Chaperone-rich Cell Lysate Research Articles

Exosomes from Dendritic Cells Loaded with Chaperone-Rich Cell Lysates Elicit a Potent T Cell Immune Response Against Intracranial Glioma in Mice.

Chaperone-rich cell lysates (CRCLs) may play an important role in the development of anti-tumor vaccines. Tumor-derived CRCLs have been reported to activate dendritic cells (DCs) to elicit potent anti-tumor activity. However, the role of DC-derived exosomes (DEXs) secreted from DCs loaded with CRCLs in the treatment of tumors has not been clearly determined. In the present study, DEXs were generated from DCs loaded with CRCLs derived from GL261 glioma cells. These DEXs, designated DEX (CRCL-GL261), were then used to treat DCs to create DEX (CRCL-GL261)-DCs. The DEX (CRCL-GL261)-DCs were found to promote cell proliferation and cytotoxic T lymphocyte (CTL) activity of CD4(+) and CD8(+) T cells in vitro compared with DEX (GL261)-DCs, which were loaded with DEXs derived from DCs loaded with GL261 tumor cell lysates. DEX (CRCL-GL261)-DCs significantly prolonged the survival of mice with tumors and inhibited tumor growth in vivo. In addition, DEX (CRCL-GL261)-DCs induced enhanced T cell infiltration in intracranial glioma tissues compared with other treatments. DEX (CRCL-GL261)-DCs induced strong production of anti-tumor cytokines, including interleukin-2 and interferon-γ. Moreover, depletion of CD4(+) and CD8(+) T cells significantly impaired the anti-tumor effect of DEX (CRCL-GL261)-DCs. Finally, DEX (CRCL-GL261)-DCs were found to negatively regulate Casitas B cell lineage lymphoma (Cbl)-b and c-Cbl signaling, leading to the activation of phosphatidyl inositol 3-kinase (PI3K)/Akt and extracellular signal-regulated kinase (ERK) signaling in T cells. In summary, we present evidence that DEX (CRCL-GL261)-DCs induce more potent and effective anti-tumor T cell immune responses and delineate the underlying mechanism by which DEX (CRCL-GL261)-DCs exerted their anti-tumor activity through modulating Cbl-b and c-Cbl signaling. These results provide novel and promising insight for the development of an anti-tumor vaccine.

Endoplasmic reticulum chaperones and their roles in the immunogenicity of cancer vaccines.

The endoplasmic reticulum (ER) is a major site of passage for proteins en route to other organelles, to the cell surface, and to the extracellular space. It is also the transport route for peptides generated in the cytosol by the proteasome into the ER for loading onto major histocompatibility complex class I (MHC I) molecules for eventual antigen presentation at the cell surface. Chaperones within the ER are critical for many of these processes; however, outside the ER certain of those chaperones may play important and direct roles in immune responses. In some cases, particular ER chaperones have been utilized as vaccines against tumors or infectious disease pathogens when purified from tumor tissue or recombinantly generated and loaded with antigen. In other cases, the cell surface location of ER chaperones has implications for immune responses as well as possible tumor resistance. We have produced heat-shock protein/chaperone protein-based cancer vaccines called “chaperone-rich cell lysate” (CRCL) that are conglomerates of chaperones enriched from solid tumors by an isoelectric focusing technique. These preparations have been effective against numerous murine tumors, as well as in a canine with an advanced lung carcinoma treated with autologous CRCL. We also published extensive proteomic analyses of CRCL prepared from human surgically resected tumor samples. Of note, these preparations contained at least 10 ER chaperones and a number of other residents, along with many other chaperones/heat-shock proteins. Gene ontology and network analyses utilizing these proteins essentially recapitulate the antigen presentation pathways and interconnections. In conjunction with our current knowledge of cell surface/extracellular ER chaperones, these data collectively suggest that a systems-level view may provide insight into the potent immune stimulatory activities of CRCL with an emphasis on the roles of ER components in those processes.

