Heat Shock Protein 105 as an Immunotherapeutic Target for Patients With Cervical Cancer.

BACKGROUND/AIM: Heat shock protein 105 (HSP105) is overexpressed in various cancers, but not in normal tissues. We investigated the expression levels of HSP105 in cervical cancer and the efficacy of immunotherapy targeting HSP105. MATERIALS AND METHODS: Previously, we established human leukocyte antigen-A*02:01 (HLA-A2) restricted HSP105 peptide-specific cytotoxic T lymphocyte (CTL) clones from a colorectal cancer patient vaccinated with an HSP105 peptide. Herein, we evaluated the expression of HSP105 in cervical cancer and cervical intraepithelial neoplasia. Moreover, we tested the effectiveness of an HLA-A2-restricted HSP105 peptide-specific CTL clone against cervical cancer cell lines. RESULTS: HSP105 was expressed in 95% (19/20) of examined cervical cancer tissues. Moreover, the HSP105 peptide-specific CTL clone recognized HSP105- and HLA-A*02:01-positive cervical cancer cell lines and also showed that cytotoxicity against the cervical cancer cell lines depends on HSP105 peptide and HLA class I restricted manners. CONCLUSION: HSP105 could be an effective target for immunotherapy in patients with cervical cancer.

Creation of Recombinant Chaperone Vaccine Using Large Heat Shock Protein for Antigen-Targeted Cancer Immunotherapy.

Large heat shock proteins (HSPs) or stress proteins, including Hsp110 and Grp170, are unique molecular chaperones with superior capability of shuttling tumor protein antigens into professional antigen-presenting cells, such as dendritic cells, for highly efficient cross-presentation and T cell priming. Reconstituted chaperone complexes of large HSP and tumor protein antigen have been demonstrated to generate a robust antigen-specific T lymphocyte response with therapeutic potency against multiple cancer types in preclinical models. Here, we describe the methods for preparing this recombinant chaperone complex vaccine and analyzing the vaccine-induced activation of antigen-specific T cells using in vitro and in vivo systems.

Localization of heat shock protein 110 in canine mammary gland tumors.

Heat shock proteins (HSPs) function as molecular chaperones in the regulation of protein folding, conformation, and assembly; in addition, they also protect cells from protein-protein aggregation resulting from cellular stress. Recently, HSPs were shown to be overexpressed in several human cancer cells compared with normal cells. HSPs are considered to be related to apoptosis-associated proteins, and inhibition of apoptosis promotes tumor growth. Canine mammary gland tumors have received a great deal of attention from researchers due to the many common biological and histological characteristics that they share with human tumors. We previously confirmed that HSP110 is a canine mammary gland tumor antigen and reported that HSP110 mRNA expression significantly increased in tumor tissue. We have now created a functional recombinant canine HSP110 protein and a rabbit anti-HSP110 polyclonal antibody. This recombinant protein can refold heat-denatured firefly luciferase at 42°C. Immunohistochemical analysis showed that HSP110 was mainly localized in the cytoplasm of epithelial and interstitial cells in canine mammary gland tumors. Extensive genomic research has revealed genetic similarities between humans and dogs; comparative oncological studies between these species have made remarkable progress. The results reported here contribute valuable oncological knowledge for the development of novel therapeutic methods in both veterinary science and human medicine.

Identification of tumor antigens that elicit a humoral immune response in the sera of Chinese esophageal squamous cell carcinoma patients by modified serological proteome analysis.

Our aim was to identify novel tumor-associated antigens from the esophageal squamous cell carcinoma (ESCC) cell line EC0156, and related autoantibodies in sera from patients with ESCC. We used modified serological proteome analysis, involving one- and two-dimensional electrophoresis, Western blot, and MALDI-TOF/TOF-MS to identify 6 ESCC-associated antigens. From these, 105 kDa heat shock protein (HSP105) and triosephosphate isomerase (TIM) were further evaluated and we determined they could induce autoantibody responses in ESCC sera and are highly expressed in ESCC tissues. Anti-HSP105 and anti-TIM autoantibodies were found in 39.1% (18/46) and 34.8% (16/46) of patients with ESCC, respectively, but only in two controls. A receiver operating characteristic curve constructed with HSP105 and TIM gave a sensitivity of 54.3% and 95% (38/40) specificity in discriminating ESCC from matched controls. Interestingly, we found that autoantibodies against TIM in ESCC serum mainly reacted with glycosylated but not deglycosylated TIM. The preliminary results suggest the potential utility of screening autoantibodies in sera for use as biomarkers for cancer diagnosis.

