Phenotype and functional characterization of long-term gp100-specific memory CD8+ T cells in disease-free melanoma patients before and after boosting immunization

PURPOSE: Effective cancer vaccines must both drive a strong CTL response and sustain long-term memory T cells capable of rapid recall responses to tumor antigens. We sought to characterize the phenotype and function of gp100 peptide-specific memory CD8+ T cells in melanoma patients after primary gp100(209-2M) immunization and assess the anamnestic response to boosting immunization. EXPERIMENTAL DESIGN: Eight-color flow cytometry analysis of gp100-specific CD8+ T cells was done on peripheral blood mononuclear cells collected shortly after the primary vaccine regimen, 12 to 24 months after primary vaccination, and after boosting immunization. The anamnestic response was assessed by comparing the frequency of circulating gp100-specific T cells before and after boosting. Gp100 peptide-induced in vitro functional avidity and proliferation responses and melanoma-stimulated T-cell CD107 mobilization were compared for cells from all three time points for multiple patients. RESULTS: The frequency of circulating gp100-specific memory CD8+ T cells was comparable with cytomegalovirus-specific and FLU-specific T cells in the same patients, and the cells exhibited anamnestic proliferation after boosting. Their phenotypes were not unique, and individual patients exhibited one of two distinct phenotype signatures that were homologous to either cytomegalovirus-specific or FLU-specific memory T cells. Gp100-specific memory T cells showed some properties of competent memory T cells, such as heightened in vitro peptide-stimulated proliferation and increase in central memory (TCM) differentiation when compared with T-cell responses measured after the primary vaccine regimen. However, they did not acquire enhanced functional avidity usually associated with competent memory T-cell maturation. CONCLUSIONS: Although vaccination with class I-restricted melanoma peptides alone can break tolerance to self-tumor antigens, it did not induce fully competent memory CD8+ T cells--even in disease-free patients. Data presented suggest other vaccine strategies will be required to induce functionally robust long-term memory T cells.     

Recombinant gp100 protein presented by dendritic cells elicits a T-helper-cell response in vitro and in vivo.

Induction of a helper T (TH)-cell response is a critical element in the generation of anti-tumor immunity. The majority of immunotherapeutic approaches have so far been concerned with the generation of cytotoxic T lymphocytes (CTLs). This also accounts for gp100, a melanoma-associated protein which induces a potent CTL response. Because of the high immunogenicity of gp100, we considered it of special interest to explore the feasibility of generating gp100-specific TH cells. Human dendritic cells (DCs) were loaded with recombinant gp100 protein, and the response of autologous TH cells was evaluated in vitro and in vivo. We have observed that gp100 peptides can be presented by DCs of certain MHC class II haplotypes, which led to proliferation and cytokine production of TH-1 cells in vitro. Furthermore, transfer of gp100 protein-loaded human DCs into SCID mice also induced proliferation of autologous, unprimed peripheral blood leukocytes (PBLs) and selective expansion of TH cells. When human T cells from the spleen of SCID mice were recovered and restimulated in vitro, they strongly proliferated in response to gp100-loaded DCs, while showing minimal proliferative activity in response to DCs loaded with a control antigen. Thus, it is possible to induce an efficient MHC class II-restricted TH response by in vitro stimulation or in vivo vaccination with DCs which have been loaded with a purified tumor-associated antigen.     

Dendritic cells genetically engineered to simultaneously express endogenous tumor antigen and granulocyte macrophage colony-stimulating factor elicit potent therapeutic antitumor immunity.

Recently, several studies have shown that vaccine therapy using dendritic cells (DCs) genetically engineered to express a surrogate tumor antigen can effectively induce antitumor immunity. In this study, murine bone marrow DCs were adenovirally transduced with murine endogenous tumor antigen gp70 expressed in CT26 cells and granulocyte macrophage colony-stimulating factor (GM-CSF), and we examined whether antigen-specific CTL responses and therapeutic immunity could be induced in mice immunized with those genetically modified DCs. The cytotoxic activity against CT26 in mice immunized with gp70-transduced DCs was significantly higher than that in control (P < 0.01) and was enhanced by GM-CSF-cotransduction (P < 0.001). GM-CSF gene transfer into DCs expressing tumor-associated antigen enhances CC chemokine receptor 7 expression on DCs, leading to improved migratory capacity of DCs to draining lymph nodes. Consequently, an effective antitumor immune response would be induced. Vaccination using gp70-transduced DCs provided remarkable therapeutic efficacy in s.c. models. Moreover, it could be sufficiently augmented by GM-CSF-cotransduction of DCs. These results support that vaccination therapy using DCs simultaneously transduced with tumor-associated antigen can elicit potent CTL response, and GM-CSF-cotransduction of DCs could optimize therapeutic response. Further investigation is needed to optimize this vaccine therapy to achieve the obvious benefit in clinical application.     

