2. BACKGROUND OF THE INVENTION In modern medicine, immunotherapy or vaccination has virtually eradicated diseases such as polio, tetanus, tuberculosis, chicken pox, measles, hepatitis, etc. The approach using vaccinations has exploited the ability of the immune system to prevent infectious diseases. Vaccination with non-live materials such as proteins generally leads to an antibody response or CD4+ helper T cell response. (Raychaudhuri and Morrow (1993) Immunology Today 14: 344). On the other hand, vaccination or infection with live materials such as live cells or infectious viruses generally leads to a CD8+ cytotoxic T-lymphocyte (CTL) response. A CTL response is crucial for protection against cancers, infectious viruses and certain bacteria. This poses a practical problem, for, the only way to achieve a CTL response is to use live agents which are themselves pathogenic. The problem is generally circumvented by using attenuated viral and bacterial strains or by killing whole cells which can be used for vaccination. These strategies have worked well but the use of attenuated strains always carries the risk that the attenuated agent may recombine genetically with host DNA and turn into a virulent strain. Thus, there is need for methods which can lead to CD8+ CTL response by vaccination with non-live materials such as proteins in a specific manner. It has been discovered that heat shock protein complexes have particular utility as vaccines against cancers and infectious diseases. (Srivastava et al., (1994) Curr. Op. Immu. 6: 728 ; Srivastava (1993) Adv. CancerRes. 62: 153).

Heat shock proteins are proteins synthesized by a cell in response to heat shock.

Heat shock proteins fall into families such as but not limited to the families HSP25/HSP27, HSP60, HSP70 and HSP90 where the numbers reflect the approximate molecular weight of the stress proteins in kilodaltons. Heat shock proteins are capable of binding proteins or peptides, with which they form complexes endogenously in cells or in vitro under the

appropriate conditions. (Nair et al. (1999) J. Immun. 162: 6426; Flynn et al. (1989) Science 245: 385; Blachere et al. (1997) J Exp. Med. 186-1315).

Immunization of mice with the gp96 complex or hsp84/86 complex isolated from a particular tumor rendered the mice immune to that particular tumor, but not to antigenically distinct tumors. Isolation and characterization of genes encoding gp96 and hsp84/86 revealed significant homology between them, and showed that gp96 and hsp84/86 were, respectively, the endoplasmic reticular and cytosolic counterparts of the same heat shock proteins (Srivastava, et al. (1988) Ifyafmunogenetics 28: 205; Srivastava, et al. (1991) Curr.

Top. Microbiol. Immunol. 167: 109). Further, hsp70 complex was shown to elicit immunity to the tumor from which it was isolated but not to antigenically distinct tumors. However, hsp70 complex depleted of peptides was found to lose its immunogenic activity (Udono and Srivastava (1993) J. Exp. Med. 178: 1391). The observations revealed that the heat shock proteins are not immunogenic per se, but are carriers of antigenic peptides that elicit specific immunity to cancers (Srivastava (1993) Adv. Cancer Res. 62: 153).

This phenomenon has been observed in both tumor and viral models with known and unknown antigens (Srivastava, et al. (1998) Immunity 8: 657; Ciupitu, et al. (1998) J.

Exp. Med 5: 685 ; Arnold et al. (1995) J. Exp. Med. 182: 885). The presence of an antigenic peptide bound to gp96, hsc70, and hsp84/hsp86 has been structurally demonstrated in cells for which the antigenic peptide is known (Nieland, et al. (1996) Proc. Nat'1. Acod. Sci. USA 93: 6135; Breloer, et al. (1998) Eur. J. Immunol. 28: 1016; Ishii et al. (1999) J. Immunol.

162: 1303). Vaccination with heat shock protein complexes is applicable for both the prophylactic (Srivatava, et al. (1986) Proc. Nat'l. Acad. Sci. USA 83: 3407; Ullrich, et al.

(1986) Proc. Nat'l. Acad. Sci. USA 83: 3121; Peng, et al. (1997) J. I. Meth. 204: 13; Basu and Srivastava (1999) J. Exp. Med. 189: 797) and therapeutic treatment of cancer (Tamura et al. (1997) Science 278: 117; Yedavelli, et al. (1999) Int. J : Mol. Med. 3: 243) and for the treatment of infectious diseases (Ciupitu et al. (1998) J. Exp. Med. 5: 685). The translation of this approach to immunotherapy of human cancer is currently under investigation using either gp96 complex (Janetzki, et al. (l998) J. Immunother. 4: 269; Amato, et al. (1999) ASCO meeting, abstract 1278; Lewis, et al. (1999) ASCO meeting abstract 1687) or hsp70 complex as an autologous vaccine and the individual patient's cancers as a source of the heat shock proteins (Menoret and Chandawarkar (1998) Semin. in Oncology 25: 654).

Previous methods for purification of the immunogenic heat shock protein complexes lead to the isolation of a unique heat shock protein species while neglecting the rest of the sample that could be used as a complementary source of heat shock protein-based vaccine.

Therefore, it is an object of the invention to provide a method for collecting multiple heat shock proteins and heat shock protein complexes from a limited sample source.

3. SUMMARY OF THE INVENTION The present invention provides methods for recovery of hsps and hsp complexes which is based on the different binding affinities of the various hsps for heparin.

In one embodiment, the methods of the invention can be used to recover one or more hsp and/or hsp complexes from a sample comprising contacting the sample with heparin such that the hsps and/or hsp complexes bind to heparin, and eluting the bound hsps and/or hsp complexes with buffer at various salt concentrations. Hsps such as hsp60, hsc70, hsp90, gp96, and hsp70 bind heparin and can be eluted at different salt concentrations. For example, the hsps can be eluted from a heparin-agarose column using a linear NaCl concentration gradient. The method thus allow the concomitant recovery of multiple hsps and hsp complexes form a single sample.

Heat shock protein-based vaccines have been shown to provoke an immune response against cancer and infectious diseases in both prophylactic and therapeutic protocols. Thus, in another embodiment, the methods of the invention can be used for preparing a vaccine composition, comprising the steps of contacting a sample comprising antigenic hsp complexes with heparin, and eluting the bound antigenic hsp complexes from heparin with buffer at the appropriate salt concentrations. The eluted hsp complexes can be used to make vaccine compositions for administration to a subject in need of treatment or prevention of a disease. Depending on the disease, the sample comprising antigenic hsp complexes can be a lysate of cancer tissues, tumor cells, infected cells from biopsies, or transformed or transfected cells from cell cultures. However, in instances where the hsp complexes are for autologous use, the source of antigentic hsp complexes is not available in large quantity, thus the methods of the invention are particularly efficient for recovering the majority of these heat shock protein complexes from a limited amount of sample.

In another embodiment, the heparin-based methods of the invention can also be combined with other processes known in the art to further purify the individual species of hsps and hsp complexes. The methods of the invention can serve to enrich for the desired species prior to the purification process.

4. BRIEF DESCRIPTION OF THE FIGURES FIGS. 1A-B. Profile of fractionation of 100,000 g supernatant by heparin-Sepharose chromatography.

FIG. 1A : Chromatographic profiles of proteins eluted from heparin-Sepharose. The elution profile of the column was developed with a linear gradient of 0-1M NaCl in 20mM Phosphate Buffer at a flow rate of 1 ml/min and 23 fractions were collected. The absorption

of the proteins was recorded at 280 nm (right axis), the salt concentration was measured in mS and reported on the left axis.

FIG. 1B : Analysis by silver staining of 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) of : the starting sample (100,000 g supernatant of MethA lysate), the material that does not bind the heparin-Sepharose column (Heparin Unbound), the fractions 6-16 (F6, F7, F8, F9, F 10, F 11, F 12, F 13, F 14, F 15, F 16) and the proteins that remain bound to the column after elution (Beads).

FIGS. 2A-I. Immunoblot analysis of the fractionation on heparin-Sepharose column. The same samples as in Fig 1B: 100,000 g supernatant, heparin unbound, fractions 6 tol6 and heparin-Sepharose beads were resolved on 10% SDS-PAGE, transferred on PVDF membrane and probed with antibodies specific for hsp40 (A), hsp60 (B), hsc70 (C), hsp84 (D), hsp86 (E), gp96 (F), hsp70 (G), calreticulin (H) and BiP (I). The membranes were probed with appropriate secondary peroxidase-labeled antibodies and developed by chemiluminescence.

