bound protein was eluted with a 0 300 mM NaC) gradient. followed by a 300 mM NaCI wash. Q-Scpharosc fractions were analyzed on coomnasiea stained SOS-PAGE Igels and the Hsp7OW 'contalniag fractions pooled. MgCI 2 (Sigma) was added to a final conlcentration of 3 mM and 'the pool was loaded onto an ATP agam-ose (Fluka) column. After 500 mnM NaCI and 20 mM NaCI washes, bound H-sp7O' protein was eluted with ATP agar clution bufer (l0 mM Tris.HCI pH 7.5,3 iM MgCl,, 10 rM ATP (Sigma), 15 mM BME.

0.1 mM PMSF. I pg/mi aprolinin, I pag/mi leupeptin). Hsp7OB' containing fraction, were poolcd and sodium acetate (Maltinekrodt) pH 5.5 added to a final concentration of 40 mM. The pool was adjusted to pH 5.5 with acetic acid (Fisher Scientific) and then loaded onre a SP 1-iTrup (Asneshamn Pharmacia) ion exchange colun. Protein was tluted with a 0- I M NaCI gradient, 24 COMS ID No: SBMI-02132445 Received by IP Australia: Time 17:45 Date 2005-12-06 06-12-'05 17:30 FROM- T-751 P032/078 F-502 WO 91142423 PCTIUSDOi3fl41 O ~Hsp7OB' conlainin 4 fractions were pooled and the Protein Preparation wam dialyzed and stored in 10 MM TriS-ilcI pH- 7.5, 150 m1M NaC) buffer, Protein concentration was determined by the oBrad ford assay (Bi o-Rad) and purity was assessed by densitometry scanning of a coomassice stained SDS gel with 0.5. 1.0 and 1 .5 lag HspJOB'r.

6 Cell Culture was performed as follows.. HeLa. (human epitheloid cervical carcinoma.

O ATCC CCL-2) cells were grtown in Eagle's minimal esential mediumn with Earle salts (ICM) sUpplemented with 0.1mM non-essential Hunino acids (Gibco). 2 mM L-glutamine (Gibto), ofeta bovine serum (Gibc), 50 pg/mI gentarnycin sulfate (Gibco), and I m.NM sodium pyruvate Jurkat (clone human acute V-cell leukemia, ATCC TIS-1 52) cells were grown in c-i iC RPM! 16.40 medium (Gibco) supplemented with 10% fetal bovine serum (Gibco), 2 mM L-gluwamine (Gibco). 1.5 g/I sodium bicarbonate (ICN), 10 mM HErBS (Oibco), 4.5 g/L glucose (Sigmna). I mMv sodium pyrivate (Gibac), and 50 glml gentazniycin sulfate (Gibco), and O~ 1.5 gIL sodiumn bicabonajte (ICN). Veto (African green Monkey, normal kidney epithelial, Cl ATCC CCL49 1) cells were grown in Eagle's minimal essential medium with Earle salts ([CN) is supplemented with 2 mM L-glutamine (Gibco), 0. 1 mM non 'essential ainino acids (Gibco), 1 atM sodium. pyruvate (Oibco), 0-1 mM mon-essential amnino acids, 50 pqWml geniarnycin sulfate .(Oibco) and 10% fetal bovinec serumn (Gibco). CHO-K I (Chinese hamster ovary epithelial.

ATCC CCL-61) cells were grown in Dulbecco's modified Eagle'sq medium (Gibeo) supplemented with 2 mM L-glutanine, 10 mM HEPES (Gibco), 50 pg/mI genamycin sulfate (Gibco) and 5% fetal bovine sertim (Gibco). N4DBK (bovine normal kidney epithelil,* ATCC CCL-22) tells were grown in Eagle's minimal essential medium with Earle salts (ICN) supplemented *With 2 mrM L-glueamine <Gibco), 0.1 mM non-essential amino acids (Gibco).

I mM sodium pyruvate (Gibee), SO pig/mi gernamycin sulfate (Glbco) and 109/6 horse: serumn (Gibto). H-lLa, Jurkat, Veto, CR0 and MDBIC cells were incubated at 37 0 C in a water-jacketd incubator with CO 2 A-431 cells were similarly incubatmed at 37 0 C, but with 10% CO 2 Treatment with Metals. Azofidine and Hea. At 900/ conflueney. HeLa, A-431I and Jurkat cells were incubated at 37 0 C for 2 hours with 100 .rMl CdCI2 (Sigma) or 250 pM ZnCl 2 (Sigma) and harvested alter a 5 hour recovery period in media Without CdCl, Or ZnICI 2 Cells were similarly treated for 5 hours with 5 mM of the proline analogue, L-azctidine-2-carboxylic acid (Sig=a), and harvested after ao2 hour recovery-period. Cells were also hecat shocked for COMS ID No: SBMI-02132445 Received by IP Australia: lime 17:45 Date 2005-12-06 06-12-'05 17:31 FROM- T-751 P033/078 F-502 WO 01142423 PCTIUSoo J33341 minutes or 2 hours at 440C and harvested after a 5 hour recovery period at 37C. Cell lysates 0 were prepared for SDS polyacrylamide gel electirophoresis and immunoblot analysis.

Vero, CHO and MDBK cells were also subjected to heat stress. Vero cells were heat stressed for 1.5 hours at 42CC and recovered at 37 0 C for 18 hours prior to harvesting. CHO cells were heat stressed for 2 hours at 42 0 C and recovered at 37 0 C for 8I hours prior To O harvesting. MDBK cells were heat stressed for 1.5 hours at 44'C and harvested after an 18 hour recovery at 37C. Cell lysates were prepared for SDS polyacrylamide gel electrophorcsis and immnunoblot analysis.

CHeat Treatment ofHeLa Cells. HeLa cells were grown to 90% confluency and heated for D lo 2 hours at 37C, 38. SC, 40'C, 41.5*C, 43CC. and 44.5 C. Cells were harvested after a 5 hour recovery period at 37"C and prepared for SDS polyacrylamrnide gel electrophoresis and inmunoblot analysis.

Recovery ofHeat Treated HeLa Cells. HeLa cells were grow-n to 90% confluency and heated for 2 hours at 44.5"C. Cells were harvested after recovering at 31C for 0. 2.5, 5, 16 and 24 hours. Control cells were maintained at 37C and were similarly harvested at the same recovery times. Cell lysates were prepared for SDS polyacrylamrnide gel electrophoresis and immunoblot analysis.

SDS Polyaerylamide Gel Elecrrophoresis and Immunoblotting. After the appropriate recovery period, cells were washed with Dulbccco's phosphate buffered saline without calcium and magnesium (ICN) and harvested with a cell scraper. Harvested cells were resuspended with lysis buffer (Dulbecco's phosphate buffered saline without calcium and magnesium (ICN), 0.05% triton-X 100 (Sigma). 0.1 mM phcnylmethylsulfonyl fluoride (Sigma), I Pg/ml leupeptin (Sigma) and 1 pg/mrl aprolinin (Sigma)) and disrupted by sonication. The protein content of the cellular extracts was determined by the Bradford method (Bio-Rad). The extract was diluted in SDS buffer containing 40 mM Tris-HC pH 6.8 (BDH), 1% SDS (Bio-Rad), 50 mM DTT (ICN).

glycerol (Anachemrnia), and 0.003% bromophenol blue (Sigma) and heated at 70'C for minutes. Cell extracts were resolved on 12.5% SDS polyacrylamide gels and separated proteins were electroblotted onto nitrocellulose membranes (Gelman Sciences) by using a mM Tris 192 mM glycine (Fisher Scientific). 20% (vv) methanol (Fisher Scientific) transfer buffer in a Trans-Blot apparatus (Bio-Rad) at 100 V for 1 hour. Blots wore blocked in 26 COMS ID No: SBMI-02132445 Received by IP Australia: Time 17:45 Date 2005-12-06 06-12-' 05 17:31 FROM- T-751 P034/78 F-5022 WO 01/42423 PCTUS00O33341 Carnation non-fat milk, 0.05% Tween-20 (Bio-Rad). 0.02% NnN3 (Fisher Scientific) or thimerosal (Fisher Scientific) in phosphate buffered saline (15.4 mM Na 2 zPO0 (Mallinckrodt), 4.6 mM NaH 2 F0O 4 (Mallinckrodt), 120 mM NaCI Blots were probed with a mouse monoclonal antibody specific for inducible Hsp7OA and lsp70'FR (StressGen Biotechnologies, SPA- 10), rabbit polyclonal antibody specific for inducible Hsp70A (StressGen O Biotechnologies, SPA-812), rat monoclonal antibody specific for cognate Hsc7O (StressGen Biotechnologies, SPA-S 15), rabbit polyclonal antibody for Hspl 10 (StressGen Biotechnologies, O SPA-I 101), mouse monoclonal antibody specific for Grp75 (StressGen Biotechnologies. SPA- 825), mouse monoclonal antibody specific for the endoplasmic reticulum KDEL retention signal 10 peptide (StressGcn Biotechnologies, SPA-827), mouse monoclonal antibody specific for E colt OnaK (Strrssien Biotrchnologics. SPA-880), mouse monoclonal antibody specific for M. rubercularis Hsp71 (StressGen Biotechnologies, SPA-885). and polyclonal antibodies raised O either against a panel of synthetic peptides derived from the linear human Hsp70' sequence or purified recombinant human lsp70B'. Blots were incubated for I hour at room tcmpeniture with It primary antibodies diluted in blocking buffer. Alkaline phosphatase or peroxidase conjugated secondary antibodies (StrcssGen Biotechnologies) were respectively diluted 1:1000 and 1:5000 in blocking buffer and incubated with the blots for I hour at room temperature. Blots using alkaline phosphatase conjugated secondary antibodies were developed with 0.15 mg/ri bromo-4-chloro-3-indolyl phospbate (Sigma) and 0.3 mg/ml nitro blue tetratolium (Sigma) in zo alkaline phosphavase buffer (0l0 mM Tris-HCI pH 9.5 (IDH), 150 mM NaCl (BDH), 10 mM MgC 2 (Fisher Scientific)). The developed blots were washed with deionized water to stop the colour reaction. Blots using peroxidase conjugated secondary antibodies were developed by enhanced ehemiluminescence (ECL, Amersham).