Murine dendritic cells transfected with human GP100 elicit both antigen-specific CD8(+) and CD4(+) T-cell responses and are more effective than DNA vaccines at generating anti-tumor immunity.

Dendritic cells (DCs) are potent inducers of cytotoxic T lymphocytes (CTLs) when pulsed with an antigenic peptide or tumor lysate. In this report, we have used liposome-mediated gene transfer to examine the ability of plasmid DNA encoding the human melanoma-associated antigen gp100 to elicit CD8(+) and CD4(+) T-cell responses. We also compared the efficacy between gp100 gene-modified DCs and naked DNA (pCDNA3/gp100)-based vaccines at inducing anti-tumor immunity. DCs were generated from murine bone marrow and transfected in vitro with plasmid DNA containing the gp100 gene. These gp100-modified DCs (DC/gps) were used to stimulate syngeneic naive spleen T cells in vitro or to immunize mice in vivo. Antigen-specific, MHC-restricted CTLs were generated when DC/gps were used to prime T cells both in vitro and in vivo. Thus, these CTLs were cytolytic for gp100-transfected syngeneic (H-2(b)) tumor MCA106 (MCA/gp) and vaccinia-pMel17/gp100-infected syngeneic B16 and MCA106, but not parental tumor MCA106 and B16, or gp100-transfected allogeneic tumor P815 (H-2(d)). Immunization with DC/gp protected mice from subsequent challenge with MCA/gp but not parental MCA106. Antibody-mediated T-cell subset depletion experiments demonstrate that induction of CTLs in vivo is dependent on both CD4(+) and CD8(+) T cells. Furthermore, DC/gp immunization elicits an antigen-specific CD4(+) T-cell response, suggesting that DC/gps present MHC class II epitopes to CD4(+) T cells. In addition, our data show that gene-modified, DC-based vaccines are more effective than the naked DNA-based vaccines at eliciting anti-tumor immunity in both prophylactic and therapeutic models. These results suggest that the use of DCs transfected with plasmid DNA containing a gene for TAA may be superior to peptide-pulsed DCs and naked DNA-based vaccines for immunotherapy and could provide an alternative strategy for tumor vaccine design.     

Immunogenicity, including vitiligo, and feasibility of vaccination with autologous GM-CSF-transduced tumor cells in metastatic melanoma patients.

PURPOSE: To determine the feasibility, toxicity, and immunologic effects of vaccination with autologous tumor cells retrovirally transduced with the GM-CSF gene, we performed a phase I/II vaccination study in stage IV metastatic melanoma patients. PATIENTS AND METHODS: Sixty-four patients were randomly assigned to receive three vaccinations of high-dose or low-dose tumor cells at 3-week intervals. Tumor cell vaccine preparation succeeded for 56 patients (88%), but because of progressive disease, the well-tolerated vaccination was completed in only 28 patients. We analyzed the priming of T cells against melanoma antigens, MART-1, tyrosinase, gp100, MAGE-A1, and MAGE-A3 using human leukocyte antigen/peptide tetramers and functional assays. RESULTS: The high-dose vaccination induced the infiltration of T cells into the tumor tissue. Three of 14 patients receiving the high-dose vaccine showed an increase in MART-1- or gp100-specific T cells in the peripheral blood during vaccination. Six patients experienced disease-free survival for more than 5 years, and two of these patients developed vitiligo at multiple sites after vaccination. MART-1- and gp100-specific T cells were found infiltrating in vitiligo skin. Upon vaccination, the T cells acquired an effector phenotype and produced interferon-gamma on specific antigenic stimulation. CONCLUSION: We conclude that vaccination with GM-CSF-transduced autologous tumor cells has limited toxicity and can enhance T-cell activation against melanocyte differentiation antigens, which can lead to vitiligo. Whether the induction of autoimmune vitiligo may prolong disease-free survival of metastatic melanoma patients who are surgically rendered as having no evidence of disease before vaccination is worthy of further investigation.     

gp100(209-2M) peptide immunization of human lymphocyte antigen-A2+ stage I-III melanoma patients induces significant increase in antigen-specific effector and long-term memory CD8+ T cells.