FIGS. 3A-C. Purification of hsp90.

FIG. 3A: Chromatogram of the elution of the HQ-poros column by a linear gradient of 200mM to 600mM NaCl in Phosphate Buffer. The absorption of protein was recorded at 280 nm on the left axis; the salt concentration was measured in mS and reported on the right axis.

FIG. 3B: Analysis by silver staining of 10% SDS-PAGE of: the starting sample (F7 to F9 of the heparin chromatography), the material that does not bind the HQ-poros column (HQ Unbound) and the fractions 4 to 15 (F4, F5, F6, F7, F8, F9, F10, Fl l, F12, F13, F14, F15).

FIG 3C: The same samples as in Fig. 3B were resolved on 10% SDS-PAGE, transferred on PVDF membrane and probed with antibodies specific for hsp86.

FIGS. 4A-D. Purification of gp96.

FIG. 4A: Analysis by silver staining of 10% SDS-PAGE of the ConA-agarose chromatography step. The starting sample correspond to the fractions 10 tol2 of the heparin chromatography and the ConA-unbound fraction is the material that is not retained on the column ; the fractions 1 to 8 were analyzed.

FIG. 4B: Immunoblot analysis of the samples described in Fig. 4A using a monoclonal antibody specific for gp96.

FIG. 4C: Analysis by silver staining of 10% SDS-PAGE of the DEAE-sephacel chromatography step. The starting sample corresponds to the fractions 1 to 6 of the ConA chromatography; the DEAE-unbound fraction is the material that is not retained on the column; the fractions 1 to 8 were analyzed.

FIG. 4D: Immunoblot analysis of the same samples described in Fig. 4C using a monoclonal antibody specific for gp96.

FIGS. 5A-D. Purification of hsc70.

FIG. 5:. Analysis by silver staining of 10% SDS-PAGE of the ADP-agarose chromatography step. The starting sample corresponds to the fractions 5 to 7 of the heparin chromatography, the ADP-unbound fraction is the material that is not retained on the column ; the fractions 1 to 8 were analyzed.

FIG. 5B : Immunoblot analysis of the samples described in Fig. SA using a monoclonal antibody specific for hsc70.

FIG. 5C : Analysis by silver staining of 10% SDS-PAGE of the DEAE-sephacel chromatography step. The starting sample corresponds to the fractions 1 to 4 of the ADP chromatography; the DEAE-unbound fraction is the material that is not retained on the column ; the fractions 1 to 8 were analyzed.

FIG. 5D : Immunoblot analysis of the same samples described in Fig. 5C using a monoclonal antibody specific for hsc70.

FIGS. 6A-B. Purification of hsp complexes produced in vitro by heparin-Sepharose chromatography. Hsc70 and gp96 (100 mg each) were complexed with the radio-labeled VSV-19 as described in section 6, applied in heparin buffer to two separate 1.5 ml heparin agarose columns, washed and eluted respectively with 350mM and 700mM NaCl.

FIG. 6A: Hsc70 containing fractions were analyzed by coomassie blue staining of 10% SDS-PAGE and by autoradiography of the same gel.

FIG. 6B: Gp96 containing fractions were analyzed by coomassie blue staining of 10% SDS-PAGE and by autoradiography of the same gel. The proteins were mixed in SDS-PAGE sample buffer but not boiled before electrophoresis.

5. DETAILED DESCRIPTION OF THE INVENTION The invention described herein provides methods for recovering heat shock proteins (hsps) and heat shock protein complexes (hsp complexes) in one step from a cell sample.

The methods of the invention can be used to recover hsps and hsp complexes that include but are not limited to species such as gp96, the hsp90 isoforms hsp86 and hsp84, the hsp70 isoforms hsp70 and hsc70, hsp60, and hsp40 and complexes thereof. The methods of the invention can also be used to prepare a vaccine composition when the hsp complexes recovered by the methods comprise immunogenic peptides.

Hsps are generally capable of associating with proteins or peptides to form hsp complexes. The term"heat shock protein-protein complex"or"hsp complex"encompasses

a molecular complex of hsp and protein; the term"protein"as used herein encompasses protein, polypeptide, denatured and improperly folded polypeptide, peptide, and fragments thereof. The protein associated with the hsp in a hsp complex can be a naturally occurring protein, a protein produced by a genetically engineered cell, or a synthetic protein. The proteins can become associated with hsps inside a cell, i. e., endogenously. Inside a cell, a protein is noncovalently associated with a hsp. Under the appropriate conditions, an hsp can also become associated with a protein in a reaction in vitro, i. e., exogenously. The methods of the invention can be used to recover hsp complexes comprising noncovalently associated proteins, as well as covalently linked proteins provided that the covalent linkage of the protein to the hsp does not alter the ability of the hsp to bind to heparin.

Hsps are among the most highly conserved proteins in existence. For example, DnaK, the hsp70 from E. coli has about 50% amino acid sequence identity with hsp70 proteins from eucaryotes (Bardwell, et al., 1984, Proc. Natl. Acad. Sci. 81: 848). The hsp60 and hsp90 families also show similarly high levels of intrafamilies conservation (Hickey, et al. (1989) MoL Cell. Biol. 9: 2615; Jindal (1989) Mol. Cell. Biol. 9: 2279). Because hsps are so highly conserved, the methods of the invention are applicable to homologous hsps in other species of organisms. Preferably, the hsps recovered by the methods of the invention are mammalian hsps. More preferably, the hsps are human hsps.

The preparation and use of a customized, autologous vaccine against the tumors of individual patients is now feasible using tumor-derived hsp complexes (Menoret and Chandawarkar (1998) Semin. in Oncol. 25: 654). Preliminary clinical trials with this approach have demonstrated that patients immunized with gp96 complex, derived from their own tumors, develop cancer-specific CD8+ T cell response (Janetzki, et al. (1998) J.

Immunother. 4: 269; Lewis, et al. (1999) ASCO meeting abstract 1687 ; Amato, et al. (1999) ASCO meeting abstract 1278). Clinical trials using autologous tumor-derived hsp70 complex are being conducted for the treatment of breast carcinoma and chronic low grade leukemia. Preliminary evidence of clinical responses to gp96 complex has also been obtained in a trial with patients with renal carcinoma (Amato, et al. (1999) ASCO meeting abstract 1278). These trials rely on a time-tested immunologic principle that anti-tumor immunity is generally private (Srivastava, et al. (1998) Immunity 8: 657; Berd, et al. (1999) Semin. Oncol. 25: 1315), justifying the use of a patient's own tumor as the source of the anti-tumor vaccine. Accordingly, the methods of the invention are particularly useful for the manufacture of vaccines comprising autologous hsp complexes.

In this context, the ability to obtain purified gp96, hsp86/84 and hsc70 complexes or other hsp complexes from the same tumor sample provides the opportunity for new clinical development. It will allow the exploration of the effect of a synergistic immunization with

multiple hsp complexes. The respective immunogenicity of each hsp complex can be tested under conditions where the different hsp-vaccines are truly comparable as they are derived from an identical source. These studies will also permit one to look comparatively and sequentially into the intracellular trafficking of proteins that are chaperoned by these hsps.

5.1. SEPARATION OF MULTIPLE HSPS AND/OR HSP COMPLEXES USING HEPARIN-AFFINITY CHROMATOGRAPHY Previously available methods for purification of immunogenic hsps and hsp complexes from a sample have lead to the isolation of a unique hsp species while neglecting the rest of the sample that might be used as a complementary source of hsp-based vaccine.

For example, Concanavalin A (ConA) column that retains gp96 as a first chromatographic step is not useful for for hsp70, hsp90 and calreticulin purification. See Menoret and Bell (2000) J. Immun. Methods 237 (1-2): 119.

