WO2001023421A9 - Stress protein compositions and methods for prevention and treatment of cancer and infectious disease - Google Patents
Stress protein compositions and methods for prevention and treatment of cancer and infectious diseaseInfo
- Publication number
- WO2001023421A9 WO2001023421A9 PCT/US2000/027023 US0027023W WO0123421A9 WO 2001023421 A9 WO2001023421 A9 WO 2001023421A9 US 0027023 W US0027023 W US 0027023W WO 0123421 A9 WO0123421 A9 WO 0123421A9
- Authority
- WO
- WIPO (PCT)
- Prior art keywords
- hspllo
- polypeptide
- tumor
- ceu
- ceus
- Prior art date
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Definitions
- the present invention relates generally to prevention and therapy of cancer and infectious disease.
- the invention is more specifically related to polypeptides comprising at least a portion of a stress protein, such as heat shock protein 110 (hspllO) or glucose-regulated protein 170 (grpl 0), complexed with an immunogenic polypeptide, and to polynucleotides encoding such stress proteins and immunogenic polypeptides, as well as antigen presenting cells that present the stress proteins and the immunogenic polypeptides.
- hspllO heat shock protein 110
- grpl 0 glucose-regulated protein 170
- Such polypeptides, polynucleotides and antigen presenting cells may be used in vaccines and pharmaceutical compositions for the prevention and treatment of cancers and infectious diseases.
- the invention further relates to increasing the efficacy of stress protein complexes, such as by heating.
- primary breast carcinomas can often be treated effectively by surgical excision. If further disease recurs, however, additional treatment options are limited, and there are no effective means of treating systemic disease. While immune responses to autologous tumors have been observed, they have been ineffective in controlling the disease.
- One effort to stimulate a further anti-tumor response is directed at the identification of tumor antigens useful for vaccines.
- a related approach takes advantage of the promiscuous peptide binding properties of heat shock proteins, such as hsp70. These molecular chaperones bind peptides and are involved in numerous protein folding, transport and assembly processes, and could be involved in the antigen presentation pathway of MHC complexes.
- the heat shock proteins of mammalian cells can be classified into several families of sequence related proteins.
- the principal mammalian hsps based on protein expression levels, are cytoplasmic/nuclear proteins with masses of (approximately) 25 kDa (hsp25), 70 kDa (hsp70), 90 kDa (hsp90), and 110 kDa (hspllO).
- hsp25 cytoplasmic/nuclear proteins with masses of (approximately) 25 kDa (hsp25), 70 kDa (hsp70), 90 kDa (hsp90), and 110 kDa (hspllO).
- a second set of stress proteins is localized in the endoplasmic reticulum (ER). The induction of these stress proteins is not readily responsive to hyperthermic stress, as are the hsps, but are regulated by stresses that disrupt the function of the ER (e.g.
- grps glucose regulated proteins
- the principal grps on the basis of expression, have approximate sizes of 78 kDa (grp78), 94 kDa (grp94), and 170 kDa (grpl70).
- Grp78 is homologous to cytoplasmic hsp70
- grp94 is homologous to hsp90.
- hsp70 While individual stress proteins have been studied for several years (in some cases intensively studied, e.g. hsp70), the largest of the above hsp and grp groups, hspllO and grpl70, have received little attention. Both have been found by sequence analysis to represent large and highly diverged relatives of the hsp70 family. It is recognized that the hsp70 family, the hspl 10 family, and the grpl70 family comprise three distinguishable stress protein groups of eukaryotic cells that share a common evolutionary ancestor.
- the invention provides a pharmaceutical composition comprising a stress protein complex.
- the stress protein complex comprises an hspllO or grpl70 polypeptide and an immunogenic polypeptide.
- the hspllO or grpl70 polypeptide is complexed with the immunogenic polypeptide, for example, by non- covalent interaction or by covalent interaction, including a fusion protein.
- the complex is derived from a tumor.
- the complex is derived from cells infected with an infectious agent.
- the immunogenic polypeptide of the stress protein complex can be associated with a cancer or an infectious disease.
- the stress protein complex of the invention can further include additional stress polypeptides, including members of the hsp70, hsp90, grp78 and grp94 stress protein families.
- the stress protein complex comprises hspllO complexed with hsp70 and/ or hsp25.
- the invention additionally provides a pharmaceutical composition
- a pharmaceutical composition comprising a first polynucleotide encoding an hspllO or a grpl70 polypeptide and a second polynucleotide encoding an immunogenic polypeptide.
- the first polynucleotide is linked to the second polynucleotide.
- the pharmaceutical compositions of the invention can further comprise a physiologically acceptable carrier and/ or an adjuvant.
- the efficacy of a pharmaceutical composition can further comprise GM-CSF-secreting cells.
- GM-CSF-secreting cells can be co-administered with a pharmaceutical composition of the invention, by aiiministration before, during or after acjjxiinistration of the pharmaceutical composition.
- the use of GM-CSF-secreting cells enhances the efficacy of the pharmaceutical composition.
- the complex is purified from a tumor or from cells infected with an infectious agent.
- the stress polypeptide, as purified is complexed with one or more immunogenic polypeptides.
- the binding of the stress polypeptide to the immunogenic polypeptide can be altered and/ or enhanced by stress, such as by exposure to heat, anoxic and/or ischemic conditions, or proteotoxic stress.
- a stress protein complex of the invention can comprise a stress polypeptide complexed with an immunogenic polypeptide, wherein the complex has been heated.
- Such heating particularly wherein the stress polypeptide comprises a heat-inducible stress protein, can increase the efficacy of the stress protein complex as a vaccine.
- heat-inducible stress proteins include, but are not limited to, hsp70 and hspllO.
- the immunogenic polypeptide is known. Where the immunogenic polypeptide is a known molecule, the immunogenic polypeptide can be provided in admixture with the stress polypeptide, or as a complex with the stress polypeptide. The hspllO or grpl 70 polypeptide can be complexed with the immunogenic polypeptide by non-covalent binding. Alternatively, the complex can comprise a fusion protein, wherein the stress polypeptide is linked to the immunogenic polypeptide. Examples of immunogenic polypeptides include, but are not limited to, antigens associated with cancer or infectious disease, such as the breast cancer antigen her2/neu or the Mycobacterium tuberculosis antigens Mtb8.4 and Mtb39. Where the immunogenic polypeptide is unknown, it can be obtained incidentally to the purification of the stress polypeptide from tissue of a subject having cancer or an infectious disease.
- compositions comprising an antigen-presenting cell (APC) modified to present an hspllO or grpl70 polypeptide and an immunogenic polypeptide.
- APC antigen-presenting cell
- the APC can be modified to present an immunogenic polypeptide obtained by purification of hspllO or grpl70 from disease cells, including cancer cells and cells infected with an infectious agent.
- the APC is a dendritic cell or a macrophage.
- the APC can be modified by various means including, but not limited to, peptide loading and transfection with a polynucleotide encoding an immunogenic polypeptide.
- compositions of the invention can be administered to a subject, thereby providing methods for inhibiting M. tuberculosis-infec ⁇ on, for inhibiting tumor growth, for inl ibiting the development of a cancer, and for the treatment or prevention of cancer or infectious disease.
- the invention further provides a method for producing T cells directed against a tumor cell.
- the method comprises contacting a T cell with an antigen presenting cell (APC), wherein the APC is modified to present an hspllO or grpl70 polypeptide and an immunogenic polypeptide associated with the tumor cell.
- APC antigen presenting cell
- Such T cells can be used in a method for killing a tumor cell, wherein the tumor cell is contacted with the T cell.
- the invention provides a method for producing T cells directed against a M. tuberculosis-infected cell, wherein a T cell is contacted with an APC that is modified to present an hspllO or grpl70 polypeptide and an immunogenic polypeptide associated with the M. tuberculosis-infected cell.
- T cells produced by this method and a pharmaceutical composition comprising such T cells.
- the T cells can be contacted with a M. tuberculosis-infected cell in a method for killing a M. tuberculosis-infected cell.
- the T cells can be CD4+ or CD8+.
- the invention also provides a method for removing tumor cells from a biological sample.
- the method comprises contacting a biological sample with a T cell of the invention.
- the biological sample is blood or a fraction thereof.
- a method for inhibiting tumor growth in a subject comprises incubating CD4+ and/ or CD8+ T cells isolated from the subject with an antigen presenting cell (APC), wherein the APC is modified to present an hspllO or grpl70 polypeptide and an immunogenic polypeptide associated with the tumor cell such that T cells proliferate.
- the method further comprises at ministering to the subject an effective amount of the proliferated T cells, and thereby inhibiting tumor growth in the subject.
- the method for inhibiting tumor growth in a subject comprises incubating CD4+ and/ or CD8+ T cells isolated from the subject with an antigen presenting cell (APC), wherein the APC is modified to present an hspl 10 or grpl70 polypeptide and an immunogenic polypeptide associated with the tumor cell such that T cells proliferate, cloning at least one proliferated cell, and administering to the patient an effective amount of the cloned T cells, thereby inhibiting tumor growth in the subject.
- APC antigen presenting cell
- Figure 1 A shows silver staining and analysis of purified hsp proteins.
- Gel staining of hspllO and hsp70 from tumor are shown in lanes 1 and 2, respectively.
- Lanes 3 and 4 show results of an immunoblot analysis with hspllO antibody and hsp70 antibody, respectively.
- Figure IB shows silver staining and analysis of purified grp proteins, with gel staining of grp 170 from tumor in lane 1, of grp 170 from liver in lane 2, grp78 from tumor in lane 3, grp78 from liver in lane 4. Results of an immunoblot analysis with grpl70 antibody and grp78 antibody, respectively, are shown in lanes 5-6 and 7-8.
- FIG. 2A shows tumor growth after immunization with purified hspl 10.
- Tumor volume in cubic millimeters, is plotted against the number of days after challenge with 20,000 colon 26 tumor cells, for mice immunized with PBS (circles), 40 ⁇ g of liver- derived hspllO (squares), 20 ⁇ g of tumor derived hspllO (upward triangles), 40 ⁇ g of tumor derived hspllO (downward triangles) and 60 ⁇ g of tumor derived hspllO (diamonds).
- Figure 2B shows tumor growth after immunization with purified grpl70.
- Tumor volume in cubic millimeters, is plotted against the number of days after challenge with 20,000 colon 26 tumor cells, for mice immunized with PBS (circles), 40 ⁇ g of liver- derived grpl70 (squares), 20 ⁇ g of tumor derived grpl70 (upward triangles), 40 ⁇ g of tumor derived grpl70 (downward triangles) and 60 ⁇ g of tumor derived grpl70 (diamonds).
- Figure 3A is a plot showing the survival of Balb/C mice bearing colon 26 tumors after immunization with tumor derived hspl 10. Percent survival is plotted as a function of days after tumor inoculation for mice immunized with PBS (control, circles), 40 ⁇ g liver-derived hspllO (squares), and 40 ⁇ g tumor derived hspllO (triangles).
- Figure 3B is a plot showing the survival of Balb/C mice bearing colon 26 tumors after immunization with tumor derived grpl70. Percent survival is plotted as a function of days after tumor inoculation for mice immunized with PBS (control, circles), 40 ⁇ g liver-derived grpl70 (squares), and 40 ⁇ g tumor derived grpl70 (triangles).
- Figure 4A is a graph depicting tumor size as a function of days after tumor challenge in mice immunized with PBS (control). Individual lines represent individual mice to show variations between animals.
- Figure 4B is a graph depicting tumor size as a function of days after tumor challenge in mice immunized with hspl 10 derived from MethA-induced tumor. Individual lines represent individual mice to show variations between animals.
- Figure 4C is a graph depicting tumor size as a function of days after tumor challenge in mice immunized with grp 170 derived from MethA-induced tumor. Individual lines represent individual mice to show variations between animals.
- Figure 5A is a graph showing results of a CTL assay targeting colon 26 tumor cells. Percent specific lysis is plotted as a function of effector:target ratio for control T cells (circles), T cells directed against hspllO derived from colon 26 tumor cells (squares), and T cells directed against hspllO derived from MethA tumor cells.
- Figure 5B is a graph showing results of a CTL assay targeting colon 26 tumor cells. Percent specific lysis is plotted as a function of effector:target ratio for control T cells (circles), T cells directed against grpl70 derived from colon 26 tumor cells (squares), and T cells directed against grp 170 derived from MethA tumor cells.
- Figure 5C is a graph showing results of a CTL assay targeting MethA tumor cells. Percent specific lysis is plotted as a function of effector:target ratio for control T cells (circles), T cells directed against hspllO derived from colon 26 tumor cells (squares), and T cells directed against hspllO derived from MethA tumor cells.
- Figure 5D is a graph showing results of a CTL assay targeting MethA tumor cells.
- Percent specific lysis is plotted as a function of effector:target ratio for control T cells (circles), T cells directed against grp 170 derived from colon 26 tumor cells (squares), and T cells directed against grpl70 derived from MethA tumor cells.
- Figure 6 is a graph showing tumor volume, in cubic millimeters, as a function of days after tumor challenge in mice itnrnunized with gr l70-pulsed dendritic cells (triangles), control dendritic cells (squares), or PBS (circles).
- Figure 7 is a graph showing tumor volume, in cubic millimeters, as a function of days after tumor challenge in mice immunized with PBS (open circles), grpl70 derived from tumors (squares), grp 170 derived from tumors of whole body heat-treated mice (upward triangles), hspllO derived from tumors (downward triangles), hspllO derived from tumors of whole body heat-treated mice (diamonds), hsp70 derived from tumors (hexagons), hsp70 derived from tumors of whole body heat-treated mice (solid circles).
- Figure 8 is a graph showing percent protein aggregation (determined by light scattering) as a function of time, in minutes, for luciferase incubated with hspllO + hsp70 + hsp25 at a molar ratio of 1:1:1:1 (squares), hspllO at 1:1 (triangles), hsp25 at 1:1 (X's), grpl70 at 1:1 (asterisks), or luciferase alone (circles).
- Figure 9A shows chromatography profiles of native hspllO separated by size exclusion column for FPLC for characterization of hspllO complex.
- HspllO was partially purified by successive chromatography on Con-A sepharose and mono Q column. Pooled fraction was loaded on the superose 6 column, proteins in each fraction were detected by immunoblotting with antibodies for hspllO, hsc70 and hsp25 (1:1000).
- Figure 9B is an immunoblot that shows composition analysis of native hspllO complex. Purified hspllO fraction was detected by antibodies for hsp90 (lane 1, 2), hsc70 (lane 3, 4), TCP-1 (lane 5, 6) and hsp25 (lane 7, 8). Total cell extracts was also used as a positive control (lane 1, 3, 5, 7).
- Figures 10A-C are immunoblots showing reciprocal immunoprecipitation between hspllO and hsp70, hsp25. Following incubation with the indicated antibodies, protein A-sepharose was added and further incubated at 4°C overnight, immunoprecipitates were examined by imniunoblotting with hspl 10, hsp70 and hsp25 antibodies. Total cell extracts was also used as a positive control (lane 1).
- Figure 10A shows results observed when cell lysates (lane 2) were incubated with antibodies for hspllO (1:100).
- Figure 10B shows results observed when cell lysates (lane 2) were incubated with antibodies for hsp70 (1:200).
- Figure 10C shows results observed when cell lysates (lane 2) were incubated with antibodies for hsp25 (1:100).
- Figure 11 A shows immunoblots prepared when luciferase and Hsps were incubated at room temperature for 30 min, and soluble fraction after centrifugation at 16,000g was loaded on Sephacryl S-300 column. The eluted fractions were analyzed by imniunoblotting with antibodies for Hsps and luciferase.
- Figure 1 IB shows immunoblots prepared when luciferase and Hsps were incubated at 43°C for 30 min, and soluble fraction after centrifugation at 16,000g was loaded on Sephacryl S-300 column. The eluted fractions were analyzed by immunoblotting with antibodies for Hsps and luciferase.
- Figure 12 shows the results of interaction analysis of hspllO mutants and hsp70, hsp25 in vitro.
- E. coli expressed full-length hspllO (lane 1, 4) and mutant #1 (lane 2, 5), mutant #2 (lane 3, 6) were incubated with hsc70 or hsp25 at 30°C for 1 hour, then anti- hsc70 or anti-hsp25 antibodies were added. Immunoprecipitates were detected by anti- His antibody.
- In vitro interaction between hsc70 and hsp25 was also analyzed by the same method described above; hsc70 antibodies were used to test immunoprecipitate (lane 8). Total cell lysate was used as a positive control (lane 7). Equal amount of protein (2 ⁇ g) for wild-type hspllO, hspllO mutants, hsc70 and hsp25 were included in each assay.
- Figure 13 shows the results of immunoprecipitation of her2/neu intracellular domain (TCD) with anti-hspllO and anti-grpl70 antibodies after formation of binding complexes in vitro.
- Lane 1 is a protein standard from 205 kDa to 7.4 kDa;
- lane 2 is hspllO + anti-hspllO antibody;
- lane 3 is hspllO + ICD;
- lane 4 is grpl70 + ICD (in binding buffer);
- lane 5 is grpl70 + ICD (in PBS);
- lane 6 is ICD; and
- lane 7 is hspllO.