Thirty-five HLA-A2(+) patients with completely resected stage I-III melanoma were vaccinated multiple times over 6 months with a modified melanoma peptide, gp100(209-2M), emulsified in Montanide adjuvant. Direct ex vivo gp100(209-2M) tetramer analysis of pre- and postvaccine peripheral blood mononuclear cells (PBMCs) demonstrated significant increases in the frequency of tetramer(+) CD8(+) T cells after immunization for 33 of 35 evaluable patients (median, 0.36%; range, 0.05-8.9%). Ex vivo IFN-gamma cytokine flow cytometry analysis of postvaccine PBMCs after brief gp100(209-2M) in vitro activation showed that for all of the patients studied tetramer(+) CD8(+) T cells produced IFN-gamma; however, some patients had significant numbers of tetramer(+) IFN-gamma(-) CD8(+)T cells suggesting functional anergy. Additionally, 8 day gp100(209-2M) in vitro stimulation (IVS) of pre- and postvaccine PBMCs resulted in significant expansion of tetramer(+) CD8(+) T cells from postvaccine cells for 34 patients, and these IVS tetramer(+) CD8(+) T cells were functionally responsive by IFN-gamma cytokine flow cytometry analysis after restimulation with either native or modified gp100 peptide. However, correlated functional and phenotype analysis of IVS-expanded postvaccine CD8(+) T cells demonstrated the proliferation of functionally anergic gp100(209-2M)- tetramer(+) CD8(+) T cells in several patients and also indicated interpatient variability of gp100(209-2M) stimulated T-cell proliferation. Flow cytometry analysis of cryopreserved postvaccine PBMCs from representative patients showed that the majority of tetramer(+) CD8+ T cells (78.1 +/- 4.2%) had either an "effector" (CD45 RA(+)/CCR7(-)) or an "effector-memory" phenotype (CD45RA(-)/CCR7(-)). Notably, analysis of PBMCs collected 12-24 months after vaccine therapy demonstrated the durable presence of gp100(209-2M)-specific memory CD8(+) T cells with high proliferation potential. Overall, this report demonstrates that after vaccination with a MHC class I-restricted melanoma peptide, resected nonmetastatic melanoma patients can mount a significant antigen-specific CD8(+) T-cell immune response with a functionally intact memory component. The data further support the combined use of tetramer binding and functional assays in correlated ex vivo and IVS settings as a standard for immunomonitoring of cancer vaccine patients.     

Interleukin-12-secreting human papillomavirus type 16-transformed cells provide a potent cancer vaccine that generates E7-directed immunity

The development of a vaccine that would be capable of preventing or curing the (pre)cancerous lesions induced by genital oncogenic human papillomaviruses (HPVs) is the focus of much research. Many studies are presently evaluating vaccines based on the viral E6 and E7 oncoproteins, both of which are continually expressed by tumor cells. The success of a cancer vaccine relies, in large part, on the induction of a tumor-specific Th1-type immunity. In this study, we have evaluated the ability of B7-related and/or interleukin-12 (IL-12)-expressing, non-immunogenic murine HPV16-transformed BMK-16/myc cells, to achieve this goal. BMK-16/myc cells engineered to express surface B7-1 or B7-2 molecules remain tumorigenic in syngeneic BALB/c mice, suggesting that expression of these molecules alone is not sufficient to induce tumor regression. In contrast, mice injected with tumor cells engineered to secrete IL-12 remained tumor-free, demonstrating that IL-12 expression is sufficient to induce tumor rejection. IL-12-secreting BMK-16/myc cells were further shown to induce potent and specific long-term tumor resistance, even after irradiation. B7-1 was found to slightly but systematically improve anti-tumor immunity elicited by IL-12-secreting BMK-16/myc cells. Injection of irradiated B7-1/IL-12+ BMK-16/myc cells generates long-lasting, Th1-type, BMK-16/myc-directed immunity in tumor-resistant mice. These mice display a memory-type, E7-specific, cell-mediated immune response, which is potentially significant for clinical applications.     