The methods described herein have several advantages over previously available methods of isolating hsps and hsp complexes. The inventor of the present invention has discovered that heparin affinity chromatography can separate the major hsps and hsp complexes from a given cell sample. The methods of the invention involve contacting a sample containing different hsps or hsp complexes under certain conditions and for a sufficient period of time for the hsps to bind to the heparin, and eluting different hsps and hsp complexes from the heparin under different conditions. The basis of the methods is the distinct affinities of various hsps for heparin under different salt concentrations. As shown in the working examples, the association of peptides with hsps is preserved during heparin affinity chromatography.

In one embodiment, the present invention provides a method for recovering hsps and hsp complexes from a mixture, for example, from a lysate of cells. In another embodiment, the present invention provides methods for separating individually or in combination hsps or hsp complexes from a mixture of different hsps and hsp complexes. According to the invention, a sample containing different hsps or hsp complexes is mixed with heparin present on a solid phase under conditions that allow the hsps to bind to the heparin, for example, in the presence of a binding buffer of various salt concentrations. The bound hsps and hsp complexes can then be eluted from the heparin with an elution buffer containing a higher concentration of salt relative to the binding buffer. If multiple hsp or hsp complexes are bound to a heparin composition, bound individual hsp and hsp complexes can be sequentially eluted from the heparin by using a set of elution buffer compositions comprising an increasing amount of salt. Unwanted materials, including unwanted hsps that are bound and that can be eluted at a lower salt concentration, can be removed by

washing the heparin with one or more washing buffer compositions. Typically, the salt concentration of the washing buffer compositions are higher than that of the binding buffer but lower than that of the elution buffer compositions. Sodium chloride is the preferred salt for the methods of the invention.

In a preferred embodiment, the methods of the invention can be used to separately recover different hsps or hsp complexes from a single sample of biological materials. The binding of hsps and hsp complexes to the heparin on a solid phase is carried out at about OmM NaCl, i. e., no salt, and the elution of the various hsps can be performed sequentially by way of a sodium chloride concentration gradient starting from a low salt concentration to a high salt concentration, e. g., 0 to 1 M NaCl ; or a series of buffer compositions having salt concentrations ranging from low to high, e. g., 0 to 1 M NaCl.

In yet another embodiment, a single species of hsps or hsp complexes can be bound to heparin on a solid phase in a binding buffer having a first salt concentration, and eluted from the heparin by applying an elution buffer having a second salt concentration. The binding buffer has a lower salt concentration than the elution buffer. Standard methods for detecting and quantitating hsps and/or hsp complexes can be used to analyze the collected fractions.

According to the invention, hsp60 binds to heparin at zero or very low salt concentration, i. e., lower than 30mM NaCl ; bound hsp60 can be eluted from heparin by a buffer composition comprising at least 30mM NaCl, and preferably at about 235mM NaCl.

If different bound hsps or hsp complexes are to be eluted sequentially from the heparin, elution buffer containing from about about 30 to about 185mM NaCl is used to elute hsp60.

Therefore, the invention provides a method for recovering hsp60 or hsp60 complex from a sample comprising hsp60 or hsp60 complex, said method comprising contacting the sample with heparin present on a solid phase with a binding buffer at a salt concentration lower than 30mM NaCl, preferably close to OmM NaCl and eluting stepwise or by a concentration gradient hsp60 or hsp60 complex with one or more buffer compositions comprising from about 30mM to about 185mM NaCl. The invention also provides another method for recovering hsp60 or hsp60 complex from a sample comprising hsp60 or hsp60 complex, said method comprising contacting the sample with a heparin composition at a salt concentration of about OmM to 30mM NaCl and preferably at about OmM NaCl, and eluting hsp60 or hsp60 complex from the heparin using a buffer comprising about 235mM NaCl.

Hsp60 or hsp60 complex prepared from a cell sample by these two methods is substantially free of hsp84 or hsp84 complex, hsp86 or hsp86 complex, gp96 or gp96 complex, hsp70 or hsp70 complex, and hsp40 or hsp40 complex.

According to the invention, hsp84 binds to heparin at a concentration of NaCl at less than about 155mM. Bound hsp84 can be eluted from heparin in the presence of a elution buffer containing at least about 185mM NaCl, and preferably about 450mM NaCl. If different bound hsps or hsp complexes are to be eluted sequentially from the heparin, one or more buffer compositions containing from about 185 to about 400mM NaCl is used to elute hsp84. Therefore, the invention provides a method for recovering hsp84 or hsp84 complex from a sample comprising hsp84 or hsp84 complex, said method comprising contacting the sample with heparin present on a solid phase with a binding buffer at a salt concentration lower than 155mM NaCl, eluting stepwise or by a concentration gradient bound proteins including other hsps and hsp complexes with one or more buffer compositions comprising less than 185mM NaCl, and eluting stepwise or by a concentration gradient hsp84 or hsp84 complex with one or more buffer compositions comprising from about 185mM to about 400mM NaCl. Hsp84 or hsp84 complex prepared from a cell sample by this method is substantially free of hsp60 or hsp60 complex, gp96 or gp96 complex, hsp70 or hsp70 complex, and hsp40 or hsp40 complex. The invention also provides another method for recovering hsp84 or hsp84 complex from a sample comprising hsp84 or hsp84 complex, said method comprising contacting the sample with a heparin composition at a salt concentration at about 115mM to 155mM NaCl and preferably at about 135mM NaCl, and eluting hsp84 or hsp84 complex from heparin using a buffer comprising about 450mM NaCl. Hsp84 or hsp84 complex prepared from a cell sample by this method is substantially free of hsp60 or hsp60 complex, hsp70 or hsp70 complex, and hsp40 or hsp40 complex.

According to the invention, hsp86 binds to heparin at a concentration of NaCl at less than about 155mM. Bound hsp86 can be eluted from heparin in the presence of a elution buffer containing at least about 185mM NaCl, and preferably about 450mM NaCl. If different bound hsps or hsp complexes are to be eluted sequentially from the heparin, one or more buffer compositions containing from about 185 to about 400mM NaCl is used to elute hsp86. Therefore, the invention provides a method for recovering hsp86 or hsp86 complex from a sample comprising hsp84 or hsp84 complex, said method comprising contacting the sample with heparin present on a solid phase with a binding buffer at a salt concentration lower than 155mM NaCl, eluting stepwise or by a concentration gradient bound proteins including other hsps and hsp complexes with one or more buffer compositions comprising less than 185mM NaCl, and eluting stepwise or by a concentration gradient hsp86 or hsp86 complex with one or more buffer compositions comprising from about 185mM to about 400mM NaCl. Hsp86 or hsp86 complex prepared from a cell sample by this method is substantially free of hsp60 or hsp60 complex, gp96 or gp96 complex, hsp70 or hsp70 complex, and hsp40 or hsp40 complex. The invention also provides another method for

recovering hsp86 or hsp86 complex from a sample comprising hsp86 or hsp86 complex, said method comprising contacting the sample with heparin present on a solid phase at a salt concentration at about 115mM to 155mM NaCl and preferably at about 135mM NaCI, and eluting hsp86 or hsp86 complex from heparin using a buffer comprising about 450mM NaCl. Hsp86 or hsp86 complex prepared from a cell sample by this second method is substantially free of hsp60 or hsp60 complex, hsp70 or hsp70 complex, and hsp40 or hsp40 complex.

According to the invention, hsc70 binds to heparin at a concentration of NaCl at less than about 30mM. Bound hsc70 can be eluted from heparin in the presence of a elution buffer containing at least about 30mM NaCl, and preferably about 350mM NaCl. If different bound hsps or hsp complexes are to be eluted sequentially from the heparin, one or more buffer compositions containing from about 30 to about 260mM NaCl is used to elute hsc70. Therefore, the invention provides a method for recovering hsc70 or hsc70 complex from a sample comprising hsc70 or hsc70 complex, said method comprising contacting the sample with heparin present on a solid phase with a binding buffer at a salt concentration lower than 330mM NaCl, eluting stepwise or by a concentration gradient bound proteins including other hsps and hsp complexes with one or more buffer compositions comprising less than 30mM NaCl, and eluting stepwise or by a concentration gradient hsc70 or hsc70 complex with one or more buffer compositions comprising from about 30mM to about 260mM NaCl. Hsc70 or hsc70 complex prepared from a cell sample by this method is substantially free of hsp60 or hsp60 complex, gp96 or gp96 complex, hsp70 or hsp70 complex, and hsp40 and hsp40 complex. The invention also provides another method for recovering hsc70 or hsc70 complex from a sample comprising hsc70 or hsc70 complex, said method comprising contacting the sample with heparin present on a solid phase at a salt concentration less than about 30mM NaCl and eluting hsc70 or hsc70 complex from heparin using a buffer comprising about 310mM to 350mM NaCl. Hsc70 or hsc70 complex prepared from a cell sample by this second method is substantially free of gp96 or gp96 complex, hsp70 or hsp70 complex, and hsp40 and hsp40 complex.