Generation ofMouse Hybridomnas. BALB/ce mice were immunized with peptidde-KI-H conjugate essentially as already described. Test bleeds were analyzed for antipeptide and anti-Hsp70B' protein serum titres by indirect EIA and mice with bigh Hsp70B' titres were selected for fusion. Four days prior to spicnectomy and cell fusion, the selected mice were boosted with antigen in the absence of adjuvaint. Mouse spleens were aseptically removed, minced with forceps and strained through a sieve. Cells were washed twice with IMDM medium and counted using a hemocytometer. Spleen cells were mixed with mouse mycloma P3x63Ag8.653 cells at a ratio of 5:1 (spleen:myeloms cells) and centrifuged. Cell pellets were 27 COMS ID No: SBMI-02132445 Received by IP Australia: Time 17:45 Date 2005-12-06 06-12-'05 17:31 FROM- T-751 P035/078 F-502 WO 01/42423 PCTIUS0C33341 O resuspended with Iml of 50% PEG (MW 1450), added dropwise over a period of 30 seconds.

C The rcsuspended cells were gently mixed for 30 seconds using a pipette and then allowed to o stand undisturbed for another 30 seconds. Sml of IMDM media was added over a period of seconds, followed immediately with another 5ml. The resulting cell suspension was left undisturbed for 5 minutes, after which the cells were pelleted and resuspcnded at 5 X cells/ml in HAT medium (IMDM containing 10% FBS, 2mM L-glutamine 0.6% 2 -mcrcaptoethanal, hypoxanthine, aminopterin, thymidine, and 10% Origen growth factor).

0 Cells were plated into 96 well plates at 10 cells/well. Plates were incubated at 37CC in a 7% en

CO

2 atmosphere with 100% humidity. Seven days after fusion, the media was removed and replaced with IMDM containing 10% FBS, 2mM L-glutamine. 0.6% 2-mcrcaptoethanol, C hypoxanthine and thymidine. 10-14 days after fusion, the supematant was taken from wells with growing hybridoma colonies and screened for anti-peptide and anti-prdtcitn antibody by indirect EIA. Hybridoma cells from positive walls were cloned by limiting dilution in 96 well plates at a Cl density of 0.25 cells per well or one cell in every fourth well. Growing colonies were tested 10-14 days later using the same assay(s) used to initially select the hybridomas. Positive clones were expanded and frozen.

Results Eight peptide sequences were chosen, one an N-terminus epilope as well as seven epitopes from the carboxyl end of the human Hsp70B' protein. Peptide sequences were selected based on minimal identity and homology with other HSP70 family members and algorithmic predictions of hydrophilicity (Kyte-Doolitle), antigenicity (Jameson-Wolf) and surface probability (Emini). These sequences, their respective residue numbers (according to the GeneBank sequence #P 17066) and epitope designations are listed in Table 1. The CB and CB2 sequences arc the same, except the CB2 pcptide was synthesized on a separate occasion.

28 COMS ID No: SBMI-02132445 Received by IP Australia: Time 17:45 Date 2005-12-06 06-12-'05 17:32 FROM- T-751 P036/078 F-5022 W 0142423 p Table 1: Summyy of Imnunoenicity of Different Hsp7OB' Epiopes -Tnl]S /33341 Epitope Location Peptide Imniuncgen Anirna Antisera Titer Animals (Ab) (PI7066) 1 Host (EA) Respondin Name

I

*CB 624-638 ICLH QARQGDFSTGPI Rabbit 3/3 33.021 >204800 *CA 561-573 KLH-(C)RDKIDEWRRKMQ Rabbit >204,001) 3/3 >204,800 191,311 "CD 561-576 RDCIPEEDRICMQDKC-(LU -Rabbit >204,000 212 >204,500 CC 546-559 KLH-AVFHVKGSLQEES Rab bit 204000 3/3 >204,800 '204,800 'NT 1-12 MQAPRJELAVCIDC)-KLH Rabbit >204,000 3/3 168.019 204,800 ECB 619-638 KLi-vpgg9Sscgtqarqg~dstgpi Rabbit >204800 3/3 5204800 TCD 624-65 KL-1-CGQARQGDP~SsT Rabbit >20480 31 >204800 >20420 CEm 553-567 KLl-t-GSLQEESLRDAPEE Rabbit' 8424 3/3 67510 4368 "2Z 624-638 KLWiCGTQARQGDPSTCPI Rabbit >204800 >204800 >204800 CR2 624-638 ICLH-CGTQARQCDPSTGPI Goat 42001/i C92 624-68 KU-ICGTQARQGDPSTGPI Mouse >204800 10/10 111432 >204800 >20400 36685 108253 151239 >204800 64358 >204800 Hsp7OB' full length recombinant Rabbit >243000 3/3 WP 1-643 human J-Jap7OB' protein 123100 222900 COMS ID No: SBMI-02132445 Received by IP Australia: Time 17:45 Date 2005-12-06 06-12-'05 17:32 FROM- T-751 P037/078 F-502 WO 01142423 PC'r 34 PCTUgM/334t O* indicates antiserasubsequently purified by peptide immunoaffinity chromatography.

(i The peptides were synthesized, conjugated to KLH and used to immunize rabbits, goals U or mice. Specific antibody responses to the immuiziog peptide were detected in every animal as assessed by indirect peptide EIA (Table Six of the nine rabbit anti-peptide antisera were s then purified by poptide immunoaffmiiy chromatography. Antibody preparations were tested fur O specificity of binding with a panel of HSP70 protein homologs as well as a series of stressed and non-stressed human and mammalian cell lysatcs (Tables 2-6).

SHsp 708' Antibody Production with Recomhinant Human isp 70B' Protein.

e In addition to generating Hsp70B antibodies via the peptide immunization route, a C, 10o Hsp70O' antibody, designated 70B' WP, was produced in rabbits using purified homogeneity) recombinant human Hsp70B' as the immunogen. Like the peptide generated antibodies, the antibody response to the immunizing protein was detected in each rabbit by o indirect EtA (Table The sera was pooled and then tested for spccificity against a panel of C" purified HSP70 homologs by indirect EIA (Table 2) and competition EIA, (Table 3) The 1i antisera was also assessed by irnmunoblotting with control and heat stressed cell lysacts (Tables and 6).

Determinano of Antibody Specificity by Relative Titre Index The specificity of selected Hsp70B' antibodies was determined by indirect EtA and expressed as an antibody titre index, relative to an irrelevant or negative control antibody (Table Titre is defined as the antibody concentration (for purified antibodies) or reciprocal dilution (for unpurified antisera) that results in 0.2 absorbance units. This cutoff value approximately represents the absorbance of the assay background 3 standard deviations. 11 is the lowest distinguishable positive signal at 95% confidence.

The relative titre index directly compares the titres of the Hsp70B' antibodies with the titre of an irelevant anlibody. It is therefore a measurement of antibody reactivity towards a particular protein or peptide above non-specific binding. The index value is directly proportional to the reactivity of the antibody. Thus, the higher the relative index, the more reactive the antibody is toward a particular protein or peptide. An index of I reflects reactivity that is on par with that of the negative control antibody and is considered as negligible binding, An index value greater than 1 is indicative of binding. If an antibody is found to react with only one COMS ID No: SBMI-02132445 Received by IP Australia: Time 17:45 Date 2005-12-06 06-12-'05 17:32 FROM- T-751 P033/0a78 F-502 WO DI/42423 PCT/JUS003341 0 protein or peptide the antibody has an index of with only one protein or peptide), Sspecificity can be defined within the context of the proteins or peptides tested.

