HK1153389A - Heat shock protein gp96 vaccination and methods of using same - Google Patents

Abstract

The invention provides a tumor cell genetically modified to express a nucleic acid encoding a secreted form of a heat shock protein (hsp) gp96 polypeptide. The invention also provides a method of stimulating an immune response to a tumor by administering a tumor cell genetically modified to express a nucleic acid encoding a secreted form of a gp96 polypeptide.

Description

Vaccination with heat shock protein GP96 and methods of use thereof

RELATED APPLICATIONS

This application claims priority to U.S. provisional application serial No. 61/038,313, filed on 20/3/2008, which is incorporated herein by reference in its entirety.

Government support

The invention was made with U.S. government support under fund numbers CA109094, CA039201 awarded by the national institutes of health and from ACGT. The united states government may have certain rights in the invention.

Technical Field

The present invention relates to the fields of medicine, immunology and oncology. More particularly, the present invention relates to methods and compositions for inducing an immune response against a tumor in an animal subject.

Background

Anti-tumor vaccination was quite effective when administered to tumor-free mice for the first time used in the experiment, resulting in protection from tumor growth after subsequent challenge. Protection was generally persistent and tumor-specific, suggesting involvement of an adaptive immune response. This scene changes fundamentally when vaccines are used for the therapeutic treatment of established tumors. The same dose of vaccine that is effective in establishing protective immunity generally does not provide therapeutic benefit. The lack of efficacy of therapeutic vaccination is believed to be due to induction of tumor-induced suppressor cells, generation of regulatory cells, induction of T cell anergy or tolerance, or a combination of these mechanisms. Regardless of the exact mechanism of tumor-directed immunosuppression, the success of vaccine therapy for cancer treatment will depend on overcoming or neutralizing these tumor-directed suppressions.

Heat shock protein (hsp) gp96, located in the Endoplasmic Reticulum (ER), is thought to act as a chaperone for peptides on their way to MHC class I and II molecules. The gp96 chaperone peptides include the full range of peptides and larger protein fragments that are produced in cells and transported to the ER. Gp96, obtained from tumor cells and used as a vaccine, induces specific tumor immunity presumably by transporting tumor-specific peptides to APCs. See J immunol.1999 Nov 15; 163(10): 5178-82.

The invention described in this application provides anti-tumor compositions. Heat shock glycoprotein (gp) 96-related peptides are cross-presented by dendritic cells to CD8 cells. Vaccination systems suitable for anti-tumour therapy have been developed. See J immunoher.2008 May; 31(4): 394-401 and the references cited therein. Transfection of gp 96-immunoglobulin (Ig) G1-Fc fusion protein into tumor cells results in secretion of gp96-Ig complexed with a chaperone tumor peptide. Parenteral administration of gp96-Ig secreting tumors elicits robust antigen-specific CD8 cytotoxic T lymphocyte expansion in combination with innate immune system activation. Gp96 secreting tumors cause recruitment of Dendritic Cells (DCs) and Natural Killer (NK) cells to sites secreting gp96 and mediate activation of DCs by binding to CD91 and Toll-like receptors-2 and-4. Endocytic uptake of gp96 and its companion peptides triggers cross-presentation of the peptides through the Major Histocompatibility Complex (MHC) class I and strong CD8 activation independent of CD4 cells. In this model system, CD8CTL amplification can be accurately quantified within 4 to 5 days of vaccination by using adoptively transferred T Cell Receptor (TCR) transgenic, Green Fluorescent Protein (GFP) -labeled CD8T cells.

SUMMARY

The present invention provides tumor cells genetically modified to express a nucleic acid encoding a secreted form of a heat shock protein (hsp) gp96 polypeptide. The invention also provides methods of stimulating an immune response to a tumor, including a cancer tumor, by administering a tumor cell genetically modified to express a nucleic acid encoding a secreted form of gp96 polypeptide. Preferably, the immune response is a protective immune response. Preferably, the tumor cell is a allogeneic tumor cell. The invention further provides methods of inhibiting tumors, including cancers, by administering tumor cells, such as cancer tumor cells, that are genetically modified to express a secreted form of gp96 polypeptide. Preferably, the tumor cell is a allogeneic tumor cell. The invention further provides a method of making a vaccine against cancer, the method comprising genetically modifying a population of cancer cells to express tumor cells encoding a nucleic acid, the tumor cells transfected with a eukaryotic expression vector comprising a nucleic acid encoding a secreted form of a heat shock protein (hsp) gp9 polypeptide. The present invention further provides a method of generating a protective immune response in a human subject, the method comprising administering to the subject an effective amount of tumor cells transfected with a eukaryotic expression vector comprising a nucleic acid encoding a secreted form of a heat shock protein (hsp) gp96 polypeptide.

According to some preferred embodiments, the gp96 polypeptide is a fusion protein comprising a gp96 polypeptide and an immunoglobulin signal peptide (IgSP). Optionally, the IgSP is selected from the group consisting of mouse IgSP, rat IgSP, porcine IgSP, monkey IgSP, human IgSP.

According to some preferred embodiments, gp96 immunization is administered frequently (e.g., once or twice daily) for a period of time from about 1 day to about 6 months. According to other preferred embodiments, the therapeutic composition is administered parenterally in a dose of 20mg to 2000mg per dose.

According to some preferred embodiments, gp96 immunization is administered in combination with one or more compounds that destroy normal and/or malignant B lymphocytes, or one or more compounds used to treat diseases characterized by having too many B cells, overactive B cells, or dysfunctional B cells. These diseases include neoplastic diseases such as leukemia or lymphoma. Preferably, the compound that destroys B cells is an antibody. In certain embodiments, the antibody is selected from the group consisting of a subhuman primate antibody, a murine monoclonal antibody, a chimeric antibody, a humanized antibody, and a human antibody. In other embodiments, the antibody binds to a B cell antigen selected from the group consisting of CD19, CD20, CD22, HLA-DR, and CD 74.

Description of the drawings

FIGS. 1A-F. gp96-Ig transfected E.G7(A) and LLC (B) reduced tumorigenicity. ■, Gp 96-Ig; o, mock transfected; and □, untransfected cells. Six mice per group were used per dose of inoculated cells. Vaccination with C-F, secreted gp96-Ig produced tumor-specific memory. At a biweekly interval 10⁶Gp96-Ig transfected E.G7(■ in all figures), 10⁶Irradiated e.g7(□) immunized twice, or not (-) C57BL/6 mice. Two weeks later, mice were challenged with the tumor cell numbers indicated in each figure (six mice per group). Mice that did not develop tumors were observed for 3 months and then judged tumor-free.

FIGS. 2A-C.A. of immunocompetent cells during the sensitization phaseDepletion (depletion) vs. rejection 10⁶The role of G7-gp 96-Ig; the control received PBS. Tumor growth curves of individual mice are shown. The exhaustion schedule is shown in a schematic way at the top. The depletion of immunocompetent cells is at vaccination 10⁶G7-gp96-Ig was completed 2 days before with anti-CD 8, anti-CD 4, or carrageenan. B, CD4 deficient mice are able to reject e.g. g 7-gp-Ig. With unirradiated 10⁶G7-gp96-Ig challenged five CD4 deficient mice subcutaneously. Tumor growth was recorded and the mean tumor diameter was reported. C, effect of depletion of immunocompetent cells on the effector phase of e.g. 7 rejection. The time schedule of immunization and immunodepletion is shown in a schematic way at the top. For immunization, 10 will be⁶Each group of six mice was inoculated twice subcutaneously with non-irradiated e.g. g7-gp 96-Ig. In use 10⁶Three days before G7 challenge, immune cells were depleted as above; the control received PBS. Tumor growth was recorded and reported as mean tumor diameter.

Gp-96-mediated cross-sensitization (cross-priming) of CD8T cells was enhanced in the absence of CD4 cells, but not affected by the deletion of CD 40L. Mice received 1 million GFP-OT-I by intravenous injection and were immunized intraperitoneally 2 days later with 4 million EG7-gp 96-Ig. Cells were harvested from the Peritoneal Cavity (PC) after another 5 days and analyzed by FACS for GFP-OT-I frequency in the CD8 gate (gate). Values are expressed as absolute numbers of GFP-OT-I in PC. A, CD4 deficient mice compared to wild type mice. B, CD40L deficient mice.

Gp96-mediated cross-sensitization of CD8T cells required CD80 and CD86 and was NKT cell independent. Mice received 1 million GFP-OT-I by intravenous injection and were immunized intraperitoneally 2 days later with 4 million EG7-gp96-Ig (A and C) or 2 million 3T3-OVA-gp96-Ig (B). Cells were harvested from Spleen (SP) or PC after another 5 days and analyzed by FACS for GFP-OT-I frequency in the CD8 gate. A, CD80 or CD86 single defects; b, CD80/CD86 double defect; and C, NKT deficiency (J)_α18Knock out).

FIGS. 5A-C efficient cross-sensitization of gp96 in the absence of lymph nodes. A, LT α defect, representative FACS data. Mice received 1 million GFP-OT-I by intravenous injection and were immunized intraperitoneally 2 days later with 2 million 3T3-OVA-gp 96-Ig. Cells were harvested from the indicated sites after another 5 days and analyzed by FACS for GFP-OT-I frequency in CD8 gate. B, the same data is presented as a histogram. Data are representative of two independent experiments, each bar representing the mean ± SE of two mice. And C, 3T3-OVA-gp96-Ig cross-sensitizes OT-I ex vivo. PECs collected from mice intraperitoneally injected three early days with 3T3-OVA-gp96-Ig, 3T3-gp96-Ig or 3T3 were incubated with CFSE-labeled OT-I for 72 hours at OT-I: PEC ratios of 1: 10, 1: 100 and 1: 1000 (a-f). As an additional control, 3T3 transfectants were also incubated directly with OT-I in vitro. Cells were stained with anti-CD 8-PE and analyzed for CFSE dilution, which was plotted in b-h.

FIG. 6A-B. Cross-sensitization ratio of gp96-OVA to CD8T cells by EG7-K^b-OVAThe direct sensitization of antigen presentation of (a) is more effective. Mice received 1 million GFP-OT-I by intravenous injection and were immunized intraperitoneally 2 days later with 2 million EG7-gp96-Ig or EG 7. A, cells were harvested from PCs before (pre-immunization) and 5 days post-immunization and analyzed by FACS for GFP-OT-I frequency in the CD8 gate. B, kinetics of GFP-OT-I amplification in PC and spleen following EG7-gp96-Ig and EG7 immunization; the total number of GFP-OT-I at a given site was plotted.

7A-B. increased recruitment of innate immune cells to PC by gp 96. One million OT-I cells were transferred intravenously, and 4 million EG7 or EG7gp96-Ig cells were injected intraperitoneally on day-2 and after day 2. Cells were harvested from PCs on the indicated days and phenotyped by flow cytometry. A, CD11c by injection of EG7-gp96-Ig⁺、NK1.1⁺And F4/80^Darkness(F4/80^dim) Recruitment of cells. F4/80^{Bright Light (LIGHT)}(F4/80^bright) Cells are present in PCs prior to immunization and the number does not change after immunization. B comparison of recruitment of cells to PC by EG7 and EG7-gp 96-Ig.

Gp96 secretion mediated proliferation of DC and CD8 cells and activated NK cells in PC. Mice received 1 million GFP-OT-I by intravenous injection and were immunized intraperitoneally 2 days later with 4 million EG7-gp96-Ig or EG 7. A, by using in4×10⁶CD11c measured by BrdU staining in PC, mesenteric and periaortic lymph nodes (dL) and Spleen (SP) 2 and 4 days after EG7 or EG7-gp96-Ig immunization⁺And (4) proliferation of the cells. CD11c⁺Cells were gated out and analyzed for BrdU by intracellular staining. Note CD11c⁺Proliferation (red streaks) was only in the next day of PC and only after EG7-gp96-Ig administration. Mice received BrdU in drinking water from the day of immunization. B, CD8 proliferation as measured by BrdU uptake was detectable only in PC at day 2 (red streaks) and only after gp96 sensitization. Strong CD8 proliferation in PC at day 4 after EG7-gp96-Ig immunization; dLN, draining lymph nodes (periaortic, mesenteric); ndLN, non-draining lymph nodes (inguinal). C, activation of NK1.1 cells in PC by EG7-gp96-Ig immunization, as measured by CD69 upregulation. A-C represent three independent experiments.

Figures 9A-b. enhanced cross-sensitization of gp 96-accompanied Ovalbumin (OVA) to CD8T cells compared to ovalbumin-free. C57BL/6 mice received 1 million GFP-OT-I by intravenous injection; after 2 days, they were immunized intraperitoneally with different numbers of isogenic EG7-gp96-Ig or isogenic 3T3-OVA or 3T3-OVA-gp96-Ig or with OVA protein in PBS. The number of cells injected intraperitoneally was adjusted to produce the amount of gp96-Ig or OVA secreted at 24 hours as indicated on the x-axis. OVA and gp96-Ig secretion were determined by ELISA in vitro, respectively. GFP-OT-I amplification was determined by flow cytometry on day 5 after immunization in PC. A, GFP-OT-I amplification in response to EG7-gp96-Ig and OVA proteins. B, GFP-OT-I amplification in response to 3T3-OVA, 3T3-OVA-gp96-Ig and OVA proteins.

Gp96 is an adjuvant for protein cross-sensitization and works most efficiently when released continuously. Mice received 1 million GFP-OT-I by intravenous injection and were immunized intraperitoneally after 2 days as indicated. GFP-OT-I amplification in vivo was measured by FACS 4 days after immunization. Secreted products from injected cells were quantified in vitro by ELISA. The amount of secreted product indicated refers to the amount secreted in the culture by the number of injected cells within 24 hours. Note that 50 μ g OVA together with 200ng gp96 secreted from 3T3-gp96-Ig cells caused less GFP-OT-I amplification than 200ng gp96-Ig containing approximately 0.1% gp96-OVA secreted from 3T3-OVA-gp 96-Ig. B, mice received 1 million GFP-OT-I by intravenous injection. After two days, they were immunized intraperitoneally with 200ng of soluble gp96-Ig collected from the supernatant 3T3-OVA gp96-Ig culture or 3T3-gp96-Ig cells endocrine for 200ng gp96-Ig over the next 24 hours. GFP-OT-I amplification in PC was determined by flow cytometry at day 4 post immunization.

FIGS. 11A-D antigen non-specific inhibition of OT-I CTL amplification by distant established tumors. A, comparison of OT-I CD8CTL frequency in the peritoneal cavity of non-immunized mice, in immunized tumor-free mice, and in immunized EG7 tumor-bearing mice. One million EG7 tumor cells were implanted subcutaneously in the flank and allowed to grow for 5 days before immunization with EG7-gp 96-Ig. One million OT-I CD8T cells were adoptively transferred intravenously 2 days prior to immunization. Mice were immunized intraperitoneally with 2 million EG7-gp 96-Ig. Peritoneal cells were analyzed by flow cytometry after 5 days. B, the inhibition of OT-I amplification by the formed tumors is antigen non-specific. EL4 and LLC, which do not express ovalbumin, formed 5 days instead of EG 7. OT-I adoptive transfer and vaccination were performed as in A. C, absolute number of OT-I accumulated in the peritoneal vaccination site in the absence or presence of formed tumors (same experiment as B). D, the total number of cells recruited to the peritoneal cavity by EG7-gp96-Ig immunization increased in the presence of the formed tumor. Representative experiments of 3 or more individual experiments are shown. N-3 to 5 mice in each group. Significance values indicated in the graph were calculated by t-test. The negative control was non-immunized mice (pre-immunization) and the positive control was mice with no peripheral tumor in the flank. CTL denotes cytotoxic T lymphocytes; gp, glycoprotein; ig, immunoglobulin; LLC, Lewis lung carcinoma.