According to the invention, gp96 binds to heparin at a concentration of NaCl at less than about 370mM. Bound gp96 can be eluted from heparin in the presence of a elution buffer containing at least about 400mM NaCl, and preferably about 650mM NaCl, If different bound hsps or hsp complexes are to be eluted sequentially from the heparin, one or more buffer compositions containing from about 400 to about 600mM NaCl is used to elute gp96. Therefore, the invention provides a method for recovering gp96 or gp96 complex from a sample comprising gp96 or gp96 complex, said method comprising contacting the sample with heparin present on a solid phase with a binding buffer at a salt concentration

lower than 370mM NaCl, eluting stepwise or by a concentration gradient bound proteins including other hsps and hsp complexes with one or more buffer compositions comprising less than 400mM NaCl, and eluting stepwise or by a concentration gradient bound gp96 or gp96 complex with one or more buffer compositions comprising from about 400mM to about 600mM NaCl. Gp96 or gp96 complex prepared from a cell sample by this method is substantially free of hsp60 or hsp60 complex, hsc70 or hsc70 complex, hsp84 or hsp84 complex, hsp86 or hsp86 complex, hsp70 and hsp70 complex, and hsp40 and hsp40 complex. The invention also provides a second method for recovering gp96 or gp96 complex from a sample comprising gp96 or gp96 complex, said method comprising contacting the sample with heparin present on a solid phase at a salt concentration at about 330mM to 370mM NaCl and preferably at about 350mM NaCl, and eluting gp96 or gp96 complex from heparin using a buffer comprising about 650mM to 700mM NaCl. Gp96 or gp96 complex prepared from a cell sample by this second method is substantially free of hsp60 or hsp60 complex, hsc70 or hsc70 complex, and hsp40 and hsp40 complex.

According to the invention, hsp70 binds to heparin at a concentration of NaCl at less than about 470mM. Bound hsp70 can be eluted from heparin in the presence of a elution buffer containing at least about 500mM NaCl, and preferably about 620mM NaCl, If different bound hsps or hsp complexes are to be eluted sequentially from the heparin, one or more buffer compositions containing from about 500 to about 570mM NaCl is used to elute hsp70. Therefore, the invention provides a method for recovering hsp70 or hsp70 complex from a sample comprising hsp70 or hsp70 complex, said method comprising contacting the sample with heparin present on a solid phase with a binding buffer at a salt concentration lower than 430mM NaCl, eluting stepwise or by a concentration gradient bound proteins including other hsps and hsp complexes with one or more buffer compositions comprising less than 500mM NaCl, and eluting stepwise or by a concentration gradient hsp70 or hsp70 complex with one or more buffer compositions comprising from about 500mM to about 570mM NaCl. Hsp70 or hsp70 complex prepared from a cell sample by this method is substantially free of hsp60 or hsp60 complex, hsc70 or hsc70 complex, hsp84 or hsp84 complex, hsp86 or hsp86 complex, and hsp40 and hsp40 complex. The invention also provides a second method for recovering hsp70 or hsp70 complex from a sample comprising hsp70 or hsp70 complex, said method comprising contacting the sample with heparin present on a solid phase at a salt concentration at about 430mM to 470mM NaCl and preferably at about 450mM NaCl, and eluting hsp70 or hsp70 complex from heparin using a buffer comprising about 610mM NaCl. Hsp70 or hsp70 complex prepared from a cell sample by this method is substantially free of hsp60 or hsp60 complex, hsc70 or hsc70 complex, hsp84 or hsp84 complex, hsp86 or hsp86 complex, and hsp40 and hsp40 complex.

Hsp40 does not bind to heparin under the conditions other hsps bind to heparin, and hence, according to the invention, hsp40 and hsp40 complex can be recovered from heparin non-binding fractions. Hsp40 or hsp40 complex prepared from a cell sample by this method is substantially free of hsp60 or hsp60 complex, hsc70 or hsc70 complex, hsp84 or hsp84 complexes, hsp86 or hsp86 complexes, gp96 or gp96, and hsp70 or hsp70 complex.

The invention further provides a method for recovering separately hsp40, hsp60, hsc70, hsp84/hsp86 (i. e., hsp90), gp96 and hsp70, and complexes thereof from a single sample comprising binding the hsp or hsp complexes to heparin and eluting with multiple sets of buffer compositions. The method comprises the steps in the order stated: binding hsps and/or hsp complexes in the sample to heparin present on a solid phase at zero or very low salt concentration, such as 0 to 30mM NaCl, washing the heparin to collect hsp40 which is not bound to the heparin; eluting stepwise or by a concentration gradient with one or more buffer compositions comprising NaCl in the range of 30mM to 185mM and collecting one or more fractions comprising hsp60 or hsp60 complex; eluting stepwise or by a concentration gradient with one or more buffer compositions comprising NaCl in the range of 30mM to 260mM and collecting one or more fractions comprising hsc70 or hsc70 complex; eluting stepwise or by a concentration gradient with one or more buffer compositions comprising NaCl in the range of 185mM to 400mM and collecting one or more fractions comprising hsp84/hsp86 (hsp90) or hsp84/hsp86 (hsp90) complex; eluting stepwise or by a concentration gradient bound with one or more buffer compositions comprising NaCl in the range of 400mM to 700mM and collecting one or more fractions comprising gp96 or gp96 complex; and eluting stepwise or by a concentration gradient with one or more buffer compositions comprising NaCl in the range of 500mM to 570mM and collecting one or more fractions comprising hsp70 or hsp70 complex. The sequential elution can be carried out conveniently by a concentration gradient.

Hsps, such as those described above, that have been genetically engineered can also be recovered by the methods of the invention provided that the modification does not abolish or interfere with the ability of the modified hsps to interact with heparin. Examples of modified hsps and complexes thereof are described in WO 99/42121 which is incorporated by reference herein in its entirety.

In yet another embodiment of the invention, any hsp or hsp complexes that are bound to heparin present on a solid phase can be eluted by contacting with a composition comprising about 100mM soluble heparin. Accordingly, elution with a composition comprising about 100mM soluble heparin can be used for recovering hsp or hsp complexes in the methods of the invention.

In yet another embodiment, heparin affinity chromatography of the invention can be combined with other methods for purification of hsps and hsp complexes. According to the invention, bound hsps or hsp complexes are eluted from heparin over a range of NaCl concentrations, thus, one or more of the eluted fractions can be used as a starting source which is enriched for a particular hsp and/or complexes thereof. It is contemplated that heparin affinity chromatography can be used in combination with methods well known in the art for the purification of hsps and/or hsp complexes to homogeneity, including but not limited to ADP affinity chromatography, Concanavalin A chromatography, or ion exchange chromatography such as anion or cation exchange chromatography or DEAE chromatography.

In one embodiment, after heparin affinity chromatography, the fractions containing hsp60 or hsp60 complex are pooled for further purification using methods well known in the art, such as ATP affinity chromatography (Vittanen (1992) J. Biochem. 267: 695).

In another embodiment of the invention, after heparin affinity chromatography, one or more fractions containing hsc70 or hsc70 complex are used, pooled if necessary, for further purification using methods well known in the art. For example, hsc70 fractions from heparin separation are pooled and applied to ADP affinity chromatography. The resulting fractions can be further purified using ion exchange chromatography. (See Menoret and Bell (2000) J. Immun. Methods 237 (1-2): 119; see also U. S. Patent No.

5,837, 251.) Therefore, the invention provides a method for purifying hsc70 or hsc70 complex from a sample comprising hsc70 or hsc70 complex, said method comprising heparin affinity chromatography followed by ADP affinity chromatography, and optionally ion exchange chromatography purification.