SBased on absolute index values in Table 2, the CB2, CE, ECB and TCB antibodies preferentially reacted with Hsp70B' by 177, 48, 41 and 29 fold respectively over other protein homologs. Under these assay conditions, the CB2, CE. ECB and TCB antibodies were Sspecific for Hsp70B' protein.

At lower dilutions, the 70B' WP antibody minimally exhibited some cross reactivity with o Hp70A, DnaK, and likely with Hsc70 and Hsp71. However, the 70B' WP antibody still reacted en 90 rold higher with Hsp70B' over Hsp70A and DnaK. SPA-81 2 is a rabbit polyclonal antibody produced to recombinant human Hsp70A protein. In addition to reacting with Hsp70A, SPA- 812 also cross reacted with Hsp70B' (and likely with Hsc70 and DnaK) at lower dilutions. For both of these whole protein antibodies, reactivity with their respective antigens was high (index o >100). Therefore, by using the antibodies at higher dilutions, cross reactivity can likely be N "diluted out" and reactivity to the intended protein still retained This essentially selects for the is population of higher affinity antibodies that recognize specific epitopes on the antigen of interest.

The 70B' WP antibody reacted 4-6 fold higher with Hsp70l' protein over the CE, ECB and TCB serum antibodies. However, the 70B' WP antibody also exhibited cross reactivity with other homologs whereas the peptide antibodies did not. This is not unexpected since the WP antibody likely consists of several antibody populations that recognize different epitopes throughout the surface of the molecule. Some of these epiropes though ate likely shared or homologous epitopes with other homologs. The peptide antibodies probably also consist of several antibody populations that recognize Hsp70B', but the epitopes are limited to the immunizing peptide sequence, thus limiting cross reactivity. This supports the peptide approach over whole protein immunizations, if specific Hsp 7 0 B' polyclonal antibodies are required.

Reactivity of peptide polyclonal antibodies with the intended protein can also be enhanced by affinity purification, as illustrated by the purified CB2 antibody. Under these assay conditions.

this antibody had a high index value >100) for Hsp70B' and was specific for Hsp70B'. Of course, the 708' WP could also be affinity purified to enhance specificity, thus increasing the usefulness of this antibody if required at lower dilutions.

The CB2 and ECB antibodies reacted with the reciprocal immunizing peptide. This was not unexpected since ECB was just an extension of CB2. Similarly, the CB2 and ECB 31 COMS ID No: SBMI-02132445 Received by IP Australia: Time 17:45 Date 2005-12-06 06-12-'05 17:33 FROM- 06-1-' 0 1733 FOM-T-751. P039/078 F-502 WO 01/42423 PCTUSOO/33341 o antibodies were expected to react with the TCB peptide, the tmuncaled version of CR2. However, O the C8 antibod did notreact with the TCI peptide and the SCB antibody had some ratvt to the TCS peptide that was beyond the lower limits of the assay. 70B WP also did not react C) with thc TCB peptide, but there was some reactivity with the CR2, ECB and CE peptides. This suggests that more antigenic epitopes on the Bsp7OR' protein are responsible for the reactivity of o 708' WP. The TCB antibody reacted with the immunizing pepTidc and both the C82 and ECE3 peptides.

Table 2: Relative Tit Index of Hsp7OB' Antibodies or ?w'ified Antibote serumMttoie ci I~pVO'Peplide C1132 CE] ECB TCB 701WF j SPA-812 Nor-zat Hspfltr t IproB' Hs2 V sp7OB' Hp7or' 7-iplA Rbbt 7 53-S07 6184i8 6zq-W5 I -M41 I54I Ser4j fir a-s73 rtt 177 ~x a~ a48 ;-41 !-29 i182 Z12 oHsc7O Protein Vfri; I I i '11 oHsp,7DA -Protein 1 iE' I I I 2 a143 t a] DnaC Protein I Ir$fl- 2 *1 I C132Peptide 779 k37, 22i 1 :7 elv-t a ti o i s s d -ss s on s ec f c i d ng a c h de n gry-F r pu f e anbdietindex vaes wer calulte bydvdnahe 57 ftereeatanioybh IrSeletHs7B antibodies weevlaed tn asss no-pfComeidion BA asanohed igry opifor assgantibody easpeifcity te Thi C dmpeitin te wasbae o the 50%len dilacem e mefth fort calcltingdy orss rua nti es (Arh e. nde C/in.s weeluaendd! by dividingthe titr 1969). A st escibd by bthae ar ontth f amouvnt ofantibodyadlbldsadr S r inutd inr thecupresencerofs rtvaryin dosesam of. Jnaee or. crosreatntl CCR)b Theunlabele 3-Y COMS ID No: SBMI-02132445 Received by IP Australia: Time 17:45 Date 2005-12-06 06-12-'05 17:33 FROM- T-751 F040/078 F-502 WO 01/42423 PCT/S00/33341 O S or CR is the "displacer" that competes with the labeled S for antibody binding sites. The C bound labeled S is then quantified at each dose of unlabeled S or CR. Displacement curves are o generated by plotting the 8/Bo against the dose of unlabeled S or CR. B is the amount of bound labeled S in the presence of added unlabeled S and Be is the amount of bound labeled S in the absence of added unlabeled S maximum bound labeled standard). In the Sdisplacement method, cross reactivity is the ratio of unlabeled S to CR doses that give displacement of bound labeled S. expressed as a percentage.

SA competition EIA was developed to determine the specificity of selected en antibodies. Labeled standard is required for the competition reaction described by Abraham.

C- 10 However, labeled Hsp70B' protein was unavailable for this study. Instead, the competition reaction was modified to measure the amount of antibody binding to a constant amount ofS bound to a solid phase, in the presence of varying amounts of free S or CR. Displacement curves Swere generated by plotting the %AJAmax against the dose of the free displacer, where A was the Cl amount of antibody bound in the presence of displacer and Amax was the amount of antibody bound in the absence ofdisplacer. Similar to the Abraham method, cross reactivity was defined as the ratio of the free standard and cross reactant doses that resulted in 50% displacement (A/Amax) of bound antibody.

Displacement curves for CB2 (Figure 1A, purified rabbit antibody), CE (Figure 1 B) and .708' WP (Figure IC) antibodies were generated with Hsp70B' protein and five homologs Hsp70A. Hsc70, Orp78, DnaK and Hsp7l. The concentration range of the free protein was 0.016 10 pg/ml whereas the range for the cross reactants was 0.8 500 Ag/ml. For all three H4sp70' antibodies, there was no distinct competition from the cross reactants when compared to Hsp70B', despite using 50 fold more cross reacrant at the highest dose. Under these assay conditions, a 50% displacement concentration could not be obtained for the cross reactants since the average A/Amax values throughout the cross reactant concentrations ranged from 93-100%. It was unsuitable to redefine the 50% displacement cutoff to 5% A/Amax of 95%) since variability of the assay could account for that level of displacement, rather than being a true competition. In any case, higher cross reactani concentrations beyond 500ng/ml are required for a more accurte assessment.

In Table 3, the percentage of cross reactivity with HSP70 homologs are presented for the CB2, CE and 70B' WP antibodies. Since the 50% displacement doses for the cross reactants 33 COMS ID No: SBMI-02132445 Received by IP Australia: Time 17:45 Date 2005-12-06 06-12-'05 17:33 FROM- T-751 F041/078 F-502 WO 01/42423 PCT/USCO/33341 Swere unobtainable, >500pg/ml was reported and S00pg/ml was used for calculating the cross Sreactivity. The cross reactivity percentages are therefore actually lower than the absolute o reported values. Based on this data and under these assay conditions, the CB2, CE and 70B' WP antibodies were more reactive with Hsp70B' by at least 758, 142 and 263 fold respectively over the tested HSP70 homologs. The CB2 antibody is notably more reactive with Hsp7OB' than the O CE and 70B' WP antibodies. This is likely because the CB2 antibody was affiniry purified, whereas CE and 70B' WP were antisera. The CB2 antibody was competed with lower am6unts Sof free standard, suggesting that this preparation contained a greater proportion of higher affinity n antibodies as compared to the CE and 708' WP antibodies.

to The CB2 and CE antibodics were considered specific for Hsp708' when expressed in C terms of a relative litre index (Table Under the competition EIA conditions, these two q antibodies can also be considered specific for lsp70B'. The 708' WPantibody. however, was o not specific for Hsp 7 0B' at lower dilutions (eg. 1:2000) by relative titre index. The competition C assay used 70B' WP at a dilution that was 16 fold higher. This indicates that cross reactivity can is essentially be "diluted out" for this antibody and perhaps should be used at higher dilutions to maximize specificity.

Displacement Curves for rabbit Hsp70B' antibodies: CB2 CE and 708' WP homologs were unable to significantly displace CB2. CE and 70B' WP antibody binding to solid phase Hsp70B'. Under these assay conditions, CB2, CE and 70B' WP are specific for Hsp7O'.