Fig. 12A-d. frequent gp96 immunization was able to overcome tumor-directed immunosuppression. A, one million EG7 tumor cells were implanted subcutaneously in the flank. Immunization by intraperitoneal administration of one million EG7-gp96-Ig or irradiated EG7 began on the same day of tumor transplantation or 2 or 4 days after tumor transplantation. Negative control-no treatment, n ═ 17; irradiated EG7 was immunized with n 15. Immunization with EG7-gp96-Ig, n 15, was performed at different schedules. B, same as in a except that intraperitoneal immunization started on day 3 and was repeated daily until day 14 (black arrow). One million EG7-gp96-Ig (n ═ 17) or one million LLC-gp96-Ig (n ═ 5) or irradiated EG7 (negative control, n ═ 5) or no treatment (negative control, n ═ 19). C, tumor formation for 5 days, followed by intraperitoneal immunization with one million EG7-gp96-Ig (black arrows) or twice daily (red arrows) from day 5 to day 16; n is 5 in each group. D, tumor formation for 7 days, followed by intraperitoneal immunization with one million EG7-gp96-Ig once daily (black arrow) or twice daily (red arrow) from day 7 to day 18; n is 5 in each group. Significance values for differences in tumor growth are plotted in the individual plots, gp representing glycoprotein; ig, immunoglobulin; LLC, Lewis lung carcinoma; ns, not significant.

FIG. 13. frequent immunizations caused a tumor growth delay in the LLC formed. LLC (105) was implanted subcutaneously in the flank and allowed to develop for 3 days. Immunization with one million LLC-gp96-Ig (n ═ 15), EG7-gp96-Ig (n ═ 5), or irradiated LLC (n ═ 5) or no treatment (n ═ 19) began on day 3 and was repeated on days 7, 10, and 14. The significance of the difference between 19 untreated tumor-bearing mice and 15 treated tumor-bearing mice is shown (P ═ 0.0234), gp representing glycoprotein; ig, immunoglobulin; LLC, Lewis lung carcinoma.

Fig. 14A-b.b cells inhibited gp 96-mediated recruitment of NK cells into the peritoneal cavity and retention of DCs in the peritoneal cavity. A, EG7-gp96-Ig immunization recruits B cells, but only modest CD5+ B cells into the peritoneal cavity. Tumor-free mice received one million of EG7-gp96-Ig intraperitoneally, after which the accumulation of CD5+ and CD5_ B cells was determined daily by flow cytometry. Representing more than 3 experiments. B, recruitment of NK cells and increased retention of NK cells and DCs in B Cell Deficient Mice (BCDM) and their reversal by adoptive transfer of B cells. Wild-type and BCDM were immunized intraperitoneally with 2 million EG7-gp96-Ig and cells harvested from the peritoneal cavity after 2 and 4 days and analyzed by flow cytometry. B cell reconstitution was performed by intravenous adoptive transfer 107 of wild type B cells 2 days prior to immunization with EG7-gp 96-Ig. Representative of 3 experiments. BCDM means B cell deficient mice, DCs, dendritic cells, gp, glycoproteins; ig, immunoglobulin; NK, natural killer cell, WT, wild type.

15A-B. gp 96-mediated OT-I CD8CTL amplifications were increased and persistent in the absence of B cells. Wild type mice and B cell deficient mice received one million GFP-OT-I, and B cell reconstituted mice additionally received ten million wild type B cells by intravenous adoptive transfer. Mice were immunized 2 days later with 4 million EG7-gp96-Ig and analyzed by collecting cells from the peritoneal cavity (A) and mesenteric and periaortic lymph nodes (dLN) (B) on the indicated days. Analysis of variance by repeated measures (ANOVA) P ═ 0.04. Four mice in each group, representing 3 experiments. ANOVA means analysis of variance; CTL, cytotoxic T lymphocytes; dLN, draining lymph nodes; GFP green fluorescent protein; gp, glycoprotein; ig, immunoglobulin; WT, wild type.

Gp96-mediated tumor rejection was enhanced in BCDM and eliminated by B cell reconstitution. A, wild type mice. B, BCDM. One million LLC-ova cells in 0.2-mL PBS were transplanted into the flank. Five days later, one million OT-I was given intravenously. Seven days after tumor implantation, mice were immunized intraperitoneally with one million LLC-ova-gp 96-Ig. Tumor size was measured with a two-dimensional caliper. N-5 in each group represents 3 experiments. BCDM denotes B cell deficient mice; gp, glycoprotein; ig, immunoglobulin; LLC, Lewis lung carcinoma; PBS, phosphate buffered saline.

Fig. 17A-c. high CTL precursor frequency and immune enhancement tumor rejection in BCDM caused by gp96 vaccine. A, BCDM was treated as in FIG. 6 except that vaccination with LLC-ova-gp96-Ig was omitted. B, as in fig. 6. OT-1 transfer was omitted. C, as in fig. 16, except that BCDM mice were reconstituted with 1 million B cells prior to tumor (LLC-ova) transplantation. N-5 to 6 mice in each group represent 2 experiments. BCDM denotes B cell deficient mice; CTL, cytotoxic T lymphocytes; gp, glycoprotein; ig, immunoglobulin; LLC, Lewis lung carcinoma.

FIG. 18: the small B cell population may suppress the immune response against LLC-ova tumor challenge. Four days after LLC-ova transplantation (one day before OT-I injection), each human CD20 transgenic mouse received 1mg Rituximab (Rituximab)) Or PBS. Seven days after treatment, the frequency of CD19+ cells in PBLs was examined by flow cytometry. Approximately 3% of the CD19+ cell population remained in the PBL after rituximab injection. Data for each bar are the mean ± s.e. of three mice.

Detailed description of the invention

According to some preferred embodiments, the present invention provides a method of generating a protective immune response in a human subject, the method comprising administering to the subject an effective amount of tumor cells transfected with a eukaryotic expression vector comprising a nucleic acid encoding a secreted form of a heat shock protein (hsp) gp96 polypeptide.

According to some preferred embodiments, a method of making a vaccine against cancer, the method comprising genetically modifying a population of cancer cells to express tumor cells encoding a nucleic acid, the tumor cells transfected with a eukaryotic expression vector comprising a nucleic acid encoding a secreted form of a heat shock protein (hsp) gp96 polypeptide.

According to some preferred embodiments, the present invention provides methods of stimulating an immune response to a tumor, the method comprising administering to the subject an effective amount of tumor cells transfected with a eukaryotic expression vector comprising a nucleic acid encoding a secreted form of a heat shock protein (hsp) gp96 polypeptide.

According to a preferred embodiment, the present invention provides a method of inhibiting tumor growth, the method comprising administering to a subject an effective amount of tumor cells transfected with a eukaryotic expression vector comprising a nucleic acid encoding a secreted form of a heat shock protein (hsp) gp96 polypeptide.

gp96 polypeptide

Preferably, the gp96 polypeptide is one of the wild-type proteins, and preferably is a wild-type human gp96 polypeptide. Gp96 polypeptides useful in the invention also include those gp96 polypeptides: which has an amino acid sequence substantially similar or identical to the gp96 polypeptide described above. Preferably, the gp96 polypeptide used is at least 70%, more preferably 85%, still more preferably 90% or even more preferably 95% identical or similar to the gp96 polypeptide described herein or known in the art. Most preferably, the gp96 polypeptide used has at least 99% similarity or identity to a wild-type human gp96 polypeptide.

The degree of homology shared by a candidate polypeptide with a gp96 polypeptide of the invention is determined by the degree of similarity or identity between the two amino acid sequences.

A high level of sequence identity indicates the likelihood that the first sequence is derived from the second sequence. Amino acid sequence identity requires an amino acid sequence that is identical between two aligned sequences. Thus, a candidate sequence sharing 70% amino acid identity with a reference sequence requires that 70% of the amino acids in the candidate sequence are identical to the corresponding amino acids in the reference sequence after alignment. Identity is determined by computer analysis, for example, without limitation, by The ClustaIX computer alignment program (Thompson J D, Gibson T J, Plewniak F, Jeanmougin F, & Higgins D G: "The ClustaIX windows interface: flexible strategy for multiple sequence alignment aided by mass analysis tools"; Nucleic Acids Res.1997, 25 (24): 4876-82) and The default parameters suggested therein. Using this procedure, the mature portion of the polypeptide encoded by an analogous DNA sequence of the invention exhibits a degree of identity to the amino acid sequence of the gp96 polypeptide sequence of at least 70%, more preferably 85%, still more preferably 90% or even more preferably 95%, most preferably at least 99%.

Gp96 polypeptides of the invention include variant polypeptides. In the context of the present invention, the term "variant polypeptide" includes polypeptides (or proteins) having an amino acid sequence that differs from a wild-type gp96 polypeptide at one or more amino acid positions. Such variant polypeptides include modified polypeptides as described above, as well as conservative substitutions, splice variants, isoforms, homologues from other species, and polymorphs.

As defined herein, the term "conservative substitution" denotes the replacement of one amino acid residue by another, biologically similar residue. Typically, the above-mentioned biological similarities reflect substitutions on wild-type sequences with conserved amino acids.

For example, conservative amino acid substitutions would be expected to have little or no effect on biological activity, particularly if they represent less than 10% of the total number of residues in the polypeptide or protein. Preferably, conservative amino acid substitutions represent less than 5%, most preferably less than 2% change in the polypeptide or protein.

The gp96 polypeptide in one embodiment includes up to 15 amino acid substitutions. In another embodiment, the gp96 polypeptide includes up to 12 amino acid substitutions. In another embodiment, the gp96 polypeptide includes up to 10 amino acid substitutions. In another embodiment, the gp96 polypeptide includes up to 8 amino acid substitutions. In another embodiment, the gp96 polypeptide comprises up to 5 amino acid substitutions. In a particularly preferred embodiment, there is a single amino acid substitution in the mature sequence, wherein the substituted amino acid and the replacement amino acid are acyclic. Other examples of specific conservative substitutions include the substitution of one hydrophobic residue for another, such as isoleucine, valine, leucine or methionine, or the substitution of one polar residue for another, such as the substitution of arginine for lysine, glutamic for aspartic acids, or glutamine for asparagine, and the like.

The term conservative substitution also includes the substitution of the unsubstituted parent amino acid residue with the substituted amino acid residue, provided that the antibody raised against the substituted polypeptide also immunoreacts with the unsubstituted polypeptide.

Such modifications of the primary amino acid sequence may result in the following proteins: which has substantially equivalent activity compared to the unmodified counterpart protein and is therefore considered as a functional analogue of the parent protein. These modifications may be deliberate, for example by site-directed mutagenesis, or they may occur naturally and include splice variants, isoforms, homologues from other species, and polymorphs. These functional analogs are also contemplated according to the present invention.

Signal peptide

The signal peptide is contained in the coding part of the chromosomal DNA and synthesized by the ribosomal apparatus as part of the protein. The signal peptide typically constitutes the N-terminus and facilitates the directing of the newly synthesized polypeptide into the rough endoplasmic reticulum. Here, the signal peptide is cleaved from the polypeptide and the mature protein is secreted into the surrounding environment. Thus, the signal peptide remains inside the cell.

Signal peptide-eukaryotic signal peptide. Eukaryotic signal peptides are peptides present on proteins destined to be a secretory component or to be a membrane component. It is usually the N-terminus of the protein. In the present context, all signal peptides identified in SignalP (version 2.0 or preferably version 3.0) are considered to be signal peptides.

Mammalian signal peptides are signal peptides derived from mammalian proteins secreted by the ER.

According to a preferred embodiment of the invention, a gp96 molecule is fused to a Signal Peptide (SP) to produce a gp96-SP fusion protein. Expression vectors according to the present invention include nucleic acids comprising a promoter sequence capable of directing expression of a nucleotide sequence encoding a signal peptide operably linked to a gp96 polypeptide.

The signal peptide may be any functional signal peptide, for example a heterologous signal peptide, such as an immunoglobulin signal peptide. The signal peptide may be from any suitable species, e.g., human, mouse, rat, monkey, pig.

In some embodiments, the immunoglobulin signal peptide (IgSP) is a small 19 amino acid peptide known to be from a large group of mammals. Preferably, the IgSP is of mouse or human origin, as mouse IgSP is known to be functional in mice, rats and humans. For use in humans, IgSP is preferably of human origin to reduce the risk of any cross species side effects.

Preferably, the IgSp is one of: human IgSP (Met Asp Cys Thr Trp Arg IleLeu Phe Leu Val Ala Ala Ala Thr Gly Thr His Ala); macaque (Macaca mulatta) (monkey) igsp (met Lys His Leu Trp Phe Leu Val Ala Pro Arg Trp Val Leu ser); common marmoset (Callithrix jacchus) (monkey) IgSP (Met Asp Trp Thr Trp Arg Ile Phe Leu Val Ala Thr Ala Thr Gly Ala His Ser); mouse (Mus musculus) (mouse) igsp (Met Lys Cys Ser Trp Val Ile Phe Leu Met Val Thr Gly Val Asn Ser); boar (Sus scrofa) (pig) igsp (met Glu Phe Arg Leu Asn trpval Leu Phe Ala Leu Gln Gly Val Gln Gly); and brown mice (rat) igsp (Met Lys Cys Ser Trp Ile Leu Phe Leu Met Ala Leu Thr Gly Val Asn Ser).

Cleavage of the signal peptide: the possibility of cleaving signal peptides such as IgSP can be examined using the state of the art prediction tools before determining the incorporation of a particular pg96 form into an expression construct. Such a preferred prediction tool is the SignalP software available at the SignalP WWW server, or preferably an updated version 3.0 available from the same server. In addition, there are several references describing tools and techniques for selecting signal peptides. These references include: henrik Nielsen, Jacob Engelbrecht, Sren Brunak and Gunnar von Heijne: identification of prokaryotic and eukaryotic signal peptides and prediction of their cleavage sites Protein Engineering, 10, 1-6 (1997). For the SignalP-HMM output model: henrik Nielsen and Anders Krogh: (iii) Prediction of signal peptides and signal anchors by a high signature Markov model (Prediction of signal peptides and signal anchors by hidden Markov model) In Proceedings of the six International Conference on Molecular Biology (ISMB6) AAAI Press, Menlo Park, Calif., page 122 and 130 (1998) Improved Prediction of signal peptides-Signal P3.0. Jarnick Dyndv Bindsen, guide Nickel, guide tension and Srun Bruns J M B (Prediction of signal peptides) In Molecular Biology Conference records of the six International Conference on Molecular Biology (ISMB 896) and signal anchors by hidden Markov model of the six International Conference on Molecular Biology (ISMB6) In Molecular Biology Conference on Molecular Biology (ISMB6) AAAI Press, Menlo Park, Calif., page 122-130, 1998. Each of the above documents is incorporated by reference herein in its entirety.

Administration of

According to some preferred embodiments, gp96 immunization is administered frequently (e.g., once daily, twice daily, three times daily, etc.) for a period of about 1 day to about 6 months. According to some embodiments, the period of administration is from about 1 day to 90 days; from about 1 to 60 days; from about 1 day to 30 days; from about 1 to 20 days; from about 1 to 10 days; from about 1 to 7 days. According to some embodiments, the period of administration is about 1 to 50 weeks; from about 1 week to 50 weeks; from about 1 week to 40 weeks; from about 1 week to 30 weeks; from about 1 week to 24 weeks; from about 1 week to 20 weeks; from about 1 week to 16 weeks; from about 1 week to 12 weeks; from about 1 week to 8 weeks; from about 1 week to 4 weeks; from about 1 week to 3 weeks; from about 1 week to 2 weeks; from about 2 weeks to 3 weeks; from about 2 weeks to 4 weeks; from about 2 weeks to 6 weeks; from about 2 weeks to 8 weeks; from about 3 weeks to 8 weeks; from about 3 weeks to 12 weeks; or from about 4 weeks to 20 weeks.