- Figure 14 is a western blot showing hspllO-ICD complex in both fresh (left lane) and freeze-thaw (center lane) samples, after immunoprecipitation of the complexes with anti-hspllO antibody.
- the right lane is ICD.
- Figure 15 is a bar graph showing hsp-peptide binding using a modified ELISA and p546, a 10-mer peptide of her-2/neu, selected for its HLA-A2 binding affinity and predicted binding to hspllO.
- the peptide was biotinylated and mixed with hspllO in vitro. Purified mixture concentrations were 1 ⁇ g/ml (white bars), 10 ⁇ g/ml (cross- hatched bars), and 100 ⁇ g/ml (dark stippled bars).
- Figure 16 shows the results of immunoprecipitation of M. tuberculosis antigens Mtb8.4 and Mtb39 with anti-hspllO antibody after formation of binding complexes in vitro, using both fresh samples and samples that had been subjected to freezing and thawing.
- Lane 1 is a protein standard from 205 kDa to 7.4 kDa; lane 2 is hspl 10 + Mtb8.4; lane 3 is hspllO + Mtb8.4 (after freeze-thaw); lane 4 is Mtb8.4; lane 5 is hspllO; lane 6 is hspllO + Mtb39; lane 7 is hspllO + Mtb39 (after freeze-thaw); lane 8 is Mtb39; and lane 9 is anti-hspllO antibody.
- Figure 17 is a bar graph showing gamma interferon (IFN-gamma) production (determined by number of spots in an ELISPOT assay) by T cells of A2/Kb transgenic mice (5 animals per group) after i.p. immunization with 25 ⁇ g of recombinant mouse hspllO-ICD complex.
- Total splenocytes or depleted cells (5 x 10 6 cells/ml) were cultured in vitro with 25 ⁇ g/ml PHA (checkered bars) or 20 ⁇ g/ml ICD (dark stippled bars) overnight and IFN-gamma secretion was detected using the ELISPOT assay.
- Figure 18 is a bar graph showing immunogenicity of hspllO-peptide complexes reconstituted in vitro, as determined by number of positive spots in an ELISPOT assay for IFN-gamma secretion.
- Recombinant hamster hspllO 100 ⁇ g was incubated with 100 ⁇ g of the 9-mer her-2/neu peptide p369, an HLA-A2 binder.
- Counts for the non-stimulated cells negative controls were ⁇ 40 and were subtracted from the counts for stimulated cells.
- Figure 19 is a bar graph showing immunogenicity of hspllO-peptide complexes reconstituted in vitro, as determined by number of positive spots in an ELISPOT assay for IFN-gamma secretion.
- Recombinant hamster hspllO 100 ⁇ g was incubated with 100 ⁇ g of the 10-mer her-2/neu peptide p546, an HLA-A2 binder.
- Eight-week old HLA-A2 transgenic mice (n 2) were immunized i.p. with either hspllO + peptide complex (group A, cross-hatched bars) or peptide alone (group B, dark stippled bars).
- Counts for the non-stimulated cells negative controls were ⁇ 40 and were subtracted from the counts for stimulated cells.
- Figure 20 is a graph showing specific anti-hspllO antibody response in A2/Kb transgenic mice following i.p. immunization with the hspllO-ICD (her2/neu) complex.
- ELISA results are plotted as optical density (OD) at 450 nm as a function of serum and antibody dilutions. Results are shown for the positive control of anti-hspllO (solid squares), the negative control of unrelated antibody (open circles), and serum at day 0 (closed circles), day 14 (open squares, dashed line), and day 28 (open squares, solid line). These results confirm that the mice did not develop an autoimmune response to hspllO.
- Figure 21 is a graph showing specific anti-ICD antibody response in A2/Kb transgenic mice following i.p. immunization with the hspllO-ICD complex.
- ELISA results are plotted as optical density (OD) at 450 nm as a function of serum and antibody dilutions. Results are shown for the positive control of anti-ICD (solid squares), the negative control of unrelated antibody (open diamonds), and serum at day 0 (closed circles), day 14 (open squares, dashed line), and day 28 (open squares, solid line).
- Figure 22 is a bar graph comparing specific anti-ICD antibody responses in A2/Kb transgenic animals 2 weeks after priming with different vaccine formulas. Results are plotted as OD at 450 nm for the various serum and antibody dilutions and bars represent data for animals primed with hspl 10-ICD (stippled bars), the positive control of ICD in complete Freund's adjuvant (checkered bars), ICD alone (cross-hatched bars), anti-ICD antibody (dark stippled bars), and the negative control of unrelated antibody (open bars).
- Figure 23 is a bar graph comparing specific anti-ICD antibody generation 2 weeks after s.c. or i.p. priming of A2/Kb transgenic with hspllO-ICD complex. Results are plotted as OD at 450 nm for the various serum and antibody dilutions and bars represent serum at day 0 (stippled bars), serum i.p. at day 14 (checkered bars), serum s.c. at day 14 (cross-hatched bars), anti-ICD antibody (dark stippled bars), and the negative control of unrelated antibody (open bars).
- Figure 24A is an immunoblot showing that colon 26 cells (CT26) transfected with a vector encoding hspllO exhibit increased hspllO expression relative to untransfected CT26 cells and CT26 cells transfected with an empty vector.
- Equivalent protein samples from CT26 (lane 1), CT26-vector (lane 2), and CT26-hspllO (lane 3) were subjected to 10% SDS PAGE and transferred onto immobilon-P membrane. Membranes were probed with antibodies for hspllO. After washing, membranes were incubated with horseradish peroxidase-conjugated goat anti-rabbit IgG or goat anti- mouse IgG diluted 1:2,000 in TBST.
- FIG. 24B shows that CT26-hspllO cells do not exhibit enhanced hsc70 expression relative to untransfected CT26 cells or CT26 cells transfected with an empty vector.
- Figure 25A is a photomicrograph showing immunofluorescence staining of hspllO in CT26 cells. Cells were seeded on the cover slips one day before the staining. Cover slips were then incubated with rabbit anti-hspllO antibody (1:500 dilution) followed by FITC-labeled dog anti-rabbit IgG staining. Normal rabbit IgG was used as negative control.
- Figure 25B is a photomicrograph showing immunofluorescence staining of hsp 110 in empty vector transfected CT26 cells. Cells were prepared and immunostained as in Figure 25A.
- Figure 25C is a photomicrograph showing immunofluorescence staining of hspllO in hspllO over-expressing cells. Cells were prepared and immunostained as in Figure
- Figure 26 is a graph demonstrating in vitro growth properties of wild type and hspl 10- transfected cell lines, plotted as cell number at 1-5 days after seeding. Cells were seeded at a density of 2x10 4 cells per well. 24 hours later cells were counted (assigned as day 0). Cells from triplicate wells were counted on the indicated days. The results are means ⁇ SD of three independent experiments using wild type CT26 cells (circles), CT26 cells transfected with empty vector (squares), and hspllO-transfected CT26 cells (triangles).
- Figure 27 is a bar graph showing the effect of hspllO over-expression on colony forming ability in soft agar. Wild-type CT26 cells, empty vector transfected CT26- vector cells and hspllO over-expressing CT26-hspllO cells were plated in 0.3 % agar and analyzed for their ability to form colonies ( ⁇ 0.2) in soft agar. P ⁇ 0.05, compared with CT26-vector, as assessed by student's t test.
- Figure 28 is a graph showing in vivo growth properties of wild-type and hspllO transfected CT26 cell line. 5 X 10 4 cells were inoculated s.c. into flank area of balb/c mice.
- Tumor growth was recorded twice a week measuring both the longitudinal and transverse diameter with a caliper.
- Tumor volume, in cubic mm, is plotted as a function of days after tumor implantation for CT26 wild type cells (circles), CT26 cells transfected with empty vector (squares), CT26 cells transfected with hspllO, 5 x 10 4 (upward triangles), and CT26 cells transfected with hs llO, 5 x 10 5 (downward triangles).
- Figure 29 is a plot showing the effect of injection with irradiated hspllO-overexpressing cells on the response to challenge with live CT26 cells.
- Mice were injected with 5x10 5 irradiated (9,000 rad) CT26-hspllO cells subcutaneously in the left flank. Two weeks later, mice were challenged on the right flank with live CT26 cells. Growth of tumor in mice without preimmunization was also shown.
- Results are plotted as percent tumor free mice as a function of days after tumor challenge for mice immunized with PBS and challenged with 5xl0 4 CT26 cells (circles); irradiated CT26 cells with empty vector/5xl0 5 CT26 cells (squares); irradiated CT26 cells with empty vector/5xl0 6 CT26 cells (upward triangles); irradiated CT26-hspllO cells/5xl0 5 CT26 cells (downward triangles); and irradiated CT26-hspllO cells/5xl0 6 CT26 cells (diamonds).
- Figure 30 is a graph showing tumor specific CTL response elicited by immunization with tumor derived hspl 10.
- Mice were injected with 5x10 5 irradiated (9,000 rad) CT26- empty vector and CT26-hsp 110 cells subcutaneously. Two weeks later, splenocytes were isolated as effector cells and re-stimulated with irradiated Colon 26 in vitro for 5 days. The lymphocytes were analyzed for cytotoxic activity using 51 Cr-labeled Colon 26 as target cells. Meth A tumor cells were also used as target in the experiment, and no cell lysis was observed. Results are plotted as percent specific lysis as a function of effectof.target ratio for control (circles), irradiated CT26 cells (squares), and irradiated CT26-hspll0 cells (triangles).
- Figure 31 is a graph showing antibody response against CT26 cells following immunization with irradiated hspllO-overexpressing cells. Mice were injected with 5xl0 5 irradiated (9,000 rad) CT26 empty vector and CT26-hspllO cells subcutaneously. Two weeks later, serum was collected and assayed for antibody response using ELISA. Results are plotted as OD at 450 nm as a function of serum dilution for control (circles), CT26-empty vector (squares), and CT26-hspl O (triangles).
- Figure 32 is a graph showing the effect of GM-CSF from bystander cells on the growth of hspllO overexpressing cells.
- Mice were injected subcutaneously with 5xl0 4 live tumor cells as follows: CT26-empty vector cells (circles), CT26-vector cells plus irradiated B78H1GM-CSF cells (2:1 ratio; squares), CT26-hspllO cells plus irradiated B78H1GM CSF cells (2:1 ratio; upward triangles), CT26-hspllO cells (downward triangles), CT26-hspllO plus irradiated B78H1 cells (2:1 ratio; diamonds).
- the B78H1GM-CSF are B16 cells transfected with CM-CSF gene, while B78H1 are wild type cells. Tumor growth was recorded by measuring the size of tumor, and is plotted as tumor volume in cubic mm as a function of days after implantation.
- Figure 33 is a graph showing the effect of co-injecting irradiated hspllO-overexpressing tumor vaccine and GM-CSF-secreting bystander cells on the response to wild-type CT26 tumor cell challenge.
- Mice were immunized subcutaneously with irradiated 5X10 5 tumor cells as follows: CT26-empty vector cells, CT26-vector cells plus B78H1 GM-CSF cells (2:1 ratio; squares), CT26-hspllO cells plus B78H1GM-CSF ceUs (2:1; upward triangles), CT26-hspll0 cells (downward triangles), CT26-hspllO plus B78H1 cells (2:1; diamonds). Also shown are results for mice immunized only with PBS (circles). Mice were challenged at a separate site with CT26 wild-type cells and monitored every other day for the tumor development. Results are plotted as percent tumor free mice at the indicated number of days after tumor challenge.
- Figure 34 is a bar graph showing that immunization with colon 26-derived hspllO or grp 170 stimulates interferon (IFN) gamma secretion.
- IFN interferon
- mice were immunized with hspl 10 or grpl70, splenocytes were isolated for ELISPOT assay.
- Phytohemagglutinin (PHA) treated lymphocytes were used for positive control.
- Figure 35 is a graph showing tumor specific CTL response elicited by immunization with B16F10 tumor derived grpl70. Mice were immunized twice with grpl70 (40 ⁇ g) at weekly intervals. One week after the second immunization, splenocytes were isolated as effector cells and restimulated with irradiated B16F10 cells in vitro for 5 days. The lymphocytes were analyzed for cytotoxic activity using 51 Cr-labeled B16F10 or Meth A cells as target cells.
- Results are plotted as percent specific lysis as a function of effector:target ratio for controls (circles), liver-derived grp 170 (squares), B16F10- derived grpl70 (upward triangles), and Meth A-derived grpl70 (downward triangles).
- Figure 36 shows immunization with B16F10-derived grpl70 stimulates IFN gamma secretion.
- mice were immunized with hspl 10 or grp 170, splenocytes were isolated for ELISPOT assay.
- Figure 37 shows lung metastases for mice in which 1 x 10 5 B16F10 cells were inoculated intravenously into the tail vein of each C57BL/6 mouse. 24 hr after tumor cell injection, mice were then treated with PBS (closed circles), liver-derived grp 170 (open circles), or tumor-derived grpl70 (40 ⁇ g). Three treatments were carried out during the whole protocol. The animals were killed 3 weeks after tumor injection, lungs were removed and surface colonies were counted.
- the present invention is based on the discovery that the stress proteins hspllO and grp 170, when complexed with tumor antigens, are remarkably effective as anti-tumor vaccines.
- the efficacy of these stress protein complexes has been demonstrated in both prophylactic and therapeutic contexts.
- the discovery of the ability of these stress proteins to facilitate an effective immune response provides a basis for their use in presenting a variety of antigens for use in prophylaxis and therapy of cancer and infectious disease. Because both hspllO and grpl70 have an enlarged peptide binding cleft and can stabilize unfolded peptide chains with greater efficiency relative to hsp70, these molecules can elicit different immunological reactions than previously obtained.
- Heat shock proteins which are produced in response to a variety of stressors, have the ability to bind other proteins in the non- native states (e.g., denatured by heating or guanidium chloride treatment), and in particular the ability to bind nascent peptides emerging from ribosomes or extruded from the endoplasmic reticulum (Hendrick and Hartl, Ann. Rev. Biochem. 62:349-384, 1993; Hartl, Nature 381:571-580, 1996).
- non- native states e.g., denatured by heating or guanidium chloride treatment
- Heat shock proteins have also been shown to serve a chaperoning function, referring to their important role in the proper folding and assembly of proteins in the cytosol, endoplasmic reticulum and mitochondria (Frydman et al., Nature 370:111-117, 1994).
- Mammalian heat shock protein families include hsp28, hsp70, hsp90 and hspllO. These primary heat shock proteins are found in the cytoplasm and, to a lesser extent, in the nucleus. An additional set of stress proteins, known as glucose regulated proteins (grps), reside in the endoplasmic reticulum. The major families of glucose regulated proteins includes grp78, grp74 and grpl70. This category of stress proteins lack heat shock elements in their promoters and are not inducible by heat, but by other stress conditions, such as anoxia.
- HspllO is an abundant and strongly inducible mammalian heat shock protein. Human hspllO is also known as KIAA0201, NY-CO-25, HSP105 alpha and HSP105 beta. Mouse hspllO is also known as HSP105 alpha, HSP105 beta, 42°C-specific heat shock protein, and hsp-E7I. HspllO has an ATP binding beta sheet and alpha helical regions that are capable of binding peptides having greater size and different binding affinities as compared to hsp70.
- HspllO has also been shown to bind shorter peptides (12mers) and a preferred consensus motif for binding to hspllO has been determined (i.e., basic, polar, aromatic/basic, proline, basic, acidic, aromatic, aromatic, basic, aromatic, proline, basic, X (no preference), basic/aromatic).
- This sequence differs from preferred sequence motifs previously identified to bind to members of the hsp70 family.
- HspllO is more efficient in stabilizing heat denatured proteins compared to hsp70, being four-fold more efficient on an equimolar basis.
- the peptide binding characteristics of hsp70 and hspllO make them effective in inhibiting aggregation of denatured protein by binding to denatured peptide chain.
- hspllO Using two different denaturing conditions, heating and guanidium chloride exposure, hspllO exhibits nearly total efficacy in inhibiting aggregation of these luciferase and citrate synthase when present in a 1:1 molar ratio.
- Hsp70 family members perform a similar function, but with significantly lower efficiency.
- Grp 170 is a strong structural homolog to hspllO that resides in the endoplasmic reticulum (Lin et al., Mol. Biol. Cell 4:1109-19, 1993; Chen et al, FEBS Lett. 380:68-72, 1996).
- Grpl70 exhibits the same secondary structural features of hspllO, including an enlarged peptide binding domain.
- Grp 170 is predicted to contain a beta sheet domain near its center, a more C-terminal alpha-helical domain, and a loop domain connecting both that is much longer than the loop domain present in hspllO (200 amino acids versus 100 amino acids in length) and absent in DnaK.
- grpl70 is likely the critical ATPase required for protein import into the mammalian endoplasmic reticulum (Dierks et al, EMBO J. 15;6931-42, 1996).
- Grpl70 is also known as ORP150 (oxygen-regulated protein identified in both human and rat) and as CBP-140 (calcium binding protein identified in mouse).