34 COMS ID No: SBMI-02132445 Received by IP Australia: Time 17:45 Date 2005-12-06 06-12-'05 17:34 FROMI- T-751 P042/078 F-502 W001/42423 FCT/J$V01f341 Cl Table 3. Specificity of Selected Hsp7OB' Antibodies in terms of Cr055 Reactivity with III P70 Itouielogs Rabbit Free flhpl'_acer Concentration of Free Cross Reactivity 11 sp701' (Standard Displacer at Antibody I o 5001 Aunax Cross Reactant)(p/) o Hsrp7O)B' (S)I 0.66 100% s7 C):500 <.3 C82 H90'0 (CR) >50WC 3 o (P'urdwtd) Frp7S (CA) >500 c~3 en naiC (CR) >50U <0.13% 1 Hsp 7 1 (CR) >500 <0.13% C'I HSp7OB' 3.52 1 N1 01 0I Ca (Antisra) p70r? (CR) 0<0. l-sc7O (C9) >500 <0.70% Grp7S (CR) >W0 t 0. DnaK (CR) >50 ca (CR >500 <0207(% Hsp7B S 1.88 ip70 (CR) '"500 <0.38%

WP

1 IHc7O 500 <0.38% (Antisera) Crp78 (CIR) ->500 <0.38% 1 DnaK ICR) >500 <0.38% l-sp7l (CR) >5W0 <0.38% Cross reactivity Was calculated by dividing h concentration of frc-c standard by the concentration o17C1700 reactant at 5W% A./Atnax; The ratio was then expi-csscd as a percentage.

Dnerm 0narion OfAnd body Specificity by Jmnninohlwelng Each of the I-sp7OB' antibodies detected their respective epitopes in the synttheuic peptide.

tbe peptide-CIJ conjugate, the rcombinant ).-sp7Ofr protein as we)ll as in the native H4sp7OB' protein present in cultured cell lysates. Antibody binding to native Hsp7OW protein in ccll lysates was determined by immuznoblouting. Several control antibodies were included in these experiments to validate the assays and induction conditions. The reactivity profiles of the control antibodies is summarized in Tables 4 to 6. The anti-)-IsclO antibody (SPA-SI 5; clone is a rat monoclonal antibody originally produced to the CH-O (hamrster) Hsc7O protein.

This antibody was found to react only with flac7O protein, no reactivity twith any of the iniducible isofonns, Grp7S, float, or Hsp7l wasi detected. Anti-l145p70A (SPA-S 12) specific antibody is a rabbit polyclonal antibody produced to purified recombinant human Hsp7OA protein. This COMS IDNo: SBMI-02132445 Received by IP Australia: Time 17:45 Date 2005-12-06 06-12-'05 17:34 FROM- 06-1-' 0 1734 POM-T-751 P043/078 F-502 WO )91/42423 PTUom antibody reacted only with [be flsp7OA protein, there was no reactivity detected'with Cl Hsp70B3 fragment, l-sc.7O, Grp~g. co/1i DnaK. and Af, rubercudasis Hsp7] proteins in o mmunoblots. The third control antibody is a mnouse mOnoclonal31 antibody (SPA-S 10; clonec C) C92FA-s) which Was ocriinally produced to human H~sc7Offlsp7o proteins purified rrom UcLa SN cells. This antibody was found to react with H-sp70A and recombinant l-SP7OD', but not with H4sc7O, H-sp7OR protein fragment. Grp7S. Dnalc or Hsp7l. Other control anti bodie s included a rabbit I-sp I110 polyclonal (SPA- I 10 a mouse Crp7S muonoclonal (SPA-82 5; clone 3 0A5), a o mouse monoclonal specific for the ER retention signal peptide KDEL (5PA4827; clone 1.013). a en mouse DaaK monoclonal (SPA-98O; clone XXX), and a maouse M-sp7l m onoclonal (SPA-S&S; clone SAg). The Usp I 10 and Cirp7S ant ibodics did not react with HsplOA. Hsp7OB', H~scOi, Orp7S. DnaK or 11 sp 71 proteins. The KDEL antibody reacted with GrpVS protein, but not wit Hsp7OA. Hsp7OB'. Ic00, DnaK Or [Isp7 I proteins. The DnaK and Hsp7 I antibodies rcactcd ruspectively with DnaK and Hsp7 I proteins, but not with Hsp7OA. l-sp7OB1', H-sc7O or 0rp75 proteins, Using these control antibodies, Hspl 10. GrplS. H-sc70. and KDEL prt*eins were detected in control and heat stressed human A431I cells. monkey Vero cells, hamnstet Cf-TO cells mid bovine MDBK cells (Table R-sp7OA was detected in control A43 1. Vero and IDIJK cells. It was not detected in control CR0 cells. )Jsp7OA was detecled at elevated levels in the heat Stressed A43 1, Veto, CHO and MDBK cells.

36 COMS ID No: SBMI-02132445 Received by IP Australia: Time 17:45 Date 2005-12-06 06-12-'05 17:34 FROM- 05-1-' 0 1734 FOM-T-751 P044/078 F-502 I3. I WO 01i42423 PCrAIue333s1 Table A. Sumry of Anti-HSP7O Antibody Reactivity Profies with Different H-S P70 Family Members Eriepe Location (A1b) Name II -;r-70 teartivity Retwrotein" Hr7 I4p7OA Hp7U Rec. ec

CA

'ECII

'CE

C92..

(rabbft).

C2g (muC)8 "B0wp 624-638 561-573 561-576 54-509 1-72 6T18-6-3 62-4-638 a 2 0 2

'C

Wi 10 2 Hs7V 3 3 3 3 IHapTQD1 Reartlyy teabuty caeiityRectvit&C:V Dnnl( HAp~l Rteactivity I Reactivity VeaCtiity pitedii' roei' tec. I fl Vre.im

K

32

ND]

ND

pp~tein P!rnsi3i' protein'

I

NO

ND

ND

0 0

NID

NID

Ism

-ND

ND

ND

ND

NID

ND

ND

0 6 2 4 4 3 8 N

DM

NU 'o ND ND 0-sPIU -0 Nnt SPA-810 437-504 -3 z aa _IHSP7OA) MuIcpf 3' 0 a 0 SP-il HepitO a 0 ND 0 f 0D 0 SPA-827 DE 0 0 0 ND 0__ SPA880 -CDE a D 0 1 SPA480~N Dea a SI'A418Q. Mfl7a 7P4S Relatiratvt 0 ND aa 0f 3 '~~keiativ. rec Iv~ Ssc5d by Western blotting a-naysis offSSdennafarcd ruwombinant proteins. intensity oflthe afugen sPIecifi bands was Lcoern on a relative scale where 0) no signal detected and 3 a very' sWong Signal. ND not determined, 'Representative Sera fTam One anirnai. hPooled SCEU "Determnincd wvith native protein in a call lysate instadof recombinant protein. Snaded I"SPJOB'atibodies are affinfity Purified.

COMS ID No: SBMI-02132445 Received by IP Australia: Time 17:45 Date 2005-12-06 06-12-'05 17:34 FROM- T-751 P045/078X F-502 WO 01/42423 PCTUS0o3341 o The nine rabbit anti-peptide antibodics generated to Hsp70B' epitopes reacted with recombinant protein in Western blot analysis (Table The antibodies also detected protein only in stressed cell lysales prepared from human cell lines (Tables 5 and 6).

C. The immunoaffinity purified rabbit anti-CB and CB2 antibody preparations (epitope Ss residues: 624-638) were found to be specific for the Hsp70B' protein; only a single 69 kDa protein is detected on Western blots analysis with complex protein mixtures from total human cell lysates. Although strong reactivity was seen with both native and recombinant there was no reactivity with recombinant and/or native Hsc70, Hsp70A. DnaK, Hsp 7 1, Hsp r) Grp75, Grp78 and other KDEL proteins (Tables 4 6).

Representative CB2 sera samples from individual mice exhibited specificity to the native protein in a lysate (Table 5) when assessed by immunoblorting. However. 3/10 CB2 immunized mice either did not detect native Hsp70B' or weakly reacted with other unknown proteins in a lysate. The goat CB2 antisera also specifically detected native Rsp70' protein, C, despite low anti-C02 peptide titres. However, the reactivity of the goat antisera with native Hsp70B' was lower, when compared to the rabbit and mouse CB2 antibodies (Table In any case, CB/CB2 is a unique epitope that can be used reproducibly separate occasions, different animals) to generate Hsp70B' specific antibodies. This epitope may be used to generate antibodies, such as monoclonals. of elquisitc specificity and useful affnity. The CBICB2 cpitope has already been used to produce mouse hybridomas that react with the CB/CB2 peptides. The reactivity of the hybridomas with Hsp70A and Hsp70B' proteins is currently being evaluated. Ideally, the monoclonal(s) will exhibit the same specificity as the polyclonal CB antibodies and will bind peptide, recombinant and native Hsp70B' with a K, in the range of 104. 10 2 The antibodies generated to this epitope are excellent candidates for inclusion in the cstablishmem of rapid screening assays.