Exosome from chaperone-rich cell lysates-loaded dendritic cells produced by CELLine 1000 culture system exhibits potent immune activity

Dendritic cells (DCs) pulsed with exosomes can stimulate efficient cytotoxic T-lymphocyte responses and anti-tumor immunity. However, the quantity of DC-derived exosomes (DCex) obtained from various culture systems is very low, which is a significant practical issue hampering progress in this research area and needs to be addressed. Gliomas were particularly aggressive, with high morbidity and mortality, indicating that this is a form of incurable highly malignant tumor of the brain with poor prognosis. In the present study, we demonstrate that the CELLine 1000 culture system can dramatically increase the production of DCex. The morphology, phenotype and immune molecules of these DCex were found to be identical to those using traditional methods. Our researches supply a cost-effective, useful method for significantly increasing the quantity of exosomes. In addition, GL261 glioma cells were chosen to separate chaperone-rich cell lysates (CRCL). The results indicate that CRCL-GL261 cell lysates can trigger the most intense expression of immune molecules on DCex or DCs, which has important implications for the research into tumor treatment and diagnosis.

Prolonged remission of advanced bronchoalveolar adenocarcinoma in a dog treated with autologous, tumour-derived chaperone-rich cell lysate (CRCL) vaccine

Purpose: This paper presents the treatment of a 12-year-old female spayed Great Dane who presented with vestibular signs (ataxia, nystagmus, hind end collapse). Thoracic radiographs revealed a discrete pulmonary nodule in the right cranial lung lobe. Ultrasound-guided fine needle aspirate detected primary bronchoalveolar adenocarcinoma, verified via computed tomography, with a second smaller nodule discovered in the right cranial lung lobe.Materials and methods: A lateral thoracotomy with right cranial lung lobectomy was performed. Histopathological analysis of the nodules and an excised lymph node identified grade III bronchoalveolar adenocarcinoma with vascular infiltration and lymph node metastasis – a grim diagnosis with a reported median survival time of 6–27 days. A 10-g sample of the tumour was processed into a chaperone-rich cell lysate (CRCL) vaccine, which was administered weekly to the patient. Imiquimod – a Toll-like receptor 7 (TLR7) agonist – was applied topically for the first 12 treatments to stimulate local Langerhans cells. A single injection of bacillus Calmette-Guerin (BCG) was administered for additional immune stimulation at week 30 of treatment.Results: The dog remained stable and in otherwise good health until diffuse relapse occurred 44 weeks after the initial treatment; following gastrointestinal bleeding, the dog was euthanised 50+ weeks post diagnosis.Conclusion: To the authors’ knowledge, this is the first report of significantly prolonged survival following a diagnosis of grade III/stage III bronchoalveolar adenocarcinoma in a canine patient. This case report suggests that CRCL vaccine combined with topical imiquimod is a safe, effective treatment for canine tumours.

Proteomic analyses of different human tumour-derived chaperone-rich cell lysate (CRCL) anti-cancer vaccines reveal antigen content and strong similarities amongst the vaccines along with a basis for CRCL's unique structure: CRCL vaccine proteome leads to unique structure

Purpose: The aim of this paper was to compare protein content of chaperone-rich cell lysate (CRCL) anti-cancer vaccines prepared from human tumours of different histological origins to evaluate the uniformity of their protein content.Materials and methods: Clinical grade CRCL was prepared under Good Manufacturing Practice (GMP) conditions from surgically resected human tumours (colorectal cancer, glioblastoma, non-small cell lung cancer, ovarian cancer). Protein samples were separated by SDS-PAGE and slices cut from gels for protease digestion followed by mass spectrometry analysis. Proteins were identified, and the content assessed by gene ontogeny/networking programmatic computation. CRCL preparations were also analysed by nanoparticle tracking analysis (NTA) and transmission electron microscopy (TEM).Results: We identified between 200 and 550 proteins in the various CRCL preparations. Gene ontogeny analysis indicated that the vaccines showed clear relationships, despite different tumour origins. A total of 95 proteins were common to all the CRCLs. Networking analyses implicated heat shock proteins in antigen processing pathways, and showed connections to the cytoskeletal network. We found that CRCL vaccines showed a particulate structure by NTA, and TEM revealed an extended fence-like structural network in CRCL, with regions that were microns in size.Conclusions: We conclude that it is feasible to prepare and characterise CRCL from a variety of different tissue sources; a substantial portion of the protein content is identical among the different CRCLs, while the overall compositions also suggest high overlaps in functional categories. The protein content indicates the presence of antigens and implies a potential structure, which we believe may play a role in CRCL's ability to stimulate innate antigen presenting cell activation.