放疗或化疗诱导淋巴细胞减少联合免疫重建和瘤苗免疫

目的利用淋巴细胞减少期T细胞发生增殖活化的原理,以全身照射或环磷酰胺引起淋巴细胞减少,联合免疫重建及肿瘤疫苗免疫,以增强机体的肿瘤特异性免疫反应。方法分别以放疗或化疗（环磷酰胺）引起小鼠淋巴细胞减少,然后输入同系小鼠的未致敏脾细胞,建立免疫重建的淋巴细胞减少小鼠模型（RLM）。用黑色素瘤细胞F10对前者行免疫．肿瘤攻击实验,并行T细胞亚群清除试验。而化疗-RLM-免疫模型的抗肿瘤免疫反应效果通过过继免疫治疗检测。免疫用瘤苗为GMCSF修饰的黑色素瘤细胞D6-G6。免疫后9～10d,采集肿瘤疫苗接种部位的引流淋巴结,制备效应T细胞,然后过继回输给荷瘤3d（D5）的小鼠。2周后处死小鼠,计数肺转移灶数目。结果63．2％的放疗-RLM-免疫组小鼠可对肿瘤攻击产生抵抗,显著高于正常-免疫组（16．7％,P〈0．0001）。CD8^＋T细胞是介导抗肿瘤保护性免疫的主要效应细胞。延长免疫重建和瘤苗接种之问的间隔可削弱保护性抗肿瘤免疫。化疗-RLM-免疫组效应T细胞的在体抗肿瘤活性显著强于正常．免疫组。结论在放、化疗引起的淋巴细胞减少期进行瘤苗免疫,有助于打破机体对肿瘤的免疫耐受,增强抗肿瘤免疫反应。     

Enhancement of DNA vaccine potency by sandwiching antigen-coding gene between secondary lymphoid tissue chemokine (SLC) and IgG Fc fragment genes

DNA vaccine has become an attractive approach for generating antigen-specific immunity. Targeting antigens to FcRs for IgG (FcgammaRs) on dendritic cells (DCs) has been demonstrated to enhance antigen presentation. Secondary lymphoid tissue chemokine (SLC) has been shown to increase immune responses not only by promoting coclustering of T cells and DCs in the lymph nodes and spleen but also by regulating their immunogenic potential for the induction of T cell responses. In this study, using HPV 16 E7 as a model antigen, we constructed a chemotactic-antigen plasmid DNA vaccine (pSLC-E7-Fc) by linking SLC and Fc gene sequences to each end of E7 and evaluated its potency of eliciting specific immune response. We found that immunization with pSLC-E7-Fc generated much stronger E7-specific lymphocyte proliferative and cytotoxic T lymphocyte (CTL) responses than control DNA. All the mice receiving pSLC-E7-Fc prophylactic vaccination remained tumor free upon subcutaneous inoculation of TC-1 cells, while those given control DNA all developed tumors. These tumor-free mice were also protected against TC-1 rechallenge. Complete tumor regression with long-term survival occurred in 72% of mice given pSLC-E7-Fc as therapeutic vaccination. In experimental lung metastasis model wherein TC-1 cells were intravenously injected, therapeutic vaccination with pSLC-E7-Fc significantly reduced the number of tumor nodules in the lung. In vivo depletion with antibodies against CD4+or CD8+ T cells both resulted in complete abrogation of the pSLC-E7-Fc-induced immunotherapeutic effect. Our data indicate that the DNA vaccine constructed by the fusion of SLC and IgG Fc fragment genes to antigen-coding gene is an effective approach to induce potent anti-tumor immune response via both CD4+ and CD8+ T cells dependent pathways.     

Dendritic cells transduced with tumor-associated antigen gene elicit potent therapeutic antitumor immunity: comparison with immunodominant peptide-pulsed DCs

Several studies have shown that vaccine therapy using dendritic cells (DCs) pulsed with specific tumor antigen peptides can effectively induce antitumor immunity. Peptide-pulsed DC therapy is reported to be effective against melanoma, while it is still not sufficient to show the antitumor therapeutic effect against epithelial solid tumors such as gastrointestinal malignancies. Recently, it has been reported that vaccine therapy using DCs transduced with a surrogate tumor antigen gene can elicit a potent therapeutic antitumor immunity. In this study, we investigated the efficacy of vaccine therapy using DCs transduced with the natural tumor antigen in comparison with peptide-pulsed DCs. DCs derived from murine bone marrow were adenovirally transduced with murine endogenous tumor antigen gp70 gene, which is expressed in CT26 cells, or DCs were pulsed with the immunodominant peptide AH-1 derived from gp70. We compared these two cancer vaccines in terms of induction of antigen-specific cytotoxic T lymphocyte (CTL) responses, CD4+ T cell response against tumor cells, migratory capacity of DCs and therapeutic immunity in vivo. The cytotoxic activity of splenocytes against CT26 and Meth-A pulsed with AH-1 in mice immunized with gp70 gene-transduced DCs was higher than that with AH-1-pulsed DCs. CD4+ T cells induced from mice immunized with gp70 gene-transduced DCs produced higher levels of IFN-gamma by stimulation with CT26 than those from mice immunized with AH-1-pulsed DCs (p < 0.0001), and it was suggested that DCs transduced with tumor-associated antigen (TAA) gene induced tumor-specific CD4+ T cells, and those CD4+ T cells played a critical role in the priming phase of the CD8+ T cell response for the induction of CD8+ CTL. Furthermore, DCs adenovirally transduced with TAA gene showed an enhancement of expression of CC chemokine receptor 7 and improved the migratory capacity to draining lymph nodes. In subcutaneous models, the vaccination using gp70 gene-transduced DCs provided a remarkably higher therapeutic efficacy than that using AH-1-pulsed DCs. These results suggested that vaccine therapy using DCs adenovirally transduced with TAA gene can elicit potent antitumor immunity, and may be useful for clinical application.     