Combination therapy

According to some preferred embodiments, gp96 is immunized with one or more compounds that destroy normal and/or malignant B lymphocytes, or one or more compounds that are used to treat a disease characterized by having too many B cells, overactive B cells, or dysfunctional B cells (e.g., Rituximab)) The administration is combined. These diseases include neoplastic diseases such as leukemia or lymphoma.

Preferably, the one or more compounds for treating a disease characterized by having too many B cells, overactive B cells, or malfunctioning B cells are B cell-targeting antibodies. See, for example, U.S. patent publication No. 2003/0133930, which is incorporated by reference herein in its entirety. Preferably, the antibody targeting the B cell is an antibody against a B cell antigen, such as CDl9, CD20, CD22, HLA-DR and CD 74. Preferably, the therapeutic composition is administered parenterally at a dose of 20mg to 2000mg per dose. According to some embodiments, the subject receives a parenteral dose of the antibody. According to some embodiments, the subject receives the antibody in repeated parenteral doses. According to some embodiments, the antibody is one of a subhuman primate antibody, a murine monoclonal antibody, a chimeric antibody, a humanized antibody, and a human antibody. According to some embodiments, the antibody is one of a murine antibody, a chimeric antibody or a humanized antibody.

According to some preferred embodiments, gp96 immunization is administered in combination with one or more anti-cancer agents. The numerous types of anti-cancer agents are those exemplary anti-cancer agents that find use in the methods of the invention. These classes of anti-cancer agents and their preferred mechanisms of action are described below:

1: an alkylating agent: a compound for administering an alkyl group to a nucleotide. The alkylated DNA is unable to replicate itself and cell proliferation ceases. Examples of such compounds include, but are not limited to: busulfan, coordination metal complexes (e.g., platinum coordination compounds such as carboplatin, oxaliplatin, and cisplatin), cyclophosphamide (cytoxan), dacarbazine, ifosfamide, mechlorethamine (mechlorethamine), and melphalan;

2. bifunctional alkylating agent: a compound having two labile mesylate groups attached to the opposite end of a four carbon alkyl chain. The mesylate group interacts with and damages DNA in cancer cells, preventing its replication. Examples of such compounds include, but are not limited to: chlorambucil and melphalan.

3. Non-steroidal aromatase inhibitors: compounds that inhibit the enzyme aromatase involved in estrogen production. Thus, blockade of aromatase leads to prevention of estrogen production. Examples of such compounds include anastrozole and exemestane.

4. Immunotherapeutic agents: an antibody or antibody fragment that targets cancer cells that produce proteins associated with malignancy. Exemplary immunotherapeutics include herceptin targeting HER2 or HER2/neu, HER2 or HER2/neu occur in high numbers in approximately 25% to 30% of breast cancers; erbitux (Erbitux) targeting Epidermal Growth Factor Receptor (EGFR) in colon cancer; avastin targeting Vascular Epidermal Growth Factor (VEGF) expressed by colon cancer; and Rituxan (Rituxan) anti-CD 20 antibody that triggers apoptosis of B cell lymphoma. Additional immunotherapeutic agents include immunotoxins in which toxin molecules such as ricin, diphtheria toxin and pseudomonas toxin are conjugated to antibodies that recognize tumor-specific antigens. Conjugation can be achieved biochemically or by recombinant DNA methods.

5. Nitrosourea compound: inhibit enzymes required for DNA repair. These agents are able to travel to the brain, so they are useful in the treatment of brain tumors as well as non-hodgkin's lymphoma, multiple myeloma and malignant melanoma. Examples of nitrosoureas include carmustine and lomustine.

6. Antimetabolites: a class of drugs interferes with the synthesis of DNA and ribonucleic acid (RNA). These agents are phase-specific (S phase) and are useful in the treatment of chronic leukemia and tumors of the breast, ovary and gastrointestinal tract. Examples of antimetabolites include 5-fluorouracil, methotrexate, Gemcitabine (GEMZAR)) Cytarabine (Ara-C) and fludarabine.

7. Anti-tumor antibiotics: a compound having antimicrobial and cytotoxic activity. These compounds can also interfere with DNA by chemically inhibiting enzymes and mitosis or altering cell membranes. Examples include, but certainly are not limited to: bleomycin, dactinomycin, daunorubicin, doxorubicin (adriamycin), idarubicin, and manumycin (e.g., manumycin A, C, D, E and G and their derivatives; see, e.g., U.S. Pat. No. 5,444,087).

8. Mitotic inhibitors: compounds capable of inhibiting mitosis (e.g., tubulin binding compounds) or inhibiting enzymes that prevent protein synthesis required for cell replication. Examples of mitotic inhibitors include taxanes such as paclitaxel and docetaxel, epothilones, etoposide, vinblastine, vincristine, and vinorelbine.

9. Radiotherapy: including but not limited to X-rays or gamma rays delivered from an external source such as a beam or delivered by an implanted small radioactive source.

10. Topoisomerase I inhibitors: agents that interfere with topoisomerase activity thereby inhibiting DNA replication. These agents include, but are not limited to CPT-11 and topotecan.

11. Hormone therapy: including but not limited to antiestrogens such as tamoxifen; GNRH agonists, such as willow (Lupron), and progestin agents, such as megestrol.

Naturally, other types of anti-cancer agents that function by a wide variety of mechanisms have application in gp96 immunization and the methods of the invention. Other such agents include, for example: folinic acid; kinase inhibitors such as Iressa (Iressa) and flavopiridon; conventional chemotherapeutic agent analogs, such as taxane and epothilone analogs; anti-angiogenic agents, such as matrix metalloproteinase inhibitors and other VEGF inhibitors, such as ZD6474 and SU 6668. Retinoids, such as Targretin, may also be employed in gp96 immunization and the methods of the invention. Signal transduction inhibitors that interfere with farnesyl (famesyl) transferase activity and chemotherapeutic drug resistance modulators such as valsevida may also be employed. Monoclonal antibodies, such as the C225 antibody and anti-VEGFr antibody, may also be used.

Cancer type

The term "tumor" is used to denote a neoplastic growth that may be benign (e.g., a tumor that does not form metastases and does not destroy nearby normal tissue) or malignant/cancerous (e.g., a tumor that invades surrounding tissue and is generally capable of producing metastases, may recur after attempted removal, and may cause death of the host if not adequately treated) (see Steadman's Medical Dictionary, 26 th edition, Williams & Wilkins, Baltimore, MD (1995)). As used herein, the terms "tumor," "tumor growth," or "tumor tissue" are used interchangeably and refer to abnormal growth of tissue resulting from uncontrolled, progressive cell multiplication and not bearing physiological functions.

Solid tumors can be malignant, e.g., prone to metastasis and life threatening, or benign. Examples of solid tumors that can be treated or prevented according to the methods of the present invention include sarcomas and carcinomas such as, but not limited to: fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma, osteogenic sarcoma, chordoma, angiosarcoma, endotheliosarcoma, lymphangiosarcoma, lymphangioendotheliosarcoma, synovioma, mesothelioma, ewing's tumor, leiomyosarcoma, rhabdomyosarcoma, colon cancer, colorectal cancer, stomach cancer, pancreatic cancer, breast cancer, ovarian cancer, fallopian tube cancer, peritoneal protocarcinoma, prostate cancer, squamous cell cancer, basal cell cancer, adenocarcinoma, sweat gland cancer, sebaceous gland cancer, papillary carcinoma, papillary adenocarcinoma, cystadenocarcinoma, medullary cancer, bronchogenic cancer, renal cell cancer, liver metastasis, bile duct cancer, choriocarcinoma, seminoma, embryonal carcinoma, thyroid cancer such as anaplastic thyroid cancer, wilms' tumor, cervical cancer, testicular tumor, lung cancer such as small cell lung cancer and non-small cell lung cancer, bladder cancer, epithelial cancer, glioma, carcinoma of the lung, colon cancer, bladder cancer, astrocytoma, medulloblastoma, craniopharyngioma, ependymoma, pinealoma, hemangioblastoma, acoustic neuroma, oligodendroglioma, meningioma, melanoma, neuroblastoma, and retinoblastoma.

In addition, tumors including abnormal proliferative changes (e.g., metaplasia and dysplasia) can be treated or prevented in epithelial tissues such as those of the cervix, esophagus and lung using gp96 immunization or the methods of the invention. Accordingly, the present invention provides a method for the treatment of conditions known or suspected of prior progression to a neoplasm or cancer, in particular a neoplasm or cancer in which non-neoplastic cell growth has occurred, including hyperplasia, metaplasia or most particularly dysplasia (for a review of such abnormal growth conditions, see robblins and Angell, 1976, Basic Pathology, 2 nd edition, w.b.saunders co., philidelphia, pages 68 to 79). Hyperplasia is a form of controlled cell proliferation involving an increase in the number of cells in a tissue or organ without significant structural or functional changes. For example, endometrial hyperplasia typically occurs before endometrial cancer. Metaplasia is a form of controlled cell growth in which one type of adult cell or fully differentiated cell replaces another type of adult cell. Metaplasia can occur in epithelial or connective tissue cells. Atypical metaplasia includes a somewhat disordered metaplastic epithelium. Dysplasia is often a precursor to cancer and is found primarily in the epithelium, which is the most disordered form of non-neoplastic cell growth, including loss of individual cellular uniformity and direction of cell architecture. Dysplastic cells typically have abnormally large, deeply stained nuclei and exhibit polymorphism. Dysplasia occurs characteristically where there is chronic irritation or inflammation, and is commonly found in the cervix, respiratory tract, oral cavity, and gallbladder. For a review of these disorders, see fisherman et al, 1985, Medicine, second edition, j.b. lippincott co., philiadelphia.

Other examples of tumors that are benign and can be treated or prevented according to the methods of the present invention include Arteriovenous (AV) malformations, particularly at intracranial sites, and myelomas.

According to some embodiments, there is provided a method for controlling solid tumor growth (e.g., tumor growth of breast, prostate, melanoma, kidney, colon, cervix) and/or metastasis, comprising administering to a subject in need thereof an effective amount of a compound of the invention. In some embodiments, the subject is a mammal. In some embodiments, the mammal is a human.

As used herein, the term "effective amount" refers to an amount sufficient to provide a desired anti-cancer or anti-tumor effect in an animal, preferably a human, suffering from cancer. Desirable anti-tumor effects include, but are not limited to: modulating tumor growth (e.g., tumor growth delay), tumor size or metastasis, reducing toxicity and side effects associated with particular anticancer agents, ameliorating or minimizing clinical damage to cancer symptoms, prolonging survival of subjects beyond what would otherwise be expected in the absence of such treatment, and preventing tumor growth in animals without any tumor formation prior to administration, i.e., prophylactic administration.

As used herein, the terms "modulate", "modulating" or "modulation" refer to altering the rate at which a particular process occurs, inhibiting a particular process, reversing a particular process, and/or preventing the initiation of a particular process. Accordingly, if the particular process is tumor growth or metastasis, the term "modulating" includes, but is not limited to: reducing the rate at which tumor growth and/or metastasis occurs; inhibiting tumor growth and/or metastasis; reversing tumor growth and/or metastasis (including tumor shrinkage and/or eradication) and/or preventing tumor growth and/or metastasis.

"synergistic effect" as used herein refers to the effect produced by the combination of two drugs and more than the single administrationAny of the drugs additionally produces a greater than additive anti-cancer effect. One measure of synergy between two drugs is the Combination Index (CI) method of Chou and Talalay (see Chang et al, Cancer Res.45: 2434-. The method calculates the degree of synergy, additivity or antagonism between two drugs at different levels of cytotoxicity. There is synergy between these two drugs at CI values less than 1. At a CI value of 1, there was an additive effect, but no synergistic effect. CI values greater than 1 indicate antagonism. The smaller the CI value, the greater the synergy. Another measure of synergy is Fractional Inhibitory Concentration (FIC). This score value is the IC of the drug that will work in combination₅₀IC expressed as drug acting alone₅₀Is determined as a function of (c). For two interacting drugs, the sum of the FIC values for each drug represents a measure of synergistic interaction. The two drugs have a synergistic effect when the FIC value is less than 1. An FIC value of 1 indicates an additive effect. The smaller the FIC value, the greater the synergistic interaction.

The term "anti-cancer agent" as used herein denotes a compound or electromagnetic radiation (in particular X-rays) capable of modulating tumor growth or metastasis. When referring to the use of such an agent having a secreted form of a gp96 polypeptide, the term refers to an agent other than the secreted form of the gp96 polypeptide. Unless otherwise indicated, the term may include one or more than one such agent. Where more than one anti-cancer agent is employed, the relative timing of administration of the secreted form of gp96 polypeptide can be selected as desired to provide a time-dependent effective tumor concentration of the one or more than one anti-cancer agent.

As used in this specification, the singular forms "a", "an" and "the" include plural referents unless the content clearly dictates otherwise. As used herein, unless otherwise specified, the word "or" is used in the "inclusive" sense of "and/or" and not in the "exclusive" sense of "or/and. In this specification and the appended claims, the singular forms "a", "an", and "the" include plural referents unless the context clearly dictates otherwise.

The term "about" is used herein to mean approximately, near …, approximately, or around …. When the term "about" is used in conjunction with a numerical range, it modifies that range by extending the boundaries above and below the numerical values set forth. In general, the term "about" is used herein to modify a numerical value within a 20% variance above and below the stated value. As used in this specification, the terms "comprises" and "comprising," whether in transitional phrases or in the subject of the claims, are to be construed to have an open-ended meaning. That is, these terms should be interpreted synonymously with the phrases "having at least" or "including at least". When used in the context of a method, the term "comprising" means that the method includes at least the recited steps, but may include additional steps. The term "comprising" when used in the context of a compound or composition means that the compound or composition includes at least the recited features or components, but may also include additional features or components.

Other definitions

The compositions and methods of the invention are useful for stimulating an immune response against a tumor. Such an immune response is useful in treating or alleviating signs or symptoms associated with a tumor. Such an immune response can ameliorate the signs or symptoms associated with lung cancer. As used herein, by "treating" is meant reducing, arresting and/or reversing symptoms in an individual administered a compound of the invention as compared to symptoms in an individual not treated according to the invention. A practitioner will understand that the compositions and methods described herein should be used in conjunction with a continuous clinical assessment given by a skilled practitioner (physician or veterinarian) to determine subsequent treatment. Therefore, a medical practitioner will evaluate any improvement in treating pulmonary inflammation following treatment according to standard methodologies. Such an assessment will aid and inform the assessment whether to increase, decrease or continue with a particular therapeutic dose, mode of administration, and the like.

The methods of the invention can therefore be used to treat tumors, including, for example, cancer. The methods of the invention can be used to inhibit the growth of a tumor, for example, by preventing further growth of the tumor, by slowing tumor growth, or by causing regression of the tumor. Thus, the methods of the invention can be used, for example, to treat cancer, such as lung cancer. It will be understood that the subject to whom the compounds of the invention are administered need not be subjected to a particular traumatic state. Indeed, the compounds of the invention may be administered prophylactically (e.g., in a patient who is remission from cancer) prior to any development of symptoms. The terms "therapeutic", "therapeutically" and variations of these terms are intended to encompass therapeutic, palliative, and prophylactic uses. Thus, as used herein, by "treating or alleviating a symptom" is meant reducing, arresting and/or reversing the symptoms of an individual to whom a therapeutically effective amount of a composition of the present invention has been administered as compared to the symptoms of an individual not receiving such administration.