In another embodiment of the invention, after heparin affinity chromatography, the fractions containing hsp84 or hsp84 complex are pooled for further purification using methods well known in the art. In another embodiment of the invention, after heparin affinity chromatography, the fractions containing hsp86 or hsp86 complex are pooled for further purification using methods well known in the art. For example, fractions containing hsp90 (which includes hsp 84 and hsp86) collected from heparin affinity chromatography are pooled and applied to anion exchange chromatography. Purified hsp90 or hsp90 complexes are eluted in the presence of buffer with a concentration of about 440 to 590mM NaCl. (See Menoret and Bell (2000) J Immun. Methods 237 (1-2): 119; see also U. S. Patent No. 5,837, 251.) Therefore, the invention provides a method for purifying hsp90 from a sample comprising hsp90 or hsp90 complex, said method comprising heparin affinity chromatography followed by anion exchange chromatography.

In yet another embodiment of the invention, after heparin affinity chromatography, the fractions containing gp96 or gp96 complex are pooled for further purification using methods well known in the art. For example, according to a preferred embodiment, gp96 fractions from heparin separation are pooled and applied to Concanavalin A (ConA) chromatography. The resulting fractions containing gp96 or gp96 complex are further purified by ion exchange chromatography. (See Menoret and Bell (2000) J. Immun.

Methods 237 (1-2): 119 ; see also U. S. Patent No. 5,837,251.) Therefore, the invention provides a method for purifying gp96 or gp96 complex to homogeneity from a sample comprising gp96 or gp96 complex, said method comprising heparin separation followed by Con A chromatography and ion exchange chromatography purification.

In yet another embodiment of the invention, after heparin affinity chromatography, the fractions containing hsp40 or hsp40 complex are pooled for further purification using methods well known in the art.

In yet another embodiment of the invention, after heparin affinity chromatography, the fractions containing hsp70 or hsp70 complex are pooled for further purification using methods well known in the art, such as ADP affinity chromatography (Peng et al. (1997) J.

Immun. Meth. 204: 13).

Preparations of three main hsp complex have been used as vaccines against tumors and infectious diseases: gp96, hsc70 and hsp86/84. The hsp complexes elute at different salt concentrations and can be easily separated from each other. Fractions containing gp96, hsc70 and hsp86/84 can be further processed independently to obtain homogenous preparations. Accordingly, in a preferred embodiment of the invention, the separate purification of gp96, hsc70 and hsp86/84 complexes to homogeneity from the same cell sample is accomplished with heparin affinity chromatography and other methods known in the art.

In yet another embodiment of the invention, a kit comprising a heparin composition, such as heparin agarose, and instructions for the application of a sample to the heparin composition and elution of hsps and hsp complexes by specified salt concentrations.

Optionally, the kit may further comprise heparin buffer, elution buffers at various salt concentrations, and cell samples comprising recoverable amounts of hsp or hsp complex.

5.1.2. HEPARIN AGAROSE AND HEPARIN SEPHAROSE CHROMATOGRAPHY In various embodiments, the invention uses heparin that is present on an inert, solid phase, such as agarose, so as to facilitate washing, and separation of bound material from the non-binding or eluted fractions. A preferred heparin composition is heparin-agarose which is stable, tolerates high pressure, can be autoclaved, has excellent flow properties and

high binding capacity. In preferred embodiments, the methods of the invention involve heparin-agarose chromatography. Accordingly, the invention encompasses a composition comprising heparin present on a solid phase, such as heparin-agarose, with bound hsps, including hsp60, hsc70, hsp84, hsp86, hsp90, gp96, hsp70, BiP and calreticulin. The contacting and elution steps of the methods can be conveniently performed in a column The flow of fluid through the column can be maintained by gravity if the column is vertically placed. Alternatively, the flow can be regulated by pressure. Accordingly, in preferred embodiments, the methods of the invention involve heparin-agarose column chromatography. The invention thus also encompasses a column comprising heparin present on a solid phase, such as heparin-agarose, with bound hsps, including hsp60, hsc70, hsp84, hsp86, hsp90, gp96, hsp70, BiP and calreticulin. In a most preferred embodiment, a sample containing hsps or hsp complexes is applied on Heparin-Sepharose which is heparin coupled to cross-linked 6% agarose (Pharmacia Biotech., Piscataway, NJ) in the presence of a buffer and under conditions specified by the manufacturer's instructions. The hsps or hsp complexes are eluted from the heparin agarose or Heparin Sepharose by one or more buffer compositions comprising the appropriate concentration of salt. A concentration gradient, preferably a linear gradient, from low to high salt concentration, such as from 0 to 1 M NaCl, can be used. Alternatively, a series of buffer compositions can be used stepwise, wherein successive buffer compositions in the series each comprises a salt concentration higher than the immediately previous buffer composition.

The methods of the invention can readily be scaled up to accommodate the recovery of hsp or hsp complexes from large-volume sample, e. g, when cell culture supernatant or fermentation broth is used, or large number of samples, e. g., when many clinical samples are to be processed rapidly or simultaneously. Optimization and/or automation of the methods for use in large scale batch or continuous processes, or high turnover parallel operations are also contemplated.

5.2. PREPARATION OF HSP COMPLEX VACCINES The association of hsps with peptides is preserved during binding and elution from heparin. Experiments described in Section 6 hereinbelow show that the interaction of the chaperoned peptides with two exemplary hsps, namely, hsc70 and gp96, is not compromised during heparin affinity chromatography. Accordingly, it is contemplated that this method can be used in the concomitant preparation of multiple vaccines based on different immunogenic hsp complexes.

In one embodiment, the invention provides a method for preparing a vaccine composition said method comprising the steps of contacting a sample, such as a lysate of

cells, comprising immunogenic hsps or hsp complexes with heparin, and eluting the desired immunogenic hsp complexes under appropriate salt concentrations. In a preferred aspect, the invention provides a method for preparing a vaccine composition comprising hsps or hsp complexes derived from the patient to whom the vaccine is administered wherein said method utilizes heparin affinity for recovery of said hsps or hsp complexes. The patient's bound hsps or hsp complexes can be eluted from the heparin under appropriate salt concentrations and administered back into the same patient. The vaccine compositions so recovered are useful for treatment or prevention of diseases, such as cancer, infectious diseases, and autoimmune diseases. Thus, multiple hsps or hsp complexes can be readily obtained from cancer cells or cells infected by an infectious agent or other antigenic cells by an efficient one-step heparin agarose chromatographic method and prepared as vaccines.

As set forth in detail in section 5.2.1 to follow, the methods of the invention can be used to recover hsps and hsp complexes from a variety of cells. The methods may be used to recover hsps or hsp complexes from any eukaryotic cells, for example, tissues, isolated cells or immortalized eukaryotic cell lines infected with a pathogen, tumor cells or tumor cell lines, and eukaryotic cells transfected with a gene encoding and expressing a tumor- specific antigen, tumor-associated antigen or an antigen of the pathogen. The recovered hsp complexes may be stored or combined with a pharmaceutically acceptable carrier to form a pharmaceutical composition suitable for administration as a vaccine. In another aspect, two or more hsp complexes recovered from a cell sample may be combined to prepare a vaccine composition. This is particularly useful when the amount of one type of hsp complexes recovered from a limited sample is insufficient. It is contemplated that the methods of the invention will facilitate the development of vaccines based on individual or multiple hsp complexes recovered from cell samples which are available only in limited amounts.

It is further contemplated that the hsp complexes typically will be isolated directly from tumor tissue excised from the animal being treated. In a preferred embodiment of the invention, the hsp complex is isolated from the cancer cells or precancerous cells of each individual patient (e. g., preferably prepared from infected tissues or tumor biopsies of the patient.) Under certain conditions, however, the amount of tumor tissue available for isolation of the complex may be limiting. Accordingly, it is contemplated that the excised tumor tissue would be used more efficiently using heparin affinity chromatography which can separate in one step the hsp complexes from such a limited sample. Accordingly, a most preferred embodiment of the invention utilizes heparin affinity chromatography in the preparation of multiple hsp-based vaccines, circumvents the sample size limitation, and provides the possibility of using multiple hsp complexes synergistically in eliciting immunity.