Identification of HLA-A2 or HLA-A24-restricted CTL epitopes for potential HSP105-targeted immunotherapy in colorectal cancer.

We previously reported that heat shock protein 105 (HSP105) is overexpressed in a variety of human cancers, including colorectal, pancreatic and esophageal cancer and has proven to be a novel biomarker for the immunohistochemical detection of these cancers. In the present study, we used HLA-transgenic mice (Tgm) and the peripheral blood mononuclear cells (PBMCs) of colorectal cancer patients to identify HLA-A2 and HLA-A24-restricted HSP105 epitopes, as a means of expanding the application of HSP105-based immunotherapy to HLA-A2- or HLA-A24-positive cancer patients. In addition, we investigated by ex vivo IFN-γ ELISPOT assay whether the HSP105-derived peptide of cytotoxic T cells (CTLs) exists in PBMCs of pre-surgical colorectal cancer patients. We found that four peptides, HSP105 A2-7 (RLMNDMTAV), HSP105 A2-12 (KLMSSNSTDL), HSP105 A24-1 (NYGIYKQDL) and HSP105 A24-7 (EYVYEFRDKL), are potential HLA-A2 or HLA-A24-restricted CTL HSP105-derived epitopes. HSP105-specific IFN-γ-secreting T cells were detected in 14 of 21 pre-surgical patients with colorectal cancer in response to stimulation with these four peptides. Our study raises the possibility that these HSP105 peptides are applicable to cancer immunotherapy in patients with HSP105-expressing cancer, particularly colorectal cancer.

[Cytotoxic activity of spleen lymphocytes in BALB/c mice immunized by HSP110-HER2/neu ICD].

OBJECTIVE: To explore the cytotoxic responses of spleen T lymphocytes (CTL) in BALB/c mice induced by recombinant HSP110-HER2/neu ICD complex. METHODS: Tumor-bearing mouse model was immunized by HSP110-HER2/neu ICD complex. The IFN-γ level secreted by activated spleen T lymphocytes was detected by enzyme linked immunospot assay (ELISPOT). The corresponding CTL activity was measured by granzyme release assay. RESULTS: The BALB/c mouse model of human mammary tumor highly expressing HER2/neu was established. HSP110-HER2/neu ICD complex immunization led to a significantly higher level of INF-γ than that in HSP110-P(789-797) immunized and HER2/neu ICD immunized mice. HSP110-HER2/neu ICD complex immunized animals also show significant CTL activity. The results of immunohistochemical staining showed that the number of blue spots in the PBS group was 4.57 ± 1.33, HSP110 group 6.83 ± 2.08, HER2/neu ICD group 16.17 ± 2.86, HSP110-P(789-797) group 43.67 ± 4.78, and SP110-HER2/neu ICD group 76.51 ± 8.17. The number of IFN-γ-secreting spleen lymphocytes in the HSP110-HER2/neu ICD group was significantly higher than that in the HSP110-P(789-797) group, and that of HSP110-P(789-797) group was significantly higher than that of HER2/neu ICD group (P < 0.01). The target cell-killing rate of the PBS group was (8.15 ± 1.27)%, HSP110 group (9.51 ± 1.51)%, HER2/neu ICD group (14.03 ± 2.45)%, HSP110-P(789-797) group (25.99 ± 3.04)% and HSP110-HER2/neu ICD group (38.15 ± 3.95)% (all P < 0.01). CONCLUSIONS: HSP110-HER2/neu ICD complex can promote the proliferation and maturation of T lymphocytes into CTLs, and might be used as anti-tumor vaccine to induce potent cytotoxic T lymophocyte immunoresponse against specific tumor cells.