- ORP150 oxygen-regulated protein identified in both human and rat
- CBP-140 calcium binding protein identified in mouse
- polypeptide includes proteins, fragments of proteins, and peptides, whether isolated from natural sources, produced by recombinant techniques or chemically synthesized. Polypeptides of the invention typically comprise at least about 6 amino acids.
- vector means a construct, which is capable of delivering, and preferably expressing, one or more gene(s) or sequence(s) of interest in a host cell.
- vectors include, but are not limited to, viral vectors, naked DNA or RNA expression vectors, plasmid, cosmid or phage vectors, DNA or RNA expression vectors associated with cationic condensing agents, DNA or RNA expression vectors encapsulated in liposomes, and certain eukaryotic cells, such as producer cells.
- expression control sequence means a nucleic acid sequence that directs transcription of a nucleic acid.
- An expression control sequence can be a promoter, such as a constitutive or an inducible promoter, or an enhancer.
- the expression control sequence is operably linked to the nucleic acid sequence to be transcribed.
- nucleic acid or “polynucleotide” refers to a deoxyribonucleotide or ribonucleotide polymer in either single- or double-stranded form, and unless otherwise limited, encompasses known analogs of natural nucleotides that hybridize to nucleic acids in a manner similar to naturally-occurring nucleotides.
- antigen-presenting cell means a cell capable of handling and presenting antigen to a lymphocyte.
- APCs include, but are not limited to, macrophages, Langerhans-dendritic cells, follicular dendritic cells, B cells, monocytes, fibroblasts and fibrocytes.
- Dendritic cells are a preferred type of antigen presenting cell. Dendritic cells are found in many non-lymphoid tissues but can migrate via the afferent lymph or the blood stream to the T-dependent areas of lymphoid organs. In non-lymphoid organs, dendritic cells include Langerhans cells and interstitial dendritic cells. In the lymph and blood, they include afferent lymph veiled cells and blood dendritic cells, respectively. In lymphoid organs, they include lymphoid dendritic cells and interdigitating cells.
- modified to present an epitope refers to antigen-presenting cells (APCs) that have been manipulated to present an epitope by natural or recombinant methods.
- APCs antigen-presenting cells
- the APCs can be modified by exposure to the isolated antigen, alone or as part of a mixture, peptide loading, or by genetically modifying the APC to express a polypeptide that includes one or more epitopes.
- tumor protein is a protein that is expressed by tumor cells. Proteins that are tumor proteins also react detectably within an immunoassay (such as an ELISA) with antisera from a patient with cancer.
- a "heat-inducible stress polypeptide” means a stress polypeptide or protein whose expression is induced by elevated temperature.
- a heat- inducible stress polypeptide comprises a stress protein that contains one or more heat shock elements in its promoter.
- immunogenic polypeptide is a portion of a protein that is recognized (i.e., specifically bound) by a B-cell and/or T-cell surface antigen receptor.
- immunogenic polypeptides generally comprise at least 5 amino acid residues, more preferably at least 10, and still more preferably at least 20 amino acid residues of a protein associated with cancer or infectious disease.
- Certain preferred immunogenic polypeptides include peptides in which an N-terminal leader sequence and/or transmembrane domain have been deleted.
- Other preferred immunogenic polypeptides may contain a small N- and/ or C-terminal deletion (e.g., 1-30 amino acids, preferably 5- 15 amino acids), relative to the mature protein.
- pharmaceutically acceptable carrier includes any material which, when combined with an active ingredient, allows the ingredient to retain biological activity and is non-reactive with the subject's immune system.
- examples include, but are not limited to, any of the standard pharmaceutical carriers such as a phosphate buffered saline solution, water, emulsions such as oil/water emulsion, and various types of wetting agents.
- Preferred diluents for aerosol or parenteral administration are phosphate buffered saline or normal (0.9%) saline.
- compositions comprising such carriers are formulated by well known conventional methods (see, for example, Remington's Pharmaceutical Sciences, Chapter 43, 14th Ed., Mack Publishing Co, Easton PA 18042, USA).
- adjuvant includes those adjuvants commonly used in the art to facilitate an immune response.
- adjuvants include, but are not limited to, helper peptide; uminum salts such as aluminum hydroxide gel (alum) or aluminum phosphate; Freund's Incomplete Adjuvant and Complete Adjuvant (Difco Laboratories, Detroit, MI); Merck Adjuvant 65 (Merck and Company, Inc., Rahway, NJ); AS-2 (Smith-Kline Beecham); QS-21 (Aquilla Biopharmaceuticals); MPL or 3d-MPL (Corixa Corporation, Hamilton, MT); LEIF; salts of calcium, iron or zinc; an insoluble suspension of acylated tyrosine; acylated sugars; cationically or anionically derivatized polysaccharides; polyphosphazenes; biodegradable microspheres; monophosphoryl lipid A and quil A; muramyl tripeptide phosphatidyl ethanolamine or an immunostearate, or sodium
- the invention provides polynucleotides, including a first polynucleotide that encodes one or more stress proteins, such as hspllO or grpl70, or a portion or other variant thereof, and a second polynucleotide that encodes one or more immunogenic polypeptides, or a portion or other variant thereof.
- the first and second polynucleotides are linked to form a single polynucleotide that encodes a stress protein complex.
- the single polynucleotide can express the first and second proteins in a variety of ways, for example, as a single fusion protein or as two separate proteins capable of forming a complex.
- Preferred polynucleotides comprise at least 15 consecutive nucleotides, preferably at least 30 consecutive nucleotides and more preferably at least 45 consecutive nucleotides, that encode a portion of a stress protein or immunogenic polypeptide.
- the first polynucleotide encodes a peptide binding portion of a stress protein and the second polynucleotide encodes an immunogenic portion of an immunogenic polypeptide.
- Polynucleotides complementary to any such sequences are also encompassed by the present invention.
- Polynucleotides may be single-stranded (coding or antisense) or double-stranded, and may be DNA (genomic, cDNA or synthetic) or RNA molecules.
- RNA molecules include HnRNA molecules, which contain introns and correspond to a DNA molecule in a one-to-one manner, and mRNA molecules, which do not contain introns. Additional coding or non-coding sequences may, but need not, be present within a polynucleotide of the present invention, and a polynucleotide may, but need not, be linked to other molecules and/or support materials.
- Polynucleotides may comprise a native sequence (i.e., an endogenous sequence that encodes a stress protein, immunogenic polypeptide or a portion thereof) or may comprise a variant of such a sequence.
- Polynucleotide variants contain one or more substitutions, additions, deletions and/or insertions such that the immunogenicity of the encoded polypeptide is not diminished, relative to a native stress protein.
- the effect on the immunogenicity of the encoded polypeptide may generally be assessed as described herein.
- Variants preferably exhibit at least about 70% identity, more preferably at least about 80% identity and most preferably at least about 90% identity to a polynucleotide sequence that encodes a native stress protein or a portion thereof.
- Two polynucleotide or polypeptide sequences are said to be “identical” if the sequence of nucleotides or amino acids in the two sequences is the same when aligned for maximum correspondence as described below. Comparisons between two sequences are typically performed by comparing the sequences over a comparison window to identify and compare local regions of sequence similarity.
- a “comparison window” as used herein refers to a segment of at least about 20 contiguous positions, usually 30 to about 75, 40 to about 50, in which a sequence may be compared to a reference sequence of the same number of contiguous positions after the two sequences are optimally aligned.
- Optimal alignment of sequences for comparison may be conducted using the Megalign program in the Lasergene suite of bioinformatics software (DNASTAR, Inc., Madison, WI), using default parameters.
- This program embodies several alignment schemes described in the following references: Dayhoff, M.O. (1978) A model of evolutionary change in proteins - Matrices for detecting distant relationships. In Dayhoff, M.O. (ed.) Atlas of Protein Sequence and Structure, National Biomedical Research Foundation, Washington DC Vol. 5, Suppl. 3, pp. 345-358; Hein J. (1990) Unified Approach to Alignment and Phylogenes pp. 626-645 Methods in Enzymology vol.
- the "percentage of sequence identity” is determined by comparing two optimally aligned sequences over a window of comparison of at least 20 positions, wherein the portion of the polynucleotide or polypeptide sequence in the comparison window may comprise additions or deletions (i.e. gaps) of 20 percent or less, usually 5 to 15 percent, or 10 to 12 percent, as compared to the reference sequences (which does not comprise additions or deletions) for optimal alignment of the two sequences.
- the percentage is calculated by determining the number of positions at which the identical nucleic acid bases or amino acid residue occurs in both sequences to yield the number of matched positions, dividing the number of matched positions by the total number of positions in the reference sequence (i.e. the window size) and multiplying the results by 100 to yield the percentage of sequence identity.
- Variants may also, or alternatively, be substantially homologous to a native gene, or a portion or complement thereof.
- Such polynucleotide variants are capable of hybridizing under moderately stringent conditions to a naturally occurring DNA sequence encoding a native stress protein (or a complementary sequence). Suitable moderately stringent conditions include prewashing in a solution of 5 X SSC, 0.5%
- alleles are endogenous genes that are altered as a result of one or more mutations, such as deletions, additions and/ or substitutions of nucleotides.
- the resulting mRNA and protein may, but need not, have an altered structure or function. Alleles may be identified using standard techniques (such as hybridization, amplification and/or database sequence comparison).
- Polynucleotides may be prepared using any of a variety of techniques known in the art.
- DNA encoding a stress protein may be obtained from a cDNA library prepared from tissue expressing a stress protein mRNA. Accordingly, human hspllO or grpl70 DNA can be conveniently obtained from a cDNA library prepared from human tissue.
- the stress protein-encoding gene may also be obtained from a genomic library or by oligonucleotide synthesis. Libraries can be screened with probes (such as antibodies to the stress protein or oligonucleotides of at least about 20-80 bases) designed to identify the gene of interest or the protein encoded by it.
- Illustrative libraries include human liver cDNA library (human liver 5' stretch plus cDNA, Clontech Laboratories, Inc.) and mouse kidney cDNA library (mouse kidney 5'-stretch cDNA, Clontech laboratories, Inc.). Screening the cDNA or genomic library with the selected probe may be conducted using standard procedures, such as those described in Sambrook et al., Molecular Cloning: ⁇ laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989). An alternative means to isolate the gene encoding hspllO or grpl70 is to use PCR methodology (Sambrook et al, supra; Dieffenbach et al., PCR Primer: A. Laboratory Manual (Cold Spring Harbor Laboratory Press, 1995)).
- the oligonucleotide sequences selected as probes should be sufficiently long and sufficiently unambiguous that false positives are miiiimized.
- the oligonucleotide is preferably labeled such that it can be detected upon hybridization to DNA in the library being screened. Methods of labeling are well known in the art, and include the use of radiolabels, such as 32 P-labeled ATP, biotinylation or enzyme labeling. Hybridization conditions, including moderate stringency and high stringency, are provided in Sambrook et al., supra.
- Sequences identified in such library screening methods can be compared and aligned to other known sequences deposited and available in public databases such as GenBank or other private sequence databases. Sequence identity (at either the amino acid or nucleotide level) within defined regions of the molecule or across the full-length sequence can be determined through sequence alignment using computer software programs, which employ various algorithms to measure homology.
- Nucleic acid molecules having protein coding sequence may be obtained by screening selected cDNA or genomic libraries, and, if necessary, using conventional primer extension procedures as described in Sambrook et al., supra, to detect precursors and processing intermediates of mRNA that may not have been reverse-transcribed into cDNA.
- Polynucleotide variants may generally be prepared by any method known in the art, including chemical synthesis by, for example, solid phase phosphoramidite chemical synthesis. Modifications in a polynucleotide sequence may also be introduced using standard mutagenesis techniques, such as oligonucleotide-directed site-specific mutagenesis (see Adelman et al., DNA 2:183, 1983). Alternatively, RNA molecules may be generated by in vitro or in vivo transcription of DNA sequences encoding a stress protein, or portion thereof, provided that the DNA is incorporated into a vector with a suitable RNA polymerase promoter (such as T7 or SP6). Certain portions may be used to prepare an encoded polypeptide, as described herein.
- a suitable RNA polymerase promoter such as T7 or SP6
- a portion may be administered to a patient such that the encoded polypeptide is generated in vivo (e.g., by transfecting antigen-presenting cells, such as dendritic cells, with a cDNA construct encoding a stress polypeptide, and administering the transfected cells to the patient).
- Any polynucleotide may be further modified to increase stability in vivo.
- flanking sequences at the 5' and/ or 3' ends Possible modifications include, but are not limited to, the addition of flanking sequences at the 5' and/ or 3' ends; the use of phosphorothioate or 2' O-methyl rather than phosphodiesterase linkages in the backbone; and/or the inclusion of nontraditional bases such as inosine, queosine and wybutosine, as well as acetyl- methyl-, thio- and other modified forms of adenine, cytidine, guanine, thymine and uridine.
- flanking sequences at the 5' and/ or 3' ends Possible modifications include, but are not limited to, the addition of flanking sequences at the 5' and/ or 3' ends; the use of phosphorothioate or 2' O-methyl rather than phosphodiesterase linkages in the backbone; and/or the inclusion of nontraditional bases such as inosine, queosine and wybutosine, as well as
- Nucleotide sequences can be joined to a variety of other nucleotide sequences using established recombinant DNA techniques.
- a polynucleotide may be cloned into any of a variety of cloning vectors, including plasmids, phagemids, lambda phage derivatives and cosmids.
- Vectors of particular interest include expression vectors, replication vectors, probe generation vectors and sequencing vectors.
- a vector will contain an origin of replication functional in at least one organism, convenient restriction endonuclease sites and one or more selectable markers. Other elements will depend upon the desired use, and will be apparent to those of ordinary skill in the art.
- polynucleotides may be formulated so as to permit entry into a cell of a mammal, and to permit expression therein. Such formulations are particularly useful for therapeutic purposes, as described below.
- a polynucleotide may be incorporated into a viral vector such as, but not limited to, adenovirus, adeno-associated virus, retrovirus, or vaccinia or other pox virus (e.g., avian pox virus). Techniques for incorporating DNA into such vectors are well known to those of ordinary skill in the art.
- a retroviral vector may additionally transfer or incorporate a gene for a selectable marker (to aid in the identification or selection of transduced cells) and/ or a targeting moiety, such as a gene that encodes a ligand for a receptor on a specific target cell, to render the vector target specific. Targeting may also be accomplished using an antibody, by methods known to those of ordinary skill in the art.
- Other formulations for therapeutic purposes include colloidal dispersion systems, such as macromolecule complexes, nanocapsules, microspheres, beads, and lipid-based systems including oil-in-water emulsions, micelles, mixed micelles, and liposomes.
- a preferred colloidal system for use as a delivery vehicle in vitro and in vivo is a liposome (i.e., an artificial membrane vesicle). The preparation and use of such systems is well known in the art.
- stress polypeptides and stress proteins comprise at least a peptide binding portion of an hspllO and/or grpl70 protein and/ or a variant thereof.
- Polypeptides as described herein may be of any length. Additional sequences derived from the native protein and/or heterologous sequences may be present, and such sequences may, but need not, possess further peptide binding, immunogenic or antigenic properties.
- the stress polypeptide further includes all or a portion of a member of the hsp70, hsp90, grp78 and grp94 stress protein families.
- Functional domains and variants of hspllO that are capable of mediating the chaperoning and peptide binding activities of hspllO are identified in Oh, HJ et al., J. Biol. Chem. 274(22):15712-18, 1999. Functional domains of grpl70 parallel those of hspllO.
- Candidate fragments and variants of the stress polypeptides disclosed herein can be identified as having chaperoning activity by assessing their ability to solubilize heat-denatured luciferase and to refold luciferase in the presence of rabbit reticulocyte lysate (Oh et al., supra).
- the immunogenic polypeptide is associated with a cancer or precancerous condition.
- an immunogenic polypeptide associated with a cancer is a her-2/neu peptide (Bargmann et al., 1986, Nature 319(6050):226-30;
- her-2/neu peptides include, but are not limited to, the intracellular domain of her-2/neu (amino acid residues 676-1255; see Bargmann et al. references above), p369 (also known as E75; KIFGSLAFL; SEQ ID NO: 6) of the extracellular domain of her-2/neu, and p546, a transmembrane region of her-2/neu (VLQGLPREYV; SEQ ID NO: 5).
- the immunogenic polypeptide is associated with an infectious disease.
- an immunogenic polypeptide associated with an infectious disease is an antigen derived from M. tuberculous, such as M.
- tuberculosis antigens Mtb 8.4 (Coler et al., 1998, J. Immunol. 161(5):2356-64) or Mtb 39 (also known as Mtb39A; Dillon et al, 1999, Infect. Immun. 67(6):2941-50).
- the immunogenic polypeptide may be known or unknown. Unknown immunogenic polypeptides can be obtained incidentally to the purification of hspllO or grpl70 from tissue of a subject having cancer or a precancerous condition or having an infectious disease. In other embodiments, the immunogenic polypeptide comprises a known antigen.
- Immunogenic polypeptides may generally be identified using well known techniques, such as those summarized in Paul, Fundamental Immunology, 4th ed., 663-665 (Lippincott-Raven Publishers, 1999) and references cited therein. Such techniques include screening polypeptides for the ability to react with antigen-specific antibodies, antisera and/or T-cell lines or clones.