The term "therapeutically effective amount" is intended to mean a dose of treatment effective to achieve the therapeutic result sought. Furthermore, the skilled person will understand that the therapeutically effective amount of a composition of the invention may be reduced or increased by fine tuning and/or by administering more than one composition of the invention (e.g. by co-administering two different genetically modified tumor cells) or by administering a composition of the invention with another compound to enhance the therapeutic effect (e.g. synergistically). The present invention thus provides a method of adapting administration/treatment to the specific, urgent needs of a given mammal. As illustrated in the examples below, a therapeutically effective amount can be readily determined, for example empirically by simultaneously assessing the beneficial effects starting at relatively low amounts and in stepwise increments. The methods of the invention can therefore be used alone or in combination with other well-known tumor therapies for treating patients with tumors. One skilled in the art will readily appreciate the advantageous use of the present invention, for example, in extending the life expectancy and/or improving the quality of life of lung cancer patients.

Vaccination methods such as those disclosed herein can be an effective means of inducing an immune response in patients with non-immunogenic tumors.

Unless defined otherwise, technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.

The methods of the invention are intended for use with any subject who may benefit from the methods of the invention. Thus, according to the present invention, "subject", "patient" and "individual" (used interchangeably) include human as well as non-human subjects, particularly domestic animals.

As used herein, "allogeneic cell" refers to a cell that is: is not obtained from the individual to whom the cell is administered, i.e., a cell having a different genetic makeup than the individual. Allogeneic cells are typically obtained from the same species as the individual to whom the cells are administered. For example, the allogeneic cells can be human cells as disclosed herein for administration to a human patient, e.g., a cancer patient. As used herein, "allogeneic tumor cell" refers to a tumor cell that is not obtained from the individual to whom the allogeneic cell is to be administered.

Generally, allogeneic tumor cells express one or more tumor antigens that are capable of stimulating an immune response against the tumor in an individual to whom the cells are administered. As used herein, "allogeneic cancer cells," such as lung cancer cells, refer to cancer cells that are not obtained from the individual to whom the allogeneic cells are to be administered. Generally, allogeneic cancer cells express one or more tumor antigens that are capable of stimulating an immune response against a cancer, such as lung cancer, in the individual to whom the cells are administered.

As used herein, "genetically modified cell" refers to a cell that is genetically modified to express an exogenous nucleic acid, e.g., by transfection or transduction.

As disclosed herein, a allogeneic whole cell vaccine can be selected because whole cell vaccines give good clinical results to date. Allogeneic cell based vaccines provide a good alternative to autologous vaccines under the following assumptions: tumor antigens are common among tumors of different patients and the antigens can be cross-presented by antigen presenting cells of the patients. See, e.g., Fong, et al, annu, rev, immunol.18: 245-273 (2000); boon, et al, annu, rev, immunol.12: 337-365(1994).

The compositions of the present invention comprising tumor cells genetically modified to express a secreted form of gp96 polypeptide can be combined with physiologically acceptable carriers useful in vaccines by including any well-known component that is immunologically useful. The components of the physiological carrier are intended to promote or enhance an immune response to the antigen administered in the vaccine. The formulation may include buffers, salts, or other components that present the antigen to the individual that maintain a preferred pH range in the composition that stimulates the immune response to the antigen. The physiologically acceptable carrier may also comprise one or more adjuvants that enhance the immune response to the antigen. The formulation may be administered subcutaneously, intramuscularly, intradermally or in any immunologically acceptable manner.

The adjuvant refers to the following substances: when added to an immunogenic agent of the invention, such as a tumor cell genetically modified to express a secreted form of gp96 polypeptide, the immune response to the agent is non-specifically enhanced or potentiated in a recipient host exposed to the mixture. Adjuvants may include, for example, oil-in-water emulsions, water-in-oil emulsions, alum (aluminium salts), liposomes and microparticles, such as polystyrene, starch, polyphosphazene and polylactide/polyglycosides.

Adjuvants may also include, for example, squalene mixtures (SAF-I), muramyl peptides, saponin derivatives, mycobacterial cell wall preparations, monophosphoryl lipid a, mycolic acid derivatives, nonionic block copolymer surfactants, Quil a, cholera toxin B subunits, polyphosphazenes and derivatives, and immunostimulatory complexes (ISCOMs), such as those described by Takahashi et al, Nature 344: 873-875 (1990). For veterinary use and for the production of antibodies in animals, Freund's adjuvant (both complete and incomplete) mitogenic components may be used. Incomplete Freund's Adjuvant (IFA) is a useful adjuvant in humans. A variety of suitable adjuvants are well known in the art (see, e.g., Warren and Chedid, CRC Critical Reviews in Immunology (CRC statement in Immunology) 8: 83 (1988); Allison and Byars in Vaccines: New Appliches to Immunology Problums (New methods for Immunological issues), Ellis, eds., Butterth-Heinemann, Boston (1992)). Additional adjuvants include, for example, Bacillus Calmette-Guerin (BCG), DETOX (containing the Mycobacterium phlei Cell Wall Skeleton (CWS) and monophosphoryl lipid A (MPL) from Salmonella minnesota), and the like (see, e.g., Hoover et al, J.Clin. Oncol., 11: 390 (1993); Woodlock et al, J.immunotherpay 22: 251-.

The compositions and methods of the invention disclosed herein are useful for treating patients having tumors. Although the specific embodiment is exemplified with lung cancer, it is to be understood that similar methods can be used to treat other types of tumors, including cancer, using suitable allogeneic cells.

It is understood that modifications that do not materially affect the activity of the various embodiments of this invention are also within the definition of the invention provided herein. Accordingly, the following examples are intended to illustrate, but not limit, the present invention. While the claimed invention has been described in detail and with reference to specific embodiments thereof, it will be apparent to one skilled in the art that various changes and modifications can be made to the claimed invention without departing from the spirit and scope thereof. Thus, for example, those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, numerous equivalents to the specific substances and methods described herein. Such equivalents are considered to be within the scope of this invention and are covered by the following claims.

Example 1: tumor secreted heat shock fusion protein causes CD8 cell rejection

The heat shock protein gp96 indigenous to the endoplasmic reticulum accompanies peptides, including those derived from tumor antigens, on their way through MHC class I presentation. The replacement of the gp96 retention signal of the endoplasmic reticulum by the Fc portion of murine IgG1 produced the secreted form gp96-Ig of gp 96. Gp96-Ig secreting tumor cells exhibit reduced tumorigenicity and increased immunogenicity in vivo and are rejected after initial growth. Rejection requires CD8T cells during the sensitization and effector phases. CD4T cells are not required for rejection at any stage. Carrageenan is a compound known to inactivate macrophages in vivo and does not reduce CD 8-mediated tumor rejection. Thus, immunization with gp96-Ig secreting tumors produced potent tumor-rejecting CD8 CTLs without the aid of CD4 or macrophages. In contrast, immunization with purified tumor-derived gp96 or with irradiated tumor cells requires both.

The secreted form gp96-Ig of gp96 was developed and tested in tumor models. Transfection of tumor cells with cDNA for gp96-Ig resulted in gp96-Ig secretion. As shown in this publication, gp 96-Ig-secreting tumor cells elicit robust immunity and tumor rejection in vivo that is exclusively dependent on CD8 cells.

Cell line: all cell lines were obtained from the American Type Culture Collection (Manassas, Va.) and cultured in medium with 10% FCS. Such as Savaraj et al, am.j.clin.oncol.20: human Small Cell Lung Cancer (SCLC) cell lines (SCLC-2 and SCLC-7) were established as described in 398. Chicken OVA, apc-NEO-OVA, cloned into an expression vector was gifted by m.bevan bosch (seattle, washington) and used to transfect Lewis Lung Cancer (LLC).

Construction of gp 96-Ig: to generate gp96-Ig fusion proteins, KDEL sequence was deleted and replaced with murine hinge, CH2 and CH3 domains; double-stranded cDNA was prepared from Jurkat DNA using the GeneAmp RNA PCR kit (Perkin-Elmer Cetus, Norwalk, CT) and amplified by PCR. The PCR primers were 5'-ATTACTCGAGGGCCGCACGCCATGAGGG-3' and 5'-GCCCGGATCCTTCAGCTGTAGATTCCTTTGC-3'. The PCR primers included an XhoI site (forward primer) and a BamHI site (reverse primer). The hinge, CH2 and CH3 domains of murine IgG1 were amplified by using murine IgG1cDNA as template and mutating the three cysteines of the hinge portion to serines. The PCR primers were 5'-GCGAGGATCCGTGCCCAGGGATTCTGGTTCTAAG-3' and 5'-CTAAGCGGCCGCAAGGACACTGGGATCATTTACCAGG-3'. The PCR primers included a BamHI site (forward primer) and a NotI site (reverse primer). Gp96 was inserted into XhoI and BamHI sites of eukaryotic expression vectors pBCMGSNeo and pBCMGHis and transfected into SCLC-2, SCLC-7, B16F10, MC57, LLC NIH3T3, EL4, E.G7 and P815. Transfected cells were selected with 1mg/ml G418 or 2.5-10mM L-histidine alcohol (Sigma, St. Louis, Mo.). See J mmunol.1999 Nov 15; 163(10): 5178-82 and references cited therein.

ELISA: this was done using antibodies against Ig tags. The Gp96-Ig producing cells were treated with 10⁶The/ml plates were plated in AIMV or IMDM with 10% FCS and culture supernatants were collected at different time points. To analyze intracellular expression of gp96-Ig, cells were lysed by three freeze-thaw cycles and centrifuged at 13,000 Xg for 60 min.

Purification of gp96-Ig fusion protein: gp96-Ig was purified by affinity chromatography on a protein A column using standard procedures (Bio-Rad, Hercules, Calif.). The concentration of gp96-Ig was determined by the Micro BCA protein assay kit (Pierce, Rockford, IL.). SDS-PAGE and Western blotting were performed using standard procedures.

FACS analysis: for membrane staining of gp96-Ig transfected SCLC, cells were stained with goat anti-mouse IgG-FITC or goat anti-rabbit IgG-FITC as a control at 4 ℃ for 15 min and analyzed by Becton Dickinson FACScan flow cytometer (San Diego, Calif.). For intracellular staining, cells were fixed with 4% paraformaldehyde and permeabilized with 1% saponin, followed by staining with goat anti-mouse IgG-FITC, goat anti-mouse IgG-PE, goat anti-rabbit IgG-FITC, or goat anti-Syrian hamster IgG-FITC for 15 min at 4 ℃ and analyzed by flow cytometry.

Tumor vaccination and vaccination: in vivo tumorigenicity was determined by injecting viable tumor cells subcutaneously in 200 μ l PBS into the flanks of mice. Tumor two-dimensional size measurements were taken twice weekly for at least 2 months. When the mean tumor growth exceeded 10mm diameter, mice were sacrificed.

Administration of 10 in the right flank by subcutaneous injection⁶Live E.G7-gp96-Ig or as a controlMice were immunized with irradiated e.g. 7 (in 200 μ l PBS). Two immunizations were given at 2 week intervals. Two weeks later, mice were challenged by subcutaneous injection of the indicated number of viable tumor cells (EL 4, e.g. g7, LLC, or LLC-OVA in 200 μ l PBS) into the left flank.

Depletion of T cells or macrophages in vivo: a total of 100. mu.g of GK1.5 (anti-CD 4) or 2.43 (anti-CD 8) in 200. mu.l of PBS was administered by intraperitoneal injection. Depletion of CD4 and CD8 cells was verified by FACS analysis. CD4 or CD8 levels remained low (> 95% depletion) for greater than 2 weeks following antibody injection (data not shown). For functional inhibition of macrophages, 1mg of carrageenan (type II; Sigma) in 200. mu.l PBS was administered by intraperitoneal injection.

Results: hsp gp96 indigenous to ER purified from tumor cells is capable of providing tumor-specific immunity. The C-terminal sequence KDEL of gp96 is used as ER retention signal. Deletion of this sequence results in secretion of gp96 together with the binding peptide from transfected tumor cells and can render the tumor more immunogenic to allow rejection of the tumor by the immune system.

Substitution of the hinge, CH2 and CH3 domains of the Ig isotype murine IgG1, which are ineffective in Fc receptor binding, for the KDEL sequence of gp96, and transfection of the cDNA into tumor cells resulted in secretion of gp96-Ig into the culture supernatant where gp96-Ig was quantified by ELISA. Protein A purified gp96-Ig migrated on SDS-PAGE, with the major band with a predicted molecular mass of 120kDa being the fusion protein and the two minor, higher molecular bands being unmodified gp96, also previously reported. Western blotting with mAb specific for gp96 confirmed the identity of the fusion protein. Only the major band was stained, suggesting that the minor band is a glycosylated variant of gp96 that is not recognized by the antibody.

The secretion of gp96-Ig resulted in a time-dependent linear accumulation in the supernatant. Intracellular gp96-Ig was detected in lysates of transfected cells at a low and constant steady-state level, indicating that it did not accumulate in the cells. FACS analysis of transfected tumor cells with intact membranes revealed no staining of anti-mouse IgG above background, indicating that the Ig portion of the fusion protein was not displayed on the outer layer of the plasma membrane. In contrast, gp96-Ig was detected intracellularly with goat anti-mouse IgG antibody after membrane permeabilization, but gp96-Ig was not detected by control goat anti-rabbit IgG antibody. The transmembrane domain of gp96 does not interfere with gp96-Ig secretion and does not cause intracellular accumulation. These data are consistent with previous reports suggesting that the transmembrane domain is not used to anchor gp96 in membranes and gp96 is not an integral membrane protein. Altmeyer et al, 1996 int.j. cancer 69: 340.

all of the murine and human cell lines transfected with gp96-Ig secreted fusion proteins. Mock-transfected cells did not secrete gp 96-Ig. G7 is an OVA transfectant of EL4 lymphoma that forms a lethal tumor in syngeneic C57BL/6 mice. Gp96-Ig transfection of E.G7 allows determination of whether E.G7-gp96-Ig is immunized in addition to E.G7^{To is directed at}EL4 parent tumor, E.G7 is OVA surrogate^AntigensTransfected tumors. As a second tumor LLC transfected with gp96-Ig or OVA was used, since it is a non-hematopoietic, low immunogenic tumor compared to e.g. g 7. Both cell lines secrete equal amounts of gp 96-Ig.

Secreted gp96-Ig caused a reduction in tumorigenicity: secretion of gp96-Ig reduced the tumorigenicity of E.G7 in C57BL/6 mice by more than 100-fold when compared to mock-transfected E.G7 or untransfected E.G7. Subcutaneous inoculation of 1 million tumor cells secreting hsps only caused tumors in 10% of the inoculated mice (fig. 1A). Similar reduction in tumorigenicity by gp96-Ig secretion was observed for transfected EL4 (data not shown). Gp96-Ig secretion by LLC resulted in a more modest, approximately 5-fold, reduction in tumorigenicity (FIG. 1B).

To determine immunogenicity and immunological memory responses, non-irradiated doses of E.G7-gp96-Ig (10. G7-gp96-Ig were administered at 2 week intervals⁶) C57BL/6 mice were immunized twice. Subsequently, mice were challenged with untransfected e.g7 or mock-transfected e.g7, parent EL4, untransfected LLC and OVA-transfected LLC (fig. 1, C-F). Mice immunized with irradiated e.g7 or non-vaccinated mice were used as controls. G7-gp96-Ig immunized mice are resistant to E.G7 tumor challenge than are irradiatedIs 10 times higher than that of the vaccinated or non-immunized mice (fig. 1C). Tumor growth in vaccinated mice is often delayed. The effect of immunization was even more pronounced when challenged with EL4, resulting in a fifty-fold increase in the dose of EL4 challenge compared to the control (fig. 1D). As expected, e.g. g7-gp96-Ig immunization did not provide protection against challenge with untransfected LLC or vector-transfected LLC (fig. 1E), whereas a modest, approximately 3-fold increase in protection was observed when OVA-transfected LLC was used as the challenge (fig. 1F). The strong protection of mice immunized with e.g. g7-gp96-Ig against EL4 challenge may be due to the sharing of multiple tumor antigens by e.g. g7 and EL 4. Weaker protection against LLC-OVA challenge depends on T cell recognition of OVA surrogate derived from T cell recognition^AntigensOr a limited number of epitopes.