Effects of interleukin-12 on the immune response to a multipeptide vaccine for resected metastatic melanoma.

PURPOSE: Forty-eight patients with high-risk re-sected stage III or IV melanoma were immunized with two tumor antigen epitope peptides derived from gp100(209-217)(210M) (IMDQVPSFV) and tyrosinase(368-376)(370D) (YMDGTMSQV) emulsified with incomplete Freund's adjuvant (IFA). Patients received peptides/IFA with or without interleukin (IL)-12 30 ng/kg to evaluate the toxicities and immune responses in either arm with time to relapse and survival as secondary end points. PATIENTS AND METHODS: Immunizations were administered every 2 weeks for 8 weeks, then every 4 weeks for 12 weeks, and then once 8 weeks later. A leukapheresis to obtain peripheral-blood mononuclear cells for immune analyses was done before and after vaccination. Skin testing with peptides and recall reagents was performed before and after vaccinations. RESULTS: Local pain and granuloma formation, fever, and lethargy of grade 1 or 2 were observed. Transient vaccine-related grade 3-but no grade 4-toxicity was observed. Thirty-four of 40 patients developed a positive skin test response to the gp100 peptide but none to tyrosinase. Immune responses were measured by release of gamma-interferon in an enzyme-linked immunosorbent assay (ELISA) by effector cells in the presence of peptide-pulsed antigen-presenting cells or by an antigen-specific tetramer flow cytometry assay. Thirty-three of 38 patients demonstrated an immune response by ELISA after vaccination, as did 37 of 42 patients by tetramer assay. Twenty-four of 48 patients relapsed with a median follow-up of 20 months, and 10 patients in this high-risk group have died. CONCLUSION: These data suggest a significant proportion of patients with resected melanoma mount an antigen-specific immune response against a peptide vaccine and indicate that IL-12 may increase the immune response and supporting further development of IL-12 as a vaccine adjuvant.     

Immunological properties and vaccine efficacy of murine dendritic cells simultaneously expressing melanoma-associated antigen and interleukin-12

Interleukin (IL)-12 is a key factor for inducing cellular immune responses, which play a central role in the eradication of cancer. In the present study, in order to create a dendritic cell (DC)-based vaccine capable of positively skewing immune response toward a cellular immunity-dominant state, we analyzed immunological characteristics and vaccine efficacy of DCs cotransduced with melanoma-associated antigen (gp100) and IL-12 gene (gp100+IL12/DCs) by using RGD fiber-mutant adenovirus vector (AdRGD), which enables highly efficient gene transduction into DCs. gp100+IL12/DCs could simultaneously express cytoplasmic gp100 and secretory IL-12 at levels comparable to DCs transduced with each gene alone. In comparison with DCs transduced with gp100 alone (gp100/DCs), upregulation of major histocompatibility complex class I, CD40, and CD86 molecules on the cell surface and more potent T-cell-stimulating ability for proliferation and interferon-gamma secretion were observed as characteristic changes in gp100+IL12/DCs. In addition, administration of gp100+IL12/DCs, which were prepared by a relatively low dose of AdRGD-IL12, could induce more potent tumor-specific cellular immunity in the murine B16BL6 melanoma model than vaccination with gp100/DCs. However, antitumor effect and B16BL6-specific cytotoxic T-lymphocyte activity in mice vaccinated with gp100+IL12/DCs diminished with increasing AdRGD-IL12 dose during gene transduction, and paralleled the decrease in presentation levels via MHC class I molecules for antigen transduced with another AdRGD. Collectively, our results suggested that optimization of combined vector dose was required for development of a more efficacious DC-based vaccine for cancer immunotherapy, which relied on genetic engineering to simultaneously express tumor-associated antigen and IL-12.     