Enhancing antigen cross-presentation and T-cell priming by complexing protein antigen to recombinant large heat-shock protein.

Large heat-shock proteins (HSPs), including hsp110 and grp170, are unique immunochaperones capable of carrying and introducing antigens into professional antigen-presenting cells for efficient cross-presentation. Therefore, reconstituted chaperone complexes of large HSPs and protein antigen may be exploited for augmentation of an antigen-specific immune response. The methods for the preparation of the recombinant protein antigen chaperone complex and characterization of its T-cell priming capability in both in vitro and in vivo settings are described.

CD204 suppresses large heat shock protein-facilitated priming of tumor antigen gp100-specific T cells and chaperone vaccine activity against mouse melanoma.

We previously reported that scavenger receptor A (SRA/CD204), a binding structure on dendritic cells (DCs) for large stress/heat shock proteins (HSPs; e.g., hsp110 and grp170), attenuated an antitumor response elicited by large HSP-based vaccines. In this study, we show that SRA/CD204 interacts directly with exogenous hsp110, and lack of SRA/CD204 results in a reduction in the hsp110 binding and internalization by DCs. However, SRA(-/-) DCs pulsed with hsp110 or grp170-reconstituted gp100 chaperone complexes exhibit a profoundly increased capability of stimulating melanoma Ag gp100-specific naive T cells compared with wild-type (WT) DCs. Similar results were obtained when SRA/CD204 was silenced in DCs using short hairpin RNA-encoding lentiviruses. In addition, hsp110-stimulated SRA(-/-) DCs produced more inflammatory cytokines associated with increased NF-κB activation, implicating an immunosuppressive role for SRA/CD204. Immunization with the hsp110-gp100 vaccine resulted in a more robust gp100-specific CD8(+) T cell response in SRA(-/-) mice than in WT mice. Lastly, SRA/CD204 absence markedly improved the therapeutic efficacy of the hsp110-gp100 vaccine in mice established with B16 melanoma, which was accompanied by enhanced activation and tumor infiltration of CD8(+) T cells. Given the presence of multiple HSP-binding scavenger receptors on APCs, we propose that selective scavenger receptor interactions with HSPs may lead to highly distinct immunological consequences. Our findings provide new insights into the immune regulatory functions of SRA/CD204 and have important implications in the rational design of protein Ag-targeted recombinant chaperone vaccines for the treatment of cancer.

Serological identification of HSP105 as a novel non-Hodgkin lymphoma therapeutic target.

We reported that the clinical efficacy of dendritic cell-based vaccination is strongly associated with immunologic responses in relapsed B-cell non-Hodgkin lymphoma (B-NHL) patients. We have now investigated whether postvaccination antibodies from responders recognize novel shared NHL-restricted antigens. Immunohistochemistry and flow cytometry showed that they cross-react with allogeneic B-NHLs at significantly higher levels than their matched prevaccination samples or nonresponders' antibodies. Western blot analysis of DOHH-2 lymphoma proteome revealed a sharp band migrating at approximately 100 to 110 kDa only with postvaccine repertoires from responders. Mass spectrometry identified heat shock protein-105 (HSP105) in that molecular weight interval. Flow cytometry and immunohistochemistry disclosed HSP105 on the cell membrane and in the cytoplasm of B-NHL cell lines and 97 diagnostic specimens. A direct correlation between HSP105 expression and lymphoma aggressiveness was also apparent. Treatment of aggressive human B-NHL cell lines with an anti-HSP105 antibody had no direct effects on cell cycle or apoptosis but significantly reduced the tumor burden in xenotransplanted immunodeficient mice. In vivo antilymphoma activity of HSP105 engagement was associated with a significant local increase of Granzyme B(+) killer cells that very likely contributed to the tumor-restricted necrosis. Our study adds HSP105 to the list of nononcogenes that can be exploited as antilymphoma targets.

Hsp110-mediated enhancement of CD4+ T cell responses to the envelope glycoprotein of members of the family Flaviviridae in vitro does not occur in vivo.