CD8 cells are required during the sensitization and effector phases: the immune mechanisms involved in rejection of e.g. g7-gp96-Ig were further examined by depletion/inactivation of immunocompetent cells in vivo. It has been reported that for effective immunization, MethA tumor-derived gp96 requires CD4 cells, CD8 cells and macrophages, whereas immunization with irradiated MethA tumor cells requires CD4 cells and CD8 cells, but does not require macrophages.

For sensitization, one million unirradiated live e.g. g7 secreting gp96-Ig are inoculated subcutaneously. This dose is sufficient to form a tumor that grows to an average diameter of about 8mm, then shrinks and is rejected. Tumor rejection was blocked in mice treated with anti-CD 8 antibody 2.43 either 2 days before (fig. 2A) or up to 3 days after tumor inoculation (not shown). Regardless of injection time, although the anti-CD 4 antibody, GK1.5, completely depleted CD4 cells for more than 14 days (data not shown), it had no effect on tumor rejection (fig. 2A). CD 4-deficient mice were able to reject e.g. g7-gp96-Ig (fig. 2B), supporting the importance of CD8 cells. E.g. g7, which does not secrete gp96-Ig, forms tumors in untreated and immunodepleted mice. Carrageenan, which inactivates macrophages in vivo, is known to have no effect on tumor rejection.

To investigate the effector phase of tumor rejection, mice were immunized twice with live e.g. g7-gp96-Ig at 14 day intervals. Eleven days later (day 25), the immune cells were depleted and 3 days later mice were challenged with untransfected e.g 7. Only CD8 cells are needed in the effector phase; depletion of CD4 cells or inactivation of carrageenans by macrophages had no effect on e.g7 rejection during the effector phase (fig. 2C).

Removal of the endoplasmic reticulum retention signal of gp96 and replacement by the Fc portion of IgG1 readily results in secretion of gp96-Ig, gp96-Ig appearing to be dimerized via the hours chain of IgG 1. Gp96 secreted by g7 is able to provide durable specific immunity, suggesting that it accompanies tumor peptides. In contrast, irradiated e.g. 7 or mock-transfected e.g. 7 failed to provide protective immunity. Corynebacterium parvum (Corynebacterium parvum) also cannot serve as an adjuvant for e.g7 immunization. Secreted gp96-Ig provided immunospecificity for the alternative antigen OVA and other EL4 antigens, but did not cross-immunize with LLC-derived tumor antigens.

This data is consistent with the following explanation: peptides associated with secreted gp96-Ig were transferred to and presented by MHC class I and stimulated tumor-specific CD8 causing tumor rejection⁺CTL response. The CD8 response appeared to be independent of CD4 help and did not require macrophages. Whether this cellular requirement is due to gp96-Ig dimerization is unknown.

It is instructive to compare the immunological mechanisms of purified tumor-derived gp96 and tumor-secreted gp 96-Ig. Udano et al, (proc.natl.acad.sci.usa 91: 3077, 1994) using gp96 purified from Meth a tumour cells for immunization, reported the need for CD8 cells and macrophages during the sensitisation phase and CD4 cells and CD8 cells and macrophages during the tumour rejection effector phase of Meth a tumours. Immunization with irradiated MethA tumors requires CD4 cells in the sensitization phase and both CD4 and CD8 cells in the effector phase. Irradiated EG7 did not produce immunity against subsequent challenge. The significant effect of tumor-secreted gp96-Ig was completely dependent on CD8 cells without CD4 help. CD8 cells are required during the sensitization and effector phases of the CTL response to tumors. No macrophages appear to be required. The role of dendritic cells or other APCs in gp 96-chaperone peptide presentation to CD8 cells is unknown, but possibilities remain. It is also possible that gp96-Ig secreting EG7 directly stimulates CD8 cells.

Example 2: molecular and cellular requirements for enhanced antigen presentation to CD8 cytotoxic T lymphocytes

This example shows that tumor-secreted heat shock protein gp 96-chaperone peptides enhance the antigen cross-sensitization efficiency of CD8 CTLs by millions of times over the cross-sensitization activity of the uncontaminated protein alone. gp96 also acts as an adjuvant for cross-sensitization by the uncontaminated protein, but gp96 is 1000 times less active in this respect than as a peptide chaperone. Mechanistically, gp96-Ig secreted in situ by transfected tumor cells recruits and activates dendritic cells and NK cells to the gp96 release site and promotes local expansion of CD8 CTLs. Gp 96-mediated cross-sensitization of CD8T cells in the absence of NKT and CD4 cells and in the absence of CD40L required B7.1/2 co-stimulation, but did not proceed unimpeded in lymph node-deficient mice. MHC I cross-sensitization of gp 96-driven CD8 CTLs in the absence of lymph nodes provides a novel mechanism for localized, tissue-based CTL production at the gp96 release site. This pathway may constitute an extremely important early detection and rapid response mechanism that is potent in parenchymal tissues that are effective against tissue-destroying antigenic agents.

Heat shock proteins are chaperone peptides that can be taken up by APCs and cross-presented to CD8 cells. Exogenous Heat Shock Proteins (HSP) are actively captured by CD91 and LOX-1 on Dendritic Cells (DCs) and cross-presented to CD8 by delivering chaperone peptides to the MHC class I pathway⁺CTLs elicit peptide-specific immune responses. Cross-sensitization by HSP-gp96 is associated with stimulation of TLR2 and TLR4 leading to a CD8CTL biased response and maturation of DCs.

Immunization with gp 96-secreting tumor cells resulted in tumor-specific and surrogate antigen-specific immunity that was independent of CD4 cells. Oizumi et al J Immunol.2007Aug 15; 179(4): 2310-7. Using this immunization method to quantify CD8 responses, this example shows that trace, femtololar (femtolator) amounts of gp96 chaperone antigen are sufficient for homologous CD8 cross-sensitization locally at the gp96 release site independent of lymph node and CD4 cells.

Mice: wild type (wt) and B7.1, B7.2, B7.1/2, CD40L, lymphotoxin alpha (LT α), and CD4 deficient mice in a C57BL/6(B6) background were obtained from Jackson laboratories. J6. alpha. 281^-/-Mice (NKT deficient, also known as J α 18 knockouts (ko)) were provided by m.lotze bosch (university of qianye, japan) under the permission of Taniguchi (university of qianye, medical center, pittsburgh, pa). GFP transgenic mice were obtained by the producer's permission. C57BL/6 OT-I mice were obtained from m.bevan bosch (washington university medical school, seattle, washington). All mice were administered antigen (antigene) at 6-12 weeks.

Cell line: enga transfected EL4 lymphoma line EG7 generously provided by doctor bevan, m.bevan was further transfected with the gp96-Ig containing vector pCMG-His as described previously. NIH3T3 cells were transfected with OVA in pAC-neo-OVA (generously provided by m.bevan bosch) and with pCMG-His containing gp 96-Ig.

Antibody: fluorescent antibodies were purchased from BD Pharmingen and eBioscience.

Purification and adoptive transfer of OT-I cells: GFP-labeled OT-I cells were purified by positive selection with anti-CD 8 using magnetic separation (> 95% pure; Miltenyi Biotec). One million GFP-OT-I cells in a 0.3ml volume of PBS were adoptively transferred through the tail vein of C57BL/6 mice.

Immunization: two days after adoptive transfer of GFP-OT-I, 2-4X 10 cells in 0.5ml volume of PBS were intraperitoneally injected⁶Non-irradiated EG7-gp96-Ig cells or control EG7 cells. For some experiments, mice were immunized intraperitoneally with 3T3-OVA-gp96-Ig, 3T3-OVA or whole OVA (Sigma-Aldrich) dissolved in PBS.

Ex vivo antigen cross-presentation and cross-sensitization of OT-I: by 2X 10⁶Groups of 3T3-OVA-gp96-Ig or 3T3-gp96-Ig were immunized intraperitoneally to B6 wild-type mice. After 3 days, peritoneal cavity exudation was collectedA cell (PEC), and 10⁵The PEC was co-cultured with purified CFSE-labeled OT-I for the first experiment in 200. mu.l tissue culture medium in round-bottom 96-well microplates at different ratios (5: 1, 10: 1, 100: 1 and 1000: 1) for 48 hours and 72 hours. CFSE-labeled OT-I was also directly co-cultured with 3T3, 3T3-OVA-gp96-Ig, and 3T3-gp 96-Ig. After the indicated time period, cells were harvested and stained with anti-CD 8-PE. OT-I amplification was measured by CFSE dilution as analyzed in an ISR II flow cytometer (BD Biosciences). By in portal-gated lymphocytes or in total CFSE⁺CFSE dilution in cells to analyze cell division and express it as total CFSE⁺Percentage of cells.

BrdU labeling and analysis: BrdU (Sigma-Aldrich) (0.8mg/ml) was administered to mice in their drinking water at immunization time. Cell samples were analyzed by staining for brdu (ebioscience) after fixation and permeabilization.

CD4 cells inhibited antigen cross-presentation to CD8CTL by HSP gp96 peptides: tumor cells transfected with the secreted form of gp96, gp96-Ig, become immunogenic and induce tumor-specific immunity in mice. Yamazaki et al, j.immunol.163: 5178-5182(1999). Tumor immunity requires CD8 cells but is independent of CD4 cells in either the afferent arm (afferent arm) or the efferent arm (efferent arm) of the immune response.

Because CD4 cells exhibit activity as both helper and regulator cells, we were interested in determining whether any of these relative functions would modulate the cross-sensitization of gp96 to CD8 cells. We used adoptive transfer of K^b-OVASpecific TCR-transgenic CD8 cells OT-I to quantify CTL expansion in response to intraperitoneal immunity to EG7-gp96-Ig or EG 7. EG7 is OVA transfected EL4 lymphoma. OT-I amplification in this system has previously been shown to be dependent on gp96 chaperone OVA peptide and not affected by Ig-Fc-tag, since gp96-myc has equivalent activity. The gp96-Ig immune system secreted by tumors also generates immunity against authentic tumor antigens. However, measuring OT-I amplification provides a more accurate and rapid readout than measuring tumor rejection.

In CD4 ko all CD4 functions (helper and regulatory) were deleted, whereas in CD40Lko mainly the helper cell function of CD4 cells was lost. We therefore compared OT-I amplification in response to gp96-OVA secreted by EG7-gp96 in CD4 ko and CD40L relative to wild type mice (figure 3). To facilitate analysis, OT-I TCR transgenic cells were also GFP-tagged by growing OT-I mice with GFP transgenic mice (GFP-OT-I). OT-I amplification in CD4 ko mice increased 100% in response to EG7-gp96-Ig in comparison to wild type mice, suggesting that the presence of CD4 cells interfered with clonal amplification of CD8 (fig. 3A). In contrast, OT-I amplification was similar in CD40L ko mice as in wild type mice (fig. 3B). The data indicate that CD4 helper function mediated via CD40L is not required for gp 96-mediated antigen cross-presentation to CD8 CTLs. In contrast CD4⁺T regulatory cells, absent in CD4 ko, normally down-regulate OT-I cross-sensitization by gp 96-OVA.

CD8 cross-sensitization by gp96 is B7.1 and B7.2 dependent and NKT cell independent: effective T cell sensitization requires maturation of DCs and upregulation of MHC and costimulatory molecules, usually mediated by CD40 signaling. However, gp 96-mediated OT-I sensitization apparently did not require assistance by CD4 through the CD40L/CD40 axis (FIG. 3). gp96 binds to CD91 and to TLR2 and TLR4, and thus may activate DCs independently of CD 40. We determined whether this cross-sensitization mechanism of CD8 cells in vivo relies on co-stimulation with B7.1(CD80) and B7.2(CD 86). B7.1 or B7.2 deficient mice alone (fig. 4A) were able to co-stimulate gp 96-mediated cross-sensitization of OT-I with an efficiency of about 50% of wild type mice. However, in the complete absence of B7.1 and B7.2, gp 96-mediated cross-sensitization of OT-I was completely abolished in double-deficient mice (fig. 4B).

NKT cells are commonly involved in anti-tumor immunity. J. the design is a square_α18ko mice lack NKT cells because they are unable to produce invariant TCRv specific for CD1 d-restricted invariant NKT cells_α14And (3) a chain. J. the design is a square_α18The ability of ko mice to support unreduced OT-I amplification (FIG. 4C) suggestsNKT cells are not required for gp 96-mediated OT-I cross-sensitization.

Cross-presentation of antigen by gp96 does not require lymph nodes: draining lymph nodes bring together APC, CD4 helper cells, CD8CTL precursors, and NK cells, promoting cellular interactions and enhancing CTL sensitization and expansion. Because of gp96 mediated^AntigensCross-sensitization does not rely on CD4 assistance, and we address the problem that OT-I amplification requires draining lymph nodes. LT α -deficient mice lack peripheral and mesenteric lymph nodes (including peyer's patches) and are impaired in an antiviral response. However, when gp96-OVA mediated OT-I amplification was analyzed, LT α -deficient mice showed almost normal OT-I amplification in the Peritoneal Cavity (PC) when compared to wild-type mice (FIG. 5, A and B). Accumulation of GFP OT-I in the spleen was reduced by approximately 50%, reflecting the absence of lymph node-based clonal expansion of OT-I. This finding suggests that lymph nodes are not required for gp 96-mediated peptide cross-sensitization, and that local cross-sensitization occurs at the gp96 release site.

To directly test lymph node-independent OT-I cross-sensitization, we isolated PECs from B6 mice on day 3 after intraperitoneal immunization with allogeneic 3T3 cells, 3T3-OVA cells, or 3T3-OVA-gp96-Ig cells. PEC was mixed with CFSE-labeled OT-I at various ratios and CFSE dilution was determined after 48 and 72 hours. PEC isolated from mice injected with 3T3-OVA-gp96-Ig was able to cross sensitize OT-I in vitro as indicated by CFSE dilution (fig. 5, C, a-d). In contrast, PEC isolated from mice injected with 3T3-gp96 or untransfected 3T3 failed to stimulate OT-I proliferation (c, e, f of fig. 5). Likewise, direct in vitro incubation of CSFE-labeled OT-I with 3T3-OVA-gp96-Ig or with 3T3-gp96-Ig did not result in CSFE dilution. This data supports a model of gp 96-OVA-induced antigen cross-presentation to cognate CD8 cells in the absence of lymph nodes in PC.

gp96 recruits DC cells and NK cells to their site of release and causes activation of these cells: minimal cross-sensitization requires APC and CD8 cells to be together, whereas CD4 cells are not necessary in our model system. We determined whether local release of gp96 in PC caused local recruitment and activation of APC and OT-I, thereby bypassing the need for lymph nodes.

OT-I expansion after gp96-Ig immunization was greatest by day 4 and 5 and most significant in PCs. Essentially starting from 0, approximately 5 million OT-I accumulated in PC on days 4 and 5, representing up to 60% of recruited CD8 cells. Also as observed by others, strong OT-I amplification was highly dependent on gp96-Ig secretion (fig. 6), and the response to EG7 was minimal. The ability of gp96 to cross-sensitize CD8 cells in wild-type mice within 4 days suggests early activation of APC and other innate cells. Gp96 is known to activate and mature DCs in vitro, and gp96 chaperone peptides are cross-presented by MHC I on DCs and macrophages in vitro and in vivo. Oizumi et al J Immunol.2007Aug 15; 179(4): 2310-7. Gp96 has been reported to activate NK cells. Oizumi et al J Immunol.2007Aug 15; 179(4): 2310-7. The fact that unimpaired OT-I activation occurs in LT α ko mice suggests that cellular recruitment and activation must occur locally at the site of gp96 release.