In a preferred aspect of the invention, the method will be used to recover hsps or hsp complexes with particular utility as vaccines for human infections or cancers. However, it is appreciated that the method described herein will also be useful in recovering hsps and hsp complexes of other mammals, for example, farm animals including: cattle; horses; goats; sheep; pigs; etc. and household pets including: cats; dogs; etc.

In a particular embodiment, hsps or hsp complexes are autologous to (derived from) the patient to whom they are administered.

5.2.1. SOURCES FOR RECOVERY OF HSPS AND HSP COMPLEXES The source from which hsps and hsp complexes are recovered may be selected on the basis of the intended use of the resulting hsps or hsp complexes. Since hsps and hsp complexes are found in all cells, any tissue or cell sample can be used as a source. Hsps and hsp complexes can also be released from cells (e. g., by necrotic cell death) into the cells'surroundings; thus body fluids, secretions, culture supernatant, fermentation broth, and the like can be a source from which hsps and hsp complexes are recovered by the methods of the invention.

For the treatment or prevention of infectious diseases or cancer, the invention provides methods for recovering antigenic or immunogenic hsp complexes from infected cells or cancer cells, wherein said hsp complexes can be administered to induce an immune response against the infected cells or the cancer cells. Infected and cancerous cells can also be prepared in vitro from noncancerous or uninfected cells (e. g., normal cells), as appropriate by methods known in the art. (See for example U. S. Patent No. 6,017,540, which is incorporated by reference herein in its entirety.) In preferred embodiments, the hsps and hsp complexes are recovered from cancer or infected tissues, cells, or cell lines, all of mammalian origin. In a most preferred embodiment, the cancer or infected tissues, cells, or cell lines are of human origin.

In one embodiment of the invention, for applications relating to treatment and prevention of infectious diseases, the antigenic hsp complexes can be recovered from any infected cell, including; whole tissues, isolated cells, and immortalized cell lines infected or transformed with an intracellular pathogen. The antigenic hsp complexes can be recovered from cells infected with an infectious agent, and in particular with an intracellular pathogen.

It has been demonstrated that a vaccine containing antigenic hsp complexes isolated from cells infected with an intracellular pathogen and then administered to a mammal can effectively stimulate cellular immune responses against cells infected with the same pathogen. Specifically, the immune response is mediated through the cytotoxic T cell cascade which targets and destroys cells containing intracellular pathogens. An intracellular

pathogen is any viable organism, including, but not limited to, viruses, bacteria, fungi, protozoa and intracellular parasites, capable of existing within a mammalian cell and causing a disease in the mammal.

In another embodiment of the invention, the hsps or hsp complexes can be recovered from cells infected with viruses including, but not limited to, hepatitis type A, hepatitis type B, hepatitis type C, influenza, varicella, adenovirus, HSV-1, HSV-II, rinderpest rhinovirus, echovirus, rotavirus, respiratory synctial virus, papilloma virus, papova virus, cytomegalovirus, echinovirus, arbovirus, huntavirus, coxsachie virus, mumps virus, measles virus, rubella virus, polio virus, HIV-I, and HIV-II. In addition, antigenic hsp complexes can also be collected from cells transfected with a viral gene.

In another embodiment of the invention, the hsps or hsp complexes can be recovered from bacteria-infected cells including, but not limited to, cells infected with bacteria causing tuberculosis, gonorrhea, typhoid, meningitis, osteomyelitis, meningococcal septicemia, endometritis, conjunctivitis, peritonitis, pyelonephritis, pharyngitis, septic arghritis, cellulitis, epiglottitis, salpingitis, otitis media, shigella dysentery, gastroenteritis, etc. In preferred embodiments, the hsps or hsp complexes may also be recovered from cells infected with intracellular bacteria, including, but not limited to, Mycobacteria, Rickettsia, Mycoplasma, Neisseria and Legionella.

In addition, hsps or hsp complexes can also be recovered from cells infected with intracellular protoza, including, but not limited to, Leishmania, Kokzidioa, and Trypanosoma. Furthermore, hsps or hsp complexes can be recovered from cells infected with intracellular parasites including, but not limited to, Chlamydia and Rickettsia. Also encompassed by the invention are the use of cell lines infected with bacteria for recovery of hsps or hsp complexes.

In another embodiment of the invention, any tissues, or cells isolated from a cancer, including cancer that has metastasized to multiple sites, can be used as a source of hsps or hsp complexes in the present method. For example, leukemic cells circulating in blood, lymph or other body fluids can also be used, solid tumor tissue (e. g., primary tissue from a biopsy) can be used.

Using the methods of the invention, hsps or hsp complexes may be recovered from tumor cells, including, but not limited to, for example, tumors that are mesenchymal in origin (sarcomas) i. e., fibrosarcomas; myxosarcomas; liposarcomas; chondrosarcomas; osteogenic sarcomas; angiosarcomas; endotheliosarcomas ; lymphangiosarcomas; synoviosarcomas; mesotheliosarcomas; Ewing's tumors; myelogenous leukemias; monocytic leukemias; malignant lymphomas; lymphocytic leukemias; plasmacytomas; leiomyosarcomas and rhabdomyosarcoma. In addition, it is contemplated that this method

can be used in the recovery of hsps or hsp complexes from tumor cells from tumors that are epithelial in origin (carcinomas) i. e., squamous cell or epidermal carcinomas; basal cell carcinomas; sweat gland carcinomas; sebaceous gland carcinomas; adenocarcinomas ; papillary carcinomas; papillary adenocarcinomas; cystadenocarcinomas; medullary carcinomas; undifferentiated carcinomas (simplex carcinomas); bronchogenic carcinomas; bronchial carcinomas; melanocarcinomas; renal cell carcinomas; hepatocellular carcinomas; bile duct carcinomas; papillary carcinomas; transitional cell carcinomas; squamous cell carcinomas ; choriocarcinomas ; seminomas; embryonal carcinomas malignant teratomas and teratocarcinomas. Hsps or hsp complexes can also be recovered from cells of leukemia, e. g., acute lymphocytic leukemia and acute myelocytic leukemia (myeloblastic, promyelocytic, myelomonocytic, monocytic and erythroleukemia) ; chronic leukemia (chronic myelocytic (granulocytic) leukemia and chronic lymphocytic leukemia) ; and polycythemia vera, lymphoma (Hodgkin's disease and non-Hodgkin's disease), multiple myeloma, Waldenstrom's macroglobulinemia, and heavy chain disease. It is also contemplated that hsps or hsp complexes may be recovered from tumor cells from tumors induced by chemical carcinogens or radiation. Chemical carcinogens include carcinogens associated with cigarette smoking, such as hydrocarbons and carcinogenic air, food, cosmetics or other pollutants. In another embodiment of the invention, the hsps or hsp complexes may be recovered from tumor cell lines.

5.2.2. FORMULATION, ADMINISTRATION AND DOSAGE Hsp and hsp complexes recovered by the methods of the invention can be used to treat or prevent a variety of diseases in animals and humans. The antigenic or immunogenic hsp complexes are capable of eliciting an immune response against the antigenic or immunogenic peptides with which the hsps are associated. Such immunogenic or antigenic hsp complexes are useful for the treament and prevention of cancer and infectious diseases. Hsp and/or hsp complexes are formulated for administration to a subject in need of such treatment or prevention by techniques known in the art.

Formulations for administration via a route but not limited to such as oral, parenteral, intravenous, intraperitoneal, or intradermal, for inhalation, nasal drops, topical gels, and slow release formulations, and preferred dosages thereof are provided for the treatment and prevention of cancer, such as primary and metastatic neoplastic diseases (U. S.

Patents No. 5,948,646,5,935,576,5,837,251,6,017,540, and 6,017,544, which are incorporated by reference herein in their entireties), the treatment and prevention of infectious diseases (U. S. Patents No. 5,961,979 and No. 6,048,530, which are incorporated by reference herein in their entireties), adoptive immunotherapy (U. S. Patents No. 5,985,270

and No. 5,830,464, which are incorporated by reference herein in their entireties), the treatment of autoimmune diseases (U. S. Patent No. 6,007,821, which is incorporated by reference herein in its entirety), prevention of graft rejection and promotion of tissue repair (PCT patent publication WO 00/23093, which is incorporated by reference herein in its entirety).