- antisera and antibodies are antigen-specific if they specifically bind to an antigen (i.e., they react with the protein in an ELISA or other immunoassay, and do not react detectably with unrelated proteins).
- antisera and antibodies may be prepared using well known techniques.
- An immunogenic polypeptide can be a portion of a native protein that reacts with such antisera and/ or T-cells at a level that is not substantially less than the reactivity of the full length polypeptide (e.g., in an ELISA and/ or T-cell reactivity assay). Such immunogenic portions may react within such assays at a level that is similar to or greater than the reactivity of the full length polypeptide.
- Such screens may generally be performed using methods well known to those of ordinary skill in the art, such as those described in Harlow and Lane, Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory, 1988.
- a polypeptide may be immobilized on a solid support and contacted with patient sera to allow binding of antibodies within the sera to the immobilized polypeptide. Unbound sera may then be removed and bound antibodies detected using, for example, 125 I-labeled Protein A.
- Stress protein complexes of the invention can be obtained through a variety of methods.
- a recombinant hspllO or grpl70 is mixed with cellular material (e.g., lysate), to permit binding of the stress polypeptide with one or more immunogenic polypeptides within the cellular material. Such binding can be enhanced or altered by stress conditions, such as heating of the mixture.
- target cells are transfected with hspllO or grpl70 that has been tagged (e.g., HIS tag) for later purification.
- This example provides a method of producing recombinant stress polypeptide in the presence of immunogenic material.
- heat or other stress conditions are used to induce hspllO or grpl70 in target cells prior to purification of the stress polypeptide. This stressing can be performed in situ, in vittv or in cell cultures).
- the invention provides a stress protein complex having enhanced immunogenicity that comprises a stress polypeptide and an immunogenic polypeptide, wherein the complex has been heated.
- heating particularly wherein the stress polypeptide comprises a heat-inducible stress protein, can increase the efficacy of the stress protein complex as a vaccine.
- heat-inducible stress proteins include, but are not limited to, hsp70 and hspllO.
- heating comprises exposing tissue including the stress protein complex to a temperature of at least approximately 38°C, and gradually increasing the temperature, e.g. by 1°C at a time, until the desired level of heating is obtained.
- the temperature of the tissue is brought to approximately 39.5°C, ⁇ 0.5°C.
- the tissue can be in vivo, in vitro or positioned within a host environment.
- a stress protein complex of the invention can comprise a variant of a native stress protein.
- a polypeptide "variant,” as used herein, is a polypeptide that differs from a native stress protein in one or more substitutions, deletions, additions and/ or insertions, such that the immunogenicity of the polypeptide is not substantially diminished.
- the ability of a variant to react with antigen-specific antisera may be enhanced or unchanged, relative to the native protein, or may be diminished by less than 50%, and preferably less than 20%, relative to the native protein.
- Such variants may generally be identified by modifying one of the above polypeptide sequences and evaluating the reactivity of the modified polypeptide with antigen-specific antibodies or antisera as described herein.
- Preferred variants include those in which one or more portions, such as an N-terminal leader sequence or transmembrane domain, have been removed.
- Other preferred variants include variants in which a small portion (e.g., 1-30 amino acids, preferably 5-15 amino acids) has been removed from the N- and/ or C-terminal of the mature protein.
- Polypeptide variants preferably exhibit at least about 70%, more preferably at least about 90% and most preferably at least about 95% identity (determined as described above) to the identified polypeptides.
- a variant contains conservative substitutions.
- a "conservative substitution” is one in which an amino acid is substituted for another amino acid that has similar properties, such that one skilled in the art of peptide chemistry would expect the secondary structure and hydropathic nature of the polypeptide to be substantially unchanged.
- Amino acid substitutions may generally be made on the basis of similarity in polarity, charge, solubility, hydrophobicity, hydrophilicity and/ or the amphipathic nature of the residues.
- negatively charged amino acids include aspartic acid and glutamic acid; positively charged amino acids include lysine and arginine; and amino acids with uncharged polar head groups having similar hydirophilicity values include leucine, isoleucine and valine; glycine and alanine; asparagine and glutamine; and serine, tlireonine, phenylalanine and tyrosine.
- variant polypeptides differ from a native sequence by substitution, deletion or addition of five amino acids or fewer.
- Variants may also (or alternatively) be modified by, for example, the deletion or addition of amino acids that have minimal influence on the immunogenicity, secondary structure and hydropathic nature of the polypeptide.
- Polypeptides may comprise a signal (or leader) sequence at the N-terminal end of the protein that co-translationally or post-translationally directs transfer of the protein.
- the polypeptide may also be conjugated to a linker or other sequence for ease of synthesis, purification or identification of the polypeptide (e.g., poly-FEs), or to enhance binding of the polypeptide to a solid support.
- a polypeptide may be conjugated to an immunoglobulin Fc region.
- Polypeptides may be prepared using any of a variety of well known techniques, including the purification techniques described in Example 1 below.
- the stress polypeptide(s) and immunogenic polypeptide(s) are co-purified from tumor cells or cells infected with a pathogen as a result of the purification technique.
- the tumor cells or infected cells are stressed prior to purification to enhance bmding of the immunogenic polypeptide to the stress polypeptide.
- the cells can be stressed in vitro by several hours of low-level heating (39.5-40°C) or about 1 to about 2 hours of high-level heating (approximately 43°C).
- the cells can be stressed in vitro by exposure to anoxic and/ or ischemic or proteotoxic conditions. Tumors removed from a subject can be minced and heated in vitro prior to purification.
- the polypeptides are purified from the same subject to whom the composition will be administered. In these embodiments, it may be desirable to increase the number of tumor or infected cells. Such a scale up of cells could be performed in vitro or in vivo, using, for example, a SCID mouse system. Where the cells are scaled up in the presence of non-human cells, such as by growing a human subject's tumor in a SCID mouse host, care should be taken to purify the human cells from any non-human (e.g., mouse) cells that may have infiltrated the tumor.
- non-human e.g., mouse
- composition in which the composition will be administered to the same subject from whom the polypeptides are purified, it may also be desirable purify both hspllO and grpl70 as well as additional stress polypeptides to optimize the efficacy of a limited quantity of starting material.
- Recombinant polypeptides encoded by DNA sequences as described above may be readily prepared from the DNA sequences using any of a variety of expression vectors known to those of ordinary skill in the art. Expression may be achieved in any appropriate host cell that has been transformed or transfected with an expression vector containing a DNA molecule that encodes a recombinant polypeptide. Suitable host cells include prokaryotes, yeast and higher eukaryotic cells. Preferably, the host cells employed are E. coli, yeast, insect cells or a mammalian cell line such as COS or CHO. Supernatants from suitable host/vector systems that secrete recombinant protein or polypeptide into culture media may be first concentrated using a commercially available filter. Following concentration, the concentrate may be applied to a suitable purification matrix such as an affinity matrix or an ion exchange resin. Finally, one or more reverse phase HPLC steps can be employed to further purify a recombinant polypeptide.
- a suitable purification matrix such as an affinity matrix or an
- Portions and other variants having fewer than about 100 amino acids, and generally fewer than about 50 amino acids, may also be generated by synthetic means, using techniques well known to ' those of ordinary skill in the art.
- polypeptides may be synthesized using any of the commercially available solid-phase techniques, such as the Merrifield solid-phase synthesis method, where amino acids are sequentially added to a growing amino acid chain. See Merrifield, J. Am. Chem. Soc. 85:2149-2146, 1963.
- Equipment for automated synthesis of polypeptides is commercially available from suppliers such as Perkin Elmer/Applied BioSystems Division (Foster City, CA), and may be operated according to the manufacturer's instructions.
- Polypeptides can be synthesized on a Perkin Elmer/Applied Biosystems Division 430A peptide synthesizer using FMOC chemistry with HPTU (O-BenzotriazoleN,N,N',N'- tetramethyluronium hexafluorophosphate) activation.
- HPTU O-BenzotriazoleN,N,N',N'- tetramethyluronium hexafluorophosphate
- a Gly-Cys-Gly sequence may be attached to the amino terminus of the peptide to provide a method of conjugation, binding to an immobilized surface, or labeling of the peptide.
- Cleavage of the peptides from the solid support may be carried out using the following cleavage mixture: trifluoroacetic acid:ethane ⁇ -Uthiol:thioanisole:water:phenol (40:1:2:2:3).
- the peptides may be precipitated in cold methyl-t-butyl-ether.
- the peptide pellets may then be dissolved in water containing 0.1% trifluoroacetic acid (TFA) and lyophilized prior to purification by C18 reverse phase HPLC.
- TFA trifluoroacetic acid
- a gradient of 0%-60% acetonitrile (containing 0.1% TFA) in water may be used to elute the peptides.
- the peptides may be characterized using electrospray or other types of mass spectrometry and by amino acid analysis.
- the polypeptide is a fusion protein that comprises multiple polypeptides as described herein, or that comprises at least one polypeptide as described herein and an unrelated sequence.
- the fusion protein comprises a stress polypeptide of hspllO and/ or grpl70 and an immunogenic polypeptide.
- the immunogenic polypeptide can comprise all or a portion of a tumor protein or a protein associated with an infectious disease.
- a fusion partner may, for example, serve as an i munological fusion partner by assisting in the provision of T helper epitopes, preferably T helper epitopes recognized by humans.
- a fusion partner may serve as an expression enhancer, assisting in expressing the protein at higher yields than the native recombinant protein.
- Certain preferred fusion partners are both immunological and expression enhancing fusion partners.
- Other fusion partners may be selected so as to increase the solubility of the protein or to enable the protein to be targeted to desired intracellular compartments.
- Still further fusion partners include affinity tags, which facilitate purification of the protein.
- Fusion proteins may generally be prepared using standard techniques, including chemical conjugation.
- a fusion protein is expressed as a recombinant protein, allowing the production of increased levels, relative to a non-fused protein, in an expression system.
- DNA sequences encoding the polypeptide components may be assembled separately, and ligated into an appropriate expression vector. The 3' end of the DNA sequence encoding one polypeptide component is hgated, with or without a peptide linker, to the 5' end of a DNA sequence encoding the second polypeptide component so that the reading frames of the sequences are in phase. This permits translation into a single fusion protein that retains the biological activity of both component polypeptides.
- a peptide linker sequence may be employed to separate the first and the second polypeptide components by a distance sufficient to ensure that each polypeptide folds into its secondary and tertiary structures.
- Such a peptide linker sequence is incorporated into the fusion protein using standard techniques well known in the art.
- Suitable peptide linker sequences may be chosen based on the folio-wing factors: (1) their ability to adopt a flexible extended conformation; (2) their inability to adopt a secondary structure that could interact with functional epitopes on the first and second polypeptides; and (3) the lack of hydrophobic or charged residues that might react with the polypeptide functional epitopes.
- Preferred peptide linker sequences contain Gly, Asn and Ser residues.
- linker sequences which may be usefully employed as linkers include those disclosed in Maratea et al., Gene 40:39-46, 1985; Murphy et al, Proc. Natl. Acad. Sci. USA 83:8258-8262, 1986; U.S. Patent No. 4,935,233 and U.S. Patent No. 4,751,180.
- the linker sequence may generally be from 1 to about 50 amino acids in length. Linker sequences are not required when the first and second polypeptides have non-essential N-terminal amino acid regions that can be used to separate the functional domains and prevent steric interference.
- the ligated DNA sequences are operably linked to suitable transcriptional or translational regulatory elements.
- the regulatory elements responsible for expression of DNA are located 5' to the DNA sequence encoding the first polypeptides.
- stop codons required to end translation and transcription termination signals are present 3' to the DNA sequence encoding the second polypeptide.
- Fusion proteins are also provided that comprise a polypeptide of the present invention together with an unrelated immunogenic protein.
- the immunogenic protein is capable of eliciting a memory response.
- examples of such proteins include tetanus, tuberculosis and hepatitis proteins (see, for example, Stoute et al., New Engl. J. Med. 336:86-91, 1997).
- an immunological fusion partner is derived from protein D, a surface protein of the gram-negative bacterium Haemophilus influenza B (WO 91/18926).
- a protein D derivative comprises approximately the first third of the protein (e.g., the first N-terminal 100-110 amino acids), and a protein D derivative may be lipidated.
- the first 109 residues of a Lipoprotein D fusion partner is included on the N-terminus to provide the polypeptide with additional exogenous T-cell epitopes and to increase the expression level in E. coli (thus functioning as an expression enhancer).
- the lipid tail ensures optimal presentation of the antigen to antigen presenting cells.
- Other fusion partners include the non-structural protein from influenzae virus, NS I (hernaglutinin). Typically, the N-terminal 81 amino acids are used, although different fragments that include T-helper epitopes may be used.
- the immunological fusion partner is the protein known as LYTA, or a portion thereof (preferably a C-terminal portion).
- LYTA is derived from Streptococcus pneumoniae, which synthesizes an N-acetyl-L-alanine amidase known as amidase LYTA (encoded by the LytA gene; Gene 43:265-292, 1986).
- LYTA is an autolysin that specifically degrades certain bonds in the peptidoglycan backbone.
- the C-terminal domain of the LYTA protein is responsible for the affinity to the choline or to some choline analogues such as DEAR This property has been exploited for the development of E.
- coli CLYTA expressing plasmids useful for expression of fusion proteins. Purification of hybrid proteins containing the C-LYTA fragment at the amino terminus has been described (see Biotechnology 10:795-798, 1992). Within a preferred embodiment, a repeat portion of LYTA may be incorporated into a fusion protein. A repeat portion is found in the C-terminal region starting at residue 178. A particularly preferred repeat portion incorporates residues 188-305.
- polypeptides including fusion proteins
- polynucleotides as described herein are isolated.
- An "isolated" polypeptide or polynucleotide is one that is removed from its original environment. For example, a naturally occurring protein is isolated if it is separated from some or all of the coexisting materials in the natural system.
- polypeptides are at least about 90% pure, more preferably at least about 95% pure and most preferably at least about 99% pure.
- a polynucleotide is considered to be isolated if, for example, it is cloned into a vector that is not a part of the natural environment.
- Immunotherapeutic compositions may also, or alternatively, comprise T cells specific for a stress protein complexed with an immunogenic polypeptide ("stress protein complex").
- stress protein complex Such cells may generally be prepared in vitro or ex vivo, using standard procedures.
- T cells may be isolated from bone marrow, peripheral blood, or a fraction of bone marrow or peripheral blood of a patient, using a commercially available cell separation system, such as the ISOLEXTM magnetic cell selection system, available from Nexell Therapeutics, Irvine, CA (see also U.S. Patent no. 5,536,475); or MACS cell separation technology from Miltenyi Biotec, including Pan T Cell Isolation Kit, CD4+ T Cell Isolation Kit, and CD8+ T Cell Isolation Kit (see also U.S.
- T cells may be derived from related or unrelated humans, non-human mammals, cell lines or cultures.
- T cells may be stimulated with a stress protein complex, polynucleotide encoding a stress protein complex and/or an antigen presenting cell (APC) that expresses such a stress protein complex.
- the stimulation is performed under conditions and for a time sufficient to permit the generation of T cells that are specific for the polypeptide.
- a stress polypeptide or polynucleotide is present within a delivery vehicle, such as a microsphere, to facilitate the generation of specific T cells.
- T cells are considered to be specific for a stress polypeptide if the T cells kill target cells coated with the polypeptide or expressing a gene encoding the polypeptide.
- T cell specificity may be evaluated using any of a variety of standard techniques. For example, within a chromium release assay or proliferation assay, a stimulation index of more than two fold increase in lysis and/or proliferation, compared to negative controls, indicates T cell specificity. Such assays may be performed, for example, as described in Chen et al, Cancer Res. 54:1065-1070, 1994.
- T cell proliferation can be detected by measuring an increased rate of DNA synthesis (e.g., by pulse-labeling cultures of T cells with tritiated ⁇ ymidine and measuring the amount of tritiated thymidine incorporated into DNA).
- a stress protein complex 100 ng/ml - 100 ⁇ g/ml, preferably 200 ng/ l - 25 ⁇ g/ml
- a stress protein complex 100 ng/ml - 100 ⁇ g/ml, preferably 200 ng/ l - 25 ⁇ g/ml
- T cells that have been activated in response to a stress polypeptide, polynucleotide or polypeptide-expressing APC may be CD4+ and/ or CD8+. T cells can be expanded using standard techniques.
- the T cells are derived from either a patient or a related, or unrelated, donor and are administered to the patient following stimulation and expansion.
- CD4+ or CD8+ T cells that proliferate in response to a stress polypeptide, polynucleotide or APC can be expanded in number either in vitro or in vivo. Proliferation of such T cells in vitro may be accomplished in a variety of ways.
- the T cells can be re-exposed to a stress polypeptide complexed with an immunogenic polypeptide, with or without the addition of T cell growth factors, such as interleukin-2, and/ or stimulator cells that synthesize a stress protein complex.
- T cell growth factors such as interleukin-2
- stimulator cells that synthesize a stress protein complex.
- one or more T cells that proliferate in the presence of a stress protein complex can be expanded in number by cloning. Methods for cloning cells are well known in the art, and include limiting dilution.