Paradoxical enhancement of CD8 T cell-dependent anti-tumor protection despite reduced CD8 T cell responses with addition of a TLR9 agonist to a tumor vaccine

Generation of antigen-specific CD8+ T cell responses is considered optimal for an effective immunotherapy against cancer. In this study, we provide a proof of principle that in vitro observed diminished CD8+ T cell response provided a strong in vivo tumor protection. Immunization with an adenovirus vaccine containing ovalbumin (OVA) gene (Ad5-OVA) strongly induces antigen-specific CD8+ T cell responses measured in vitro using various immunological assays. However, in an attempt to augment the antigenic CD8+ T cell response, coinjection of a TLR9 agonist CpG ODN with the viral vaccine unexpectedly reduced the CD8+ T cell responses measured in vitro but provided a remarkably enhanced tumor protection compared to the CD8+ T cell response generated by Ad5-OVA vaccine alone. Interestingly, despite reduced ex vivo/in vitro CD8+ T cell responses following Ad5-OVA+CpG immunization, immunodepletion studies revealed that the augmented anti-tumor immunity was primarily dependent on CD8+ T cells. The magnitude and effector function of anti-OVA CD8+ T cells remain low following primary and secondary antigenic challenge, presenting a dichotomy between in vitro CD8 T cell responses and in vivo anti-tumor immunity. To examine the impact of CpG ODN, we observed that presence of CpG suppresses the CD8+ T cell proliferation both in vitro and in vivo. These data demonstrate that coadministration of adenovirus vaccine with a TLR9 agonist can generate potentially effective tumor-reactive CD8+ T cells in vivo. In addition, the results indicate that widely used standard immune parameters may not predict the vaccine efficacy containing a TLR9 agonist as adjuvant.     

Toxicity, immunogenicity, and induction of E75-specific tumor-lytic CTLs by HER-2 peptide E75 (369-377) combined with granulocyte macrophage colony-stimulating factor in HLA-A2+ patients with metastatic breast and ovarian cancer.

To determine the toxicity and immunogenicity of the HER-2/neu, HLA-A2-restricted peptide E75 in patients with metastatic breast and ovarian cancer, 14 patients were vaccinated with escalating amounts of E75 (100, 500, and 1000 microg) mixed with 250 microg granulocyte macrophage colony-stimulating factor as adjuvant. Each vaccine dose was administered in a total volume of 1.5 ml divided into four intradermal injections and administered weekly for 4 weeks, followed by monthly boosts for a total of 10 injections. Vaccinations were well tolerated without significant toxicity. Blood was drawn before, at 8 weeks, and up to 13-16 months after vaccination for measurement of cellular immunity. Seven of 8 patients tested had significant delayed type hypersensitivity to E75 defined as >5 mm induration. Peripheral blood mononuclear cells from 5 of 9 patients tested proliferated to E75 with a stimulation index of > or = 2.0. Of 8 vaccinated patients tested for induction of a CTL response, 4 responded to stimulation by autologous dendritic cells plus cytokines by eliciting E75-specific lytic activity consistent with the presence of activated/memory cells, 2 others after in vitro stimulation with E75 + interleukin-12 +/- anti-CD152(33KD), whereas 2 others did not respond. Four patients with E75-specific CTLs present specifically recognized E75 on indicator tumors as demonstrated by cold-target inhibition of tumor lysis. These 4 patients showed E75-specific IFN-gamma production. peripheral blood mononuclear cell from 3 of these patients proliferated to E75, but stimulation indices were higher in the prevaccine samples. All 4 of the patients showed DTH responses to E75. These results demonstrate that vaccination with E75+ granulocyte macrophage colony-stimulating factor can induce both peptide-specific IFN-gamma and epitope specific CTLs, which lyse HER-2/neu+ tumors in stage IV patients.     

Function but not phenotype of melanoma peptide-specific CD8(+) T cells correlate with survival in a multiepitope peptide vaccine trial (ECOG 1696)