The peptide immunogcns for the ECB and TCB antibodies were based on the CB/CB2 epitopes. The ECB peptide was an extended version of CB/CB2 by six amino acids at the N-terminal end. When ECB sera from three individual rabbits were analyzed by immunoblots, all three rabbits produced antibody that reacted extremely well with purified recombinant Hsp70B' (Thble 4) and native Hsp70B' in a lysate (Table However. 1/3 antisera 3 specifically detected Hsp7B' in a lysate. The remaining 2 antisera reacted very weakly with other unknown non-inducible proteins in the cell lysates. This antibody (from 3/3 rabbits), 38 COMS ID No: SBMI-02132445 Received by IP Australia: Time 17:45 Date 2005-12-06 06-12-'05 17:35 FROM- T-751 P046/0~78 F-5022 i* I WO 01/42423 PCT/USoo3as4 O however, did not react with native Hsp70A, Hsc70, Hspl 10, Grp75, Grp78 or other KDEL CN proteins. Any weak binding to the unknown lysate proteins can likely be "diluted out" or the antibody could be affinity purified to improve specificity. If successful, the ECB antibody could be used in conjunction with other Hsp70B' antibodies, such as CE, CA, or CC. for immunoassay SOs development. The ECB epitope would also be a good candidate for a monoclonal antibody O development.

Anti-TCB was generated with a CB/CB2 peptide that was truncated by three amino acids Sat the C-terminal. Although the TCB antisera from 3/3 rabbits reacted well to the immunizing peptide (Table only 1/3 rabbits produced antibody that reacted with recombinant (Table 4) $c 10 and native Hsp70B' (Table and an unknown non-inducible 40kDa protein in immunoblots.

I The reactivity with the non-inducible protein can likely be "diluted out" or the antibody can be purified to improve specificity. The sera from the two other TCB imrriunized rabbits reacted o with just recombinant Hsp70B' and not the native form (Table suggesting differences in Cprotein folding and surface exposure between the recombinant and native proteins. These two is antisera also reacted with other unknown non-inducible proteins. The antisera from all three rabbits did not react with Hsp70A or Hsc70. The lower success ratio 1/3 rabbits detected native Hsp70B') with the TCB peptide suggests an importance for the three omitted GPI amino acids present on the CB/CB2 and ECB epitopes. Based on relative titr index, the CB2, WP and ECB antibodies exhibited no to low reactivity with the TCB peptide (Table but these three antibodies reacted with both recombinant and native Hsp70B'. This supports the importance of the GPI residues for Hsp70B' antibody production, despite predictions of low antigenicity and surface probability. Taken together. TCB polyclonals that recognize native may be problematic to produce and resupply for industrial immunoassay uses.

The anti-"CA" antibody (epitope residues: 561-573) recognizes the Hsp70B protein but not the Hsc70, Hsp70A nor the Hsp70B (fragment) purified proteins. This antibody does, however, identify other unknown proteins of lower and higher molecular weight. As long as the interpretation of binding reactivity of this antibody on immunoblots is restricted to a window of 80 kDa, this antibody will act as a sensitive and specific probe in immunoblot analysis for the Hsp70B' protein. None of the other proteins seen on immunoblots with this antibody are stress inducible under the conditions of these experiments. The non-Hsp70B' proteins are detected equally well in both stressed and unstressed cell lysates. Although this antibody can be 39 COMS ID No: SBMI-02132445 Received by IP Australia: Time 17:45 Date 2005-12-06 06-12-'05 17:35 FROM- T-751 P047/78 F-502 WO 01/4223 CT/USOO34 O used in immunoblot analysis for the specific detection of the Hsp70B' protein the cross- C] reactivity of this antibody for other cellular proteins decreases the usefulness of the antibody in immunoassay rapid screening tests. However, in combination with another specific antibody such as this antibody may be a useful component in immunossays.

The anti-"CD" antibody (epitope residues; 561-576) recognizes both recombinant and Snative Hsp70B' as well as the constitutive Hsc70 protein. The inducible Hsp70A protein is also weakly recognized by this antibody. In addition, this antibody will detect, on immunoblor o analysis, some unknown lower molecular weight constitutively expressed proteins. The large variation in "CA" and "CD" antibody is surprising as the peptides used to generate these antibodics were very similar. The "CD" peptide is identical to the "CA" peptide with three exceptions; the "CD" peptide lacks the additional N-terminal cysleine residue used to facilitate coupling of the "CA" peptide to KLH, the coupling to KLH was modified to O coupling through the natural cysteine sequence at the carboxy-terminus. and three additional (DKC) residues were added to the carboxyl-terminus of the "CD" epitope. These three IS additional residues are conserved in Hsc70, Hsp70A as well as in Hsp70B' sequences and are predicted to lie within a hydrophobic region which is predicted in the Emini analyses to be antigenic. As the molecular weights of the 3 proteins are different; 69. 72 and 73 for the Hsp703 Hsp70A and Hsc70 proteins respectively, expression patterns of the different Hsp's may be differentiated with this antibody on immnunoblot analysis. However, due to the crossreactivity of this antibody for other Hsp family members as well as other cellular proteins this antibody would not be useful in rapid screening immunoassays.

The anti-"CC" antibody (epirope residues: 546-559) recognizes both Hsp70B (fragment) and Hsp70B recombinant proteins. This antibody does not recognize either the Hsc70 cognate protein nor the stress inducible Hsp70A protein. Although some additional unknown higher and lower molecular weight proteins are detected on immunoblots with this antibody, these proteins are also not inducible under these conditions and do not fall within the 60-80 kDa window for evaluation. This antibody is, therefore, useful for immunoblot analysis but due to cross.

reactivity with other cellular proteins the utility of this antibody in immunoassay analysis is decreased. However, in combination with another specific antibody such as this antibody may be a useful component in immunossays.

COMS ID No: SBMI-02132445 Received by IP Australia: Time 17:45 Date 2005-12-06 06-12-'05 17:35 FROM- T-751 P048/078 F-502 WO 142423 PCTUS00O33341 o 'he CE antibody was made from a peptide which combined seven amino acids from the C C-terminal of CC and seven amino acids from the N-terminal of CA, linked via a leucine residue.

Specific Hsp70B' binding was observed with CE antisera from 1/3 rabbits in immunoblots (Table There was no reactivity with Hsp70A, Hsc70, Hspl 10, Grp75, DnaK, Hspl. Grp78 \0 5 or other KDEL proteins. The CA and CC antibodies both detected non-inducible proteins in addition to Hsp70B'; the CE antisera from this particular rabbit did not. Like the CB2 antibody, the CE antisera from this rabbit only detected human Hsp70B' in lysates. There was no O reactivity with any proteins in monkey, hamster or bovine cell lysates. CE antisera from another Srabbit reacted with recombinant and to a lesser degree with native Hsp70B' (Table but also C to weakly with other non-inducible proteins. Interestingly, the more specific CE antisera had an C, eight fold lower anti-CE peptide titre than the non specilic CE antisera (Table The CE antisera from the remaining rabbit reacted with several non-inducible Proteins in a lysate and O very minimally with recombinant Hsp70B'. Reactivity of this antisera with native protein was inconclusive since many proteins were detected in the expected 70 kDa molecular weight range.

Although the CE antibody has a lower success ratio, a monoclonal antibody may be beneficial if it exhibits the same reactivity as the specific polyclonal. Because the locations of the CE and CBICB2 or ECB peptides are distinct, specific monoclonal antibodies to these epitopes would be choice components for two-site immunoassay development.

The "NT" antibody (epitope residues; 1-12) is an area of sequence of relative sequence homology within the Hsp70 family. This epitope has an overall homology of less than 50% with other HSP70 family members, however, the areas of sequence identity are sequential. The anti- "NT" antibody is useful as an HSP70 family marker, but has no utility in the differential detection of different Hsp 70 family members.

The 70B' WP antibody specifically detected both recombinant and native Hsp70B' in immunoblots. When the antibody was used at higher dilutions 1:20000), no cross reactivity with other HSP70 homologs or other lysate proteins was observed. This antibody may be a useful component in immunoassays if used at higher dilutions, purified or in conjunction with another specific antibody such as CB/C82, ECB or CE, This antibody had relatively low titre index values with the CB, ECB, TCB and CE peptides), but a high value with Hsp7OB' (Table 2).

This suggests that other specific epitopes, perhaps conformational, exist for Hsp70B' monoclonal antibody production that require whole protein immunizations to exploit.

41 COMS ID No: SBMI-02132445 Received by IP Australia: Time 17:45 Date 2005-12-06 06-12-'05 17:36 FROM- 06-1-' 0 1736 FOM-T-751 P049/078 F-502 WO 01/dl4f3 PcrfUSGoa334 Cl ~~Summary of Hsp7OB'Annbody Specificir. T~l UMmaie h seset specifichty for the rabbit H-sp7OB' antibodics, with respect to the three methods performed: rmlattve titre index, competition FIA and immunoblouing. Only one antibody, CB2 was considered specific fodr Hsp7OBit, all three methods of assessment, Since CB was generated O with the samne peptide sequence. it is likely that this antibody would also be considered l-sp7OB' specific for all three assessments methods. The EC 1 CE and 7DB' WP antibodies could likely O l~c purified or used at higher dilutions to maximize specificity, The Cl)andiNT antibodies were huot useful for distinguishing Hlsp70B' from other lISP7O hornologs, illustrating that the peptide Cl 10 approach is not 100%:succesful for generating HspJOW antibodies.

