Autologous Tumor Vaccine Research Articles

Phase I/IIa trial of autologous formalin-fixed tumor vaccine concomitant with fractionated radiotherapy for newly diagnosed glioblastoma

The objective of the present study was analysis of results of the prospective clinical trial directed toward the evaluation of therapeutic efficacy of the administration of autologous formalin-fixed tumor vaccine (AFTV) concomitant with fractionated radiotherapy in cases of newly diagnosed glioblastoma multiforme. Twenty-four patients were enrolled into the clinical trial, while 2 cases were excluded from the final analysis of results. The treatment protocol included aggressive tumor resection, fractionated radiotherapy up to a total dose of 60 Gy, and 3 concomitant courses of AFTV administered with an interval of one week at the late stage of irradiation. Two delayed-type hypersensitivity (DTH) tests were done--one 48 hours before the initial course of vaccination (DTH-1) and one 2 weeks after the third (DTH-2). All but one of the patients received salvage therapy at the time of tumor progression. The defined primary end point was overall survival; secondary end points were progression-free survival and safety of concomitant treatment. The median duration of overall survival was 19.8 [corrected] months (95% CI 13.8-31.3 months). The actuarial 2-year survival rate was 40%. The median duration of progression-free survival was 7.6 months (95% CI 4.3-13.6 months). Overall survival showed a statistically significant association with recursive partitioning analysis class (p < 0.05); progression-free survival showed a statistically significant association with p53 staining index (p < 0.05) and size of DTH-2 response (p < 0.001). AFTV injection concomitant with fractionated radiotherapy was well tolerated by all patients and in no case did treatment-related adverse effects exceed Grade 1 toxicity; adverse effects were limited to local erythema, induration, and swelling at the site of injection. The results of this study demonstrate that AFTV treatment concomitant with fractionated radiotherapy may be effective in patients with newly diagnosed glioblastoma. Further clinical testing is warranted.

A phase I/II clinical trial investigating the adverse and therapeutic effects of a postoperative autologous dendritic cell tumor vaccine in patients with malignant glioma

Previous clinical trials of dendritic cell (DC)-based immunotherapy in patients with glioblastoma multiforme (GBM) have reported induction of systemic immune responses and prolonged survival. From 2003 to 2005, we performed a clinical trial in which patients with malignant glioma underwent surgery for maximal cytoreduction followed by a 6-month 10-injection course of autologous DC-tumor vaccine therapy, each injection containing 1–6 × 10 7 DC. Of the 17 treated patients (16 with World Health Organization grade IV and one with grade III glioma), eight (47.1%) had an initial transient elevation in aspartate aminotransferase (AST)/alanine aminotransferase (ALT). Vaccination caused some tumor shrinkage and increased concentration of tumor-infiltrating CD8(+) lymphocytes. Median survival and 5-year survival were 525 days and 18.8%, respectively, for 16 patients with grade IV glioma (381 days and 12.5% for eight newly diagnosed; 966 days and 25% for eight relapsed patients) compared to 380 days and 0% for 63 historical control patients. We concluded that autologous DC-tumor immunotherapy benefits patients with malignant glioma but may cause transient but reversible elevation of serum AST/ALT levels.

Abstract 5522: Increase of Th1/Treg ratio in peripheral blood of breast cancer patients with autologous formalin-fixed tumor vaccine

Abstract Introduction: Autologous formalin-fixed tumor vaccine (AFTV) containing formalin-fixed and/or paraffin-embedded tumor fragments is able to induce tumor specific CTL and suppresses recurrence of hepatocellular carcinoma after resection (Clin Cancer Res 2004; 10: 1574-9). We have used delayed-type hypersensitivity (DTH) test as a parameter of cellular immune response. We also found that slight but consistent population change has occurred in peripheral blood T lymphocytes after AFTV treatment. Method: Thirty two breast cancers were operated in our hospital. AFTV was injected intradermally. DTH response to the tumor fragments without any adjuvant was determined if erythema was 10mm or more. Results: After operation, standard chemotherapy, and enough time have passed to recover immunological function, we examined change of peripheral blood T-cell population in 32 cases with breast cancer. Among them, 27 cases turned DTH response from negative to positive after AFTV injection. In these patients, WBC counts increased 1.11 times; lymphocytes, 1.09 times; Th1 in CD4+ T cells, 1.07 times; regulatory T cells (Treg) in CD4+ T cells, 0.95 times, and Th1/Treg ratio, 1.17 times (statistically significant, 95%CI was 1.060-1.284). Conclusion: AFTV probably strengthen the cellular immune response against cancer by improving Th1/Treg ratio in peripheral blood. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 5522. doi:10.1158/1538-7445.AM2011-5522