6. EXAMPLES 6.1. RECOVERY OF MULTIPLE HSPS ON HEPARIN-SEPHAROSE 6.1.1. MATERIALS AND METHODS Tumor cell line Methylcholanthrene-induced fibrosarcoma MethA (BALB/cJ origin) was maintained in ascites form in BALB/cJ mice obtained from the Jackson Laboratory (Bar Harbor, ME) by weekly passage of five million cells per mouse intraperitoneally.

The rabbit polyclonal antibody SAP-804, specific for the mouse hsp60; the mouse monoclonal antibody SAP-810 (clone C92F3A-5), specific for the inducible hsp70 ; the rat monoclonal antibody SPA-815 (clone 1B5) specific for the constitutive hsc70; the rabbit polyclonal SPA-400 specific for the hsp40; and the rabbit polyclonal antibody SPA-826, specific for BiP (grp78) ; were purchased from the StressGen Biotechnologies Corp.

(Victoria, BC, Canada). The rat monoclonal antibody specific for gp96 (clone 9G10) and the polyclonal antibodies RB-118-P and RB-119-P specific for hsp84 and hsp86 respectively, were purchased from Neomarkers (Fremont, CA). The goat anti-rabbit IgG horseradish-conjugate antibody was purchased from Bio-Rad (Hercules, CA). The goat anti-mouse IgG horseradish-conjugate and the goat anti-rat IgG horseradish-conjugate antibody were purchased from SIGMA (St. Louis, MI).

A 10 ml cell pellet of MethA cells was homogenized in 40 ml hypotonic buffer (lOmM NaHC03, 0.5 mM PMSF, pH 7.0) by dounce homogenization, and a 100, 000g supernatant was obtained. The sample buffer was changed to the heparin buffer (20mM Sodium Phosphate, 2mM MgC12, 0. 5mM DTT, pH 7.2) with PD-10 column (Sephadex G-25, Pharmacia Biotech., Piscataway, NJ). The sample was applied at room temperature on a 20 ml Heparin Sepharose (Pharmacia Biotech., Piscataway, NJ) equilibrated with the heparin buffer and washed for 100 min at a flow rate of 1 ml/min. The proteins were subsequently eluted by a linear gradient from 0 tol M NaCl in heparin buffer and fractions were collected and analyzed by SDS-PAGE and immunoblotting.

Proteins were resolved on 10% SDS-PAGE following denaturation by boiling for 5 minutes in SDS sample buffer. After electrophoresis, proteins were either silver stained or transferred onto polyvinyldenefluoride (PVDF) membrane (Millipore Corp., Bedford, MA) and probed with appropriate serum and antibodies as described above (Peng et al. (1997) J.

I. Meth. 204: 13). Detection was performed using ECL chemiluminesence kit from Amersham (Arlington Heights, IL).

6.1.2. RESULTS The separation of the 100, 000g soluble proteins contained in the tumor cell lysate was performed using heparin-affinity chromatography as previously described in section 6.1.1. Under the chromatographic parameters used, most of the proteins eluted before 0.8M NaCl (FIG. 1A), with only a few found in the unbound fraction (FIG. 1B, Heparin Unbound lane). At the end of the chromatographic procedure, the heparin-Sepharose beads were boiled in SDS-PAGE sample buffer and analyzed by 10% SDS-PAGE. The absence of detectable protein by silver-staining of the gel (FIG. 1B, Beads lane), indicates that only a negligible quantity of protein remained on the heparin agarose column after elution with 1M NaCl.

The distribution of nine hsps during the heparin chromatography was tested by immunoblot using antibodies specific for gp96, hsp86, hsp84, hsc70, hsp70, hsp60, hsp40, BiP and calreticulin (FIG. 2). Except for hsp40 that was found entirely in the heparin unbound fraction (FIG. 2A, Heparin Unbound lane), the eight other hsps detected eluted at different salt concentration revealing their distinct affinity for heparin. The first hsps to elute are the hsp60 in fractions 7 to 8 between 30 to 185mM NaCI (FIG. 2B) and the hsc70 in fractions 7 to 9 between 30 to 260mM NaCl (FIG. 2C). The two members of the hsp90 family, hsp84 (FIG. 2D) and hsp86 (FIG. 2E) co-elute in fractions 9 tol 1 between 185mm and 400mM NaCl. Gp96 elutes in fractions 12 tol4 between 400mM and 600mM NaCl (FIG. 2F). Another hsp of 70 kDa, the inducible hsp70, was detected only in fraction 13 between 500mM and 570mM NaCl (FIG. 2G). The weak signal obtained for the inducible hsp70 is due to low expression in non-stressed cells. Hsc70 has been described as a heparin-binding protein, whereas the non-denatured hsp70 has not (Hansen et al. (1995) Bioch. Biophys. Acta. 1252: 135). The difference of interaction with heparin could be due to a single amino-acid substitution in a fibronectin-like domain. According to the invention, hsp70 and hsc70 bind heparin agarose differently, and unlike Hansen et al. (1995), experiments show, using specific monoclonal antibodies, that hsp70 binds heparin-agarose more tightly than hsc70. Interestingly, heparin chromatography failed to separate ADP-purified hsc70/hsp70 preparation whereas they are well separated if the cell lysate is

first applied to heparin-agarose column (FIGS. 2C and 2G). These observations indicate a complex and conformation dependent interaction of hsp70 and hsc70 with heparin that may reflect their differential function in the cell. Previous reports indicate that the hsp70, but not the hsc70, co-segregates with the immunogenicity of tumor cells (Menoret et aL (1995) J.

Immunology 155: 740). Accordingly, the invention's facilitation of the separation and purification of these two proteins on a large scale is a key step in the study of the respective immunogenicity of hsp70 and hsc70.

Contrary to the elution of the seven different hsps discussed supra, BiP (grp78), and calreticulin were poorly resolved by heparin agarose chromatography. BiP was detected by immunobloting in fractions 7 to 12, and to a lesser extent in fractions 13 to 16 (FIG. 2H).

Calreticulin was detected mainly in the heparin unbound fraction, and in the fractions 7 to 16, as well as remaining bound on the agarose beads elution (FIG. 2I). The biological significance of the interaction of heparin with hsps remains unknown, it may participate in mediating hsp function by association with intracellular heparin sulfate or other polyanionic structures that resemble heparin.

6.2. PURIFICATION OF HSP90, GP96 AND HSC70 TO HOMOGENEITY The three main hsp complex preparations that have been used successfully as vaccines against tumors and infectious diseases, gp96, hsc70 and hsp90 (hsp86/84), elute at different salt concentration and can be easily separated from each other. Fractions containing p96, hsc70 and hsp86/84 can be further processed independently to obtain homogenous preparations.

By the following procedures, approximately 200-300 mg of hsp86/84,50-120 mg of hsc70 and 20-80 mg of gp96 were recovered per gram of tissue. These experiments demonstrate the concomitant separation and purification of the main immunogenic hsps, gp96, hsc70 and hsp86/84 from the same tumor sample.

6.2.1. PURIFICATION OF HSP90 6.2.1.1. MATERIALS AND METHODS Fractions eluted from the heparin column that contained hsp86/84 were pooled and purified as described thereafter. The buffer of the hsp86/84 containing fractions was changed to HQ-A buffer (20mM Sodium Phosphate, lmM EDTA, 200mM NaCl, pH 7.4) using PD-10 column (Sephadex G-25, Pharmacia Biotech., Piscataway, NJ) and was applied on a HQ-poros HPLC column equilibrated with buffer HQ-A. The column was

washed for 5 min, or until the absorbance at 280 nm dropped near zero and was stable, at flow rate of 4 ml/min. The elution was carried out with a linear gradient from HQ-A buffer to HQ-B buffer (20mM Sodium Phosphate, 1mM EDTA, 600mM NaCl, pH 7.4). Fractions were collected and analyzed by SDS-PAGE and immunoblotting.