The ‘peptidome’ of tumour-derived chaperone-rich cell lysate anti-cancer vaccines reveals potential tumour antigens that stimulate tumour immunity

Tumour-derived chaperone-rich cell lysate (CRCL) when isolated from tumour tissue or when embedded with peptide antigens is a potent anti-cancer vaccine consisting of numerous chaperone/heat shock proteins, including the highly immunogenic Hsp70, Hsp90, glucose regulated protein 94, and calreticulin. We have previously documented that CRCL provides both a source of tumour antigens and danger signals triggering antigen presenting cell activation. In this report we describe the ‘peptidome’ of potential antigens extracted from CRCL prepared from a murine tumour. Using mass spectrometry techniques we identify almost 60 different proteins of origin for the CRCL peptides; we determine that the parental proteins come from essentially all parts of the cell, and are involved in a broad range of functions. Further in silico analysis demonstrates that the parental proteins are components of major signalling networks of vital importance for cancer cell survival, proliferation, and migration. In many instances the peptides identified possess amino acid sequences that would allow their putative binding and display by murine major histocompatibility complex class I and II molecules, and there are also predicted binding motifs for Hsp70-type chaperones. By mixing fractionated pools of peptides with antigen-free (normal liver) CRCL, we were able to reconstitute effective anti-tumour activity of the vaccine, showing that the peptides are indeed the major purveyors of CRCL vaccines’ efficacy.

Allogeneic effector/memory Th-1 cells impair FoxP3+ regulatory T lymphocytes and synergize with chaperone-rich cell lysate vaccine to treat leukemia

AbstractTherapeutic strategies combining the induction of effective antitumor immunity with the inhibition of the mechanisms of tumor-induced immunosuppression represent a key objective in cancer immunotherapy. Herein we demonstrate that effector/memory CD4+ T helper-1 (Th-1) lymphocytes, in addition to polarizing type-1 antitumor immune responses, impair tumor-induced CD4+CD25+FoxP3+ regulatory T lymphocyte (Treg) immunosuppressive function in vitro and in vivo. Th-1 cells also inhibit the generation of FoxP3+ Tregs from naive CD4+CD25−FoxP3− T cells by an interferon-γ–dependent mechanism. In addition, in an aggressive mouse leukemia model (12B1), Th-1 lymphocytes act synergistically with a chaperone-rich cell lysate (CRCL) vaccine, leading to improved survival and long-lasting protection against leukemia. The combination of CRCL as a source of tumor-specific antigens and Th-1 lymphocytes as an adjuvant has the potential to stimulate efficient specific antitumor immunity while restraining Treg-induced suppression.

Signaling pathways induced by a tumor-derived vaccine in antigen presenting cells

We have previously reported on the anti-tumoral potential of a chaperone-rich cell lysate (CRCL) vaccine. Immunization with CRCL generated from tumors elicits specific T and NK cell-dependent immune responses leading to protective immunity in numerous mouse tumor models. CRCL provides both a source of tumor antigens and danger signals leading to dendritic cell activation. In humans, tumor-derived CRCL induces dendritic cell activation and CRCL-loaded dendritic cells promote the generation of cytotoxic T lymphocytes in vitro. The current study was designed to identify the signaling events and modifications triggered by CRCL in antigen presenting cells. Our results indicate that tumor-derived CRCL not only promotes the activation of dendritic cells, but also significantly fosters the function of macrophages that thus appear as major targets of this vaccine. Activation of both cell types is associated with the induction of the MAP kinase pathway, the phosphorylation of STAT1, STAT5 and AKT and with transcription factor NF-κB activation in vitro and in vivo. These results thus provide important insights into the mechanisms by which CRCL-based vaccines exert their adjuvant effects on antigen presenting cells.