Pathological Changes After Autologous Formalin-Fixed Tumor Vaccine Therapy Combined With Temozolomide for Glioblastoma -Three Case Reports-

Temozolomide (TMZ), an alkylating agent widely used for patients with glioblastoma multiforme (GBM), has the potential to enhance the acquired immune response to GBM. Here, we describe 3 cases of GBM patients treated with autologous formalin-fixed tumor vaccine (AFTV) combined with TMZ. All cases demonstrated pathological changes associated with the therapy. After a 4-week break from the standard initial treatments, 1 patient with primary GBM and 2 patients with secondary GBM received adjuvant TMZ for 5 days combined with AFTV injection and were subsequently treated with multiple cycles of adjuvant TMZ for 5 days every 28 days (AFTV/TMZ therapy). Adverse effects related to AFTV plus TMZ were very minor in all patients. Magnetic resonance imaging revealed partial response in 2 patients. CD3(+)CD8(+) lymphocytes were frequently detected in surgical specimens and MIB-1 labeling index in 2 cases decreased after AFTV/TMZ therapy. AFTV/TMZ therapy is suitable for larger scale clinical trials.

Phase I/IIa trial of autologous formalin-fixed tumor vaccine for newly diagnosed glioblastoma.

2069 Background: Autologous formalin-fixed tumor vaccine (AFTV) has been shown to have a potential role in effective adjuvant therapy for hepatocellular carcinoma and recurrent malignant glioma. We tried to determine the efficacy and safety of AFTV concomitant with radiation for newly diagnosed GBM patients from two institutions. Methods: Patients with supratentorial GBM who underwent debulking surgery were eligible. The AFTV was injected intradermally 3 times, with 7 days between injections, starting when the cumulative radiation dose reached 32-36 Gy. Results: A total of 24 patients were treated. Two patients were excluded by the radiation protocol or by the patient's consent. Although grade 1 dermal toxicity was observed at the injected sites in all patients, no grade 2 or higher hematologic or other toxicity was observed. The trial met the prospectively-defined primary endpoints, with 21.4 months median survival time (range 6.1 to 42 months), 40.0% 2-year survival, and 7.6 months median progression-free survival time (PFS, range 2.2 to 25.6 months). The PFS of the patients with greater delayed type hypersensitivity (DTH) reaction (13.9 months) was statistically longer (p < 0.01) than that of the patients with weaker DTH reaction (4.3 months). Conclusions: The AFTV immunotherapy concomitant with radiation is a safe treatment for patients with newly diagnosed GBM and seems to have a positive effect on the PFS. Additional testing of AFTV in combination regimens is warranted. Author Disclosure Employment or Leadership Position Consultant or Advisory Role Stock Ownership Honoraria Research Funding Expert Testimony Other Remuneration Cell-Medicine, Inc. Cell-Medicine, Inc.

Abstract 5631: Nitric oxide-based immune response modifications: A novel paradigm in tumor vaccine development against melanoma