COMS ID No: SBMI-02132445 Received by IP Australia: Time 17:45 Date 2005-12-06 2005242130 06 Dec 2005 Atfoamey Docket No.: 12071-006001

S

t~.0

IN)

Table S. Reactivity of Hsp7*', Hsp7QA and IHsc7O An tihoodits with 6SkDA (Hsp7GB t Protein (rcim Unstressed and Heat 0- 0 Cn z p 0,

(D

U

CD

'C

Antibody Antgcn Eptlape Mml lieu HrLa HS Jurkai Jurltat 145 1 4,11 A431 11$ Designuion jNumber I Co 624-638 N/A +t 3 U 3Cl- CA 561-573 N/ A 00 0 .5 a CD 361-576 N/A 0 '3 0 -3 0 Not tetedt cc 546-559 W/A 3 (3 +5 a M T 1-12 3 43 1 *3 No: fwd Not tested *ECB 61 -63 1 Ci3 Not testd Not :eswdA Not lested Not tnted 2 Ct3 NtmIested Not estwcl 'Vol toled 1%10 Nezted 3 03 WoN tWSWe Not keSted Not lowtd Not tested *TCO 624-635 1 0 2 NoL lested Not tested N14m tested Not tested 2 0 Nut testcd Nlot Nested Not Bibed Net testd 1 Ct Not tested Not tL-deIdL Nt hteid Not tested sCE SWO56 1 0 1 Notltested: Nottiested 0 2 (t 1 Ntot testedl No ettstid Not twsed Not IeWte 3 M1t: tsk Nottted prW INtolied "-Jt tested 2 2 C82 (Imbl) 624-639 NJA Notteeled Not te~ed 03.C3 O (Roil) 2441 1 at I1 No tesed No: tesed Noin oted Net tesed &C62 (murio) 643 1 0 3 Nut tested Not tested NNt woed Not Jested 2 03 Not tesitd No! toted No: Beste Nut testd 3 02 Nut tested Noft itetd Poi tested Met tested 4 1 Not boiled Not tesled No: lesbeJl Not tesked a tt oN teted Not tested Not lotted Nol teSted Y03 WI' I SP708 N/A Not totted Not tested Not tested NO? tesed 0 2 1 4'4 SWA-St0 Nsp7DA N/A *2 .3 0142' 437-(X4: SPAI~l Hsp7CA N/A .113* 4 2' Multi]* S1'A-Nl Hr7t N/ A 4+3*34 3' Unknown t

C

a.

ed Sd 0

C,

-i 9,

C

C

Ia Ia 06-12-'05 17:36 FROM- T-751 P051/078 F-502 WO 01142423 Ir PCTR/USO n3341

O

0 In Table 5. reactivity levels were assessed by imnunoblotting analysis of 10 Slysate. Intensity of antigen specific bands was scored on a relative scale where 0 no signal and 3 a very strng signal. Relative reactivity levels are reported for unpooled NO serum antibodies from individual animals. Relative reactivity levels are reported for O 5/10 mice. Relative reactivity levels are reported for pooled serum antibodies. Shaded areas represent inconclusive binding to Hsp70B'. N/A in the Animal Number column indicalcs purified antibody.

0 Ci- 44 COMS ID No: SBMI-02132445 Received by IP Australia: Time 17:45 Date 2005-12-06 2005242130 06 Dec 2005 Attorney Docket No.: 12071-006001 Table 6. Reac ity of HsP Ant lbodics "it h Control an~d Heal S1lressnd' Marma ll9n Cell L, sates Antibody Locatiosi A431 A431 Ve-ro V-110 CHlO CHO MS MDUK PMDIX H1 +3I ah7B 0 0 0 0 0 624Q68 CE HSP7CF, 0 +3 0 0 0 0 0 0 553-567 1__ 708, WP Hsp7OB' 0 +2 a0 0 0 a1 a 1-643 SPA- IUl HsplI0626- +2 1 +2 +2 -2 +2 SPA-870 HspMO +2 +2 +3 0 +2 +1+3 437-5"4 I I

I_

SPA-812 HsIp7ICA +2 +3 +2 +3 0 41 +12 Multiple HWsc7C +3 3 +3 +33 +3 +3 3 Unplnown

I

SPA-825 (vr,75 +3 +3 +3 +3 +3 +,1 Uniknown SPA-827 KDEL +1 +2 *2 +3 +1 649-654 SPA-MO 0aL a 0 0 UnknownI SPA-895 1 11SP71 0 0 0. 0 1Unknown 1 Reactivity levels Were assessed by inimunoblotting analysis of 10 20mg, lysate. Iitnsity of anfigen specific bands was scored onI a relative scale where 0 no signal and 3 a vct strong signial. "Relative re activity levels are reported for unpooled strum antibodies fronm individual animnals, J Relative reactivity levels art reported for 5110 inice. 1 Rcaive reactivity levels are reporteil for pooled scrumn antibodies. Shiaded areas reprsen~st inconclusive binding to Hsp7OB.' N/A in the Animal Number column indicates purified antibody,

U,

171 2005242130 06 Dec 2005

SI

"3

I

SI

Lii -23 1.11 0 Table 7: Summary of Rabbit flsp70B'Anibody Specificity Antibody Name Location 14sr,7031 Antiody ocaton ethod for Seecificity Assmessit Ftelative 7itre Index Cwnpelition EIA Ionmurloblot CB 624-638 NI) ND SpCdi for reconibinaulI and native 1-sp7CD' CA 561-573 ND) ND detects other proteins in cell lysates; i. e. not spEc ific CD 561-576 ND) ND0 cross reactivity with other H-SP70 family mrembes arid cellular proteins; ILe. not specific cc 546-559 NID ND detects other proteins in cellilyates; i.e. ntoH specific Nr 1-12 ND ND detects other, HSP71 family mnembers iCe. not specific ECS 63843m Specific for HSP7OB' ND One blee specific for J-sp7QB'; other bleedts weakly detect other proteins. but can likely be diluted out iTC 624-6M5 SpeCifiC for i-SP70B0 ND) Not specific; 2/3 bleeds did mat detect native C. 553-567 Specific for Hsjp7OB Specific for Ilisp7QW One bleed a eciFic for Msp705;, 2/3 bWeeds not spelific CBZ 624-63 Specffc far Hsp7OW' Speci for H-sp7OW Specific for Hsp>7l' 705' WI 1-643 Not specific for Hsp7]W at Specific forHsp70B' when Specific for Hs 2 208' when used at a high dilution I lowerdiations usdaah iuin Relative reactivity levels were assessed by Western blouig analysis of iOjisg of total cell lysate. Immune bls were probed with the H-spOB antibodies, C82, CE and 7W3I WP, as well as anti-I-Isp antibodies speri fic for HsplI 10, Hsp7OA, 0rp75, KIJEL. flaK and H-sp71. The CE atibody used for this study was the scre specific Foy HsplOBW from one rabbit.

Immunoblots were developed by ECL. Intensity of the antiasnspecific bands was scored -on a relative scale where 0 =no signal detected and I3 a very strong signal.

06-12-'025 17:37 FROM-T-5.P5/7F-0 T-751 P054/078 F-502 WO 01/42423 IPCTAUSOOMMIe Hisp 70B'Profrein:- Disribuion andlducaion Conditions. As can be seen in Tables S.,6 and 8, ihe l-spo7OD' antibodies (NT, CA, CB, CC, CID, ECB. TCB, CB2, CE anid WE') detect the 'Hsp7OB' protein Drnly in stressed human tissues, no reactivity is detected in non-stressed cells or tissues.

Table 8. Reactivity of Anti-Hsp7OR' Antibody with a Panel of Different Cell Cell Line Description Species Reactivity Ratvt Designtion (nkon- (stressed) stressed) HeLa Epatluelaid rarcinrna, cervixt H-uman Negative +13 A431 Epidermoid carcinoma Human Negative 1+3 Juricat AdultT-elleuke-wi, ua egle 1-I Neurogliomna, brain Human Negative +3J MCFY Breast adenocareinoma, Pleural effusion Hiuman NegRative +(weak) HMCMammairy epithelial cells (normal) Human Negative Not tested HRERenal cortical epithelial cells (normnal) Human Negative Not tested RPTZEC Renal proximal tubie epithellal calls Human Negative Not tested (normal) NSMBE Bronchial epithelial cells (normal) Human fNegative Not tested Prostate epithelialicells (normal) Human TNegative Nat tested NHEK-Ad Epidermal keratiaocytes; adult (normall) Human lNegative Not tested M-EI-Neo Epidermal keratinocyes neo (normal) Human fNegative INttse lNtHEF-Neo Epidermal keratiuiocytes flea pool Human Negative Not tested pool (normal) NHDP-Ad Dermal fibroblast adult (normal) Human Negative Not lae NHDP-Neo Dermal fibroblasr.o (niormal) H kuman INegative Not tested COMS ID No: SBMI-02132445 Received by IP Australia: Time 17:45 Date 2005-12-06 06-12-' 05 17:37 FROM- T71 P5/7 -0 T-751 F055/078 F-502

I.