PECs were collected on days 1-4 following intraperitoneal injection of EG7-gp96-Ig or EG7 as a control, and activation was analyzed phenotypically and by uptake of BrdU. The largest proportion of EG7-gp96-Ig recruited cells, about 80-90%, was F4/80^DarknessMonocytes/macrophages. The resident peritoneal macrophages present prior to immunization are F4/80^{Bright Light (LIGHT)}And the number did not change after injection of EG7-gp 96-Ig. CD11c⁺DC and NK1.1⁺NK cells each constitute about 5-10% of the cells recruited into PCs within the first 2 days. The number of B cells and CD4T cells in PC was found to increase starting on day 3 and further increased on days 4 and 5 (data not shown). Intraperitoneal injection of EG7-gp96-Ig doubled the total number of cells recruited into PC within the first 2 days in comparison to EG7 (fig. 7A). This effect required that the injected cells secrete at least 60ng of gp96-Ig within 24 hours as measured by ELISA. Oizumi et al J Immunol.2007Aug 15; 179(4): 2310-7. If this number of injected cells secrete lower amounts of gp96-Ig, the effect on cell recruitment and CD8 cross-sensitization is rapidA rapid decay suggesting that there is a threshold level of sensitivity to cross-sensitized stimuli (data not shown). Gp96 secreted by EG7 causes recruitment of F4/80, as compared to EG7 which does not secrete gp96-Ig^DarknessThe total number of cells doubled and the number of DC and NK cells increased 2-fold (fig. 7B). DC recruited to PC by gp96 incorporated large amounts of BrdU within the first 2 days, indicating activation of DC. In contrast DCs isolated from draining paraaortic, mesenteric and spleen lymph nodes were BrdU negative (fig. 8A). This finding strongly suggests that DCs are activated and proliferate locally at the gp96 secretion site. BrdU positive DCs were also found in the lymph nodes and spleen shortly. EG7, which does not secrete gp96, does not allow BrdU uptake by DC in PC during the first 2 days. Interestingly, however, DC recruited by EG7 were weakly positive for BrdU at day 4 (fig. 8A), indicating delayed and weaker activation by EG7 compared to earlier and stronger activation by EG7-gp 96. Delayed activation of DCs by the wild-type tumor EG7 was associated with only minimal CD8 amplification.

CD8 cells appeared in PC by day 2 in the EG7-gp96-Ig group showed a large uptake of BrdU, while CD8 cells in draining lymph nodes and spleen were still BrdU negative (fig. 8B). This finding is consistent with the proliferation of CD8 starting locally in the peritoneum rather than in the lymph nodes. By day 4, gp 96-dependent uptake of BrdU by CD8 cells was very evident in PC and still very significantly higher than in lymph nodes or spleen (fig. 8B).

NK cells in the gp96 group, but not the EG7 group, were activated by day 4 as indicated by upregulation of CD69 (fig. 8C) and 2B4 (data not shown). NK activation occurred only in PEC (fig. 8C) and not in lymph nodes or spleen as measured by up-regulation of CD69 (data not shown), antigen (antipigenain) suggesting local activation.

These data show that local gp96 release in PC can deliver signals leading to local recruitment and activation of innate and adaptive immune cells, providing a cellular mechanism of CD8 cross-sensitization independent of lymph nodes and CD4 cells. The cross-sensitization mechanism was independent of the particular anatomy of the PC, as subcutaneous administration of EG7-gp96-Ig or 3T3-OVA-gp96-Ig was equally effective at OT-I cross-sensitization (data not shown).

High-efficiency cross-sensitization of CD8CTL by gp 96-chaperone peptides: gp96-Ig secretion of EG7-gp96-Ig resulted in a dramatic increase in OT-I expansion when compared to EG7, even though both cell lines secreted equal amounts of OVA (approximately 80ng/24 hr. times.10)⁶Cells) (fig. 6). Similar differences in OT-I amplification were seen when comparing OT-I amplification in the response to the isogenic 3T3-OVA and 3T3-OVA-gp 96-I. Oizumi et al J Immunol.2007Aug 15; 179(4): 2310-7. Gp96-Ig secreted from OVA-transfected cells contained a small fraction (about 0.1% or less) of gp96 molecules (gp96-OVA) that accompany the OVA peptide, and these gp96 molecules are believed to be responsible for OT-I cross-sensitization. However, secreted gp96 may also act as a non-specific adjuvant for recruitment and activation of DCs, and thereby enhance uptake and cross-sensitization of OVA proteins. Finally, it is possible that gp96-Ig and OVA proteins are secreted as separate molecules and form a gp96-Ig-OVA complex extracellularly. Several experiments were performed to distinguish between these possibilities.

First, we compared the dose-response curves for the efficiency of OT-I amplification in PC and spleen following intraperitoneal injection of 3T3-OVA, 3T3-OVA-gp96-Ig or EG7, EG7gp96-Ig and pure OVA protein (FIG. 9). The secretion rates of OVA and gp96-Ig were determined in vitro by ELISA as nanograms secreted per 24 hours, respectively. By injecting different cell numbers, dose ranges of secreted OVA and gp96-Ig were obtained as shown in FIG. 9. OT-I amplification was measured 4 days after stimulation. OT-I was not amplified by 3T3-OVA cells that secreted only OVA at a rate of 80-800ng per 24 hours. Clearly, this amount of OVA does not cross sensitize OT-I even in the presence of allogeneic activation of the immune system. Similarly, even though EG7 cells express K^b-OVAIsogenic EG7 cells that singly secreted OVA only minimally amplified OT-I (FIG. 6), suggesting that direct sensitization of OT-I is very inefficient. In contrast, 80-800ng gp96 per 24 hours effectively cross-sensitizes OT-I when gp96 is secreted by OVA-containing tumor cells, and causes them to expand locally as well as in the spleen. In contrast, effective cross-sensitization of OT-I by OVA protein requires 3-10mg of protein. In pair of OVA eggsThe sensitivity difference in OT-I amplification in response to gp96 secreted by white and OVA-containing cells was about 10,000 fold by weight (fig. 9). Given the molecular weight and the fact that secreted gp96 molecules were associated with OVA peptides at most 0.1%, gp96-OVA was about 2 million-fold different in OT-I cross-sensitizing activity compared to OVA protein on a molar basis.

Adjuvant activity of gp96 on non-chaperone induced CD8-CTL cross sensitization: the data presented in fig. 9 does not address the following possibilities: that is, gp96-Ig and OVA, secreted as individual molecules rather than as the gp96-OVA complex, are responsible for the efficient cross-sensitization of OT-I. To test this possibility, OT-I amplification was studied under conditions where gp96 and OVA were intentionally administered as separate molecules. Gp96 alone but not OVA secreting 3T3-gp96 cells were injected intraperitoneally alone or together with OVA protein and OT-I expansion was quantified by the usual procedure. As shown in FIG. 10A, the allogeneic 3T3-gp96-Ig cells secreting 200ng gp96-Ig every 24 hours did not cause non-specific OT-I expansion. Likewise, 200ng and 50 μ g OVA injected alone did not mediate OT-I amplification. In contrast, when 50 μ g of OVA was co-injected with 3T3-gp96-Ig cells secreting 200ng gp96-Ig every 24 hours, nearly optimal OT-I expansion was observed, indicating that gp96 acts as an adjuvant to OVA cross-sensitization of OT-I. The effect of gp96 acting in trans with OVA increased OT-I cross-sensitization by 1000-fold over OVA alone, while gp96 (cis) with companion OVA increased cross-sensitization by more than 1 million-fold (relative to OVA alone). As a negative control, 3T3-gp96-Ig had no effect on OT-I amplification in the absence of OVA, although it had an allotype (allotype). Furthermore, the combination of 3T3-gp96-Ig secreting 200ng gp96-Ig with co-injected 200ng OVA protein failed to cross sensitize OT-I, precluding the possibility of gp96-Ig and OVA forming an extracellular complex.

This data suggests that adjuvant effects of gp96 are mediated by stimulation of activation and pinocytosis of DCs, resulting in increased uptake of OVA protein and cross presentation by MHC I to OT-I. Although gp96 shows substantial adjuvant activity for cross-sensitization to non-chaperone OVA, internalization of the gp96-OVA complex by the CD91 receptor is even more effective in obtaining gp96 chaperone peptides for MHC class I presentation and thereby further enhancing cross-sensitization efficacy.

Continuous secretion of gp96-Ig provided the greatest CD8 cross-sensitizing activity for adoptively transferred transgenic CD8 cells and endogenous CD8 cells: the model system for secretion of gp96 from tumor cells raises a problem: how continuously secreted gp96 was compared to bolus gp96 in its effect on OT-I cross-sensitization. Since OVA and OT-I are artificial test systems, it is also important to ensure that the data obtained with OT-I are applicable to endogenous, non-transgenic CD8 cells. Importantly, EG7-gp96-Ig immunization of B6 mice provided 50 to 100-fold improved CD 8-dependent protection against subsequent maternal EL4 cell challenge rather than against Lewis lung cancer compared to preimmunized mice, suggesting gp 96-dependent cross-sensitization against endogenous tumor antigens. Yamazaki et al, 1999, j.immunol.163: 5178-5182. Endogenous non-transgenic OVA-specific CD8 cells present at a low frequency (0.005%) of CD8 cells of about 1/20,000 were immune expanded against EG7-gp96-Ig to a frequency of 1-3% in the CD8 gate, indicating similar expansion to OT-I starting from lower frequencies (data not shown). Together, these data indicate that gp 96-mediated cross-sensitization is not limited to TCR-transgenic OT-I cells, but also plays a role in endogenous tumor-specific and OVA-specific CD8 cells.

Comparing the effect of intraperitoneal injection of 200ng of serum-free gp96-Ig-OVA harvested from 3T3-OVA-gp96-Ig culture with the effect of injection of 3T3-OVA-gp96-Ig cells secreting 200ng within 24 hours in vivo, a significant increase in OT-I expansion was observed when gp96-OVA was continuously secreted by bolus injection of gp 96-OVA. This observation indicates that continuous release of gp96, which may occur, for example, due to ongoing cell death caused by injection, is the most optimal stimulus for homologous CD8 cross-sensitization without the assistance of CD4 and without the need for lymph nodes.

This study revealed that the gp96 chaperone peptide surprisingly enhanced cross-sensitization activity by more than 1 million fold compared to the purified protein alone. This finding is significant because it provides a highly sensitive mechanism for generating CD8 CTLs against antigenic peptides released from dying cells.

In our analysis of the efficiency of antigen cross-presentation, OT-I amplification was used as a sensitive and quantitative readout for antigen cross-presentation mediated by gp96-OVA, by gp96+ OVA, or by OVA alone. The observed differences in OT-I amplification can only be explained by the efficiency of the cross-presentation activity of different forms of OVA. gp96 companion OVA apparently were most active in cross presentation, followed by OVA plus gp96 as adjuvant, followed by OVA alone, which was more than 1 million fold less active than companion OVA in cross sensitization.

However, this gp 96-mediated cross-sensitization mechanism may be physiologically important when cells die due to infection or necrosis, a process that may be accompanied by the release of gp 96-accompanied antigenic peptide from an infection that causes cell death^Factor(s)And (4) obtaining. The attraction and activation of DC cells and NK cells to the site of infection, cell death, and gp96 release provide an efficient route for cross-presenting antigenic gp96 chaperone peptides to CD8 cells and for generating lymph node independent in situ CTLs. These CTLs are then used to clear adjacent infected cells, thereby limiting the spread of infectious agents.

Defense systems based on stimulation of the innate immune system by heat shock proteins have apparently been present in early vertebrate phylogeny in amphibians. With the evolution of adaptive immunity, it appears that the role of gp96 extends from its adjuvant function to that of a carrier for specific antigens for efficient MHC class I cross-presentation and cross-sensitization of CD8 CTLs.

In support of this model and hypothesis, we provide evidence that in situ secretion of gp96 results in local recruitment and activation of a large number of DC cells and NK cells capable of locally activating homologous CD8 cells. DCs in secretory response to gp96 proliferate in PC but not elsewhere; similarly, NK cells become activated only in PC. Syngeneic CD8 cells showed the earliest and most active proliferation in PCs; later, however, CD8 proliferation was also spread to other sites including the spleen. As suggested in our model in PC, the interpretation of local cross-sensitization of CD8 cells by gp96 predicts and requires that the cross-sensitization process should be able to function in the absence of lymph nodes. This was confirmed in LT α ko mice. Importantly, efficient CD8 cross-presentation by gp96-Ig is not limited to PC. Equally effective cross-presentation of CD8 and generation of systemic immunity was also observed following subcutaneous immunization of gp96-Ig secreting tumors. The peritoneal site was chosen for analysis because it is readily available and there is no heterogeneous population of cells found elsewhere.

The cross-presentation of lymph node independent CD8 cells by gp96 chaperone peptides is consistent with its CD40L and CD4 independent help. DC activation, however, appears to be mediated by gp96 binding to CD91 and TLR2/4 as previously shown by others. In preliminary experiments, we were able to demonstrate that anti-CD 91 antibodies completely block gp 96-mediated CD8 cross-sensitization. However co-stimulation of CD8 cells by CD80 and CD86 is absolutely required for cross-sensitization of CD8 by gp 96.

These studies also show that gp96 can act as an adjuvant for CTL production by enhancing cross-sensitization of antigenic proteins that colonize the extracellular environment. The release of heat shock proteins from dying cells may serve as a "danger signal" for activating innate immune responses by activating DCs, stimulating the pinocytosis of DCs on extracellular proteins, and their MHC I cross-presentation. Adjuvant activity of gp96 also activates NK cells, thereby eliciting Th1 responses and enhancing clearance of extracellular infectious agents.

An important factor for the outstanding cross-sensitizing activity of gp96 is its continuous, sustained release through secretion. In our model system, allogeneic or syngeneic tumor cells secrete gp96, allowing analysis of the secretion and non-secretion of a single variable gp96 in an in vivo system. This method does not require fractionation of the cells and purification of the antigen or gp96, thereby avoiding potential problems associated with biochemical purification steps. The data show that sustained (24h) release (secretion) of a small amount of gp 96-peptide complex (about 200ng/24h) is much more effective in CD8 cross sensitization than the same amount of gp 96-peptide complex as a bolus. It is evident that continuous stimulation of the immune system over a period of time, similar to that observed in ongoing infections, is much stronger than immune stimulation by rapid dilution or by bolus ingestion by phagocytic cells. Preliminary data suggest that gp 96-secreting, live, allogeneic 3T3 fibroblasts injected intraperitoneally survive 5-7 days before being cleared. Irradiation of gp96 secreting tumor cells or treatment with mitomycin C neither reduced their gp96 secretion nor their in vivo cross-sensitizing activity (data not shown), indicating that enhanced CD8 cross-sensitization does not require cell proliferation.

In addition to revealing potentially important lymph node and CD4 dependent immune defense mechanisms, these studies provide the basis for designing effective cellular vaccine strategies.

Example 3: immune disruption of tumor-induced immunosuppression in the absence of B cells or frequent vaccination

This example demonstrates that tumor-induced immunosuppression is antigen-nonspecific and can be overcome by frequent immunization or by the absence of B cells. The resulting tumors inhibit clonal expansion of CD8T cells in vivo, which is typically observed in tumor-free mice following vaccination with antigen-specific glycoprotein (gp) 96-chaperone. The resulting tumor inhibits CD8T cell expansion independent of tumor-associated expression of antigens recognized by CD8-T cell receptors. Vaccination of tumor-bearing mice was accompanied by increased recruitment of cells to the vaccine site compared to tumor-free mice. However, rejection of established suppressive tumors requires frequent (daily) vaccination with gp 96. B cells are known to attenuate T helper-1 responses. We found that in B cell deficient mice, tumor rejection of established tumors can be achieved by a single vaccination. Thus, clonal expansion of cognate CD8 cytotoxic T lymphocytes is enhanced in response to vaccination with the gp96 chaperone in tumor-free B cell deficient mice. Frequent vaccination with a cell vaccine and simultaneous B-cell depletion may greatly enhance the activity of anti-cancer vaccine therapy in patients.