Abstract Although of cardinal importance, the specific role of nitric oxide (NO) in the immune response to cancer remains elusive. NO is generally considered to act as a terminal effector molecule that will promote direct cell death due to its cytotoxic and/or cytostatic properties. Conversely, NO has been also found to be associated with a cytoprotective effect due to its ability to interfere with the apoptotic machinery. We have previously shown that NO is capable to immuno-sensitized tumor cells by regulating the expression of apoptosis-related genes and modifying the structure of many protein targets. Thus, we hypothesize that NO-based modification of an autologous tumor vaccine preparation may improve the immunological priming against tumor cells and potentiate the specific anti-tumor immune response. In order to test this hypothesis we used the well-characterized B16-C57BL/6 syngeneic mouse model of melanoma. B16-F0 melanoma cells were cultured in the presence or absence of a non-cytotoxic concentration of a slow rate release NO donor for 18 hours, and later homogenized into a crude lysate. These preparations were used to vaccinate mice previously challenged with B16-F0 melanoma cells on the contralateral dorsal flank one week before, in order to frame a therapeutic paradigm of treatment in cancer. Additional doses of vaccine were applied every 7 days for a period of 3 weeks and tumor volume was determined every 3 days. Our results demonstrate that the NO-modified autologous cancer vaccine decreases the rate of tumor growth by approximately 85% (p &amp;lt;0.001) by day 25 when compared with the control groups. In addition, this vaccination scheme increases the survival rate by approximately 60% (p &amp;lt;0.001) of the experimental group when compared with controls. NO can exert a myriad of modifications at the genetic, epigenetic and post-translational level in the tumor cells promoting a robust and effective therapeutic immune response against cancer cells. Currently, we are defining the molecular and immunological mechanisms responsible for the significant increase in survival and anti-tumor immune response triggered by the NO-mediated modifications of the melanoma tumor cells. Our results suggest a new role of NO as an immune response modifier that can be exploited in cancer immunotherapy and serves as a model for vaccine development. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 5631.

Phase I study of autologous dendritic cell tumor vaccine in patients with non-small cell lung cancer

Background A dendritic cell vaccine has been developed as a novel strategy for generating antitumor immunity in the treatment of cancer. The purpose of this study was to assess the maximal tolerated dose, safety, and immunologic response of a new dendritic cell vaccine (DC-Vac) into which tumor lysate was loaded by electroporation and pulse in patients with advanced non-small cell lung cancer (NSCLC). Patients and methods Fifteen patients with inoperable stage III or IV NSCLC were assigned to cohorts that received 3, 6, or 12 × 10 6 DC-Vac intradermally 3 times at 2 week intervals. We also evaluated immunologic and tumor responses. Results The maximum dose of DC-Vac (12 × 10 6) was shown to be safe. In 5 of 9 patients, the vaccine resulted in increased interferon (IFN)-γ production by CD8+ cells after exposure to tumor lysate. Additionally, there were mixed responses which do fulfill progressive disease definition but demonstrate some clinical benefit in two patients. Conclusion The administration of tumor lysate-loaded autologous dendritic cells by electroporation and pulse was non-toxic and induced immunologic responses to tumor antigens. The two mixed tumor responses which were achieved may represent a potential benefit of this new DC-Vac.

Recent findings in immunological monitoring for high-grade glioma patients treated with autologous dendritic cell-based tumor vaccination: CD107a expression as effector cell degranulation marker and correlation between CD127dim and Foxp3 expression in CD

Recent findings in immunological monitoring for high-grade glioma patients treated with autologous dendritic cell-based tumor vaccination: CD107a expression as effector cell degranulation marker and correlation between CD127dim and Foxp3 expression in CD

Selective Loss of a Putative Precursor Population of B-Chronic Lymphocytic Leukemia Cells Following Immunization with hCD40L/IL-2 Expressing Autologous Tumor Cells