- the invention provides stress protein complex polypeptides, polynucleotides, T cells and/ or antigen presenting cells that are incorporated into pharmaceutical compositions, including immunogenic compositions (i.e., vaccines).
- Pharmaceutical compositions comprise one or more such compounds and, optionally, a physiologically acceptable carrier.
- Vaccines may comprise one or more such compounds and an adjuvant that serves as a non-specific immune response enhancer.
- the adjuvant may be any substance that enhances an immune response to an exogenous antigen.
- adjuvants include conventional adjuvants, biodegradable microspheres (e.g., polylactic galactide), immunostimulatory oligonucleotides and liposomes (into which the compound is incorporated; see e.g., Fullerton, U.S. Patent No. 4,235,877).
- Vaccine preparation is generally described in, for example, M.F. Powell and M.J. Newman, eds., "Vaccine Design (the subunit and adjuvant approach),” Plenum Press (NY, 1995).
- Pharmaceutical compositions and vaccines within the scope of the present invention may also contain other compounds that may be biologically active or inactive. For example, one or more immunogenic portions of other tumor antigens may be present, either incorporated into a fusion polypeptide or as a separate compound, within the composition or vaccine.
- a pharmaceutical composition or vaccine can contain DNA encoding one or more of the polypeptides as described above, such that the polypeptide is generated in situ.
- die DNA may be present within any of a variety of delivery systems known to those of ordinary skill in the art, including nucleic acid expression systems, bacteria and viral expression systems. Numerous gene delivery techniques are well known in the art, such as those described by Rolland, Crit. Rev. Therap. Drug Carrier Systems 15:143-198, 1998, and references cited therein. Appropriate nucleic acid expression systems contain the necessary DNA sequences for expression in the patient (such as a suitable promoter and terminating signal).
- Bacterial delivery systems involve the administration of a bacterium (such as Bacillus-Calmette-Guetrin) that expresses an immunogenic portion of the polypeptide on its cell surface or secretes such an epitope.
- the DNA may be introduced using a viral expression system (e.g., vaccinia or other pox virus, letrovirus, or adenovirus), which may involve the use of a non-pathogenic (defective), replication competent virus.
- a viral expression system e.g., vaccinia or other pox virus, letrovirus, or adenovirus
- vaccinia or other pox virus e.g., vaccinia or other pox virus, letrovirus, or adenovirus
- a viral expression system e.g., vaccinia or other pox virus, letrovirus, or adenovirus
- a viral expression system e.g., vaccinia or other pox virus, letrovirus, or adenovirus
- Suitable systems are disclosed, for example, in Fisher-Hoch et al., Proc. Natl. Acad. Sci. USA 86:317- 321, 1989; Flexner et al, Ann.
- compositions of the present invention may be formulated for any appropriate manner of administration, including for example, topical, oral, nasal, intravenous, intracranial, intraperitoneal, subcutaneous or intramuscular administration.
- the carrier preferably comprises water, saline, alcohol, a fat, a wax or a buffer.
- any of the above carriers or a solid carrier such as mannitol, lactose, starch, magnesium stearate, sodium saccharine, talcum, cellulose, glucose, sucrose, and magnesium carbonate, may be employed.
- Biodegradable microspheres e.g., polylactate polyglycolate
- Suitable biodegradable microspheres are disclosed, for example, in U.S. Patent Nos. 4,897,268 and 5,075,109.
- compositions may also comprise buffers (e.g., neutral buffered saline or phosphate buffered saline), carbohydrates (e.g., glucose, mannose, sucrose or dextrans), mannitol, proteins, polypeptides or amino acids such as glycine, antioxidants, chelating agents such as EDTA or glutathione, adjuvants (e.g., aluminum hydroxide) and/or preservatives.
- buffers e.g., neutral buffered saline or phosphate buffered saline
- carbohydrates e.g., glucose, mannose, sucrose or dextrans
- mannitol proteins
- proteins polypeptides or amino acids
- proteins e.glycine
- antioxidants e.g., antioxidants, chelating agents such as EDTA or glutathione
- adjuvants e.g., aluminum hydroxide
- preservatives e.g., aluminum hydroxide
- adjuvants may be employed in the vaccines of this invention.
- Most adjuvants contain a substance designed to protect the antigen from rapid catabolism, such as aluminum hydroxide or mineral oil, and a stimulator of immune responses, such as hpid A, Bortadella pertussis or Mycobacterium tuberculosis derived proteins.
- Suitable adjuvants are commerciaHy available as, for example, Freund's Incomplete Adjuvant and Complete Adjuvant (Difco Laboratories, Detroit, MI); Merck Adjuvant 65 (Merck and Company, Inc., Rahway, NJ); aluminum salts such as aluminum hydroxide gel (alum) or aluminum phosphate; salts of calcium, iron or zinc; an insoluble suspension of acylated tyrosine acylated sugars; cationicaUy or anionically derivatized polysaccharides; polyphosphazenes biodegradable microspheres; monophosphoryl Hpid A and quil A. Cytokines, such as GM CSF or interleukin-2, -7, or -12, may also be used as adjuvants.
- the adjuvant composition is preferably designed to induce an immune response predominantly of the Thl type.
- High levels of Thl-type cytokines e.g., IFN- , IL-2 and IL-12
- Th2-type cytokines e.g., IL-4, IL-5, IL-6, IL-10 and TNF- ⁇
- a patient will support an immune response that includes Thl- and Th2-type responses.
- the level of Thl -type cytokines wiH increase to a greater extent than the level of Th2-type cytokines.
- the levels of these cytokines may be readily assessed using standard assays. For a review of the famiHes of cytokines, see Mosmann and Coffman, Ann. Rev. Immunol. 7:145-173, 1989.
- Preferred adjuvants for use in eHciting a predominantly Thl-type response include, for example, a combination of monophosphoryl Hpid A, preferably 3-de-O-acylated monophosphoryl Hpid A (3D-MPL), together with an aluminum salt.
- MPL adjuvants are available from Corixa Corporation (Hamilton, MT) (see U.S. Patent Nos. 4,436,727; 4,877,611; 4,866,034 and 4,912,094).
- CpG-containing oHgonucleotides in which the CpG dinucleotide is unmethylated also induce a predominantly Thl response. Such oHgonucleotides are weU known and are described, for example, in WO 96/02555.
- Another preferred adjuvant is a saponin, preferably QS21, which may be used alone or in combination with other adjuvants.
- an enhanced system involves the combination of a monophosphoryl Hpid A and saponin derivative, such as the combination of QS21 and 313MPL as described in WO 94/00153, or a less reactogenic composition where the QS21 is quenched with cholesterol, as described in WO 96/33739.
- Other preferred formulations comprises an oil-in-watei emulsion and tocopherol.
- a particularly potent adjuvant formulation involving QS21, 3D-MPL and tocopherol in an oil-in-water emulsion is described in WO 95/17210.
- Another adjuvant that may be used is AS-2 (Smith-KHne Beecham). Any vaccine provided herein may be prepared using well known methods that result in a combination of antigen, immune response enhancer and a suitable carrier or excipient.
- a stress polypeptide of the invention can also be used as an adjuvant, eHciting a predominantly Thl-type response as weH.
- the stress polypeptide can be used in conjunction with other vaccine components, including an immunogenic polypeptide and, optionaUy, additional adjuvants.
- compositions described herein may be administered as part of a sustained release formulation (i.e., a formulation such as a capsule or sponge that effects a slow release of compound following administration).
- a sustained release formulation i.e., a formulation such as a capsule or sponge that effects a slow release of compound following administration.
- Such formulations may generally be prepared using weU known technology and administered by, for example, oral, rectal or subcutaneous implantation, or by implantation at the desired target site.
- Sustained- release formulations may contain a polypeptide, polynucleotide or antibody dispersed in a carrier matrix and/or contained within a reservoir surrounded by a rate controlling membrane.
- Carriers for use within such formulations are biocompatible, and may also be biodegradable; preferably the formulation provides a relatively constant level of active component release.
- the amount of active compound contained within a sustained release formulation depends upon the site of implantation, the rate and expected duration of release and the nature of the condition to be treated or prevented.
- deHvery vehicles may be employed within pharmaceutical compositions and vaccines to faciHtate production of an antigen-specific immune response that targets tumor ceHs or infected ceHs.
- DeHvery vehicles include antigen presenting ceHs (APCs), such as dendritic cells, macrophages, B ceHs, monocytes and other ceHs that may be engineered to be efficient APCs.
- APCs antigen presenting ceHs
- ceHs may, but need not, be geneticaHy modified to increase the capacity for presenting the antigen, to improve activation and/or maintenance of the T ceH response, to have anti-tumor or anti- infective effects per se and/ or to be immunologicaUy compatible with the receiver (i.e., matched BLA haplotype).
- APCs may generaUy be isolated from any of a variety of biological fluids and organs, including tumor and peritumoral tissues, and may be autologous, aUogeneic, syngeneic or xenogen
- Dendritic ceHs are highly potent APCs (Banchereau and Steinman, Nature 392:245-251, 1998) and have been shown to be effective as a physiological adjuvant for eHciting prophylactic or therapeutic antitumor immunity (see Timmerman and Levy, Ann. Rev. Med. 50:507-529, 1999).
- dendritic ceUs may be identified based on their typical shape (steUate in situ, with marked cytoplasmic processes (dendrites) visible in vitro) and based on the lack of differentiation markers of B ceUs (CD19 and CD20), T ceUs (CD3), monocytes (CD14) and natural kiuer ceUs (CD56), as determined using standard assays.
- Dendritic ceHs may, of course, be engineered to express specific ceU surface receptors or Hgands that are not commonly found on dendritic ceHs in vivo or ex vivo, and such modified dendritic ceUs are contemplated by the present invention.
- antigen-loaded dendritic ceUs may be used within a vaccine (see Zitvogel et al., Nature Med. 4:594-600, 1998).
- Dendritic ceHs and progenitors may be obtained from peripheral blood, bone marrow, tumor-infiltrating ceUs, peritumoral tissues-infiltrating ceUs, lymph nodes, spleen, skin, umbiHcal cord blood or any other suitable tissue or fluid.
- dendritic ceHs may be differentiated ex vivo by adding a combination of cytokines such as GM-CSF, IL-4, IL-13 and/or TNFoc to cultures of monocytes harvested from peripheral blood.
- CD34 positive ceHs harvested from peripheral blood, umbiHcal cord blood or bone marrow may be differentiated into dendritic ceUs by adding to the culture medium combinations of GM-CSF, IL-3, TNFoc, CD40 Hgand, LPS, flt3 Hgand and/ or other compound(s) that induce maturation and proHferation of dendritic ceUs.
- Dendritic ceUs are conveniently categorized as "immature” and "mature" ceUs, which aUows a simple way to discriminate between two weU characterized phenotypes. However, this nomenclature should not be construed to exclude aU possible intermediate stages of differentiation.
- Immature dendritic ceUs are characterized as APC with a high capacity for antigen uptake and processing, which correlates with the high expression of Fc ⁇ receptor, mannose receptor and DEC-205 marker.
- the mature phenotype is typicaHy characterized by a lower expression of these markers, but a high expression of ceU surface molecules responsible for T ceU activation such as class I and class II NMC, adhesion molecules (e.g., CD54 and CD 11) and costimulatory molecules (e.g., CD40, CD80 and CD86).
- APCs may generaUy be transfected with a polynucleotide encoding a stress protein (or portion or other variant thereof) such that the stress polypeptide, or an immunogenic portion thereof, is expressed on the ceU surface. Such transfection may take place ex vivo, and a composition or vaccine comprising such transfected ceHs may then be used for therapeutic purposes, as described herein. Alternatively, a gene deHvery vehicle that targets a dendritic or other antigen presenting ceU may be administered to a patient, resulting in transfection that occurs in vivo.
- dendritic ceUs may generaUy be performed using any methods known in the art, such as those described in WO 97/24447, or the gene gun approach described by Mahvi et al., Immunology and CeU Biology 75:456-460, 1997.
- Antigen loading of dendritic ceUs may be achieved by incubating dendritic ceUs or progenitor ceUs with the stress polypeptide, DNA (naked or within a plasmid vector) or RNA; or with antigen- expressing recombinant bacterium or viruses (e.g., vaccinia, fowlpox, adenovirus or lentivirus vectors).
- the polypeptide Prior to loading, the polypeptide may be covalently conjugated to an immunological partner that provides T ceU help (e.g., a carrier molecule).
- a dendritic ceU may be pulsed with a non-conjugated immunological partner, separately or in the presence of the polypeptide.
- the stress protein complexes and pharmaceutical compositions of the invention can be acdministered to a subject, thereby providing methods for inhibiting M. tuberculosis- infection, for inhibiting tumor growth, for inhibiting the development of a cancer, and for the treatment or prevention of cancer or infectious disease.
- Treatment includes prophylaxis and therapy.
- Prophylaxis or tiierapy can be accompHshed by a single direct injection at a single time point or multiple time points to a single or multiple sites. Acln inistration can also be nearly simultaneous to multiple sites.
- Patients or subjects include mammals, such as human, bovine, equine, canine, feline, porcine, and ovine animals.
- the subject is preferably a human, and may or may not be afflicted with cancer or disease.
- the condition to be treated or prevented is cancer or a precancerous condition (e.g., hyperplasia, metaplasia, dysplasia).
- a precancerous condition e.g., hyperplasia, metaplasia, dysplasia
- Example of cancer include, but are not limited to, fibrosarcoma, myxosarcoma, Hposarcoma, chondrosarcoma, osteogenic sarcoma, chordoma, angiosarcoma, endothehosarcoma, lymphangiosarcoma, pseudomyxoma peritonei, lymphangioendotheHosarcoma, synovioma, mesotheUoma, Ewing's tumor, leiomyosarcoma, rhabdomyosarcoma, colon carcinoma, pancreatic cancer, breast cancer, ovarian cancer, prostate cancer, squamous ceU carcinoma, basal ceU carcinoma, adenocarcinoma, sweat gland
- the condition to be treated or prevented is an infectious disease.
- infectious disease include, but are not limited to, infection with a pathogen, virus, bacterium, fungus or parasite.
- viruses include, but are not Hmited to, hepatitis type B or type C, influenza, variceUa, adenovirus, herpes simplex virus type I or type II, rinderpest, rhinovirus, echovirus, rotavirus, respiratory syncytial virus, papiUoma virus, papova virus, cytomegalovirus, echinovirus, arbovirus, hantavirus, coxsachie virus, mumps virus, measles virus, rubeUa virus, poHo virus, human immunodeficiency virus type I or type II.
- bacteria examples include, but are not limited to, M. tuberculosis, mycobacterium, mycoplasma, neisseria and legioneUa.
- parasites examples include, but are not Hmited to, rickettsia and chlamydia.
- compositions and vaccines may be used to prevent the development of a cancer or infectious disease or to treat a patient afflicted with a cancer or infectious disease.
- a cancer may be diagnosed using criteria generaUy accepted in the art, including the presence of a maHgnant tumor.
- Pharmaceutical compositions and vaccines may be administered either prior to or foUowing surgical removal of primary tumors and/or treatment such as a ⁇ lministration of radiotherapy or conventional chemotherapeutic drugs.
- immunotherapy may be active immunotherapy, in which treatment reHes on the in vivo stimulation of the endogenous host immune system to react against tumors or infected ceUs with the administration of immune response- modifying agents (such as polypeptides and polynucleotides disclosed herein).
- immune response- modifying agents such as polypeptides and polynucleotides disclosed herein.
- immunotherapy may be passive immunotherapy, in which treatment involves the deHvery of agents with estabHshed tiimor-i mune reactivity (such as effector ceUs or antibodies) that can directly or indirectly mediate antitumor effects and does not necessarily depend on an intact host immune system.
- agents with estabHshed tiimor-i mune reactivity such as effector ceUs or antibodies
- effector ceUs examples include T ceUs as discussed above, T lymphocytes (such as CD8+ cytotoxic T lymphocytes and CD4+ T-helper tumor-infiltrating lymphocytes), kiUer ceUs (such as Natural KiUer ceHs and lymphokine-activated kiUer ceUs), B ceUs and antigen-presenting ceUs (such as dendritic ceUs and macrophages) expressing a polypeptide provided herein.
- dendritic ceUs are modified in vitro to present the polypeptide, and these modified APCs are administered to the subject.
- T ceU receptors and antibody receptors specific for the polypeptides recited herein may be cloned, expressed and transferred into other vectors or effector ceUs for adoptive immunotherapy.
- the polypeptides provided herein may also be used to generate antibodies or anti-idiotypic antibodies (as described above and in U.S. Patent No. 4,918,164) for passive immunotherapy.
- Effector ceUs may generaUy be obtained in sufficient quantities for adoptive immunotherapy by growth in vitro, as described herein.
- Culture conditions for expanding single antigen-specific effector ceHs to several biUion in number with retention of antigen recognition in vivo are weU known in the art.
- Such in vitro culture conditions typicaUy use intermittent stimulation with antigen, often in the presence of cytokines (such as IL-2) and non-dividing feeder ceUs.
- immunoreactive polypeptides as provided herein may be used to rapidly expand antigen-specific T ceU cultures in order to generate a sufficient number of ceUs for immunotherapy.