A chaperone protein-enriched tumor cell lysate vaccine generates protective humoral immunity in a mouse breast cancer model

We have documented previously that a multiple chaperone protein vaccine termed chaperone-rich cell lysate (CRCL) promotes tumor-specific T-cell responses leading to cancer regression in several mouse tumor models. We report here that CRCL vaccine generated from a mouse breast cancer (TUBO, HER2/neu positive) is also capable of eliciting humoral immunity. Administration of TUBO CRCL triggered anti-HER2/neu antibody production and delayed the progression of established tumors. This antitumor activity can be transferred through the serum isolated from TUBO CRCL-immunized animals and involved both B cells and CD4(+) T lymphocytes. Further evaluation of the mechanisms underlying TUBO CRCL-mediated humoral immunity highlighted the role of antibody-dependent cell-mediated cytotoxicity. These results suggest that tumor-derived CRCL vaccine has a wider applicability as a cancer vaccine because it can target both T-cell- and B-cell-specific responses and may represent a promising approach for the immunotherapy of cancer.

Chaperone-rich tumor cell lysate-mediated activation of antigen-presenting cells resists regulatory T cell suppression

CD4(+)CD25(+) regulatory T lymphocytes (Tregs) critically contribute to the mechanisms of cancer-induced tolerance. These cells suppress anti-tumoral CD8(+) and CD4(+) T lymphocytes and can also restrain the function of APCs. We have previously documented the immunostimulatory effects of a chaperone-rich cell lysate (CRCL) anti-cancer vaccine. Tumor-derived CRCL induces tumor immunity in vivo, partly by promoting dendritic cell (DC) and macrophage activation. In the current study, we evaluated the effects of CD4(+)CD25(+)forkhead box P3(+) Tregs isolated from mice bearing 12B1 bcr-abl(+) leukemia on DC and macrophages that had been activated by 12B1-derived CRCL. CRCL-activated DC and macrophages resisted Treg suppression, as the production of proinflammatory cytokines, the activation of transcription factor NF-kappaB, and their immunostimulatory potential was unaffected by Tregs. Our results thus highlight CRCL as a powerful adjuvant endowed with the capacity to overcome tumor-induced Treg-inhibitory effects on APCs.

Differential capacity of chaperone-rich lysates in cross-presenting human endogenous and exogenous melanoma differentiation antigens

The goal of immune-based tumor therapies is the activation of immune cells reactive against a broad spectrum of tumor-expressed antigens. Vaccines based on chaperone proteins appear promising as these proteins naturally exist as complexes with various protein fragments including those derived from tumor-associated antigens. Multi-chaperone systems are expected to have highest polyvalency as different chaperones can carry distinct sets of antigenic fragments. A free-solution isoelectric focusing (FS-IEF) technique was established to generate chaperone-rich cell lysates (CRCL). Results from murine systems support the contention that CRCL induce superior anti-tumor responses than single chaperone vaccines. We established an in vitro model for human melanoma to evaluate the capacity of CRCL to transfer endogenously expressed tumor antigens to the cross-presentation pathway of dendritic cells (DC) for antigen-specific T cell stimulation. CRCL prepared from human melanoma lines contained the four major chaperone proteins Hsp/Hsc70, Hsp90, Grp94/gp96 and calreticulin. The chaperones within the melanoma cell-derived CRCL were functionally active in that they enhanced cross-presentation of exogenous peptides mixed into the CRCL preparation. Superior activity was observed for Hsp70-rich CRCL obtained from heat-stressed melanoma cells. Despite the presence of active chaperones, melanoma cell-derived CRCL failed to transfer endogenously expressed melanoma-associated antigens to DC for cross-presentation and cytotoxic T cell (CTL) recognition, even after increasing intracellular protein levels of tumor antigen or chaperones. These findings reveal limitations of the CRCL approach regarding cross-presentation of endogenously expressed melanoma-associated antigens. Yet, CRCL may be utilized as vehicles to enhance the delivery of exogenous antigens for DC-mediated cross-presentation and T cell stimulation.