Side-population (SP) cells, defined on the basis of their ability to rapidly expel Hoechst dye, may be precursor cells for normal and malignant tissues and are more resistant to chemotherapy. We have now characterized a malignant SP subset in the peripheral blood of B-CLL patients and show how vaccination with CD40L/IL-2 expressing autologous tumor cells can induce a T cell immune response that reduces or eliminates this tumor subpopulation. Hoechst 33342 staining combined with CD5/CD19 antibody labeling of peripheral blood mononuclear cells showed a distinct CD5+CD19+ SP phenotype (median=0.22%, range; 0.2–2.17%) in 85% (18/21) of B-CLL patients (Stage I–III). We sorted SP and non-SP (NSP) cells and extracted RNA and performed a comprehensive gene expression analysis by microarray (n=9 donor SP and NSP pairs), finding 85 genes overexpressed (>2-fold log ratio) in SP compared to NSP, and 228 genes higher in NSP versus SP. The analysis showed that compared to NSP cells from the same patient, the SP subset consistently and significantly overexpressed genes associated with cellular activation, proliferation and survival including eIF4A1 (p<.05), Wnt10a (p<.05), Bcl2L12 (p<.05) and Rel A (p=.07), a difference confirmed by qPCR. Conversely, NSP significantly overexpressed GSK3β (p<.05) compared to SP. CD40 mediated activation of B-CLL tumor cells further enhanced relative expression of these genes in the SP fraction, and induced overexpression of the proliferation-associated genes Ki-67 and PCNA in SP compared to NSP cells by 8-fold and 132-fold, respectively. We found SP cells were more resistant to exposure to chemotherapeutic agents including 50 mM fludarabine than NSP. Analysis of results in patients who had received an autologous B-CLL cell tumor vaccine expressing CD40L and IL-2, however, showed that B-CLL SP cells were vulnerable to a T cell mediated immune response. Hence, 3–8 subcutaneous injections CD40L/IL-2 B-CLL cells had no immediate effect on total B-CLL cell numbers in the peripheral blood, but Hoechst analysis showed a disappearance of SP cells in 7/8 immunized patients, associated with a rise in B-CLL-reactive T cells. CD8+ T cell clones isolated and expanded with CD40-activated autologous B-CLL tumor cells specifically responded to autologous SP cells, suggesting that SP depletion is mediated vaccine induced B-CLL-specific T cells. In 6/7 patients SP cells returned within a few weeks of completing the course of vaccination, coincident with a decline in T cell immunity. In one patient, however, immunity was longer lived and in this patient control of SP B-CLL cells persisted for more that 12 months. In this individual there was a delayed (by 6 months), but progressive disappearance of the bulk (non-SP) tumor cells. These results are consistent with the existence of an SP tumor precursor population of B-CLL, that can be targeted by immune T cells in vivo, and removal of which leads to later disappearance of their non-SP progeny.

Non‐transmissible Sendai virus encoding granulocyte macrophage colony‐stimulating factor is a novel and potent vector system for producing autologous tumor vaccines

The recent clinical application of granulocyte macrophage colony-stimulating factor (GM-CSF)-transduced autologous tumor vaccines revealed substantial antitumor activity and valuable clinical results. However, for these vaccines to be optimally effective, the antitumor efficacies must be improved. Recently, Sendai virus (SeV) vectors, which are cytoplasmic RNA vectors, have emerged as safe vectors with high gene transduction. In the current study, the in vivo therapeutic antitumor efficacies of irradiated GM-CSF-transduced mouse renal cell carcinoma (RENCA) vaccine cells mediated by either fusion gene-deleted non-transmissible SeV encoding mouse GM-CSF (SeV/dF/G) or adenovirus (E1, E3 deleted serotype 5 adenovirus) encoding mouse GM-CSF (AdV/G) (respectively described as irRC/SeV/GM or irRC/AdV/GM) were compared in RENCA-bearing mice. The results showed that the antitumor effect was equivalent between irRC/SeV/GM and irRC/AdV/GM cells, even though the former produced less GM-CSF in vitro. The cell numbers of activated (CD80(+), CD86(+), CD80( (+) )CD86(+)) dendritic cells in lymph nodes from mice treated with irRC/AdV/GM or irRC/SeV/GM cells were increased significantly compared with those of mice treated with the respective controls, at both the earlier and later phases. In an in vitro cytotoxicity assay, splenocytes harvested from mice treated with both irRC/SeV/GM and irRC/AdV/GM cells showed tumor-specific responses against RENCA cells. The restimulated splenocytes harvested from mice treated with irRC/SeV/GM or irRC/AdV/GM cells produced significantly higher levels of interleukin-2, interleukin-4, and interferon-gamma compared with their respective controls (P < 0.05). Furthermore, vaccination with irRC/AdV/GM or irRC/SeV/GM cells induced significantly enhanced recruitment of the cytolytic effectors of CD107a(+)CD8(+) T cells and CD107a(+) natural killer cells into tumors compared with those induced by their respective controls (P < 0.05). Taken together, our results suggest that the SeV/dF/G vector is a potential candidate for the production of effective autologous GM-CSF-transduced tumor vaccines in clinical cancer immune gene therapy.

Regressing and progressing metastatic lesions: resistance to immunotherapy is predetermined by irreversible HLA class I antigen alterations.