Mice: c57BL/6J (B6) mice were purchased from Jackson laboratories (Bar Harbor, ME) or Charles River laboratories (Frederick, MD). Ig-m-chain deficient mice with a C57BL/6J background [ B Cell Deficient Mice (BCDM) ] were purchased from Jackson laboratories.

GFP mice are available through the producer's friendly license. C57BL/6J oxytocin-1 (OT-1) mice (obtained from m.bevan, university of washington, seattle, washington) expressed transgenic TCRs (va2vb5.1.2) specific for H-2Kb restricted chicken ovalbumin-derived peptides 257 to 264 (SIINFEKL). In the animal facility at the university of Miami, GFP mice were crossed with OT-1 mice according to institutional guidelines to produce GFP-OT-1 mice. Progeny mice expressing the ova-TCR gene were screened by polymerase chain reaction and GFP by fluorescence. All mice were administered antigen (antigene) between 6 and 12 weeks.

Cell line: EG7 cell line (obtained from m.bevan) was transfected with the vector pCMG-His containing the gp 96-Ig. Control cells were transfected with vector alone. Lewis Lung Carcinoma (LLC) cells were obtained from the American type culture Collection and were transfected with ovalbumin in pAC-neo-ova or with both ovalbumin vector and pCMG-His containing gp 96-Ig. All cells were cultured in Iscove's modified Dulbecco's medium (GIBCO, Carlsbad, Calif.) with 10% fetal bovine serum and Gentamicin (GIBCO). To maintain transfected cells, antibiotics for selection (G418 or L-histaminol, Sigma, St Louis, Mo.) were added to the cultures.

Antibody: the following antibodies were used for staining; anti-CD 16/32(2.4G2), cytochrome-anti-CD 3e (145-2C11), cytochrome-anti-CD 5(UCHT2), cytochrome-anti-CDga (53-6.7), PE-CD19(4G7), PE or FITC-anti-NK 1.1(PK136), and PE or FITC-anti-CD 11C (HL3) were purchased from BD PharMingen (San Diego, Calif.).

Purification and adoptive transfer of GFP-OT-1 cells and CD19+ B cells: mixed single cell suspensions of splenocytes and lymph node cells were obtained from GFP-OT-1 mice and depleted of erythrocytes by lysis with ammonium chloride. According to the manufacturer's instructions byGFP-OT-1 cells were sorted using positive column selection against CD8a magnetic microbeads and a MACS column (Miltenyi Biotec, Auburn, Calif.). Isolated OT-1 cells were more than 95% pure as determined by flow cytometry analysis. Expression of Va2 and vb5.1.2 on purified cells was quantified by flow cytometry. For purification of B cells, CD19+ cells were purified with anti-CD 19 microbeads (Miltenyi Biotec, Auburn, CA). To reconstitute B cells in BCDM mice, 10 days before tumor cell transplantation will be performed⁷Purified cells were adoptively transferred via tail vein.

Analysis of CD8CTL amplification in vivo: to measure the amplification of CD8+ CTL, 10 was used⁶GFP-OT-1 adoptive transfer of mice, and 2 days later by intraperitoneal injection of 1X 10⁶To 4X 10⁶Mice were immunized with non-irradiated EG7-gp96-Ig cells. Following a timed interval following immunization, peri-aortic lymph nodes [ draining lymph node (dLN) were removed from the peritoneal cavity, mesentery, and at designated times]And collecting cells from peripheral blood. Erythrocytes were removed from the samples by lysis with ammonium chloride. One million cells were incubated with anti-CD 16/32 monoclonal antibody in Phosphate Buffered Saline (PBS) containing 0.5% bovine serum albumin (phenylboronic acid) at 4 ℃ for 10 minutes to block FcR binding. Thereafter, cells were incubated with the indicated antibodies for 30 minutes. Samples were analyzed on a FACScan (Becton Dickinson) with CELL Quest software (BD Bioscience). The total number of immune cells indicated for each tissue is calculated from the percentage of target cells and the total number of cells in each tissue.

Tumor inoculation and treatment protocol: unirradiated EG7, LLC or LLC-ova cells were injected subcutaneously into syndromes of mice in 200-mL PBS. Five days after LLC-ova cell inoculation (day 5), 10 in 0.3-mL volume of PBS was injected via tail vein⁶Purified GFP-OT-1. Two days later, according to the schedule indicated in the figure, 10 in PBS by an intraperitoneal injection of 0.5-mL volume⁶Mice were immunized with unirradiated LLCova-gp96-Ig or EG7-gp96-Ig cells. Control mice were treated with PBS or with EG7 or LLC-ova. Tumor size in the flank was measured twice weekly in a two-dimensional manner for at least 20 days.

Statistical analysis: significance was assessed by analysis of variance of repeated measures and by Wilcoxon signed rank test. A value of P < 0.05 was considered to indicate statistical significance.

Established tumors inhibit TCR-independent Gp 96-mediated CD8CTL amplification: transfection of the heat shock fusion protein gp96-Ig into tumor cells results in secretion of gp96-Ig along with a gp96 chaperone peptide. gp96-Ig is a fusion protein produced by substituting the Fc portion of IgG1 for the endoplasmic reticulum retention signal (KDEL) of gp 96. Injection of mice with gp96-Ig secreting tumor cells resulted in the induction of tumor specific immunity and memory and protection from subsequent challenge with the same, but untransfected, tumor. Tumor immunity generated by secreted gp96-Ig is specific to gp 96-chaperone peptides, including peptides derived from tumor-endogenous antigens such as EL 4-specific antigens, and alternative antigens such as ovalbumin transfected into EL4(EG7) or LLC (LLC-ova). Ovalbumin replacement antigens provide a means to accurately determine CD8CTL expansion in vivo by adoptive transfer of ovalbumin-specific, OT-1 TCR transgenic CD8 cells.

Established tumors are known to be inhibitory to CTL amplification. To measure CTL responses in the presence or absence of established tumors, we used the TCR transgenic OT-1 system, in which the transgenic CD8CTL responds to either isogenic or heterogenic tumors secreting gp96-Ig-ova transfected ovalbumin. As a transplantable tumor model we used EG7 derived from EL4 transfected with ovalbumin, EG7 being classified as immunogenic and highly tumorigenic. In addition, we also used LLC and LLC-ova, which are believed to be less immunogenic and highly tumorigenic. The division rates of both cell lines were very fast with doubling times of 8 to 12 hours in culture.

In one million EG7-gp96-Ig cells (endocrine 60 to 80-nggp96-Ig/10 in 24 hours)⁶Cells) after a single intraperitoneal immunization, OT-1CD 8T cells expanded from lower preimmune levels (B0.2%) in the CD8 gate to high frequencies (15% to 40%) in tumor-free mice (figure)11A) In that respect The administration of irradiated EG7 without gp96-Ig secretion did not result in significant OT-1 amplification. However, the presence of EG7 tumors formed subcutaneously at a distal site in the flank significantly inhibited gp 96-vaccine-induced OT-1 expansion in the peritoneal cavity (FIGS. 11A-C) and systemically in the spleen and lymph nodes (not shown). EG7 tumors secreted ovalbumin and expressed Kb-ova. Thus, it is likely that adoptively transferred OT-1 becomes anergic after recirculation through the tumor bed or tumor dLN, as signals are received through their Kb-ova specific TCRs, but not co-stimulatory signals. To test this hypothesis, syngeneic tumors EL4 and LLC, both of which do not express ovalbumin, were formed subcutaneously at the distal site. Subsequently, OT-1 was adoptively transferred by intravenous injection and mice were immunized intraperitoneally with EG7-gp96-Ig as before. The established EL4 and LLC was as effective as the established EG7 in inhibiting OT-1 amplification by secreted gp96-ova, indicating inhibition of the appropriate TCR antigen Kb-ova in tumor independent (fig. 11B, C). Although OT-1 amplification was inhibited in the peritoneal cavity and systemically by the presence of the distal sites LLC and EL4, surprisingly, total cell recruitment following immunization into the peritoneal cavity upon intraperitoneal immunization with EG7-gp96-Ig was actually increased when compared to tumor-free mice (fig. 11D).

The data indicate that established tumors are capable of eliciting induction of antigen-non-specific inhibition of CTL expansion. The induction of this inhibition is associated with increased recruitment of cells to the vaccine site within the peritoneal cavity. It is being investigated whether this increase in cell recruitment is responsible for the inhibition of CD8T cells.

To overcome antigen non-specific immunosuppression, these experiments tested whether frequently repeated antigen-specific stimulation of CD8 CTLs by vaccination could counteract the inhibitory activity found in tumor-bearing mice.

Rejection of established tumors requires frequent Gp96-Ig immunization: although many vaccination strategies, including secreted gp96-Ig, are capable of establishing protective immunity in mice against tumors and tumor antigens, it is more difficult to reject already established tumors by therapeutic vaccination. Given the observation of antigen-non-specific inhibition of CD8 expansion, we analyzed how different vaccination schedules affected tumor rejection and/or tumor growth.

We initially analyzed the effect of therapeutic vaccination by starting vaccination on the same day as tumor transplantation. One million EG7 tumor cells were transplanted subcutaneously in the flank of syngeneic mice. On the same day (day 0), 60 to 80ng/10 will be used⁶One million gp96-Ig secreting EG7 vaccine cells (EG7-gp96-Ig) secreting gp96-Ig at cell x 24 hours rate were administered intraperitoneally as a vaccine and vaccination was repeated on days 3, 7, 10 and 14. Vaccination with 4 EG7-gp96-Ig initiated on the same day of tumor transplantation significantly reduced tumor growth (P ═ 0.0078) compared to untreated mice (fig. 12A). The therapeutic effect is gp96 and antigen dependent. Irradiated EG7 (FIG. 12A) which does not secrete gp96-Ig or LLC-gp96-Ig (FIG. 12B) which does not express EG7 antigen but secretes gp96-Ig at the same rate as EG7-gp96-Ig, when administered intraperitoneally as a vaccine at the same dose and schedule as EG7-gp96-Ig, did not retard tumor growth. When vaccination with EG7-gp96-Ig was started 2 days or 2 days after EG7 vaccination, the therapeutic effect was substantially reduced using the same vaccination schedule (fig. 12A). These data show that even after 2 days, tumors formed by vaccination are more difficult to control than freshly transplanted tumors.

It was also tested whether tumors that developed for 3 or more days could be controlled by a more frequent vaccination schedule. One million EG7 tumor cells were implanted subcutaneously in the flank and allowed to form for 3 to 7 days, allowing at least 7-fold or more tumor cells to multiply. During this time, visual vascularization of the tumor nodules occurred. Mice were then inoculated daily intraperitoneally with one million EG7-gp96-Ig cells, or LLC-gp96-Ig cells or irradiated EG7 cells at the same schedule and dose, or no inoculation, in a specific control. Daily vaccination with EG7-gp96-Ig vaccine significantly (P ═ 0.0078) and effectively controlled the growth of EG7 that had formed for 3 days (fig. 12B), whereas daily vaccination with irradiated EG7 or with LLC-gp96-Ig had no effect on the growth of EG7 that had formed (fig. 12B). In a further study, we allowed the formation of transplanted EG7 tumors 5 and 7 days before the start of vaccination with EG7-gp 96-Ig. As shown in fig. 12C and 12D, two vaccinations per day were required to delay tumor growth in the late stages of tumor formation. This data shows that frequent immunizations are able to examine tumor growth in mice over a 24 day period. Further studies will be needed to determine if a sustained long-term vaccination schedule can completely eradicate the tumor.

To validate the data obtained with immunogenic EG7 lymphoma, the experiment was repeated with less immunogenic, established LLC (fig. 13). Repeated intraperitoneal immunizations with LLC-gp96-Ig, starting on day three after tumor transplantation (days 3, 7, 10 and 14) resulted in a significant (P ═ 0.0234) delay in LLC tumor progression. Daily LLC immunity is not more effective in tumor delay. The effect of immunization is tumor specific, since EG7-gp96-Ig vaccination cannot control LLC tumor growth. Tumor growth control also cannot be achieved by irradiated LLC, but is dependent on gp96-Ig secretion.

These data suggest that frequent DC cell and NK cell activation coupled with cross-antigen presentation by secreted gp96-Ig and its chaperone peptides can overcome established tumor-induced antigen non-specific immunosuppression.

gp 96-mediated recruitment of DC cells and NK cells and CD8CTL expansion were enhanced in BCDM: several groups have reported that the anti-tumor response of T helper-1 is enhanced in BCDM when compared to Wild Type (WT) mice. We therefore investigated the role of B cells in gp 96-mediated CTL expansion and anti-tumor immunity. The peritoneal cavity accumulated CD5+ CD19+ B cells and CD5+ CD19+ B1-B cells, the latter producing IgM antibodies and not undergoing isotype switching upon activation (fig. 14A). Upon intraperitoneal immunization with EG7-gp96-Ig, the CD5+ CD19+ population increased approximately 5-fold by day 4 post immunization, while CD5+ B1-B cells increased only modestly (FIG. 14A). gp 96-mediated OT-1 expansion was greatest at day 4 and day 5 post-immunization. gp 96-mediated OT-1 expansion was preceded by recruitment to and activation of DC cells and NK cells within the peritoneal cavity at the site of vaccination. As previously shown, NK cells are important promoters of gp 96-Ig-mediated CD8CTL expansion. In BCDM, recruitment of DCs into the peritoneal cavity (vaccination site) was similar to that in wild type mice on day 2 post-vaccination. However, although DC numbers decreased by 50% in wild-type mice by day 4 post-vaccination, DC numbers remained at the same high frequency in B cell deficient mice (fig. 14B). NK cell recruitment in BCDM increased on days 2 and 4 (fig. 14B). The differences did not reach significance, but were reproducible in 3 independent experiments. Adoptive transfer of wild type B cells to BCDM abolished increased DC retention and recruitment of NK cells. This finding suggests that B cells influence gp 96-induced recruitment of innate immune cells, and that B cells may also be involved in mediating or inhibiting CD8CTL expansion.

Thus, it was also tested whether amplification of GFP-labeled OT-1CD 8CTL was increased in BCDM as an immune response to gp 96. As shown in fig. 15, OT-1 amplification following gp96 immunization in BCDM was significantly enhanced at day 5 compared to wild type mice. Importantly, OT-1 continued to be significantly more frequent (P ═ 0.04) (fig. 15A) at days 7 and 12 after immunization in the peritoneal cavity in dLN (fig. 15B), the expansion and retention of OT-1 also increased, however, without reaching significance. Adoptive transfer of wild type B cells to BCDM prior to immunization reduced OT-1 amplification to levels equal to or lower than those seen in wild type mice (fig. 15A, B). The inhibition of OT-1 amplification by the presence of B cells is not mediated by the production of Interleukin (IL) -10, since IL-10 deficient mice exhibit OT-1 amplification similar to wild-type mice, rather than the enhanced amplification seen in BCDM.

gp 96-mediated rejection of established non-immunogenic tumors is enhanced in the absence of B cells: as shown above, growth control of the established EG7 tumor in wild type mice minimally requires daily gp96 immunization. Similarly, LLC evolution may be delayed by frequent immunizations. EG7 and EL4 cells were rejected in BCDM and did not form tumors; however, although LLC and LLC-ova have slower growth rates than in wild type mice, they can be formed in BCDM. LLC-ova was formed subcutaneously in the flank for 5 days in BCDM and wild type mice. OT-1 was adoptively transferred intravenously, and 2 days later, one million LLC-ova-gp96-Ig was administered intraperitoneally in a single dose and tumor growth was monitored in the flank. In wild type mice, a single immunization with LLC-ova-gp96-Ig caused a significant delay in tumor progression in the flank, but failed to reject the tumor (FIG. 16A). In contrast, in BCDM, a single immunization resulted in complete rejection of established, 7-day LLC-ova tumors in 3 mice and significant tumor shrinkage in 2 mice (fig. 16B). In the absence of treatment, LLC-ova grew progressively in BCDM (FIG. 16B), although at a slower rate than in wild type mice (FIG. 16A). B-cell reconstitution in BCDM (fig. 17C) resulted in a similar effect of vaccination as seen in wild type mice (fig. 16A), i.e. a delay in progression. It would be of interest to determine whether complete or partial B cell depletion by antibodies has a similar effect as a B cell deficiency. Preliminary studies in progress appear to support this approach.