- antigen-presenting ceUs such as dendritic, macrophage, monocyte, fibroblast and/ or B ceHs
- antigen-presenting ceUs can be transfected with a polynucleotide having a promoter appropriate for increasing expression in a recombinant virus or other expression system.
- Cultured effector ceUs for use in therapy must be able to grow and distribute widely, and to survive long term in vivo. Cultured effector ceUs can be induced to grow in vivo and to survive long term in substantial numbers by repeated stimulation with antigen supplemented with LL-2 (see, for example, Cheever et al., Immunological Reviews 157:177, 1997).
- a vector expressing a polypeptide recited herein can be introduced into antigen presenting ceHs taken from a patient and clonaUy propagated ex vivo for transplant back into the same patient.
- Transfected ceUs may be reintroduced into the patient using any means known in the art, preferably in sterile form by intravenous, intracavitary, intraperitoneal or intratumoral administration. Administration and Dosage
- compositions are administered in any suitable manner, often with pharmaceuticaUy acceptable carriers.
- Suitable methods of administering ceUs in the context of the present invention to a subject are avaUable, and, although more than one route can be used to administer a particular ceU composition, a particular route can often provide a more immediate and more effective reaction than another route.
- the dose administered to a patient should be sufficient to effect a beneficial therapeutic response in the patient over time, or to inhibit infection or disease due to infection.
- the composition is administered to a subject in an amount sufficient to eHcit an effective immune response to the specific antigens and/ or to aUeviate, reduce, cure or at least partiaUy arrest symptoms and/or compHcations from the disease or infection.
- An amount adequate to accompHsh this is defined as a "therapeutically effective dose.”
- compositions and vaccines may be administered, by injection (e.g., intracutaneous, intratumoral, intramuscular, intravenous or subcutaneous), intranasaUy (e.g., by aspiration) or oraUy.
- injection e.g., intracutaneous, intratumoral, intramuscular, intravenous or subcutaneous
- intranasaUy e.g., by aspiration
- oraUy e.g., between 1 and 10 doses may be administered over a 52 week period.
- 6 doses are administered, at intervals of 1 month, and booster vaccinations may be given periodicaUy thereafter.
- Alternate protocols may be appropriate for individual patients.
- 2 intradermal injections of the composition are administered 10 days apart.
- a suitable dose is an amount of a compound that, when administered as described above, is capable of promoting an anti-tumor immune response, and is at least 10-50% above the basal (i.e., untreated) level.
- Such response can be monitored, for example, by measuring the anti-tumor antibodies in a patient or by vaccine-dependent generation of cytolytic effector ceUs capable of killing the patient's tumor ceUs in vitro.
- Such vaccines should also be capable of causing an immune response that leads to an improved clinical outcome (e.g., more frequent remissions, complete or partial or longer disease- free survival) in vaccinated patients as compared to nonvaccinated patients.
- the amount of each polypeptide present in a dose ranges from about 100 ⁇ g to 5 mg per kg of host.
- Suitable volumes wiU vary with the size of the patient, but wiU typicaUy range from about 0.1 mL to about 5 mL.
- an appropriate dosage and treatment regimen provides the active compound(s) in an amount sufficient to provide therapeutic and/or prophylactic benefit.
- a response can be monitored by estabUshing an improved clinical outcome (e.g., more frequent remissions, complete or partial, or longer disease-free survival) in treated patients as compared to non-treated patients.
- Increases in preexisting immune responses to a tumor protein generaUy correlate with an improved clinical outcome.
- Such immune responses may generaUy be evaluated using standard proHferation, cytotoxicity or cytokine assays, which may be performed using samples obtained from a patient before and after treatment.
- a ce peUet or tissue was homogenized in 5 vol. of hypotonic buffer (30 mM sodium bicarbonate, pH7.2, 1 mM PMSF) by Dounce homogenization. The lysate was centrifuged at 4500g and then 100,000g to remove unbroken ceUs, nuclei, and other tissue debris. The supernatant was further centrifuged at 100,000g for 2 hours. Supernatant was appHed to concanavalin A-sepharose beads (1 ml bed volume/ml of original material), previously equUibrated with 20mM Tris-HCl, 50mM NaCl, 1 mM MgC12, 1 mM CaC12, 1 mM MnCl 2 . The bound proteins were eluted with binding buffer A containing 15% a-D-methylmannoside (a-D-MM).
- ConA-sepharose unbound material was appHed to a Mono Q (Pharmacia) 10/10 column equUibrated with 20mM Tris-HCl, pH 7.5, 200 mM NaCl.
- the bound proteins were eluted with the same buffer by a linear salt gradient up to 500mM sodium chloride (FR:3 ⁇ n/min, 40%-60%B/60min).
- Fractions were coUected and analyzed by SDS-PAGE foUowed by imniunoblotting with an anti- hspl 10 antibody.
- Grpl70 Con A-sepharose bound material, eluted by 10% ⁇ methylmannoside, was first appHed on MonoQ column equUibrated with 20 mM Tris HC1, pH 7.5, 150mM NaCl and eluted by 150 ⁇ 500mM NaCl gradient. Grpl70 was eluted between 300mM ⁇ 350 mM NaCl. Pooled fractions were concentrated and appHed on the Superose 12 column. Fractions containing homogeneous grpl70 were coUected, and analyzed by SDS-PAGE foUowed by immunoblotting with an anti- grpl70 antibody.
- ADP-agarose column (Sigma Chemical Co., St. Louis, MO) equUibrated with binding buffer B (20 mM Tris-acetate, pH 7.5, 20mM NaCl, 15 mM ⁇ -mercaptoethanoL 3 mM
- the elute was resolved on a FPLC system using MonoQ column and eluted by a 20-
- Grp78 was present in fractions eluted between 200 mM-400 mM salt.
- the 100,000g supernatant was first appHed to a blue sepharose column (Pharmacia) to remove albumin.
- AU protein was quantified with a Bradford assay (BioRad, Richmond, CA), and analyzed by SDS- PAGE foUowed by immunoblotting with antibodies to grp78 obtained from StressGen Biotechnologies Corp. (Victoria, BC, Canada).
- Proteins hspllO, grpl70 and grp78 were purified simultaneously from tumor and Hver. Homogeneous preparations for these three proteins were obtained and they were recognized by their respective antibodies by immunoblotting. The purity of the proteins was assessed by SDS-PAGE and sUver staining (Fig. 1).
- BALB/cJ mice (viral antigen free) were obtained from The Jackson Laboratory (Bar Harbor, ME) and were maintained in the mouse facilities at RosweH Park Cancer Institute. Methylcholanthrene-induced fibrosarcoma (Meth A) was obtained from Dr. Pramod K. Srivastava (University of Connecticut School of Medicine, Farmington, Connecticut) and maintained in ascites form in BALB/cJ mice by weekly passage of 2 miUion ceUs.
- mice (6-8-week-old females; five mice per group) were immunized with PBS or with varying quantities of tumor or Hver derived hspllO or grp 170, in 200 ⁇ l PBS, and boosted 7 days later. Seven days after the last immunization, mice were injected subcutaneously on the right flank with 2 x 10 4 colon 26 tumor ceHs (viabiHty > 99%). The colon 26 tumor exempHfies a murine tumor model that is highly resistant to therapy. In other experiments, the mice were challenged 7 days after the second immunization with intradermal injections of MethA tumor ceUs. Tumor growth was monitored by measuring the two diameters.
- mice that were irrrmunized with PBS and Hver derived hspllO or grpl70 developed rapidly growing tumors.
- mice irrrmunized with tumor derived hspllO and grp 170 showed a significant tumor growth delay.
- hspllO or grp 170 that is complexed with tumor proteins significantly inhibits tumor growth.
- mice immunized with 20 ⁇ g (per injection) of hspllO or grpl70 showed sHght or no inhibition of colon 26 tumor growth, whUe those immunized with 40 or 60 ⁇ g of hspllO or grp 170 showed increasingly significant tumor growth delay.
- the mean volumes of the tumors that developed in mice immunized with hspllO and grpl70 at doses of 40 and 60 ⁇ g were significantly smaUer than those of control mice (p ⁇ 0.01, student's t test).
- the differences in the mean volumes of the groups injected with PBS or Hver derived hsp preparations did not reach statistical significance.
- grpl70 appears to be more immunogenic than hspl 10.
- the immunogenicity of grp78 was also tested by injecting 40 ⁇ g of protein, but no tumor growth delay was observed. These results indicate that grp78 is either not immunogenic, or is so at a low level only.
- the immunogenicity of hspllO and grp 170 were tested in the methylcholanthrene-induced (MethA) fibrosarcoma. Based on the immunization data in colon 26 tumor model, mice were rr unized twice with 40 ⁇ g hspllO or grpl70, and chaUenged with 100,000 MethA ceUs introduced by intradermal injection.
- FIGs. 4A-4C show the kinetics of tumor growth in each individual animal. Notable differences between individuals in tumor growtii in response to immunization was observed in the grpl70 group. Mice irnmunized with PBS developed MethA tumors (Fig. 4A). However, mice immunized with hspllO (Fig. 4B) or grp 170 (Fig. 4C) were protected. WhUe most animals initiaUy developed tumors, the tumors later disappeared. In the mice that were immunized with grpl70, two of five mice completely faUed to develop a palpable tumor (Fig. 4C).
- the aggressive colon 26 tumor was also examined in a therapy model. Tumor ceUs (500,000) were injected into the flank area and mice (10 per group) were vaccinated two times (separated by 7 days) with Hver or colon 26 derived hspllO or grpl70, starting when the tumor was visible and palpable (e.g., day 6). The survival of mice was recorded as the percentage of mice surviving after the tumor chaUenge at various times.
- Figs. 3A and 3B The results are shown in Figs. 3A and 3B.
- Tumor bearing mice treated with autologous hspllO (Fig. 3A) or grpl70 (Fig. 3B) preparations showed significantly longer survival times compared to the untreated mice or mice immunized with Hver derived hspl 10 or grp 170.
- AU the control animals died within 30 days, but approximately one-half of each group survived to 40 days, and 20% of grpl70 treated mice survived to 60 days.
- cytotoxic T lymphocyte (CTL) assay was performed to analyze the abUity of tumor derived hspllO or grpl70 preparations to eHcit a CD8+ T ceU response. The results show that vaccination with tumor derived hspllO or grp 170 eHcits an effective tumor specific CTL response.
- CTL cytotoxic T lymphocyte
- mice were immunized twice as described above. Ten days after the second immunization, spleens were removed and spleen ceUs (1 x 10 7 ) were co-cultured in a mixed lymphocyte-tumor culture (MLTC) with irradiated tumor ceUs (5 x 10 5 ) used for immunization for 7 days, supplemented with 10% FCS, 1% pe ciUin/streptomycin, 1 mM sodium pyruvate and 50 ⁇ M 2-mercaptoefhanol. Splenocytes were then purified by FicoU-Paque (Pharmacia) density centrifugation and utilized as effector ceUs. CeU- mediated lysis was determined in vitro using a standard 51 Chromium.-release assay. Briefly, effector ceHs were seriaUy diluted in 96 V-bottomed weH plates (Costar,
- Target ceUs (5 x 10 6 ) were labeled with 100 ⁇ Ci of sodium [ 51 Cr]chromate at 37°C for 1-2 h. 51 Cr-labeled tumor ceUs (5,000) were added to a final volume of 200 ⁇ l/weU.
- This example demonstrates the capacity of antigen presenting ceUs to play a role in the anti-tumor response eHcited by hspl 10 or grpl70 immunization.
- the results show the abUity of dendritic ceUs (DCs) to represent the hspllO or grpl70 chaperoned peptides.
- DCs dendritic ceUs
- immunotherapy with hspllO or grpl70 pulsed DC was more efficient than direct immunization with protein.
- Bone marrow was flushed from the long bones of the Hmbs and depleted of red ceUs with ammonium chloride.
- the medium was replaced on days 3 and 6. On day 8, the ceUs were harvested for use.
- the quaHty of DC preparation was characterized by ceU surface marker analysis and morphological analysis.
- DCs (1 x 10 7 /ml) were pulsed with tumor derived hspl 10 or grpl70 (200 ⁇ g) for 3 hrs at 37°C.
- the ceUs were washed and resuspended in PBS (10 6 pulsed DCs in 100 ⁇ l PBS per mouse) for intraperitoneal injection. The entire process was repeated 10 days later, for a total of two immunizations per treated mouse. Ten days after the second immunization, mice were chaUenged with colon 26 tumor ceHs (2 x 10 4 ).
- mice were first inoculated subcutaneously with 100,000 colon 26 tumor ceUs on the flank area. After the tumors reached a size of approximately 1/1 cm, WBH was carried out as described before. Briefly, mice were placed in microisolater cages preheated to 38 °C that contained food, bedding and water. The cages were then placed in a gravity convection oven (Memmert model BE500, East Troy, WI) with preheated mcoming fresh air. The body temperature was graduaUy increased 1 °C every 30 minutes until a core temperature of 39.5°C ( ⁇ 0.5C) was achieved. Mice were kept in the oven for 6 hours. The core temperature of the mice was monitored with the Electric laboratory Animal Monitoring system Pocket Scanner (Maywood, NJ).
- Tumors were removed on the next day for purification of hspl 10, grpl70 and hsp70. Immunizations were performed as above, twice at weekly intervals, using PBS, 40 ⁇ g hspl 10 derived from tumors, 40 ⁇ g hspllO derived from WBH-treated tumor, 40 ⁇ g grpl70 derived from tumors, 40 ⁇ g grpl70 derived from WBH-treated tumor, 40 ⁇ g hsp70 derived from tumors, or 40 ⁇ g hsp70 derived from WBH-treated tumor. Mice were then chaUenged with 20,000 Hve colon 26 tumor ceUs. Tumor volume, in mm 3 , was measured at 0, 3, 6, 9, 12, 15, 18 and 21 days after tumor chaUenge.
- fever-like exposures can influence the antigen presentation pathway and/ or peptide binding properties of these two (heat inducible) hsps purified from colon 26 tumors but not a heat insensitive grp.
- the vaccine potential of hsp70 and hspllO are significantly enhanced foUowing fever level therapy. This could result from enhanced proteosome activity, enhanced peptide binding of the hsp, altered spectrum of peptides bound to the hsp, or other factors. Because the hsps were purified 16 hours after the 8-hour hyperthermic exposure, the effect is maintained for some time at 37 °C.
- the abUity of the stress proteins to prevent protein aggregation induced by heat treatment was assessed by the suppression of the increase in Hght scattering obtained upon heat treatment in the presence of a reporter protein, firefly luciferase. Luciferase was incubated with equimolar amounts of hspllO or grpl70 at 43°C for 30 minutes. Aggregation was monitored by measuring the increase of optical density at 320 nm. The optical density of the luciferase heated alone was set to 100%.
- HspllO and grpl70 both appear to exhibit a peptide binding cleft.
- hspllO and grp 170 differ dramaticaUy from the hsp70s in their C-terminal domains which, in the case of hsp70 proteins, appears to function as a Hd for the peptide binding cleft and may have an important influence on the properties of the bound pepti.de/protein and/ or the affinity for the associated peptide/protein.
- Both hspllO and grpl70 appear to be more significantly efficient in binding to and stabilizing thermaUy denatured proteins relative to hsc70.
- hsp70 and hspllO are approximately simUar in vaccine efficiency, they may bind differing subsets of peptides, i.e. hspllO may carry antigenic epitopes that do not readUy bind to hsc70, i.e. they may exhibit differing vaccine potential if not differing (mass) efficiencies.
- a simUar argument can be made for grp 170.
- the rabbit anti-hspllO antibody has been characterized by Lee-Yoon, D. et al., 1995, J. Biol. Chem. 270, 15725-15733.
- Affinity purified mouse anti-hsc70 monoclonal antibody, rabbit anti-murine hsp25 antibody, rat anti-hsp90 antibody and rat anti-TCP- la monoclonal antibody, as weH as recombinant hsc70 and murine hsp25 were aU obtained from StressGen Biotechnological Corp (Victoria, Canada).
- Anti-His Antibody was purchased from Amersham. Colon 26 tumor ceUs were cultured in DMEM supplemented with 10% calf serum in 5% CO z incubator.
- CeHs were washed with phosphate-buffered saline and homogenized with a Teflon homogenizer with 5 volumes of buffer (30 mM NaHC0 3 , pH7.5, ImM phenylmethylsulfonyl fluoride). The homogenates were centrifuged for 20 min at 12,000xg, supernatant were further centrifuged for 2 h at 100,000xg. CeU extracts were first appHed to Con A-sepharose column, unbound proteins were coUected and loaded on ion exchange column (Mono Q, Pharmacia) equUibrated with 20 mM Tris-HCl, pH 7.5, 200mM NaCl, O.lmM dithiothreitol.
- Bound proteins were eluted with a linear salt gradient (200mM ⁇ 350mM NaCl). HspllO pooled fractions were concentrated using centricon 30 (Amicon) and appHed to size exclusion column (superose 6, Pharmacia) for high performance chromatography (HPLC) equUibrated with 20mM Tris-HCl, pH8.0, 150mM NaCl, ImM DTT), then eluted with at a flow rate of 0.2 ml/min. ThyroglobuHn (669 kDa), ferritin (440 kDa), catalase (158 kDa), albumin (67 kDa) and ovalbumin (43 kDa) were used as protein markers.