Despite the significant efforts to enhance immune reactivity against malignancies the clinical effect of anti-tumor vaccines and cancer immunotherapy is still below expectations. Understanding of the possible causes of such poor clinical outcome has become very important for improvement of the existing cancer treatment modalities. In particular, the critical role of HLA class I antigens in the success of T cell based immunotherapy has led to a growing interest in investigating the expression and function of these molecules in metastatic cancer progression and, especially in response to immunotherapy. In this report, we illustrate that two types of metastatic lesions are commonly generated in response to immunotherapy according to the pattern of HLA class I expression. We found that metastatic lesions, that progress after immunotherapy have low level of HLA class I antigens, while the regressing lesions demonstrate significant upregulation of these molecules. Presumably, immunotherapy changes tumor microenvironment and creates an additional immune selection pressure on tumor cells. As a result, two subtypes of metastatic lesions arise from pre-existing malignant cells: (a) regressors, with upregulated HLA class I expression after therapy, and (b) progressors with resistance to immunotherapy and with low level of HLA class I. Tumor cells with reversible defects (soft lesions) respond to therapy by upregulation of HLA class I expression and regress, while tumor cells with structural irreversible defects (hard lesions) demonstrate resistance to immunostimulation, fail to upregulate HLA class I antigens and eventually progress. These two types of metastases appear independently of type of the immunotherapy used, either non-specific immunomodulators (cytokines or BCG) or autologous tumor vaccination. Similarly, we also detected two types of metastatic colonies in a mouse fibrosarcoma model after in vitro treatment with IFN-gamma. One type of metastases characterized by upregulation of all MHC class I antigens and another type with partial IFN-gamma resistance, namely with lack of expression of L(d)-MHC class I molecule. Our observations may shed new light on the understanding of the mechanisms of tumor escape and might have implications for improvement of the efficacy of cancer immunotherapy.

Immune monitoring of antibody responses to complex immunotherapies using custom protein microarrays

3028 Background: Multiple autologous and allogeneic cellular tumor vaccines, as well as targeted immunotherapies such as CTLA-4 antibodies, are in current clinical trials. Vaccination can induce co...

An effective tumor vaccine optimized for costimulation via bispecific and trispecific fusion proteins

T cell costimulation has great therapeutic potential if it can be optimized and controlled. To achieve this, we engineered T cell-activating fusion proteins and immunocytokines that specifically attach to viral antigens of a virus-infected tumor vaccine. We employed the avian Newcastle Disease Virus because this agent is highly efficient for human tumor cell infection, and leads to introduction of viral hemagglutinin-neuraminidase (HN) molecules at the tumor cell surface. Here, we demonstrated the strong potentiation of the T cell stimulatory activity of such a vaccine upon attachment of bispecific or trispecific fusion proteins which bind with one arm to viral HN molecules of the vaccine, and with the other arm either to CD3 (signal 1), to CD28 (costimulatory signal 2a), or to interleukin-2 receptor (costimulatory signal 2b) on T cells. A vaccine with a combination of all three signals triggered the strongest activation of naïve human T cells, thereby inducing the most durable bystander antitumor activity in vitro. Adoptive transfer of such polyclonally activated cells into immunodeficient mice bearing human breast carcinoma caused tumor regression. Furthermore, tumor-reactive memory T cells from draining lymph nodes of carcinoma patients could be efficiently reactivated in a short-term ELISpot assay using an autologous tumor vaccine with optimized signals 1 and 2, but not with a similarly modified vaccine from an unrelated tumor cell line. Our data describe new bioactive molecules which in combination with an established virus-modified tumor vaccine greatly augments the antitumor activity of T cells from healthy donors and cancer patients.