WO O1/4Z423 IPCT/US00133342 Cell Line Description SpcelM VEC-d Ad Microvas9cular end othelial adult Human HNIVEC-d Microvascufa 1 endothijal neo (normal) [-uinan Reactivity (non- ::::sed Reactivity (stressed) Not tested Nt teste d NHiEM MlnctsMo(oml I- I

I

l-PAEC TPulmonary artery endothelial cells I(normal) Human Negative Negative Not tested Not tested HAEC Coronary artery endotheial cells- Human (normal)I HLEC J Liac artery OndothelVol cells (normal) 4 IHuman Negative N-01 tested HAC Aertie endotheial cells (normal) HMVE-C.*L Lung microvascular endotheliol cells (normal) Not tested Human Negative Negative HLNLEC tUmbilical Vein CJIColhediJI cells (normal) lHuman JNegative Not tested No.t tested No -tted Not tested1 liUA Umbilical artery enldothelial cells ESC (normal) AoSMC Aortic artery smooth muscle cells (normal) 1IS M C B o n c h i l ftr a h ia l s m o th m u s c le c e lls (normal) Hluman Negative i fmi Human Negative Nut tested i I1 Hluman Negative Not tested 4CASMC j 1 Coronaruy artery smooth muscle Cells 4 4 Human Negative (normal) PASMC Pulmonary artery smoothiflmuscle cells- Iuma. Negative Not tested NottMated (normal) COMS ID No: SBMI-02132445 Received by IP Australia: Time (Him) 17:45 Date 2005-12-06 06-12-'05 17:37 FROM- T71P5/7 -0 T-751 F056/078 F-502 1. WVO 01142423 Cell Line Designation U AS MC UtSVC PcrfUSOW33i) Uterie smooth muscjl cells (normal) Skeletal Muscle cells (normal) Liver (normal tfssue%) Species

HUMA,'

Human Human Human Lung (normal tissut) Human Brain (nrmrnal tissue) Human Kidney (normal tissue) 1-iwman Tesmis (normal tissue) Human Ovary (normnal titSue) -ua Heart (normal tisue) Human.

Spleen (nornial t i ssue)i Humanm VERO Kidney Monkey L929 Connective tissue Mouse 3T3 Embryo Mouse aO Ernbryo Rat GPC-16 Colon. adenocercinoman Guinea pig MDDK Kidney Sheep MF4DBK Kidney Cow ESIC4 Kidntey (Pig Reactivity (Ponstressed) Negative Negative Negative Negative Negative Negativie Negative Negative Negative Negative Negative, Negative Negative Negative Negative Negative Negative Negative Negative teactivity (sfrcssed) Not tested Not ktstd Not tested Nttested Not tested Not tested Not tested Not tested Not tested Not tested Not tested Negative Negative Negative Negative Negative Negative Negafive Negativej COMS ID No: SBMI-02132445 Received by IP Australia: Time (I-tm) 17:45 Date 2005-12-06 06-12-'05 17:37 FROM- T-751 P057/78 F-502 WO 01142423 PCTJUSO/33341 O As can be seen in Tables 3 and 4 the Hsp70B' antibodies (NT, CA, CB. CC and CD) detect the Hsp70B' protein only in stressed human tissues, no reactivity is detected in non- Sstressed cells or tissues. Eight different normal human tissues. 27 samples of cell line lysates derived from normal human sources, five samples of cell line lysates derived from ncoplastic O 5 tissues and seven other mammalian species were evaluated for the expression of the Hsp7OB' 0 protein. The Hsp70B' protein was not detected by the anti-Hsp70B' antibody "CB" in the 48 samples obtained from "normal" or "unstressed" cells. If these cells are exposed to elevated Stemperarures, the Hsp7OB' protein is expressed in all of the heat-shocked human cell lines evaluated. These results are consistent with earlier findings at the mRNA level, using specific l 10o oligonucleotide probes (Leung et al., Biouhem. 267:125-132, 1990; Leung et al., Genomics /2:74-79, 1992). These investigations did not detect the presence of any hsp70B' mRNA in the t) unstressed cells.

o The temperature threshold ofHsp70B' protein expression in heat treated C HcLa cells following recovery for 0, 2.5, 5, 16 and 24 hours at 370C was is investigated. In this study the level of expression of the Hsp70B' protein was greatest at the 16 hour limepolnt. Therefore if a signilicant level of Hsp7DB' protein was detected, this would imply that the cells or individual tested was cither currently or had recently been exposed to a significant stressor and not due to a stressful situation occurring far in the past. The stress response does, however, persist long enough to be potentially useful as a diagnostic probe. The persistence of the Hsp70B' protein in cells following stress is similar to the results described for Hsp70A protein in recovering peripheral blood lymphocytes (Bratton er r.

J. Hyperlhermia 13(2):157-168, 1997). These investigators found that the protein persisted for at least 48 hours and reached maximal expression at 12 hours.

COMS ID No: SBMI-02132445 Received by IP Australia: Time 17:45 Date 2005-12-06 2005242130 06 Dec 2005 Attorney Docket No.: 12071-006001 Table 9. Investigation oF the Induction" of MscOO Hsp70A and 1-sp7OB' Protins$ in Human Cell Lines Under Differeit Induction Conditions Cell Line HeIR Cel1-c JurkatCelb A431 C~lls Antigen Hsc7O I4sp7GA/ I-bp7O HsIp7'0B HsOcO 1-isp7GA/ H5V70 H-sp7VCU MOcO Hqp7rJA/ Hsp7D Hsp7CB* A A 1, A Non-stessed 4,3 t3 0 +3 +2 +2 (1 (430C) 20 nin. +3 +2 +2 +1 +3 t-2 +2 +3 +3 *l-3 +2 +3 Heat (430C) 120 miun. 13 +3 +3 4-3 +3 +2 +3 +3 03 +2 4+3 Azetidine (5pM +3 43 +3 +7 1,3 +2 +2 147 -4-3 +2 CdCIZ ('I0 1 i m, 2 +3 1-3 +3 i+2 +2 +2 +3 +3 +3 4-2 -'1 hours) ZmuCI2 (250 pM, 2 +3 +3 +3 +1 +3 +1 +2 +1 +3 43 +2 1+2 PRelative proteirf levels as asss by Westcrm blotting analysis of 2Oug of toial cdAl lysafe. imeiisity orthe antigeti specifie bands was scored on. a relative scale where O= no signal deiected and 3= a very strong signal 00 *11 06-12-' 05 17:32 FROM- T71 P5/7 -0 T-751 F059/078 F-502 WO 01/42423 PCTUSOO/3334j o J nObIots were caoried out Using the following antibodies. SPA-S 15 Cl-sc70), SPA- 810 C1-sp7OA and H-sp7OB'), SPA-RU2 (Hsp7OA, Hsp7OBW) and CB (Hsp7OB'). Polyclonsi antibodies to the CA and CC Hsp70BW epitopes were also checked for reactivity with these cell lines under these conditions. The relative levels of the Hsp7w3' proteins measured with these INOs antibodies agree in all cases with the profiles seen in ininunoblots with the anti-GB antibody.

0 Table 10. Dose Response Cur" Showing the Relative Induction of Human Hsp7G Family Members in licLa Cells N-eat Stressed at Differ-ent Temnperatures.

I-eatshock H-sc7O H-sp70A H-sp7OA 14-p7OB'/ I4 703sp7OB' temtperature /11sp70B' H-sp7OB /Hsp701l Hsp7OB S _____SPA-815 SPA-S10 SPA-B12 CB CA CC Cl37 3 3 2 0 a0 33.S 3 3 2 0 0 0 3. 3 2 a lo 0 41.5 33 2 2' 2 43 3 3 2 3 33 4453 3 2 333 Relative protein levels as assessed by Western blotting analysis of 20t12 of total cell lysaze. Intensity of the antigen specific bands was scored on a relative scale where 0- no signal 1o detcted anid 3- a very strong signal) The relative induction pf the 1-se7O, Hsp7OA and H-sp703' proteins under different conditions were evaluated in HeLa, .lurkat and A-431I human cell lines (Tables 9 and 10). It can he seen that the induction of the H-sp7OBW protein was different than that seen for other HSP7O isobna. l-spOB3'protein was not present in unstressed cells. and was induced only iii respause to cellular stress. The expression of thr consitutive H-se7O protein is not affected by exposure to the praline analogue azetidine, to the heavy metals CdCla or ZnC12 or to increascd tzmperaiurc.