ECOG 1696 was a Phase II multi-center trial testing vaccination with melanoma peptides, gp100, MART-1 and tyrosinase delivered alone, with GM-CSF, IFN-α2b or both cytokines to HLA-A2(+) patients with metastatic melanoma. Here, the frequency of circulating CD8(+) tetramer(+) (tet(+) ) T cells and maturation stages of responding T cells were serially monitored and compared with baseline values in a subset of patients (n = 37) from this trial. Multiparameter flow cytometry was used to measure the frequency of CD8(+) T cells specific for gp100, MART-1, tyrosinase and influenza (FLU) peptides. Expression of CD45RA/CCR7 on CD8(+) tet(+) T cells and CD25, CD27, CD28 on all circulating T cells was determined. Vaccine-induced changes in the CD8(+) tet(+) T cell frequency and phenotype were compared with results of IFN-γ ELISPOT assays and with clinical responses. The frequency of CD8(+) tet(+) T cells in the circulation was increased for the melanoma peptides (p < 0.03-0.0001) but not for FLU (p < 0.9). Only gp100- and MART-1-specific T cells differentiated to CD45RA(+) CCR7(-) effector/memory T cells. In contrast to the IFN-γ ELISPOT frequency, previously correlated with overall survival (Kirkwood et al., Clin Cancer Res 2009;15:1443-51), neither the frequency nor differentiation stage of CD8(+) tet(+) T cells correlated with clinical responses. Delivery of GM-CSF and/or IFN-α2b had no effects on the frequency or differentiation of CD8(+) tet(+) , CD8+ or CD4+ T cells. Phenotypic analyses of CD8(+) tet(+) T cells did not correlate with clinical responses to the vaccine, indicating that functional assessments of peptide-specific T cells are preferable for monitoring of anti-tumor vaccines.     

Adenovirus Mediated Gene Therapy in a Glioblastoma Vaccine Model; Specific Antitumor Immunity and Abrogation of Immunosuppression

Clinical trials are being performed using tumor genetically engineered to produce cytokines as a vaccine. The design of such a vaccine may be made more effective by further study using in-vitro as well as in-vivo models. We studied an in-vitro tumor vaccine model in glioblastoma. We have demonstrated high efficiency transfection of the Interleukin-2 (IL-2) gene into glioblastoma cell lines using adenoviral vectors. Glioblastoma cell lines transduced with this vector could produce high levels of IL-2 for up to 2 weeks, long enough to elicit an antitumor immune response. We studied tumor/effector cell interactions using cytotoxicity assays coupled with flow cytometric analysis. Activation of CD8+ and expansion of CD3+/CD16+ effector cell subpopulations were observed, suggesting the generation of a specific anti-tumor response and the potential for systemic immunity. We demonstrated that glioblastoma produce immunosuppressive factors which reduce the antitumor response by peripheral blood effector cells. These immunosuppressive factors could be neutralized to improve antitumor response. A better understanding of tumor/effector cell interactions may improve the design of gene therapy trials.     

Agonist anti-GITR antibody enhances vaccine-induced CD8(+) T-cell responses and tumor immunity

Immunization of mice with plasmids encoding xenogeneic orthologues of tumor differentiation antigens can break immune ignorance and tolerance to self and induce protective tumor immunity. We sought to improve on this strategy by combining xenogeneic DNA vaccination with an agonist anti-glucocorticoid-induced tumor necrosis factor receptor family-related gene (GITR) monoclonal antibody (mAb), DTA-1, which has been shown previously both to costimulate activated effector CD4(+) and CD8(+) T cells and to inhibit the suppressive activity of CD4(+)CD25(+) regulatory T cells. We found that ligation of GITR with DTA-1 just before the second, but not the first, of 3 weekly DNA immunizations enhanced primary CD8(+) T-cell responses against the melanoma differentiation antigens gp100 and tyrosinase-related protein 2/dopachrome tautomerase and increased protection from a lethal challenge with B16 melanoma. This improved tumor immunity was associated with a modest increase in focal autoimmunity, manifested as autoimmune hypopigmentation. DTA-1 administration on this schedule also led to prolonged persistence of the antigen-specific CD8(+) T cells as well as to an enhanced recall CD8(+) T-cell response to a booster vaccination given 4 weeks after the primary immunization series. Giving the anti-GITR mAb both during primary immunization and at the time of booster vaccination increased the recall response even further. Finally, this effect on vaccine-induced CD8(+) T-cell responses was partially independent of CD4(+) T cells (both helper and regulatory), consistent with a direct costimulatory effect on the effector CD8(+) cells themselves.     