- CeUs were washed with PBS and lysed in 50 mM Tris-HCl, pH 7.5, 150 mM NaCl, 2mM EDTA, 1% Triton X-100 and protease inl ibitors. After incubation on ice for 30 min, ceU extracts were boUed with equal volume of SDS sample buffer (50 mM Tris- HCl, pH 6.8, 5% ⁇ -mercaptoethanol, 2% SDS, 10% glycerol) for 10 min and centrifuged at 10,000g for 20 min. Equivalent protein samples were subjected to 7.5- 10% SDS-PAGE and electro-transferred onto immobUon-P membrane (MilHpore Ltd., UK).
- Membrane were blocked with 5% non-fat milk in TBST (20 mM Tris-HCl, pH 7.4, 137 mM NaCl, 0.05% Tween-20) for lh at room temperature, and then incubated for 2 h with primary antibodies diluted 1:1000 in TBST. After washing, membranes were incubated with horseradish peroxidase-conjugated goat anti-rabbit IgG or goat anti-mouse IgG diluted 1 :2,000 in TBST. Immunoreactivity was detected using the Enhanced ChemUuminescence detection system (Amersham, Arlington Heights, IL).
- CeUs were washed 3 times with cold PBS and lysed in Buffer (lOmM Tris-HCl, pH 7.4, 150 mM NaCl, 2 mM EDTA, 0.5% Sodium Deoxycholate, 0.1% SDS, 1% NP40, 10 ⁇ g/ml leupeptin, 25 ⁇ g/ml aprotinin, 1 mM ABESF, 0.025% NaN3).
- the lysates were centrifuged and supernatant was presorbed with 0.05 volume preimmune serum together with 30ml protein A beads for lh.
- lysates were incubated overnight at 4°C with hspllO antibody (1:100) or hsc70 antibody (1:200) or hsp25 antibody (1:100).
- hspllO antibody (1:100)
- hsc70 antibody (1:200)
- hsp25 antibody (1:100).
- recombinant wUd-type hspl 10 and hspl 10 mutants first were incubated with hsc70 or hsp25 at 30 °C. Then hsc70 antibody or hsp25 antibody were added and further incubated overnight at 4°C.
- Immune complex were precipitated with Protein A-agarose (30 ⁇ l) for 2h. Precipitates were washed 3 times with 50 mM Tris-HCl, pH 7.5, 150 mM NaCl, 0.1% SDS, 0.5% sodium deoxycholate, 1% NP40, 30-40 ⁇ l SDS sample buffer was added and boHed for 5 min. Supernatant were loaded to 7.5-12% SDS-PAGE and analyzed by inimunoblotting.
- Luciferase (Boehringer Mannheim) was incubated with hspllO, hsc70 and hsp25 (150 nM each) in 25 mM Hepes, pH 7.9, 5 mM magnesium acetate, 50 mM KC1, 5 mM b- mercaptoe hanol, and 1 mM ATP at room temperature or 43°C for 30 min.
- the solution was centrifuged at 16,000g for 20 min, the supernatant was loaded on the Sephacryl S-300 column (Pharmacia) equUibrated with 20 mM Tris-HCl, pH 7.8, 150 mM NaCl and 2 mM DTT.
- the protein was eluted at the flow rate of 0.24 nU/min at 4 °C. Fractions were coUected and analyzed by western blotting.
- hspllO was eluted as one broad peak of high molecular mass, it is reasonable that this large in situ hspllO complex might also contain additional components, potentiaUy including other molecular chaperones and/ or ceUular substrates that may interact with hspllO.
- potentiaUy including other molecular chaperones and/ or ceUular substrates that may interact with hspllO.
- the purified hspllO fraction derived from both ion exchange and size exclusion columns was examined by immunoblotting for other HSPs using avaUable antibodies. As shown in Figure 9B, antibodies for hsp90, hsc70, T-complex polypeptidel (TCP-1) and hsp25 were used.
- luciferase was added as a potential substrate to this mixture. It has been shown that hspllO can solubUize this reporter protein foUowing heat denaturation. Luciferase, with hspllO, hsc70 and hsp25 mix (at 1:1 molar ratio) were incubated at room temperature or at 43°C for 30 minutes.
- the soluble fractions were loaded onto a Sephacryl S-300 column, eluted fractions were run on SDS-PAGE and analyzed by immunoblotting with antibodies for hspllO, hsc70, hsp25 and luciferase.
- This mutant has been shown to be fuUy functional in its abUity to stabilize heat denatured luciferase in a folding competent state.
- the second mutant used here (#2) again lacked the ATP binding domain as weH as the adjacent beta sheet (peptide binding) domain, but contained the remaining C terminal sequence (size: 62 kDa and containing amino acids 508-858).
- This mutant has recently been shown to be tcapable of performing the chaperoning function of sustaining heat denatured luciferase in a soluble state.
- Mutant #1 (no ATP binding domain) was observed to co-precipitate with both hsp70 (lane 2) and hsp25 (lane 5), indicating that these interactions do not involve its ATP binding domain.
- mutant #2 (lacking both the ATP region and the peptide- binding region of hspllO) was observed to only associate with hsp70 (lane 3). This indicates that hsp25 and hsp70 can interact with hspllO at different sites and that the association of hspllO with hsp25 requires the peptide-binding domain of hspllO.
- hsc70-hspll0 binding occurs in the absence of the hspllO peptide- binding domain suggests that hsc70 may be actively binding to hspl 10 through its (i.e. hsc70's) peptide-binding domain, but does not exclude the possibUity that the two proteins interact via the involvement of other C-terminal domains. These interactions between hspllO and hsc70 raise possibUities as to how these proteins may function cooperatively.
- hsc70 may piggy-back hspllO in a manner that aUows transfer of substrate from hspl 10 to hsc70 with subsequent folding in conjunction with DnaJ homologs and other chaperones.
- HspllO has not yet been shown to have a folding function in conjunction with DnaJ co-chaperones, as is the case with hsc70 (Oh, H.J. et al., 1997, J. Biol. Chem. 272, 31696-31640; Oh, H.J. et al., 1999, J. Biol. Chem. 274, 15712-15718).
- hspllO exhibits different ATP bmding properties than do the hsp70s, and possible co- chaperones of hspllO may be awaiting discovery.
- whUe sHSPs e.g.
- HspllO and sHSPs may act in the differential bmding of a broad variety of substrates for subsequent shuttling to hsp70-DnaJ containing chaperone machines.
- That d ese three chaperones interact may represent a general phenomenon.
- Plesofsky- . Vig and Brambl have recently shown that the smaU HSP of Ne mspora crassa, caUed hsp30, binds to two ceUular proteins, hsp70 and hsp88. Cloning and analysis of hsp88 has shown that it represents the hspllO of Neurospora crassa (Plesofsky-Vig, N. and Brambl, R, 1998, J. Biol. Chem. 273, 11335-11341), suggesting that the interactions described here are phylogeneticaUy conserved.
- Figure 13 shows the results of immunoprecipitation of her-2/neu intraceUular domain (ICD) with anti-hspllO and anti-grpl70 antibodies after formation of binding complexes in vitro.
- Lane 1 is a protein standard from 205 kDa to 7.4 kDa;
- lane 2 is hspllO + anti-hspllO antibody;
- lane 3 is hspllO + ICD;
- lane 4 is grpl70 + ICD (in binding buffer);
- lane 5 is grpl70 + ICD (in PBS);
- lane 6 is ICD; and
- lane 7 is hspl 10.
- Figure 14 is a western blot showing hspllO-ICD complex in both fresh (left lane) and freeze-ti aw (center lane) samples, after immunoprecipitation of the complexes with anti-hspllO antibody.
- the right lane is ICD.
- Figure 15 is a bar graph showing hsp-peptide binding using a modified ELISA and p546, a 10-mer peptide (VLQGLPREYV; SEQ ID NO: 5) of a her-2/neu transmembrane domain, selected for its HLA-A2 binding affinity and predicted binding to hspllO.
- the peptide was biotinylated and mixed with hspllO in vitro (60 ⁇ g peptide and 60 ⁇ g hspl 10 in 150 ⁇ l PBS). The mixtures were incubated at 43°C for 30 minutes and then at 37°C for 1 hour. The mixtures were purified using a Centricon-10 centrifuge to remove the unbound peptide.
- BSA (1%) was also incubated with 100 ⁇ g of the biotinylated peptide at the same conditions, and purified.
- WeUs were coated with different concentrations of the purified mixtures, biotinylated peptide (positive control), or BSA (negative control) in a coating buffer. After incubation at 4°C overnight, weUs were washed 3 times with PBS-Tween 20 (0.05%) and blocked with 1% BSA in PBS for 1 hour at room temperature. After washing, 1:1000 streptavidin-HRP was added into the weUs and plates were incubated at room temperature for 1 hour. The color was developed by adding the TMB substrate and reading the absorbance at 450 nm. Purified mixture concentrations were 1 ⁇ g/ml (white bars), 10 ⁇ g/ml (cross-hatched bars), and 100 ⁇ g/ml (dark stippled bars).
- Figure 16 shows the results of immunoprecipitation of . tuberculosis antigens Mtb8.4 and Mtb39 with anti-hspllO antibody after formation of binding complexes in vitro, using both fresh samples and samples that had been subjected to freezing and thawing.
- Lane 1 is a protein standard from 205 kDa to 7.4 kDa; lane 2 is hspllO + Mtb8.4; lane 3 is hspllO + Mtb8.4 (after freeze-thaw); lane 4 is Mtb8.4; lane 5 is hspllO; lane 6 is hspllO + Mtb39; lane 7 is hspllO + Mtb39 (after freeze-thaw); lane 8 is Mtb39; and lane 9 is anti-hspllO antibody.
- hspllO complexed with a peptide from her-2/neu including the intraceUular domain (ICD; amino acid residues 676-1255), extraceUular domain (ECD; p369; KIFGSLAFL; SEQ ID NO: 6), or transmembrane region (p546) of her-2/neu, is immunogenic, as determined by gamma interferon (IFN-gamma) production by stimulated CTLs.
- ICD intraceUular domain
- ECD extraceUular domain
- ECD extraceUular domain
- p369 KIFGSLAFL
- p546 transmembrane region
- Figure 17 is a bar graph showing IFN-gamma production (determined by number of spots in an ELISPOT assay) by T ceUs of A2/Kb transgenic mice (5 animals per group) after i.p. mimunization with 25 ⁇ g of recombinant mouse hspl 10-ICD complex. These mice are transgenic for a hybrid human/mouse class I molecule such that the animals are capable of HLA-A2 presentation, as weU as retaining the murine poly- ⁇ 3 domain, providing for additional ceU surface protein interactions. Animals were boosted after 2 weeks, and sacrificed 2 weeks thereafter. Control groups were injected with 25 ⁇ g of ICD or hspllO, or not immunized.
- CD8 T ceUs were depleted using Dynabeads coated with anti-CD8 antibody and magnetic separation.
- Total splenocytes or depleted ceUs (5 x 10 6 ceUs/ml) were cultured in vitro with 25 ⁇ g/ml PHA (checkered bars) or 20 ⁇ g/ml ICD (dark stippled bars) overnight and IFN-gamma secretion was detected using the ELISPOT assay.
- Figure 18 is a bar graph showing immunogenicity of hspllO-peptide complexes reconstituted in vitro, as determined by number of positive spots in an ELISPOT assay for IFN-gamma secretion.
- Recombinant hamster hspl 10 100 ⁇ g was incubated with 100 ⁇ g of the 9-mer her-2/neu peptide p369, an HLA-A2 binder, at 43°C for 30 minutes, foUowed by incubation at room temperature for 60 minutes.
- the complex was purified using a Centricon-10 centrifuge to remove unbound peptides.
- Figure 19 is a bar graph showing immunogenicity of hspllO-peptide complexes reconstituted in vitro, as determined by number of positive spots in an ELISPOT assay for IFN-gamma secretion.
- Recombinant hamster hspllO 100 ⁇ g was incubated with 100 ⁇ g of the 10-mer her-2/neu peptide ⁇ 546, an HLA-A2 binder, at 43°C for 30 minutes, foUowed by incubation at room temperature for 60 minutes.
- the complex was purified using a Centricon-10 centrifuge to remove unbound peptides.
- Eight-week old HLA-A2 transgenic mice (n 2) were immunized i.p.
- Figure 20 is a graph showing specific anti-hspl 10 antibody response in A2/Kb transgenic mice foUowing i.p. immunization with the hspl 10-ICD (her2/neu) complex.
- ELISA results are plotted as optical density (OD) at 450 nm as a function of serum and antibody dUutions. Results are shown for the positive control of anti-hspl 10 (soHd squares), the negative control of unrelated antibody (open circles), and serum at day 0
- FIG. 21 is a graph showing specific anti-ICD antibody response in A2/Kb transgenic mice foUowing i.p. immunization with the hspl 10-ICD complex.
- ELISA results are plotted as optical density (OD) at 450 nm as a function of serum and antibody dUutions.
- mice developed a specific antibody response to ICD of her2/neu after iinrnunization with the stress protein complex.
- Figure 22 is a bar graph comparing specific anti-ICD antibody responses in A2/Kb transgenic animals 2 weeks after priming with different vaccine formulas. Results are plotted as OD at 450 nm for the various serum and antibody dUutions and bars represent data for animals primed with hspl 10-ICD (stippled bars), the positive control of ICD in complete Freund's adjuvant (CFA; checkered bars), ICD alone (cross- hatched bars), anti-ICD antibody (dark stippled bars), and the negative control of unrelated antibody (open bars).
- Figure 23 is a bar graph comparing specific anti-ICD antibody generation 2 weeks after s.c. or i.p. priming of A2/Kb transgenic with hspl 10-ICD complex. Results are plotted as OD at 450 nm for the various serum and antibody dUutions and bars represent serum at day 0 (stippled bars), serum i.p. at day 14 (checkered bars), serum s.c. at day 14 (cross-hatched bars), anti-ICD antibody (dark stippled bars), and the negative control of unrelated antibody (open bars).
- CT26 colon 26 ceUs
- CT26-hspllO ceUs vector encoding hspllO
- CT26-hspllO ceHs overexpress hspllO, as demonstrated by both immunoblot and immunofluorescence staining.
- Figure 24A is an immunoblot showing that CT26-hspllO ceUs exhibit increased hspllO expression relative to untransfected CT26 ceUs and CT26 ceUs transfected with an empty vector (CT26-vector).
- Equivalent protein samples from CT26 (lane 1), CT26- vector (lane 2), and CT26-hspllO (lane 3) were subjected to 10% SDS PAGE and transferred onto immobUon-P membrane. Membranes were probed with antibodies for hspl 10. After washing, membranes were incubated with horseradish peroxidase- conjugated goat anti-rabbit IgG or goat anti-mouse IgG dUuted 1 :2,000 in TBST. Immunoreactivity was detected using the Enhanced ChemHuminescence detection system.
- Figure 24B shows that CT26-hspllO ceUs do not exhibit enhanced hsc70 expression relative to untransfected CT26 ceUs or CT26 ceUs transfected with an empty vector.
- Equivalent protein samples from CT26 (lane 1), CT26-vector (lane 2), and CT26- hspl 10 (lane 3) were prepared as for Figure 24A, except that membranes were probed with antibodies for hsc/hsp70.
- Figure 25A is a photomicrograph showing itrrmunofluorescence staining of hspllO in CT26 ceUs. CeUs were seeded on the cover sHps one day before the staining. Cover sHps were then incubated with rabbit anti-hspllO antibody (1:500 dUution) foUowed by FITC-labeled dog anti-rabbit IgG staining. Normal rabbit IgG was used as negative control.
- Figure 25B is a photomicrograph showing irnrnunofluorescence staining of hsp 110 in empty vector transfected CT26 ceUs. CeUs were prepared and immunostained as in Figure 25A.
- Figure 25C is a photomicrograph showing immunofluorescence staining of hspl 10 in hspl 10 over-expressing ceUs. CeUs were prepared and immunostained as in Figure 25A.
- This example provides data characterizing the in vivo and in vitro growth properties of CT26-hspll0 ceUs.
- Figure 26 is a graph demonstrating in vitro growth properties of wild type and hsp 110- transfected ceU lines, plotted as ceU number at 1-5 days after seeding. CeUs were seeded at a density of 2x10 4 ceUs per weU. 24 hours later ceUs were counted (assigned as day 0). CeUs from tripHcate weUs were counted on the indicated days. The results are means ⁇ SD of three independent experiments using wUd type CT26 ceUs (circles), CT26 ceUs transfected with empty vector (squares), and hspllO-transfected CT26 ceUs (triangles).
- Figure 27 is a bar graph showing the effect of hspllO over-expression on colony forming abUity in soft agar.
- WUd-type CT26 ceUs, empty vector transfected CT26- vector ceUs and hspllO over-expressing CT26-hspll0 ceUs were plated in 0.3 % agar and analyzed for their ability to form colonies ( ⁇ 0.2) in soft agar.
- P ⁇ 0.05 compared with CT26-vector, as assessed by student's t test.