Vaccination with Allogeneic GM-CSF Gene-Modified Lung Cancer Cells: Antitumor Activity Comparing with that Induced by Autologous Vaccine

The aim of this study was to investigate the whole allogeneic (differing tissue-type) tumor cells as vaccine in the mouse lung cancer model. The immunogenic and antitumor activity of allogeneic vaccine was compared with that of autologous cancer cell vaccine. C57/BL mice inoculated with Lewis lung cancer (LLC) cells were used as the animal model to test the effects of allogeneic vaccination. LA795 and LLC lung cancer cell lines, which were transfected with the mouse granulocyte-macrophage colony-stimulating factor (GM-CSF) gene, were administered as allogeneic and autologous tumor vaccine, respectively. The irradiated tumor cells were administered as subcutaneous vaccines before the tumor challenge. The immunity of cancer vaccine was tested by mouse interferon-gamma (IFN-gamma) enzyme-linked immunospot (ELISPOT) lactate dehydrogenase (LDH) assays. The serum level of IFN-gamma and interleukin (IL)-4 was tested using the enzyme-linked immunosorbent assay method. Prophylactic vaccination with allogeneic LA795 cells protected against the LLC tumor challenge in C57/BL. The tumor growth was inhibited and the survival was accordingly prolonged. The cytotoxicity of the spleen cells or the purified CD(8)(+) T-cells against LLC cells in the mice immunized with either the autologous or allogeneic cancer cell vaccine was significantly increased, relative to that of the control, untreated group (p<0.05). ELISPOT IFN-gamma assays showed that spleen cells from mice immunized with LA795 cells could be activated after coculture with irradiated LLC cells. In addition, the serum level of Th1-king cytokine IFN-gamma significantly increased after vaccination; however, no statistically difference was found in Th2-kind cytokine IL-4. The allogeneic cancer vaccine could induce immune responses and protection against lung cancer, which had no significant difference with that of autologous vaccine.

TARC and RANTES Enhance Immunological Antitumor Effect Induced by GM-CSF Transduced Leukemia Vaccine in Mouse Model.

Granulocyte-macrophage colony stimulating factor (GM-CSF) has been postulated to be a critical mediator in the initial tumor immune response and obvious antitumor immunomodulationg activity of autologous GM-CSF gene-transduced tumor vaccine therapy for various tumors including leukemia has been reported. Previously we demonstrated GM-CSF gene transduction into murine monocytic leukemia cell line of WEHI3B eliminated the tumorigenesity in vivo and the tumor regression started reproducibly around 10 days after tumor injection followed by disappearance within a week. We analyzed the gene expression of the transiently established tumor masses by the serial analysis of gene expression (SAGE) method to identify molecules associated with the antitumor effect. We identified that chemokine genes of TARC and RANTES were preferentially expressed in the GM-CSF-transduced tumors and the contribution of the two genes to the regression of GM-CSF-transduced tumors was suggested. To confirm the finding, we observed in vivo tumor formation of WEHI-3B cells transduced with a retroviral vector expressing murine GM-CSF, TARC, RANTES, GM-CSF+TARC or GM-CSF+RANTES, respectively. One million cells of each transduced cell group and non-transduced cell group were inoculated subcutaneously into syngeneic BALB/c mice. Then tumorigenecity and survival rate were observed. Both of the combination of GM-CSF with TARC or RANTES gene transduction profoundly inhibited tumor formation. In addition, the 2 groups demonstrated significantly longer survival than GM-CSF transduced group. To examine their therapeutic potency, 3×10^6 of irradiated WEHI3B cells retrovirally transduced with murine GM-CSF cDNA in combination with either recombinant TARC or RANTES were subcutaneously inoculated every 4 days for 4 times into syngeneic BALB/c mice preliminarily inoculated subcutaneously with 1×10^5 of wild type WEHI3B cells. Vaccinations were well tolerated without any major adverse events. The mice treated with the GM-CSF transduced cells with chemokines demonstrated significantly longer survival than the mice treated with GM-CSF transduced cells. Splenocytes harvested from mice treated with the former vaccines produced higher levels of IL-4, IL-6, IFN-γ, and TNF-α, suggesting a reinforcement of innate immunity as well as adaptive immunity. Immunohistochemical findings of the tumor section after vaccine treatment revealed more significant contribution of CD4+, CD8+ T cells to tumor repression in the combined vaccine groups than GM-CSF vaccine group. In coclusion, TARC and RANTES enhance immunological antitumor effect induced by GM-CSF in mouse WEHI3B tumor models and would be useful as the second generation GM-CSF immune gene therapy targeting leukemia.