The use of heat shock proteins (HSP) to enhance activation of the immune response to chaperoned antigen is being explored for immunotherapy. Hsp110 chaperones large protein substrates more effectively than Hsp70, offering the potential to use complex antigens containing multiple epitopes in HSP-based vaccines. In this study, we investigated the ability of recombinant bovine Hsp110 to chaperone E2 glycoprotein, the major envelope protein of bovine viral diarrhea virus (BVDV) and the dominant target of neutralizing antibodies. Hsp110 formed complexes with E2, as demonstrated by immunoprecipitation. When monocytes from BVDV-immunized cattle were stimulated with these complexes and incubated with autologous CD4(+) T cells, enhanced levels of proliferation were observed. To determine the ability of these complexes to improve immunogenicity in vivo, cattle were vaccinated with either Hsp110-E2 complex or E2 only, combined with Quil-A adjuvant. In contrast to the in vitro data, cellular and humoral responses to E2 were greater in the E2-only vaccination group, indicating that complex formation had actually reduced the immunogenicity of E2. This study highlights the need for further understanding of the means by which HSP complexes are endocytosed and processed in vivo to enable the design of successful vaccine strategies.

Superior antitumor response induced by large stress protein chaperoned protein antigen compared with peptide antigen.

Our previous studies have demonstrated that the natural chaperone complexes of full-length tumor protein Ags (e.g., gp100) and large stress proteins (e.g., hsp110 and grp170) with exceptional Ag-holding capabilities augment potent tumor protective immunity. In this study, we assess the peptide-interacting property of these large chaperones and, for the first time, compare the immunogenicity of the recombinant chaperone vaccines targeting two forms of Ags (protein versus peptide). Both hsp110 and grp170 readily formed complexes with antigenic peptides under physiologic conditions, and the peptide association could be further stimulated by heat shock. The large chaperones displayed similar but distinct peptide-binding features compared with hsp70 and grp94/gp96. Immunization with hsp110- or grp170-tyrosinase-related protein 2 (TRP2(175-192)) peptide complexes effectively primed CD8(+) T cells reactive with TRP2-derived, MHC class I-restricted epitope. However, the tumor protective effect elicited by the TRP2(175-192) peptide vaccine was much weaker than that achieved by full-length TRP2 protein Ag chaperoned by grp170. Furthermore, immunization with combined chaperone vaccines directed against two melanoma protein Ags (i.e., gp100 and TRP2) significantly improved overall anti-tumor efficacy when compared with either of the single Ag vaccine. Lastly, treatment of tumor-bearing mice with these dual Ag-targeted chaperone complexes resulted in an immune activation involving epitope spreading, which was associated with a strong growth inhibition of the established tumors. Our results suggest that high m.w. chaperones are superior to conventional chaperones as a vaccine platform to deliver large protein Ags, and provide a rationale for translating this recombinant chaperoning-based vaccine to future clinical investigation.

Heat shock protein 110 improves the antitumor effects of the cytotoxic T lymphocyte epitope E7(49-57) in mice.

Several strategies have been used to enhance the vaccine-induced immunity of peptide vaccines and effective therapeutic benefits, including the utilization of heat shock proteins (HSP), especially the HSP70 family. HSP110 exhibits a higher binding affinity with protein and is capable of enhancing the immunogenicity of protein antigens; however, whether HSP110 can also increase the efficiency of peptide vaccine remains unclear. Here, we investigated mHSP110 as a chaperone immunoadjuvant to enhance the immune response to HPV16 oncoprotein E7-derived CTL epitope E7(49-57) in a mouse model. We developed the HSP110-E7(49-57) complex and demonstrated that mHSP110 could form complexes with peptide E7(49-57) using FITC-labeled E7(49-57) as the tracer. Inoculation of the mHSP110-E7(49-57) complex was capable of priming strong epitope-specific immune response as determined by its ability to elicit an epitope-specific splenocytes proliferation and a cytotoxic T cell response, and IFNgamma production in splenocytes. Results also showed that immunization with the mHSP110-E7(49-57) complex completely protected mice against subsequent challenge with tumor cells. More importantly, immunization of this complex also significantly inhibited the growth of established tumors and prolonged the survival time of the tumor-bearing animals. Thus, mHSP110-E7(49-57) complex vaccine represents a potentially powerful approach for use in the immunotherapy of cervical cancer associated with HPV16 infection. More importantly, the multi-epitopes derived from E7 and other E proteins can be applied to the strategy described in this study to form a multi-antigenic vaccine to induce an improved antitumor immune response to cervical cancer in the future.