Chaperone‐rich cell lysate embedded with BCR‐ABL peptide demonstrates enhanced anti‐tumor activity against a murine BCR‐ABL positive leukemia

Chaperone proteins are effective antitumor vaccines when purified from a tumor source, some of which are in clinical trials. Such vaccines culminate in tumor-specific T cell responses, implicating the role of adaptive immunity. We have developed a rapid and efficient procedure utilizing an isoelectric focusing technique to obtain vaccines from tumor or normal tissues called chaperone-rich cell lysate (CRCL). Tumor-associated peptides, the currency of T cell-mediated anticancer immunity, are believed to be purveyed by chaperone vaccines. Our purpose was to demonstrate our ability to manipulate the peptide antigen repertoire of CRCL vaccines as a novel anticancer strategy. Our methods allow us to prepare "designer" CRCL, utilizing the immunostimulation activity and the carrying capacity of CRCL to quantitatively acquire and deliver exogenous antigenic peptides (e.g., derived from the oncogenic BCR/ABL protein in chronic myelogenous leukemia). Using fluorescence-based and antigen-presentation assays, we determined that significant quantities of exogenously added peptide could accumulate in "designer" CRCL and could stimulate T cell activation. Further, we concluded that peptide-embedded CRCL, devoid of other antigens, could generate potent immunity against pre-established murine leukemia. Designer CRCL allows for the development of personalized vaccines against cancers expressing known antigens, by embedding antigens into CRCL derived from normal tissue.

Human ovarian tumour-derived chaperone-rich cell lysate (CRCL) elicits T cell responses in vitro

Tumour-derived chaperone-rich cell lysate (CRCL), which is made up of numerous heat shock proteins, has been used successfully to generate tumour-specific T cell responses and protective immunity against a wide range of murine tumours. In this study, we have investigated the potency of human ovarian cancer-derived CRCL to activate dendritic cells (DC) and to generate tumour-specific T cells in vitro. CRCL was generated from primary ovarian cancers and SKOV3-A2, a HER2/neu, Wilm's tumour gene 1 (WT1) and human leucocyte antigen (HLA)-A2 positive human ovarian tumour cell line. Peripheral blood mononuclear cells from both HLA-A2(+) healthy donors and HLA-A2(+) ovarian cancer patients were stimulated weekly with autologous DC loaded with ovarian tumour-derived CRCL. After four to six stimulations in vitro, specific cytokine secretion and cytotoxicity were measured. CRCL promoted interleukin (IL)-12 secretion and enhanced the immunostimulatory capacity of DC. T cells from healthy controls and from ovarian cancer patients secreted higher amounts of interferon-gamma following in vitro restimulation with ovarian cancer-derived CRCL than with HER2/neu or WT1 peptide-pulsed DC. We were also able to generate cytotoxic T lymphocyte activity against cancer-specific antigens such as HER2/neu and WT1 from all healthy donors, but from only one of the four ovarian cancer patients with bulky disease. These preliminary results substantiate further the concept that CRCL may prove to be a potent adjuvant for women suffering from ovarian cancer and that this personalized vaccine may be a promising approach for active immunotherapy.

Natural killer cells play a key role in the antitumor immunity generated by chaperone‐rich cell lysate vaccination

Tumor derived chaperone-rich cell lysate (CRCL) when isolated from tumor tissues is a potent vaccine that contains at least 4 of the highly immunogenic heat shock proteins (HSP) such as HSP70, HSP90, glucose related protein 94 and calreticulin. We have previously documented that CRCL provides both a source of tumor antigens and danger signals triggering dendritic cell (DC) activation. Immunization with tumor derived CRCL elicits tumor-specific T cell responses leading to tumor regression. In the current study, we further dissect the mechanisms by which CRCL simulates the immune system, and demonstrate that natural killer (NK) cells are required for effective antitumor effects to take place. Our results illustrate that CRCL directly stimulates proinflammatory cytokine and chemokine production by NK cells, which may lead to activation and recruitment of macrophages at the tumor site. Thus, this report provides further insight into the function of CRCL as an immunostimulant against cancer.