POD-03.01: Development and application of mixed vaccines in renal cell carcinoma: combining autologous tumor cells with dendritic cells derived from autologous or allogeneic origin

Purpose: To evaluate the effects of autologous tumor vaccine alone or in combination with dendritic cell vaccines, as a method of stimulating antigen-presenting cells in patients with a locoregionally confined renal cell carcinoma (RCC) or metastatic disease. Materials and Methods: Twenty-seven patients with RCC pathological stages II to IV were treated with autologous tumor cell vaccine, either with or without dendritic cell vaccine. Interleukin 2 (IL-2) based immunotherapy was also applied to the patients with metastatic disease. Immunomagnetic beads were used to isolate CD14+ monocytes from patient or donor in dendritic cell preparations. IL-4 and granulocyte-macrophage colony stimulating factor (GM-CSF) were used for maturation of dendritic cells. Flow cytometry evaluations were performed for dendritic cell maturation and changes in the immunological profiles following our treatment. Results: Both the isolation of CD14+ monocyte, using Immunomagnetic beads, and the maturation of dendritic cells, using IL-4 and GM-CSF stimulation, were effective. Tumor immunological profiles showed increased CD3 and CD56 populations after treatment. Side effects related with vaccine were minimal and tolerable. Patients were stratified by the purpose for the vaccination; 8 patients for post-nephrectomy adjuvant therapy and 19 for adjuvant immunotherapy of a metastatic disease. All 8 patients in the former showed a disease free state, while only one of the 19 in the latter group remained in complete remission, while 6 showed short-term responses. Conclusions: Autologous RCC vaccine, combined with or without dendritic cell vaccine, might be effective in the suppression of tumor recurrence in locoregionally confined RCC, although a longer follow-up will be required. These vaccines should be further developed to reach their therapeutic purpose in metastatic RCC. (Korean J Urol 2007;48:111-119)

Imiquimod Enhances the Systemic Immunity Attained by Local Cryosurgery Destruction of Melanoma Lesions

Melanoma lesions can be frozen in vivo, resulting in necrotic death of malignant cells and in tumor antigen release suitable for cross-presentation by professional antigen-presenting cells. Imiquimod is a small molecule with adjuvant pro-inflammatory effects that can be topically delivered as a cream. Local cryosurgery of B16/ovalbumin (OVA)-derived subcutaneous tumor nodules leads to curative destruction of the lesions. If imiquimod is repeatedly applied on the cryo-treated lesion, a conspicuous, leukocyte-rich inflammatory infiltrate appears during the days following treatment. Mice treated by cryosurgery plus imiquimod rejected rechallenges of B16/OVA in 90% of the cases, whereas cryosurgery alone failed to prevent tumor grafting in 70% of the cases. The combination treatment of B16/OVA tumors was also able to protect 60% of the mice against outgrowth of a lethal dose of non-transfected B16 tumor cells. Addition of imiquimod to cryosurgery results in increases of the cellular immune response against tumor antigens as measured by in vitro IFN-gamma production and T-cell proliferation in response to OVA. The potent memory response is not only directed against the OVA epitope, but also toward a broader range of B16 antigens. Our data indicate that these combined treatments turn the treated tumor lesion into an autologous tumor vaccine, which is even able to cause vitiligo in several cases. These preclinical data and the simplicity of the procedures warrant the design of a pilot clinical trial.

Xenograft-like destruction of tumors and their conversion into vaccines by intratumoral injection of α-gal glycolipids