Development of the hsp110-heparanase vaccine to enhance antitumor immunity using the chaperoning properties of hsp110.

The purpose of the study is to identify the novel approach of hsp110-heparanase vaccine in virtue of the chaperoning properties of hsp110, and to explore the antitumor potential. To test it, the recombinant hsp110 and heparanase proteins form into complex vaccine during heat shock, and the antitumor immunity is observed. We demonstrate that hsp110 and heparanase can form into complex vaccine, and the vaccine can enhance the specific IFN-gamma production and cytotoxic T cell response. Additionally, immunization of the vaccine can also significantly suppress the established tumor growth and prolong the life span of tumor-bearing mice. Thus, the use of the recombinant hsp110 and heparanase proteins to form into complex vaccine represents a novel and promising way for the therapeutical strategy of tumor.

Regression of intestinal adenomas by vaccination with heat shock protein 105-pulsed bone marrow-derived dendritic cells in Apc(Min/+) mice.

Heat shock protein (HSP) 105 is overexpressed in various cancers, but is expressed at low levels in many normal tissues, except for the testis. A vaccination with HSP105-pulsed bone marrow-derived dendritic cells (BM-DC) induced antitumor immunity without causing an autoimmune reaction in a mouse model. Because Apc(Min/+) mice develop multiple adenomas throughout the intestinal tract by 4 months of age, the mice provide a clinically relevant model of human intestinal tumor. In the present study, we investigated the efficacy of the HSP105-pulsed BM-DC vaccine on tumor regression in the Apc(Min/+) mouse. Western blot and immunohistochemical analyses revealed that the tumors of the Apc(Min/+) mice endogenously overexpressed HSP105. Immunization of the Apc(Min/+) mice with a HSP105-pulsed BM-DC vaccine at 6, 8, and 10 weeks of age significantly reduced the number of small-intestinal polyps accompanied by infiltration of both CD4(+) and CD8(+) T cells in the tumors. Cell depletion experiments proved that both CD4(+) and CD8(+) T cells play a critical role in the activation of antitumor immunity induced by these vaccinations. These findings indicate that the HSP105-pulsed BM-DC vaccine can provide potent immunotherapy for tumors that appear spontaneously as a result of the inactivation of a tumor suppressor gene, such as in the Apc(Min/+) mouse model.

Hsp110 and Grp170, members of the Hsp70 superfamily, bind to scavenger receptor-A and scavenger receptor expressed by endothelial cells-I.

Heat shock protein 110 (hsp110) and glucose-regulated protein (grp170) act as anti-cancer vaccines when complexed to tumor antigens by heat shock. It has been proposed that receptors on antigen-presenting cells contribute to HSP-mediated immune responses. Here, we show that hsp110 binds in a receptor-mediated manner to RAW264.7 macrophages, as does grp170. This hsp110/grp170 binding is inhibited by scavenger receptor ligands, suggesting a role for scavenger receptors as binding structures. We examined scavenger receptor class A (SR-A) and scavenger receptor expressed by endothelial cells-I (SREC-I). We show that hsp110/grp170 binds to both SR-A- and SREC-I-expressing CHO cells in a saturable manner and scavenger receptor ligands inhibit binding. Hsp110 also saturably binds mouse bone marrow-derived dendritic cells (bmDC) and is inhibited by scavenger receptor ligands. When an hsp110-rat neu (intracellular domain) heat shock complex vaccine is used to pulse mouse bmDC in vitro, an induction of IFN-gamma secretion is observed by CD8+ T lymphocytes isolated from vaccine-immunized mice. This immune response is inhibited by the application of scavenger receptor ligands to bmDC. Thus, SR-A and SREC-I appear to contribute to the binding of hsp110 and grp170 on APC. Scavenger receptors, in general, contribute to the cross-presentation of hsp110-chaperoned protein antigen.

