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

We have previously reported that chaperone rich 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 BCRABL leukemias are likely to chaperone BCR-ABL–derived fusion peptides and that DCs pulsed with 12B1 CRCL could crosspresent BCR-ABL fusion peptides to T cells. We found that splenocytes from mice vaccinated with BCR-ABL leukemia-derived CRCL secreted interferon- (IFN-) when restimulated with a BCR-ABL peptide, GFKQSSKAL, indicating that BCR-ABL peptides are chaperoned by leukemia-derived CRCL. We next eluted peptides from 12B1leukemia-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- 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.

(Blood. 2005;105:2016-2022)

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

Abstract: CD4CD25 regulatory T cell 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 CD4CD25forkhead box P3 Tregs isolated from mice bearing 12B1 bcr-abl leukemia on DC and macrophages that had been activated by 12B1-derived CRCL, and CRCL-activated DC and macrophages resisted Treg suppression, as the production of proinflammatory cytokines, the activation of transcription factor NF-B, 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. J. Leukoc. Biol. 83: 000–000; 2008.

Molecular Cancer Therapeutics

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

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 tumorspecific 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. 

2006 Wiley-Liss, Inc.

Tumor-derived, chaperone-rich cell lysate activates dendritic cells and elicits potent antitumor immunity

We have utilized a free-solution isoelectric focusing technique (FS-IEF) to obtain chaperone-rich cell lysate (CRCL) fractions from clarified tumor homogenates and have previously reported on their vaccinating potential. To better understand the underlying mechanisms as well as to improve on the immunizing efficacy of tumor-derived chaperone complexes, in the present study we examined the effects of CRCL-loaded dendritic cells (DCs) against 12B1, an aggressive bcrabl murine leukemia tumor. We found that DCs incubated with 12B1-derived CRCL had higher expression of CD40 and major histocompatibility complex class II (MHC-II) on their cell surface, produced more interleukin-12 (IL-12), and had superior immunostimulatory capacity in a mixed leukocyte reaction (MLR) when compared with DCs exposed to unfractionated tumor lysate or purified heatshock protein 70 (HSP70). Vaccination of mice with 12B1 CRCL–pulsed DCs significantly prolonged their survival, with more than 80% of mice rejecting their tumors following a lethal challenge with live 12B1 compared with those immunized with tumor lysate or HSP70-loaded DCs. The protective immunity generated was tumor specific, long lasting, and both CD4 and CD8 T-cell dependent. Moreover, immunization with CRCL-loaded DCs resulted in a 75% cure rate in mice with pre-existing 12B1 tumors. Our findings indicate that CRCL has prominent adjuvant effects and is a very effective source of tumor antigen for pulsing DCs. FS-IEF– derived CRCL-pulsed DCs are a promising anticancer vaccine that warrants clinical research and development. (Blood. 2003;101:4485-4491)

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

Autologously derived cancer vaccines offer a promising avenue for the treatment of many malignancies. Based on a free solution-isoelectric focusing (FS-IEF) technique, we have developed chaperone-rich cell lysate (CRCL) vaccines – capable of eliciting potent anti-tumour immune responses against a wide variety of murine model tumour types [1–8]. This has been demonstrated in vivo in murine models, and in in vitro settings with both murine and human cells [3,6].

Immunotherapy for Invasive Aspergillosis in Immunocompromised Post‐Engraftment Allogeneic Bone Marrow Transplant Patients Abstract.

Invasive aspergillosis (IA) is a dangerousinfection that is common in immunocompromised patients. IA is a major cause of mortality in bone marrow transplant(BMT) patients due to steroid‐induced immunosuppression and chemotherapy‐induced neutropenia. In normal individuals, Aspergillus is controlled by a Type 1 immune response. However, immunocompromised patients have a decreased ability to mount a Type 1 immune response. BMT patients are treated with glucocorticoids to suppress the Type 1 immune response which is associated with graft versus hostdisease (GVHD) toxicity. Therefore it is a complex problem to develop strategies to enhance Type 1 immunity without also causing GVHD. To overcome this problem, we propose that multiple intradermal injections of activated allogeneic Th1 memory cells will create a pool of alloantigen‐specific Th1 memory cells in the circulation. Intradermal allogeneic injections are expected to be rejected and thus not cause GVHD. Additional intradermal allogeneic Th1 cell injections should activate the anti‐alloantigen memory cells in circulation causing them to migrate to the sites of fungal infection and produce Type 1 cytokines. This Type 1 cytokine production in the microenvironment of the fungal infection shouldserve as an adjuvant to the stimulation of innate immune responses against the fungus and the development of Type 1 anti‐fungal adaptive immunity.

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

Therapeutic 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 CD4CD25FoxP3 regulatory T lymphocyte (Treg) immunosuppressive function in vitro and in vivo. Th-1 cells also inhibit the generation of FoxP3 Tregs from naive CD4CD25FoxP3 T cells by an interferon-–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. (Blood. 2011;117(5):1555-1564)

Allogeneic CD3/CD28 cross-linked Th1 memory cells provide potent adjuvant effects for active immunotherapy of leukemia/lymphoma

The breaking of peripheral T-cell tolerance toward self-antigens expressed by tumor cells and the subsequent establishment of an effective tumor protective immune response remains a major challenge for cancer immunotherapy. We report that both protective and therapeutic anti-tumor immune responses can be achieved in a mouse leukemia/lymphoma tumor model through the strong adjuvant effects provided by allogeneic CD3/CD28 cross-linked Th1 memory cells. The adjuvant effect of these cells is mediated by their ability to produce a variety of ‘danger signals’ which serve to deviate native non-protective Th2 anti-leukemia immune responses to effective Th1 immune responses.

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Treatment strategy designed to use the power of the human immune system to kill tumors and prevent their recurrence.
No requirement for a matched donor or chemotherapy/radiation conditioning prior to treatment.
Innovative technology – proven and non-toxic.
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Therapeutic anti-tumor vaccine developed from core break-through technology called the "Mirror Effect™“ which opens a pathway to treating patients with metastatic cancer that have failed all available therapy options.
Elicits a GVT-like mechanism without the GVHD toxicity.
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