Peritransplantation Vaccination with Chaperone-Rich Cell Lysate Induces Antileukemia Immunity

We have reported that chaperone-rich cell lysate (CRCL) is an effective anticancer vaccine in immunocompetent mice. In this study, we explored the therapeutic applicability of CRCL in the context of syngeneic hematopoietic cell transplantation (HCT) to treat preexisting leukemia. Our results demonstrate that tumor growth is significantly delayed in mice receiving syngeneic HCT from 12B1 tumor CRCL-immunized donors compared with animals receiving HCT from nonimmunized donors. CRCL immunization after immune HCT further hindered tumor growth when compared with immune HCT without posttransplantation vaccination. The magnitude of the immune response was consistent with the antitumor effects observed in vivo. Rechallenge of surviving mice with 12B1 or A20 cells in opposite groins confirmed that mice had developed long-term tumor-specific immunity against 12B1 tumor cells. In addition, we documented that both T cells and natural killer cells contributed to the antitumor effect of CRCL vaccination, because depletion of either subset hampered tumor growth delay. Thus, our results indicate that CRCL is a promising vaccine capable of generating specific immune responses. This antitumor immunity can be effectively transferred to a host via HCT and further enhanced after HCT with additional tumor CRCL immunizations.

Chaperone-rich cell lysates, immune activation and tumor vaccination.

We have utilized a free-solution-isoelectric focusing technique (FS-IEF) to obtain chaperone-rich cell lysates (CRCL) fractions from clarified tumor homogenates. The FS-IEF technique for enriching multiple chaperones from tumor lysate is relatively easy and rapid, yielding sufficient immunogenic material for clinical use. We have shown that tumor-derived CRCL carry antigenic peptides. Dendritic cells (DCs) uptake CRCL and cross-present the chaperoned peptides to T cells. Tumor-derived CRCL induce protective immune responses against a diverse range of murine tumor types in different genetic backgrounds. When compared to purified heat shock protein 70 (HSP70), single antigenic peptide or unfractionated lysate, CRCL have superior ability to activate/mature DCs and are able to induce potent, long lasting and tumor specific T-cell-mediated immunity. While CRCL vaccines were effective as stand-alone therapies, the enhanced immunogenicity arising from CRCL-pulsed DC as a vaccine indicates that CRCL could be the antigen source of choice for DC-based anti-cancer immunotherapies. The nature of CRCL's enhanced immunogenicity may lie in the broader antigenic peptide repertoire as well as the superior immune activation capacity of CRCL. Exongenous CRCL also supply danger signals in the context of apoptotic tumor cells and enhance the immunogenicity of apoptotic tumor cells, leading to tumor-specific T cell dependent long-term immunity. Moreover, CRCL based vaccines can be effectively combined with chemotherapy to treat cancer. Our findings indicate that CRCL have prominent adjuvant effects and are effective sources of tumor antigens for pulsing DCs. Tumor-derived CRCL are promising anti-cancer vaccines that warrant clinical research and development.

Induction of BCR-ABL–specific immunity following vaccination with chaperone-rich cell lysates derived from BCR-ABL+ tumor cells

AbstractWe have previously reported that chaperonerich cell lysates (CRCL) derived from the BCR-ABL+ 12B1 leukemia activate dendritic cells (DCs) and stimulate leukemia-specific immune responses. Because CRCL contain a variety of heat shock/chaperone proteins, we theorized that CRCL obtained from BCR-ABL+ leukemias are likely to chaperone BCR-ABL–derived fusion peptides and that DCs pulsed with 12B1 CRCL could cross-present BCR-ABL fusion peptides to T cells. We found that splenocytes from mice vaccinated with BCR-ABL+ leukemia-derived CRCL secreted interferon-γ (IFN-γ) when restimulated with a BCR-ABL peptide, GFKQSSKAL, indicating that BCR-ABL peptides are chaperoned by leukemia-derived CRCL. We next eluted peptides from 12B1 leukemia-derived CRCL and used high-pressure liquid chromatography (HPLC) fractions to restimulate splenocytes harvested from mice vaccinated with DC/GFKQSSKAL or DC/12B1 CRCL. We found that the same peptide fractions derived from 12B1 CRCL and from “refractionated” GFKQSSKAL stimulated IFN-γ production, suggesting the presence of BCR-ABL peptides in the peptide repertoire of 12B1 CRCL. We also demonstrated that immunization with DCs loaded with leukemia-derived CRCL induced BCR-ABL–specific cytotoxic T lymphocytes (CTLs) in vivo. Moreover, mice immunized with DCs pulsed with 12B1-derived CRCL had superior survival (60%) when compared with those immunized with DCs pulsed with BCR-ABL peptide (20%), indicating that CRCL vaccines provide additional immune stimulus over and above individual peptide vaccination.