- Figure 28 is a graph showing in vivo growth properties of wUd-type and hspllO transfected CT26 ceU line. 5 10 ceUs were inoculated s.c. into flank area of balb/c mice. Tumor growth was recorded twice a week measuring both the longitudinal and transverse diameter with a caHper. Tumor volume, in cubic mm, is plotted as a function of days after tumor implantation for CT26 wild type ceUs (circles), CT26 ceUs transfected with empty vector (squares), CT26 ceHs transfected with hspllO, 5 x 10 4 (upward triangles), and CT26 ceUs transfected with hspllO, 5 x 10 5 (downward triangles).
- mice immunized with irradiated hspl 10 over- expressing CT26 ceUs are protected against subsequent chaUenge with Hve CT26 ceHs.
- immunization with CT26-hspllO ceUs eHcits tumor specific CTL and antibody responses.
- Figure 29 is a plot showing the effect of injection with irradiated hspl 10-overexpressing ceUs on the response to chaUenge with Hve CT26 ceUs.
- Mice were injected with 5xl0 5 irradiated (9,000 rad) CT26-hspllO ceUs subcutaneously in the left flank. Two weeks later, mice were chaUenged on the right flank with Hve CT26 ceUs. Growth of tumor in mice without preimmunization was also shown.
- Results are plotted as percent tumor free mice as a function of days after tumor chaUenge for mice immunized with PBS and chaUenged with 5xl0 4 CT26 ceUs (circles); irradiated CT26 ceUs with empty vector/5xl0 5 CT26 ceUs (squares); irradiated CT26 ceUs with empty vector/5xl0 6 CT26 ceUs (upward triangles); irradiated CT26-hspllO ceUs/5xl0 5 CT26 ceUs (downward triangles); and irradiated CT26-hspllO ceUs/5xl0 6 CT26 ceHs (diamonds).
- Figure 30 is a graph showing tumor specific CTL response eHcited by immunization with tumor derived hspllO.
- Mice were injected with 5x10 5 irradiated (9,000 rad) CT26- empty vector and CT26-hsp 110 ceUs subcutaneously. Two weeks later, splenocytes were isolated as effector ceUs and re-stimulated with irradiated Colon 26 in vitro for 5 days. The lymphocytes were analyzed for cytotoxic activity using 51 Cr-labeled Colon 26 as target ceHs. Meth A tumor ceHs were also used as target in the experiment, and no ceU lysis was observed. Results are plotted as percent specific lysis as a function of effector:target ratio for control (circles), irradiated CT26 ceHs (squares), and irradiated CT26-hsp 110 ceUs (triangles) .
- Figure 31 is a graph showing antibody response against CT26 ceHs foUowing immunization with irradiated hspl 10-overexpressing ceUs. Mice were injected with 5xl0 5 irradiated (9,000 rad) CT26 empty vector and CT26-hspllO ceUs subcutaneously. Two weeks later, serum was coUected and assayed for antibody response using ELISA. Results are plotted as OD at 450 nm as a function of serum dilution for control (circles), CT26-empty vector (squares), and CT26-hspllO (triangles).
- This example demonstrates tiiat ceUs transfected with a GM-CSF gene, when co- injected with CT26-hspl 10 ceUs, provide enhanced protection against tumor chaUenge that leaves aU mice treated with the combined therapy free of tumors.
- Figure 32 is a graph showing the effect of GM-CSF from bystander ceUs on the growth of hspl 10 overexpressing ceUs.
- Mice were injected subcutaneously with 5x10 4 Hve tumor ceUs as foUows: CT26-empty vector ceUs (circles), CT26-vector ceUs plus irradiated B78H1GM-CSF ceUs (2:1 ratio; squares), CT26-hspllO ceUs plus irradiated B78H1GM CSF ceUs (2:1 ratio; upward triangles), CT26-hspllO ceUs (downward triangles), CT26-hspllO plus irradiated B78H1 ceUs (2:1 ratio; diamonds).
- the B78H1 GM-CSF are Bl 6 ceUs transfected with CM-CSF gene, whUe B78H1 are wUd type ceUs. Tumor growth was recorded by measuring the size of tumor, and is plotted as tumor volume in cubic mm as a function of days after implantation.
- Figure 33 is a graph showing the effect of co-injecting irradiated hspllO-overexpressing tumor vaccine and GM-CSF-secreting bystander ceUs on the response to wild-type CT26 tumor ceU chaUenge.
- mice were immunized subcutaneously with irradiated 5X10 5 tumor ceUs as foUows: CT26-empty vector ceUs, CT26-vector ceUs plus B78H1GM-CSF ceUs (2:1 ratio; squares), CT26-hspll0 ceUs plus B78H1GM-CSF ceUs (2:1; upward triangles), CT26-hspllO ceUs (downward triangles), CT26-hspllO plus B78H1 ceUs (2:1; diamonds). Also shown are results for mice irrrmunized only with PBS (circles). Mice were chaUenged at a separate site with CT26 wUd-type ceUs and monitored every other day for the tumor development. Results are plotted as percent tumor free mice at the indicated number of days after tumor chaUenge.
- Example 15 Immunization With Tumor-Derived Stress Protein Complexes Stimulates CeUular Immunity and Inhibits Metastatic Tumor Growth
- Tumor-derived stress protein complexes of the invention can be used to stimulate ceUular immunity and inhibit metastatic tumor growth.
- Interferon-gamma secretion was stimulated by immunization with colon 26 tumor-derived hspllO and grpl70, as weU as with B16F10-derived grpl70.
- Immunization with B16F10-derived grpl70 was also shown to eHcit a tumor-specific CTL response and a reduction in lung metastases.
- Figure 34 is a bar graph showing that immunization with colon 26-derived hspllO or grpl70 stimulates interferon (IFN) gamma secretion.
- IFN interferon
- splenocytes were isolated for ELISPOT assay.
- Phytohernagglutinin (PHA) treated lymphocytes were used for positive control.
- Figure 35 is a graph showing tumor specific CTL response eHcited by immunization with B16F10 tumor-derived grpl70. Mice were immunized twice with grpl70 (40 ⁇ g) at weekly intervals.
- splenocytes were isolated as effector ceUs and restimulated with irradiated B16F10 ceUs in vitro for 5 days.
- the lymphocytes were analyzed for cytotoxic activity using 51 Cr-labeled B16F10 or Meth A ceUs as target ceUs. Results are plotted as percent specific lysis as a function of effector:target ratio for controls (circles), Hver-derived grpl70 (squares), B16F10- derived grpl70 (upward triangles), and Meth A-derived grpl70 (downward triangles).
- Figure 36 shows immunization with B16F10-derived grpl70 stimulates IFN gamma secretion.
- mice were immunized with hspl 10 or grpl70, splenocytes were isolated for ELISPOT assay.
- Figure 37 shows lung metastases for mice in which 1 x 10 5 B16F10 ceUs were inoculated intravenously into the taU vein of each C57BL/6 mouse. 24 hr after tumor ceU injection, mice were then treated with PBS (closed circles), Hver-derived grpl70 (open circles), or tumor-derived grpl70 (40 ⁇ g). Three treatments were carried out during the whole protocol. The animals were kUled 3 weeks after tumor injection, lungs were removed and surface colonies were counted.
Abstract
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PCT/US2000/027023 WO2001023421A2 (en) | 1999-09-30 | 2000-09-29 | Stress protein compositions and methods for prevention and treatment of cancer and infectious disease |
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US (2) | US6984384B1 (en) |
EP (1) | EP1216055A2 (en) |
JP (1) | JP2003510334A (en) |
AU (1) | AU7743100A (en) |
CA (1) | CA2386032A1 (en) |
WO (1) | WO2001023421A2 (en) |
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US5750119A (en) * | 1994-01-13 | 1998-05-12 | Mount Sinai School Of Medicine Of The City University Of New York | Immunotherapeutic stress protein-peptide complexes against cancer |
WO2002036141A2 (en) * | 2000-11-02 | 2002-05-10 | Immunex Corporation | Method of enhancing lymphocyte-mediated immune responses |
ES2433915T3 (en) | 2002-05-21 | 2013-12-13 | Irun R. Cohen | DNA vaccines encoding heat shock proteins |
AU2003260426A1 (en) * | 2002-08-16 | 2004-03-11 | Glycotope Gmbh | Process for the production of temperature-induced tumor cell lysates for use as immunogenic compounds |
PT1654353E (en) | 2003-08-18 | 2013-08-23 | Glycotope Gmbh | Tumour cell lines nm-f9 (dsm acc2606) and nm-d4 (dsm acc2605), uses threreof |
US7619058B2 (en) | 2004-01-20 | 2009-11-17 | Aichi Prefecture | Epitope/peptide recognized by HLA-A2402-restricted Ep-CAM-specific CTL and use of the same |
JP2008048601A (en) * | 2004-06-15 | 2008-03-06 | Nippon Kosei Kagaku Kenkyusho:Kk | Method for producing therapeutically useful product and system |
EP1812580B1 (en) | 2004-11-16 | 2014-12-17 | Crucell Holland B.V. | Multivalent vaccines comprising recombinant viral vectors |
US20060120995A1 (en) * | 2004-12-02 | 2006-06-08 | Shah Maulik R | Neoadjuvant genetic compositions and methods |
US7585945B2 (en) | 2006-08-25 | 2009-09-08 | Health Research, Inc. | Use of recombinant heat shock protein complexed to kidney cancer antigen |
JP5767779B2 (en) | 2006-09-10 | 2015-08-19 | グリコトープ ゲーエムベーハー | Use of human cells of myeloid leukemia origin for antibody expression |
EP1920782A1 (en) * | 2006-11-10 | 2008-05-14 | Glycotope Gmbh | Carboyhdrate specific cellular immunity inducing microorganisms and fractions thereof |
AU2014201488B2 (en) * | 2007-06-01 | 2016-06-30 | The Henry M. Jackson Foundation For The Advancement Of Military Medicine, Inc | Vaccine for the Prevention of Breast Cancer Relapse |
MX2009012858A (en) * | 2007-06-01 | 2010-02-03 | Jackson H M Found Military Med | Vaccine for the prevention of breast cancer relapse. |
RU2497543C2 (en) * | 2007-11-02 | 2013-11-10 | Хелт Рисерч Инк. | Compositions and methods for using ca9 protein for immune system stimulation |
GB0816242D0 (en) * | 2008-09-05 | 2008-10-15 | Immunobiology Ltd | Method of purifying protien complexes |
EP2413953B1 (en) * | 2009-04-03 | 2017-11-08 | Agenus Inc. | Methods for preparing and using multichaperone-antigen complexes |
CN103814123A (en) | 2011-08-22 | 2014-05-21 | 葛莱高托普有限公司 | Microorganisms carrying a tumor antigen |
US9822154B2 (en) | 2012-12-14 | 2017-11-21 | Virginia Commonwealth University | Immune modulator for immunotherapy and vaccine formulation |
CN111601606A (en) * | 2017-11-29 | 2020-08-28 | 菲格内有限责任公司 | Interaction of fibroblasts with immune cells for activation and uses thereof |
US20210221910A1 (en) | 2018-05-18 | 2021-07-22 | Glycotope Gmbh | Anti-muc1 antibody |
WO2023056334A1 (en) * | 2021-09-30 | 2023-04-06 | The Children's Medical Center Corporation | Tlr4 agonist for modulating immune response |
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US5066579A (en) * | 1986-12-31 | 1991-11-19 | Genelabs Incorporated | HTLV-I peptide antigen and assay |
DK0474727T3 (en) * | 1989-05-19 | 1998-01-12 | Genentech Inc | HER2 extracellular domain |
US6268213B1 (en) * | 1992-06-03 | 2001-07-31 | Richard Jude Samulski | Adeno-associated virus vector and cis-acting regulatory and promoter elements capable of expressing at least one gene and method of using same for gene therapy |
US5801005A (en) * | 1993-03-17 | 1998-09-01 | University Of Washington | Immune reactivity to HER-2/neu protein for diagnosis of malignancies in which the HER-2/neu oncogene is associated |
PT700445E (en) | 1993-06-04 | 2002-07-31 | Whitehead Biomedical Inst | STRESS PROTEINS AND THEIR USES |
US5750119A (en) | 1994-01-13 | 1998-05-12 | Mount Sinai School Of Medicine Of The City University Of New York | Immunotherapeutic stress protein-peptide complexes against cancer |
US5550214A (en) * | 1994-02-10 | 1996-08-27 | Brigham And Women's Hospital | Isolated antigenic oncogene peptide fragments and uses |
US5961979A (en) | 1994-03-16 | 1999-10-05 | Mount Sinai School Of Medicine Of The City University Of New York | Stress protein-peptide complexes as prophylactic and therapeutic vaccines against intracellular pathogens |
US6015507A (en) * | 1994-11-08 | 2000-01-18 | Minolta Co., Ltd. | Liquid crystal element having composite layer |
CA2229543A1 (en) | 1995-08-18 | 1997-02-27 | Sloan-Kettering Institute For Cancer Research | Heat shock protein-based vaccines and immunotherapies |
US6331299B1 (en) * | 1995-08-18 | 2001-12-18 | Sloan-Kettering Institute For Cancer Research | Method for treatment of cancer and infectious disease and compositions useful in same |
US5985270A (en) * | 1995-09-13 | 1999-11-16 | Fordham University | Adoptive immunotherapy using macrophages sensitized with heat shock protein-epitope complexes |
EP0851765A4 (en) | 1995-09-13 | 2000-01-19 | Univ Fordham | Therapeutic and prophylactic methods using heat shock proteins |
US5837251A (en) * | 1995-09-13 | 1998-11-17 | Fordham University | Compositions and methods using complexes of heat shock proteins and antigenic molecules for the treatment and prevention of neoplastic diseases |
WO1997026910A2 (en) | 1996-01-27 | 1997-07-31 | Max-Delbrück-Centrum für Molekulare Medizin | Tumour vaccine for immunotherapy of malignant tumours |
US5747332A (en) * | 1996-09-20 | 1998-05-05 | University Of New Mexico | Methods for purifying and synthesizing heat shock protein complexes |
EP0950061A4 (en) * | 1996-09-20 | 2000-04-12 | Univ New Mexico | Heat shock protein complexes |
US7157089B1 (en) * | 1996-11-26 | 2007-01-02 | Stressgen Biotechnologies Corporation | Immune responses using compositions containing stress proteins |
DE69735376T2 (en) * | 1996-11-26 | 2006-12-14 | Stressgen Biotechnologies Corp., Victoria | FUSION PROTEINS INCLUDING STRESS PROTEINS TO INDICATE AN IMMUNE RESPONSE |
US6017540A (en) * | 1997-02-07 | 2000-01-25 | Fordham University | Prevention and treatment of primary and metastatic neoplastic diseases and infectious diseases with heat shock/stress protein-peptide complexes |
US5830464A (en) * | 1997-02-07 | 1998-11-03 | Fordham University | Compositions and methods for the treatment and growth inhibition of cancer using heat shock/stress protein-peptide complexes in combination with adoptive immunotherapy |
EP1047451A1 (en) | 1997-02-18 | 2000-11-02 | Whitehead Institute For Biomedical Research | Use of heat shock proteins to deliver moieties into cells |
US5891432A (en) * | 1997-07-29 | 1999-04-06 | The Immune Response Corporation | Membrane-bound cytokine compositions comprising GM=CSF and methods of modulating an immune response using same |
KR100843109B1 (en) | 1997-08-05 | 2008-07-02 | 엔벤타 바이오파마슈티컬스 코포레이션 | Immune responses against HPVhuman papillomavirus antigens elicited by compositions comprising a fusion protein comprising an HPV antigen and a stress protein |
US5888795A (en) * | 1997-09-09 | 1999-03-30 | Becton, Dickinson And Company | Thermostable uracil DNA glycosylase and methods of use |
IL144371A0 (en) | 1999-01-29 | 2002-05-23 | Corixa Corp | Her-2/neu fusion proteins |
-
2000
- 2000-09-29 JP JP2001526571A patent/JP2003510334A/en active Pending
- 2000-09-29 CA CA002386032A patent/CA2386032A1/en not_active Abandoned
- 2000-09-29 WO PCT/US2000/027023 patent/WO2001023421A2/en not_active Application Discontinuation
- 2000-09-29 EP EP00967198A patent/EP1216055A2/en not_active Withdrawn
- 2000-09-29 AU AU77431/00A patent/AU7743100A/en not_active Abandoned
- 2000-09-29 US US09/676,340 patent/US6984384B1/en not_active Expired - Lifetime
-
2005
- 2005-04-11 US US11/103,222 patent/US7976846B2/en not_active Expired - Fee Related
Also Published As
Publication number | Publication date |
---|---|
WO2001023421B1 (en) | 2001-12-06 |
AU7743100A (en) | 2001-04-30 |
EP1216055A2 (en) | 2002-06-26 |
WO2001023421A3 (en) | 2001-10-25 |
US6984384B1 (en) | 2006-01-10 |
WO2001023421A2 (en) | 2001-04-05 |
US20050202035A1 (en) | 2005-09-15 |
US7976846B2 (en) | 2011-07-12 |
JP2003510334A (en) | 2003-03-18 |
CA2386032A1 (en) | 2001-04-05 |
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