Optimal tumor control of established LLC in BCDM by a single immunization relies on a sufficiently high number of tumor-specific CTL precursors (OT-1) and on antigen-specific immunity (LLCova-gp 96-Ig). In BCDM, the presence of one million adoptively transferred OT-1 without gp96 immunization did not result in rejection of tumors in most mice (fig. 17A). Also, gp96 immunization alone was not as effective as combination without OT-1 metastasis (FIG. 17B).

It is well understood that established tumors suppress anti-tumor immunity. Tumor-specific T cells become anergic in the presence of established tumors. Anergy to the B cell lymphoma used in this study was antigen specific, MHC restricted, and dependent on the presence of MHC-matched myeloid-derived antigen presenting cells. In other studies, antigen-non-specific myelosuppressive and T-regulatory cells were associated with suppression of anti-tumor immunity. Our studies show that inhibition of CTL responses in vivo can be achieved by established tumors via an antigen-independent pathway. OT-1CD 8CTL expansion in response to gp96-ova vaccination was inhibited by established tumors independent of tumor-expressed ovalbumin. This type of suppression can be achieved by T regulatory cells or by other suppressor cells such as myelosuppressive cells or M2 macrophages. According to this hypothesis, the inhibitory activity in preliminary experiments was transferable to tumor-free mice by transferring peritoneal cells primed by gp96 vaccination in tumor-bearing mice.

Although the OT-1 response to gp96-ova immunity is strongly suppressed in the presence of established tumors, it is generally not blocked, suggesting that there is a balance between immunosuppression by established tumors and vaccine-induced CD8CTL activation through antigen cross-presentation of activated DCs stimulated by secreted gp 96-ova. We have previously shown that in tumors, innate mouse gp96-ova leads to recruitment and activation of NK cells and DCs, followed by expansion of OT-1. Established tumors although in fact cell recruitment into the peritoneal cavity was enhanced by vaccination with LLC-gp96-Ig, they inhibited OT-1 expansion, suggesting that many of the recruited cells may be suppressor cells in the presence of established tumors. This hypothesis predicts that frequent immunization with gp96-ova can overcome inhibitory activity by shifting this balance from inhibition to increased immune activation through repeated gp 96-mediated stimulation of DC cells and NK cells, increased antigen cross-presentation, and CTL sensitization. In fact, frequent immunizations have a significant effect on delaying tumor progression. In the case of established EG7, one or two daily vaccinations were much more effective at stopping tumor progression than one every two or three days. For LLC, immunization every other day or every third day is sufficient, and daily immunization is not more effective. These tumor-specific differences may be related to the rate of suppressor cells produced by the presence of peripheral tumors. Alternatively, this may depend on the mechanism by which the tumor mediates the induction of suppressor cells or the nature of the induced suppressor cells.

By studying the intraperitoneal immune response of OT-1 to tumors secreting gp96-ova, we noted that a large number of B cells were recruited into the peritoneal cavity, which is the site of the vaccine. B cells have been reported to be inhibitory against tumor immunity, posing problems with their role in gp 96-mediated OT-1 expansion. With BCDM, it became immediately clear that NK cell and DC recruitment and retention in the peritoneal cavity increased, and OT-1 expansion was enhanced following gp96-ova immunization. B-cell reconstituted BCDM responded to gp 96-ova-mediated OT-1 expansion as in wild-type mice, excluding the possibility of: that is, a B cell deficiency alters the responsiveness of BCDM to gp96-ova immunity in a manner unrelated to the absence of B cells. B cell deficiency not only caused enhanced OT-1 expansion, but also promoted a strong enhancement of tumor rejection in the formation of 7-day LLC-ova tumors after a single gp96-Ig immunization. This data suggests that tumor-mediated induction of suppressor cells is greatly reduced in the absence of B cells or that B cells themselves act as "suppressor cells". Further investigation is required whether B cells are involved in the induction of suppressor cells or whether B cells themselves are immunosuppressive to CTL responses; however, IL-10 does not appear to be involved in the suppression of B-cell mediated tumor immunity. In ongoing studies, we have found that OX40-L deficient B cells exhibit a reduced ability to inhibit anti-tumor immune responses.

These studies provide a model by which antigen-independent immunosuppression can be studied and further determined. The specific role of B cells in this process would be of great interest. In addition, these studies are directed to methods that can make anti-tumor vaccines more effective. With antibodies and subsequent frequent vaccination, for example with gp 96-secreting tumor vaccines, depletion of B cells may lead to more effective control of tumor growth than seen with conventional vaccination methods.

Example 4: the anti-tumor effect caused by vaccination with heat shock protein gp96 was enhanced in the absence of B cells.

The antitumor activity of gp96 was increased in the absence of B cells: immunological tumor rejection generally relies on the generation of cytotoxic CD8 cells during a Th1 biased anti-tumor immune response. Tumor escape strategies typically include an immunological bias towards a Th2 biased humoral response, including production of Th2 cytokines. Because of the feedback inhibition of Th2 response with B cell activation and TH1 polarizationIn connection, we tested whether the anti-tumor immune response against gp96 was affected by the absence of B cells. As a tumor system we used LLC-ova as a surrogate antigen, a spontaneously transplantable lung cancer transfected with ovalbumin. LLC-ova is non-immunogenic, rapidly growing (16 hours division time), and fatal in about four weeks. LLC-ova was further transfected with gp96-Ig to produce LLC-ova-gp96-Ig, LLC-ova-gp96-Ig was a tumor secreting gp96-Ig at a rate of 1 million cells secreted in 24 hours (Song). LLC-gp96-Ig mediated strong homologous CD8-CTL activation and generated anti-tumor immunity. This immune model was used to assess the effect of the absence of B cells on anti-tumor responses. LLC-ova was transplanted subcutaneously in the flank of wild type mice and B cell deficient (pMT) mice and allowed to develop for 7 days, after which time (day 0) 1 million viable LLC-ova-gp96-Ig cells were injected intraperitoneally. Two days before immunization with LLC-gp96-Ig were administered intravenously (10)⁶Cell) TCR transgenic OT-I cells that detect the ovalbumin-derived peptide SIINFEKL presented by ICb. LLC-ova in wild type mice grew progressively in the absence of immunity even in the presence of OT-I (FIG. 16). One million LLC-ova-gp96-Ig cells injected intraperitoneally at a time delayed tumor growth but failed to mediate complete tumor rejection. LLC-ova also forms progressive tumors in B cell deficient mice in all mice, but tumor progression is slower than in wild type mice. Immunization of B-cell deficient mice with gp 96-secreting LLC-ova resulted in complete tumor rejection. Tumors did not recur during the 6 week follow-up study.

It is evident that B-deficient mice in this model tumor system are able to mount tumor rejection responses to gp96 immunization in the presence of an elevated frequency of tumor-specific precursor CTL (OT-I). Tumor rejection was dependent on both components, as the anti-tumor response to LLC-ova-gp96-Ig was significantly reduced in the absence of adoptively transferred OT-I (FIG. 17B). Similarly, OT-I alone failed to reject LLC-ova without immunization (FIG. 17A). Reconstitution of B cells by transferring wild type B cells in B cell deficient mice abolished gp 96's ability to reject established tumors (fig. 17C). The absence of normal B cells is clearly responsible for the enhanced tumor rejection response in B-deficient mice.

Enhanced CD8CTL clonal expansion in B-deficient mice: gp 96-based immunization the increased ability to reject established LLC-ova tumors in B-deficient mice suggests an increase in CD8CTL activation. Using gfp-labeled OT-I cells we compared clonal expansion of OT-I cells after immunization of B-deficient mice and wild-type mice. OT-I cells were adoptively transferred intravenously, and mice were injected with LLC-ova-gp96-Ig after a two-day equilibration period. The frequency of gfp-OT-I was determined in the peritoneal cavity as well as in draining mesenteric and periaortic lymph nodes on days 5, 7 and 12 post-immunization. There was virtually no OT-I in the peritoneal cavity prior to immunization, and their frequency in draining lymph nodes was 0.5% in the CD8 phylum. As previously reported, gp 96-secreting tumors mediate strong CD8CTL amplification in wild-type mice, with a maximum at day 5. LLC-ova, which does not secrete gp96-Ig, does not amplify OT-I. Amplification was followed by the next week of contraction (fig. 15). In B-deficient mice, CD8CTL amplification consistently increased to approximately twice the number seen in wild-type mice.

Reconstitution of B cell deficient mice with wild type B cells resulted in a CD8 response that was phenotypically indistinguishable from wild type mice. Intraperitoneal immunization with gp 96-secreting tumor cells results in the recruitment of a large number of immune cells, including B cells, dendritic cells, and NK cells. In wild type mice, B cell accumulation was kinetically consistent with CD8CTL expansion, both occurring maximally between day 3 and day 5. DC cells and NK cells are recruited into the peritoneal cavity during the first 48 hours after gp96-Ig immunization. In the absence of B cells, recruitment of DC cells and NK cells increased, while reconstitution of B cell deficient mice with wild-type B cells restored wild-type levels of recruitment of DC cells and NK cells.

Purification and adoptive transfer of OT-I cells and CD19+ B cells: a mixed single cell suspension of spleen cells and lymph node cells was obtained from gfp-OT-I mice and depleted of red blood cells by lysis with ammonium chloride. gfp-OT-I cells were sorted by positive column selection using anti-CD 8a magnetic microbeads and MACS (Miltenyi Biotec, Auburn, CA) according to the manufacturer's instructions. Isolated OT-I cells were more than 95% pure CD8 positive as determined by flow cytometry analysis. Expression of Va2 and VP5.1.2 on purified cells was quantified by flow cytometry prior to injection. To purify B cells, CD19+ cells were purified with anti-CD 19 microbeads under the same procedure. To reconstitute B cells in pMT mice, 10' purified cells were adoptively transferred through the tail vein 2 days before inoculation with LLC-ova cells.

Tumor inoculation and treatment protocol: non-irradiated LLC or LLC-ova cells were injected subcutaneously into syndromes of mice in 200 plPBS. Five days after LLC-ova cell inoculation (day 5), 10 in 0.3ml volume of PBS was injected via tail vein⁶Purified OT-I. On day 7, mice were immunized with non-irradiated 1o6 LLC-ova-gp96-Ig cells injected intraperitoneally in 0.5ml volume of PBS. As a non-treatment control, mice were treated with PBS on days 5 and 7. Tumor size was measured in two dimensions twice a week for at least 20 days. To investigate OT-I amplification, adoptive transfer 10 was performed⁶Mice were immunized with 4X 10-non-irradiated EG7-gp96-Ig cells intraperitoneally after gfp-OT-I. To evaluate tumor growth in rituximab (rituximab) treated human CD20 transgenic mice, mice were treated on day 4 with 1mg of rituximab intraperitoneally injected in 0.5ml PBS or PBS alone. The experimental details were according to the same protocol as mentioned above, except for rituximab treatment.

Flow cytometry analysis: after timed intervals, cells were collected from mesenteric and periaortic lymph nodes (dLN) and peritoneal cavity at designated times. To test in RituximabDepletion of human CD20 expressing B cells after treatment, peripheral blood cells were obtained one week after injection. Erythrocytes were removed from the samples by lysis with ammonium chloride. First, one million cells were incubated with anti-CD 16132mAb in PBS (PBA) containing 0.5% BSA at 4 deg.CIncubate for 10 min to block FcR binding. Thereafter, cells were incubated in the indicated antibodies for 30 minutes. Samples were analyzed on a FACScan (Becton Dickinson) with CELL Quest software (BD Bioscience). The total number of immune cells indicated for each tissue is calculated from the percentage of target cells and the total number of cells in each tissue. Statistically analyzed significant differences in tumor growth were assessed by repeated analysis of variance tests, and values of p < 0.05 were considered to indicate statistical significance.

Claims (24)

1. A pharmaceutical composition comprising a tumor cell transfected with a eukaryotic expression vector comprising a nucleic acid encoding a secreted form of a gp96 polypeptide.

2. The composition of claim 1, wherein the secreted form of a gp96 polypeptide is a fusion protein comprising a gp96 polypeptide and an immunoglobulin signal peptide (IgSP).

3. The composition of claim 2, wherein the IgSP is selected from the group consisting of mouse IgSP, rat IgSP, porcine IgSP, monkey IgSP, human IgSP.

4. The composition of claim 2, wherein the IgSP is mouse IgSP.

5. The composition of claim 2, wherein the IgSP is human IgSP.

6. The composition of claim 1, further comprising at least one antibody directed against a B cell antigen.

7. The composition of claim 6, wherein the antibody is selected from the group consisting of subhuman primate antibodies, murine monoclonal antibodies, chimeric antibodies, humanized antibodies, and human antibodies.

8. The composition of claim 6, wherein the antibody is a murine antibody, a chimeric antibody, or a humanized antibody.

9. The composition of claim 6, wherein the B cell antigen is selected from the group consisting of CD19, CD20, CD22, HLA-DR, and CD 74.

10. A method of generating a protective immune response in a human subject, said method comprising administering to said subject an effective amount of tumor cells transfected with a eukaryotic expression vector comprising a nucleic acid encoding a secreted form of gp96 polypeptide.

11. The method of claim 10, wherein the secreted form of a gp96 polypeptide is a fusion protein comprising a gp96 polypeptide and an immunoglobulin signal peptide (IgSP).

12. The method of claim 11, wherein the IgSP is selected from the group consisting of mouse IgSP, rat IgSP, porcine IgSP, monkey IgSP, human IgSP.

13. The method of claim 11, wherein the IgSP is mouse IgSP.

14. The method of claim 11, wherein the IgSP is human IgSP.

15. The method of claim 10, wherein gp96 immunization is administered twice daily for a period of about 1 week to about 6 weeks.

16. The method of claim 10, wherein gp96 immunization is administered once daily for a period of about 1 week to about 6 weeks.

17. The method of claim 10, further comprising administering to the subject a therapeutic composition comprising a pharmaceutically acceptable carrier and at least one antibody directed against a B cell antigen.

18. The method of claim 17, wherein the therapeutic composition is administered parenterally at a dose of 20mg to 2000mg per dose.

19. The method of claim 17, wherein the subject receives the antibody in repeated parenteral doses.

20. The method of claim 17, wherein the antibody is selected from the group consisting of a subhuman primate antibody, a murine monoclonal antibody, a chimeric antibody, a humanized antibody, and a human antibody.

21. The method of claim 17, wherein the antibody is a murine antibody, a chimeric antibody, or a humanized antibody.

22. The method of claim 17, wherein the B cell antigen is selected from the group consisting of CD19, CD20, CD22, HLA-DR, and CD 74.

23. A method of generating a protective immune response in a human subject, the method comprising administering to the subject an effective amount of tumor cells transfected with a eukaryotic expression vector comprising a nucleic acid encoding a secreted form of a heat shock protein (hsp) gp96 polypeptide.

24. A method of making a vaccine against cancer, the method comprising genetically modifying a population of cancer cells to express tumor cells encoding a nucleic acid transfected with a eukaryotic expression vector comprising a nucleic acid encoding a secreted form of a heat shock protein (hsp) gp96 polypeptide.