The inducible Hsp7OA stress protein appears to be expressed at high levels basal levels in 52 COMS ID No: SBMI-02132445 Received by IP Australia: Time (I-tm) 17:45 Date 2005-12-06 06-12-'05 17:38 FROM- T-751 P060/078 F-502 WO 01/42423 PCT/USO03334 Sunstressed cellsas has been found previously by other investigators (Turman et al., Biochemical C< and Molecular Medicine 60:49-58, 1997). Changes to the level of Hsp70A protein expression U was found to be obscured in these investigations by the high basal level of expression of this protein. The induction as a result of stress treatment is consistent with published findings. The \N 5 extent to which Hsp70A expression is induced by heat shock is inversely correlated with initial O levels of Hsp70A (Tunnan et al., Biochemical and Molecular Medicine 60:49-58. 1997).

Therefore, in human cells, high basal expression of Hsp70A may prevent further induction of after heat shock.

f The Hsp70B' protein was not present in unstressed cells. Slight temperature increases up C to to 400C did not elicit a response. Once the threshold of 41.5C was reached, all three human cell Cl lines (Hela. Jurkat and A-431) responded by expressing die Hsp70B' protein. This induction threshold of Hsp7OB' expression is different than the threshold described previously in which mRNA levels were measured using specific oligonuclcotides (Leung et al., Biochem. J.

267:125-132, 1990). These investigators reported that hsp70B' mRNA was strongly induced at is 45"C and was not detectable after 42"C treatment. Furthermore, they found only trace amounts of hp70B' mRNA after CdCI 2 trcaunent in contrast to the protein data reported here (Table The Hsp70B' protein was shown to be induced by the proline analog azetidine as well as the heavy metals CdCl2 and ZnCI 2 all known inducers of sress proteins. This discrepancy in the induction threshold as determined at the genomic and protein levels is likely due to the inherent technical differences in the two techniques. Previous studies looking at regulation of the hp7OA and hsc70 genes have not yielded a consistent set of results (]lansen el al. Exp. Cell Research 192:587-596, 1991; Mangurten et al., Cell Sress Chaperones 2(3):168-174, 1997).

Depending on the particular system and at what level expression was examined, expression can either increase or decrease following treatment with.agents to induce differentiation. In studies of the differential expression of Hsp70A in wound healing, it was found that whilst hsp7OA mRNA did not show significant correlation with healing, a strong correlation was seen between well healing wounds and expression of the Hsp70A protein (Oberringer et al., Biochemical and Biophysical Research Communications 214(3):1009-1014, 1995). It would be preferable to measure the protein, since the protein response persists whereas the mRNA has been reported to have a very short half-life (Bratton ct al.. Int. J. Hyperihermia 13(2):157-168, 1997).

53 COMS ID No: SBMI-02132445 Received by IP Australia: Time 17:45 Date 2005-12-06 06-12-'05 17:38 FROM- T-751 P061/078 F-502 WO 01/4243 PCT/USI/333 I O At the level of transcription the hsp70A gene is regulated through ranscription factors C' other than HSF's under non-stressed conditions and therefore, the Hsp70A protein is detected even in the physiological state (Hansen et al., Exp Cell Research 192:587-596, 1991). The gene is regulated exclusively by the association of HSFs and the heat shock clement, \O S allowing no constitutive expression (Suzuki et al. Radiation Research 49:195-201, 1998). The 0 promoter regions of/hp70B' and hsp70B genes differ extensively from the hsp70A gene in that they lack TATA and CAAT boxes which are believed to contribute to the basal expression of 0 Hsp70A (Wu et al., Proc. Natl. Acad. Sci., USA 83(3):629-633, 1986; Greene et al., Mol. Cell CBiot 7(10):3646-55, 1987). The two hsp70B DNA homologs display differences in their I regions as well as several changes within key promoter sequences. The hsp7OB' gene has been cl shown to have a 19-nucleotide-residue insertion in the hsp70B gene that lies within the heatshock element of the hsp70B DNA sequence (Leung et al., Genomics 12:74-79. 1992). Elevated O mRNA levels do not always translate into increased protein levels due to regulation at the transcriptional and/or post-transcriptional levels (Obeninger et al., Biochemical and Biophysical Research Communications 214(3):1009-1014, 1995). Using the anti-Hsp70B' antibodies in immunochemical testing procedures, therefore, allows a more sensitive, longer lasting quantitative evaluation of Hsp70B' protein levels than that found using specific oligonuclcotides to evaluate mRNA levels.

The Hsp70B' protein has been shown to be expressed only following significant stress on the cell or organism, whether this stress is caused by elevated heat or exposure to heavy metals or toxic chemicals. The Hsp70B' antibodies of the present invenion offer a unique opportunity to use these naturally occurring biomarkers to evaluate the stress on a system. The nature of the sress need not be known. Monitoring the Hsp70B' biomarker provides a prognostic indicator of the general "wellness" of the cell or organism and indicate when a significant perturbation has occurred. As biomarkers, Hsp's provides sensitive early-warning of toxicity, perhaps allowing intervention at an earlier more tractable stage of the problem.

All publications and patent applications mentioned in this specification are herein incorporated by reference to the same extent as if each individual publication or patent application was specifically and individually incorporated by reference. From the foregoing, it so will be evident that, although specific embodiments of the invention have been described herein 54 COMS ID No: SBMI-02132445 Received by IP Australia: Time 17:45 Date 2005-12-06 06-12-'05 17:39 FROM- T-751 P062/078 F-502 flrifTIhyiel ,.ig _4M| 55 for purposes of illustration, various modifications may be made without deviating from the spirit and scope of the invention.

Throughout this specification and the claims which follow, unless the context requires otherwise, the word "comprise", and variations such as "comprises" and "comprising", will be understood to imply the inclusion of a slated integer or step or group of integers or steps but not the exclusion of any other integer or step or group of integers or steps.

The reference to any prior art in this specification is not, and should not be taken as. an acknowledgment or any form of suggestion that that prior art forms part of the common general knowledge in Australia.

€9 r a k *r *r COMS ID No: SBMI-02132445 Received by IP Australia: Time 17:45 Date 2005-12-06

Claims (11)

1. 06-12-'05 17:39 FROM- T-751 P063/078 F-502 o-56- -THE CLAIMS DEFINING THE INVENTION ARE AS FOLLOWS: Va O 1. An antibody that specifically binds a peptide consisting of the amino acid sequence GSLQEESLRDKIPEE (SEQ ID NO: 2, The antibody of claim 1, wherein the antibody is a monoclonal antibody. 3. A kit comprising the antibody of claim 1. 4. The kit of claim 3, further comprising an Hsp70B' protein or an Hsp70B' peptide. A method of obtaining the antibody of claim 1, the method comprising administering to an animal a peptide consisting of the amino acid sequence GSLQEESLRDKIPEE (SEQ ID NO: I0). 6. A method of obtaining the antibody of claim 1, the method comprising administering to an animal a peptide consisting of the amino acid sequence GSLQEESLRDKIPEE (SEQ ID NO: 10) and a carrier that enhances the immunogenicity of the peptide and, optionally, a linker between the peptide and the carrier.
2. 7. The method of claim 5, wherein the antibody is a monoclonal antibody.
3. 8. A method of determining whether a cell has been exposed to a stressful environment or a stressful substance, the method comprising performing an immunoassay in which proteins in or on the cell or proteins extracted from the cell are exposed to the antibody of claim 1, wherein binding between a protein in or on the cell or a protein extracted from the cell and the antibody indicates that the cell has been exposed to a stressful environment or a stressful substance.
4. 9. The kit of claim 3, wherein the antibody is a monoclonal antibody. COMS ID No: SBMI-02132445 Received by IP Australia: Time 17:45 Date 2005-12-06 06-12-'I05 17:39 FROM- r.PEP%70I241Owv cIim-d&-191 MJ0 T-751 P064/078 F-502 -57- The kit of claim 3, further comprising instructions for use. 11, The kit of claim 4, further comprising instructions for use.
5. 12. The kit of claim 4, wherein the antibody is a monoclonal antibody.
6. 13. The kit of claim 10, wherein the antibody is a monoclonal antibody.
7. 14. The kit of claim 11, wherein the antibody is a monoclonal antibody. The method of claim 6, wherein the carrier is keyhole limpet hemocyanin.
8. 16. The method of claim 6, wherein the antibody is a monoclonal antibody. 17, The method of claim 15, wherein the antibody is a monoclonal antibody.
9. 18. The method of claim 8, wherein the antibody is a monoclonal antibody.
10. 19. The method of claim 8, wherein the antibody has a relative titre index greater than one. The method of claim 5, further comprising collecting blood from the animal.
11. 21. The method of claim 20, further comprising purifying the antibody by immunoaffinity. COMS ID No: SBMI-02132445 Received by IP Australia: Time 17:45 Date 2005-12-06