Identification of an overexpressed gene, HSPA4L, the product of which can provoke prevalent humoral immune responses in leukemia patients.

OBJECTIVE: To identify leukemia-associated antigens, we applied the serological identification of antigens by the recombinant expression cloning (SEREX) method to a chronic myelogenous leukemia (CML) patient who achieved a cytogenetic response to interferon-alpha. MATERIALS AND METHODS: Immunoscreening of the cDNA library was performed with sera from a CML patient. Two isolated antigens were used to evaluate the expression pattern using Northern blot analysis and quantitative reverse transcriptase polymerase chain reaction. Western blotting and enzyme-linked immunosorbent assay were also performed for serological analysis. RESULTS: We identified 14 positive clones, representing five different antigens. Of these, two genes were further validated. One (clone 70) was the human polyribonucleotide nucleotidyltransferase 1 (PNPT1), which is the type I interferon (alpha/beta-responsive gene). The mRNA of clone 70 was ubiquitously expressed in normal human tissues. The other gene (clone 57) was the heat shock 70-kDa protein 4-like (HSPA4L), which is a member of the heat shock protein 110 family, whose mRNA is strongly expressed in normal human testis and overexpressed in leukemia cells. Seroactivity against HSPA4L was detected in 6 of 9 acute myeloid leukemia patients, 4 of 10 acute lymphoblastic leukemia patients, 9 of 11 CML patients, and none of 10 healthy volunteers. Leukemia patients had higher titer of the antibodies against the protein than healthy volunteers. CONCLUSIONS: These results suggest that HSPA4L, a member of heat shock protein, is highly expressed by leukemia cells, and elicit humoral immune responses in leukemia patients, and it might be a potential target for antileukemia therapy and an antigen-specific immunotherapy for leukemia.

Evaluation of renal cell carcinoma vaccines targeting carbonic anhydrase IX using heat shock protein 110.

Carbonic anhydrase IX (CA9) is a renal cell carcinoma (RCC)-specific tumor protein that is targeted using heat shock protein 110 (hsp110). The chaperoning ability of hsp110 can be utilized to form a complex with CA9 (hsp110 + CA9) in vitro, which can be administered as a highly concentrated tumor vaccine. In a tumor prevention model, hsp110 + CA9 prevented the growth of RENCA tumors in BALB/c mice, and produced IFN-gamma response measured using ELISPOT and an antibody response measured using ELISA. To test a second vaccine strategy, hsp110 complexed to a previously described CA9 peptide prevented tumor growth and produced a very weak IFN-gamma response, but no antibody response. A plasmid vector containing grp170, a member of the hsp110 family, linked to CA9 did not produce an antitumor response and produced no IFN-gamma response or antibodies. In a model of metastatic RCC, RENCA cells were injected intradermally prior to vaccination. Hsp110 + CA9 decreased tumor growth compared to control vaccinations. These studies suggest that recombinant hsp110 complexed to CA9 should be evaluated for treatment of RCC.

Immunization with heat shock protein 105-pulsed dendritic cells leads to tumor rejection in mice.

Recently, we reported that heat shock protein 105 (HSP105) DNA vaccination induced anti-tumor immunity. In this study, we set up a preclinical study to investigate the usefulness of dendritic cells (DCs) pulsed with mouse HSP105 as a whole protein for cancer immunotherapy in vivo. The recombinant HSP105 did not induce DC maturation, and the mice vaccinated with HSP105-pulsed BM-DCs were markedly prevented from the growth of subcutaneous tumors, accompanied with a massive infiltration of both CD4+ T cells and CD8+ T cells into the tumors. In depletion experiments, we proved that both CD4+ T cells and CD8+ T cells play a crucial role in anti-tumor immunity. Both CD4+ T cells and CD8+ T cells specific to HSP105 were induced by stimulation with HSP105-pulsed DCs. As a result, vaccination of mice with BM-DCs pulsed with HSP105 itself could elicit a stronger tumor rejection in comparison to DNA vaccination.