Tumor-Derived Chaperone-Rich Cell Lysate (CRCL) Can Be Effectively Combined with Syngeneic Bone Marrow Transplantation to Treat Bcr-abl+ Leukemia in a Murine Model.

We have previously reported that purified tumor derived chaperone-rich cell lysate (CRCL) containing multiple heat shock proteins (HSPs) is a promising cancer vaccine, capable of generating tumor-specific T cell responses and protective immunity in different animal tumor models. In this study, we have explored the therapeutic applicability of CRCL in the context of syngeneic bone marrow transplantation (BMT) to treat preexisting leukemia. BALB/c mice received a 10-fold LD100 dose of bcr-abl+ leukemia cells (12B1) subcutaneously (s.c.) on day -7. On day -1, 900cGy of total body irradiation (TBI) was given followed by syngeneic bone marrow transplantation (BMT) on day 0. Transplanted mice received 20X106 bone marrow cells along with 50X106 splenocytes (SPC) intravenously (i.v.). We have demonstrated that BMT/SPC prolonged survival of mice by a median time of 7 days (median survival time [MST] from tumor inoculation of no BMT versus BMT/SPC =19 vs 26 days), however, all mice eventually died of disseminated leukemia. When recipients received BMT and SPC from syngeneic donors that had been previously immunized with 12B1-derived CRCL (immune BMT/SPC) the MST was increased to 29 days with 26.1% of transplanted mice surviving without tumor (p<0.01 immune BMT/SPC vs naive BMT/SPC). Vaccination of immune BMT/SPC recipients with 20 mg 12B1-derived CRCL in the early post-transplant period (day +1 and day +6) increased MST to 32 days (p<0.01 vs naïve BMT/SPC control) but did not significantly improve overall survival (26.3%) when compared to immune BMT/SPC mice not receiving post transplant 12B1-CRCL vaccine. Eleven to 17 weeks later, mice with no evidence of disease were re-challenged with 12B1 cells in one groin and A20 B cell leukemia cells in the opposite groin. Eighty percent of the mice demonstrated long term tumor specific immunity by rejecting the 12B1 rechallenge while 100% of the mice developed A20 tumors. Our results indicate that CRCL is a promising vaccine that can be used to generate specific anti-tumor immunity that can be effectively transferred to a host via BMT.

Human Ovarian Tumor-Derived Chaperone-Rich Cell Lysates Activate Dentdritic Cells and Elicit T Cell Responses In Vitro

s for the 19th Annual Scientific Meeting of the International Society for Biological Therapy of Cancer, San Francisco, California, November 4-7, 2004: Dendritic Cells and Vaccines

Imatinib mesylate effectively combines with chaperone-rich cell lysate-loaded dendritic cells to treat bcr-abl+ murine leukemia.

Imatinib mesylate has become an effective agent for the treatment of chronic myeloid leukemia (CML). However, the development of drug resistance has led to examination of combination therapies. In this study, we investigated the effects of combining imatinib with immunotherapy against a murine bcr-abl(+) leukemia, 12B1. We have previously shown that multiple chaperone proteins may be enriched into a vaccine form from tumor cell lysates by a free-solution isoelectric focusing method. We refer to these vaccines as chaperone-rich cell lysates (CRCLs) and have found that they are potent immunologic agents against a variety of murine tumors, including 12B1. We now demonstrate that the combination of imatinib with dendritic cells loaded with 12B1-derived CRCL yields high activation of anti-12B1-specific T cells and potent antitumor activity, resulting in tumor-free survival in up to 63% of mice with bcr-abl(+) 12B1 tumors. Our data suggest that immunotherapy can be effectively combined with imatinib for the treatment of CML.