The boosting effect of co-transduction with cytokine genes on cancer vaccine therapy using genetically modified dendritic cells expressing tumor-associated antigen

The T-helper 1 (Th1) immune reaction is most important in dendritic cell (DC)-based immunotherapy. Interleukin 12 (IL-12) and granulocyte macrophage colony-stimulating factor (GM-CSF) play a pivotal role in inducing Th1 and cytotoxic T lymphocyte (CTL) responses. In this study, DCs expressing the natural tumor antigen gp70 of BALB/c-derived CT26 were adenovirally transduced with the IL-12 gene and/or GM-CSF gene, and it was examined whether vaccinations using these genetically engineered DCs can induce strong therapeutic antitumor immunity. Mice were immunized once by subcutaneous (s.c.) injection with genetically modified DCs. The cytotoxic activity of splenocytes against CT26 was assayed in a 51Cr-release assay 14 days after immunization. The therapeutic efficacy of the vaccination was examined in s.c. tumor models. The cytotoxic activity of CTLs against CT26 in mice immunized with DCs expressing gp70 (DC-AxCAgp70) was significantly augmented by co-transduction with the GM-CSF/IL-12 gene (p<0.0001) and remarkably reduced by the depletion of CD4+ or CD8+ cells (p<0.01). The cytotoxic activity against CT26 of the plain spleen cells in mice immunized with DC-AxCAgp70/GM-CSF/IL-12 was significantly higher than that in mice immunized with DC-AxCAgp70 (p<0.0001), and this activity decreased to almost 50% upon the depletion of NK cells. Vaccinations using DC-AxCAgp70/GM-CSF/IL-12 or DC-AxCAgp70/IL-12 could elicit potent therapeutic immunity in s.c. tumor models; tumor-free mice were observed in these vaccination groups. However, there was no significant difference between these two groups. A vaccination therapy using DCs co-transduced with the TAA gene and Th 1-type cytokine genes, especially the IL-12 gene, is ideal for immunotherapy in terms of the activation of DCs, NK cells, CD4+ T cells and CD8+ T cells, and may be useful in the clinical application of a cancer vaccine therapy.     

Effectiveness of a Simply Designed Tumor Vaccine in Prevention of Malignant Melanoma Development

We investigated the efficacy of a simple syngeneic tumor vaccine to induce specific antitumor immunity in female C57B1/6 mice. Tumor vaccine was prepared by mixing irradiated B-16 melanoma tumor cells with the pleiotropic biological response modifier—maleic anhydride divinyl ether (MVE-2). Experimental animals were pretreated with the vaccine in order to prevent the development of intraperitoneal (i.p.) B-16 melanoma tumors after inoculation of viable tumor cells. More than 40% of prevaccinated animals challenged i.p. with 5×10⁵ viable tumor cells were completely protected from tumor development and remained tumor-free 100 days after tumor cell inoculation. The percentage of tumor-free animals (survivors) rose to as much as 90% when the application of tumor vaccine was repeated two weeks after the first vaccination (i.e. one week after the inoculation of viable tumor cells). The induced antitumor response depended predominantly upon macro-phage function, since vaccinated animals which were depleted of peritoneal macrophages died within the same time range as animals in the control group. Also, tumor-type specificity of the vaccine was confirmed by the fact that the animals vaccinated with B-16 melanoma vaccine were not protected from the development of another type of tumor. In conclusion, comparison of the experimental data with the data from the literature suggests that our simple tumor vaccine may be as effective as genetically engineered tumor vaccines. At the same time, this kind of vaccine is easier to control and thus safer to apply in humans when compared to genetically engineered vaccines.     

Effectiveness of a simply designed tumor vaccine in prevention of malignant melanoma development.

We investigated the efficacy of a simple syngeneic tumor vaccine to induce specific antitumor immunity in female C57Bl/6 mice. Tumor vaccine was prepared by mixing irradiated B-16 melanoma tumor cells with the pleiotropic biological response modifier-maleic anhydride divinyl ether (MVE-2). Experimental animals were pretreated with the vaccine in order to prevent the development of intraperitoneal (i.p.) B-16 melanoma tumors after inoculation of viable tumor cells. More than 40% of prevaccinated animals challenged i.p. with 5 x 10(5) viable tumor cells were completely protected from tumor development and remained tumor-free 100 days after tumor cell inoculation. The percentage of tumor-free animals (survivors) rose to as much as 90% when the application of tumor vaccine was repeated two weeks after the first vaccination (i.e. one week after the inoculation of viable tumor cells). The induced antitumor response depended predominantly upon macrophage function, since vaccinated animals which were depleted of peritoneal macrophages died within the same time range as animals in the control group. Also, tumor-type specificity of the vaccine was confirmed by the fact that the animals vaccinated with B-16 melanoma vaccine were not protected from the development of another type of tumor. In conclusion, comparison of the experimental data with the data from the literature suggests that our simple tumor vaccine may be as effective as genetically engineered tumor vaccines. At the same time, this kind of vaccine is easier to control and thus safer to apply in humans when compared to genetically engineered vaccines.