3074 Background: We describe a novel immunotherapy exploiting the natural anti-Gal antibody to destroy tumors and convert them into endogenous vaccines. Anti-Gal constitutes 1% of immunoglobulins in humans and interacts specifically with a-gal epitopes (Gala1- 3Galβ1–4GlcNAc-R) on glycolipids and glycoproteins. a-Gal epitopes are abundant in nonprimate mammals. Binding of anti-Gal to a-gal epitopes on pig cells mediates xenograft rejection. We inject glycolipids carrying a-gal epitopes (a-gal glycolipids) into solid tumors. Methods: a-Gal glycolipids extracted from rabbit RBC have carbohydrate chains capped with a-gal epitopes. Efficacy of treatment was studied in a1,3galactosyltransferase knockout mice bearing B16 melanoma. These mice are unique since they lack a-gal epitopes and can produce anti- Gal, like humans. Also B16 cells lack a-gal epitopes. B16 lesions (∼5 mm) were injected with 1mg a-gal glycolipids. Results: Intratumoral injection of a-gal glycolipids results in local inflammation mediated by anti-Gal binding to the ∼2x1016 a-gal epitopes on these glycolipids, activation of complement and generation of chemotactic factors. a-Gal glycolipids insert spontaneously into tumor cell membranes. Binding of anti-Gal to a-gal epitopes on such tumor cells induces lesion destruction, similar to xenograft rejection. Anti-Gal further opsonizes tumor cells for effective uptake by inflammation recruited dendritic cells (DC), via Fcγ receptors of these APC. The DC transport internalized tumor Ags to draining lymph nodes and present tumor Ag peptides, thus activating tumor specific T cells and eliciting an immune response against micrometastases. Conclusions: Injected a-gal glycolipids effectively destroy lesions and convert them into vaccines. This treatment may be even more effective in humans, since complement activity is much higher than in mice. This treatment may also be considered as neo-adjuvant immunotherapy for converting primary tumor into autologous tumor vaccine that elicits a protective immune response against micrometastases, during the period preceding resection of the tumor. No significant financial relationships to disclose.

A clinical trial of autologous formalin-fixed tumor vaccine (AFTV) for patients with glioblastoma multiforme

2085 Background: This clinical pilot study is to investigate the safety, feasibility, plus clinical response of autologous formalin- fixed tumor vaccine (ATFV) in primary glioblastoma multiforme (GBM) patients. Methods: Eleven (8 recurrent and 3 initially treated) primary GBM patients were evaluated. AFTV was prepared from formalin-fixed tumor tissue or paraffin-embedded tissue and premixed with original adjuvant materials. A delayed-type hypersensitivity test (DTH) was performed before and after each course of ATFV treatment that comprised 3 vaccinations at a 5-site intradermal inoculation. In addition, immunohistochemical analysis of MIB-1, p53, and MHC class-I complex was performed on the tumor tissue to analyze the difference in the response to the treatment. Results: The treatment was well tolerated with only local induration and low-grade fever. Among the 11 patients, the best responses were 1 complete remission, 2 partial response, 1 no change, and 7 progressive disease. In this series, the median survival period was 7 months from the initiation of the AFTV treatment, and 3 of the 4 responders survived for more than 20 months after AFTV inoculation. DTH reactions, immunohistological analysis of p53 and MHC class-I complex, and patient status may be useful to predict the efficacy of this therapy. Conclusion: This study demonstrated that AFTV treatment is safe, feasible, and potentially beneficial. Further clinical investigation is highly desirable in order to improve the outcome of GBM patients. No significant financial relationships to disclose.

Clinical trial of autologous formalin‐fixed tumor vaccine for glioblastoma multiforme patients

A pilot study was performed to investigate the safety and feasibility of autologous formalin-fixed tumor vaccines (AFTV) and the clinical responses to these vaccines by glioblastoma multiforme (GBM) patients. Twelve primary GBM patients were recruited. Eight had recurrent disease while four had been treated for primary disease but retained a visible tumor mass. AFTV were prepared from formalin-fixed and/or paraffin-embedded tumor tissue obtained upon surgery and premixed with original adjuvant materials. The patients were given three five-site intradermal inoculations at weekly intervals. A delayed-type hypersensitivity test was performed before and after each vaccination. In addition, the tumor tissues were subjected to immunohistochemical analysis to determine whether MIB-1, p53, and major histocompatibility complex (MHC) class-I complex expression could predict the response to the treatment. The treatment was well tolerated, with only local erythema, induration, and low-grade fever being reported. Of the 12 patients, one showed a complete response, one showed a partial response, two showed minor responses, one had stable disease, and seven exhibited progressive disease. The median survival period was 10.7 months from the initiation of the AFTV treatment but three of the five responders survived for 20 months or more after AFTV inoculation. Low p53 and high MHC class-I expression by the tumor may help predict the efficacy of this therapy. Thus, the AFTV is safe and feasible, and could significantly improve the outcome of GBM. Further clinical investigations to confirm this are highly desirable.