6.2.1.2. RESULTS The fractions 9 and 10 containing hsp90 (hsp86/84) were pooled and purified as described in section 6.2.1.1 and analyzed by SDS-PAGE and immunoblotting (FIG. 3). The linear salt elution from the HQ-poros HPLC anion exchange column revealed a symmetrical peak, fractions 10-14, that elutes between 440-590mM NaCl (FIG. 3A). Analysis of these fractions by silver staining showed an homogenous preparation migrating at 90 kDa (Fig.

3B). The minor band migrating around 85kDa in fractions 11 and 12 is a spontaneous degradation product as showed by the immunoblotting with an antibody specific for hsp84 (FIG. 3C); no hsp84 was detected in the unbound fraction of the HQ column (FIG. 3C, lane HQ Unbound). Immunoblot using the anti-hsp86 antibody, instead of the anti-hsp84 antibody, revealed the same elution profile. These data show that heparin separation followed by anion exchange chromatography results in highly purified hsp90 preparation.

6.2.2. PURIFICATION OF GP96 TO HOMOGENEITY 6.2.2.1. MATERIALS AND METHODS Fractions eluted from the heparin column that contain gp96 were pooled and purified as described thereafter. Gp96 containing fractions were brought to 50% ammonium sulfate saturation by adding the appropriate amount of ammonium sulfate very slowly at 40°C under constant gentle agitation and store at 40°C for at least 1 hour. The solution was centrifuged at 6000 rpm and the supernatant from this step was brought to 80% ammonium sulfate saturation very slowly at 40°C under constant gentle agitation, store at 40°C for at least 1 hour and centrifuged as before. The pellet from this step was dissolved carefully in ConA-PBS (5mM Sodium Phosphate, 1. 3mM KC1, 1mM MgC12, 1mM CaC12, 160mM NaCl, pH 7.4). We empirically use 0.5 ml of bed volume of Con-A Sepharose for each ml of starting cell pellet. The solution was applied on a Con A-Sepharose (Pharamacia Biotechnology, Uppsala, Sweden) previously equilibrated with ConA-PBS, washed with 10 column-volume of the same buffer or until the absorbance at 280 nm dropped near zero and was stable. Proteins were eluted with a solution of ConA-PBS containing 10% a-methylmannoside. The gp96-containing fractions were pooled and transferred to 20mM

Phosphate, 0.3 M NaCl, pH 7.0 by PD-10 columns. For this step the same volume of DEAE-Sephacel as ConA-agarose was used. The solution was applied on a DEAE-Sephacel (Pharamacia Biotechnology, Uppsala, Sweden), washed with 10 column-volume of the same buffer or until the absorbance at 280 nm dropped near zero and was stable. The proteins were eluted with a solution of 0.7 M NaCl, 20mM Phosphate, pH 7.0. Fractions were collected and analyzed by SDS-PAGE and immunoblotting.

6.2.2.2. RESULTS Gp96-containing fractions (F12-F14) were pooled and processed as described in section 6.2.2.1. and analyzed after elution of the Con A-Sepharose column by silver staining and immunoblotting. Figure 4A shows that only 2 proteins of 96 and 75 kDa were eluted from the column. The upper band was identified by immunoblotting as gp96 whereas the lower band remained unidentified (FIG. 4B). The 75 kDa contaminant was removed by re-purifiyng the gp96-containing fractions onto a DEAE-Sephacel column as described in section 6.2.2.1. The DEAE-purified material was once again analyzed by silver staining (FIG. 4C) and immunoblotting (FIG. 4D) to reveal an homogenous gp96 purification.

6.2.3. PURIFICATION OF HSC70 TO HOMOGENEITY 6.2.3.1. MATERIALS AND METHODS Fractions eluted from the heparin column that contain hsc70 were pooled and purified as described thereafter. The sample buffer was changed to buffer D (20mM Tris-acetate, 20mM NaCl, 15mM b-mercaptoethanol, 3mM MgC12, 0.5mM PMSF, pH 7.4) with PD-10 column. One ml of bed volume of ADP-agarose was empirically used for each ml of starting cell pellet and the ADP-agarose beads were extensively washed to avoid ADP shedding during electrophoresis. The sample was applied to an ADP-agarose column (Sigma Chemical Co., St Louis, MO) previously equilibrated in buffer D. The column was first washed with 10 column-volume of buffer D, then with 10 column-volume of buffer D containing 0.5 M NaCl, and finally with 10 column-volume of buffer D or until the absorbance at 280 nm dropped near zero and was stable. The ADP-binding proteins were eluted with buffer D containing 3mM ADP. The buffer of the hsc70-containing fractions was changed to 20mM Sodium Phosphate, 20mM NaCl, pH 7.0 with PD-10 columns and applied on a DEAE column previously equilibrated in 20mM Sodium Phosphate, pH 7.0.

One ml of DEAE-Sephacel was used per 4 ml of ADP-agarose. The column was washed with 10 column-volume of the equilibration buffer or until the absorbance at 280 nm

dropped near zero and was stable. The proteins were eluted with a solution of with 20mM Sodium Phosphate, 150mM NaCl, pH 7.0. Fractions were collected and analyzed by SDS-PAGE and immunoblotting.

6.2.3.2. RESULTS Hsc70-containing fractions (F7-F8) were pooled and processed as described in section 6.2.3.1. and analyzed after elution from the ADP-agarose column by silver staining and immunoblotting. Figure 5A shows, that similarly to gp96, hsc70 co-purified with an unidentified lower molecular weight protein that is not hsp40. Interestingly, the 70 kDa protein elutes earlier than the 40 kDa protein suggesting a difference in their ability to bind ADP. The anti-hsc70 immunoblot revealed that the 70 kDa protein is indeed the constitutively expressed hsc70 (FIG. 5B). The 40 kDa contaminant can be removed using a similar step to gp96 as shown on figures 5C and 5D.

6.3. DEMONSTRATION THAT HSP COMPLEXES REMAIN INTACT DURING HEPARIN CHROMATOGRAPHY Hsp complexes remain intact during heparin chromatography. An important feature of hsps is their ability to chaperone immunogenic peptides. Using an in vitro peptide binding assay (Blachere, et al. (1997) R Exp. Med. 186: 1315) in which hsps can be non covalently complexed to synthetic peptides and retain them during electrophoresis, the stability of gp96 and hsc70-peptide complexes during heparin electrophoresis was tested.

6.3.1. MATERIALS AND METHODS Purified hsc70 and 125I-labeled VSVl9 peptide (SLSDLRGYVYQGLKSGNVS, Genemed Synthesis Inc., South San Francisco, CA) using Iodo-beads iodination reagent (Pierce, Rockford, IL), were complexed in 20mM Sodium Phosphate, 150mM NaCl, pH 7.0 for 1 hour at 37° C at a peptide/protein molar ratio of 10: 1, followed by 30 minutes incubation at room temperature. Purified gp96 was complexed with l25I-labeled VSV19 peptide in of 0.7 M NaCl, 20mM Phosphate, pH 7.0 for 10 minutes at 50° C, followed by 30 minutes incubation at room temperature. Free peptides were removed by extensive wash on Ultrafree-10 concentrator (Millipore, Bedford, MA).

6.3.2. RESULTS Hsc70 and gp96 complexed to radio-labeled peptide as described in section 6.3.1. were purified on a heparin-agarose column under the similar conditions as described in

section 6.1 and analyzed by SDS-PAGE (FIG. 6). Autoradiography of the same gels revealed that radio-labeled material at 70 kDa and gp96 kDa bands corresponding respectively to the hsc70 and gp96 purified on heparin agarose. The data indicate that, as previously reported for other chromatographic methods (Peng, et al. (1997) I. Meth.

204: 13); Flachere (1997) J. Exp. Med. 186 : 1315; Menoret et al. (1999) Bioch. Biophys.

Res. Com. 262: 813), both hsc70 and gp96 remain complexed to peptides during heparin chromatography.

The present invention may be embodied in other specific forms without departing from the spirit or essential characteristics thereof. The present embodiments are therefore to be considered in all respects as illustrative and not restrictive, the scope of the invention being indicated by the appended claims rather than by the foregoing description, and all changes which come within the meaning and range of equivalency of the claims are therefore intended to be embraced therein.

Various publications are cited herein, the disclosures of which are incorporated by reference in their entireties.