HER-2/neu-gene engineered dendritic cell vaccine stimulates stronger HER-2/neu-specific immune responses compared to DNA vaccination

HER-2/neu is a candidate for developing breast cancer-targeted immunotherapeutics. Although DNA-based and HER-2/neu transgene-modified dendritic cell (DC)-based vaccines are potent at eliciting HER-2/neu-specific antitumor immunity, there has been no side-by-side study comparing them directly. The present study utilizes an in vivo murine tumor model expressing HER-2/neu antigen to compare the efficacy between adenovirus (AdVneu)-transfected dendritic cells (DC(neu)) and plasmid DNA (pcDNAneu) vaccine. Our data showed that DC(neu) upregulated the expression of immunologically important molecules and inflammatory cytokines and partially converted regulatory T (Tr)-cell suppression through interleukin-6 (IL-6) secretion. Vaccination of DC(neu) induced stronger HER-2/neu-specific humoral and cellular immune responses than DNA vaccination, which downregulated HER-2/neu expression and lysed HER-2/neu-positive tumor cells in vitro, respectively. In two HER-2/neu-expressing tumor models, DC(neu) completely protected mice from tumor cell challenge compared to partial or no protection observed in DNA-immunized mice. In addition, DC(neu) significantly delayed breast cancer development in transgenic mice in comparison to DNA vaccine (P<0.05). Taken together, we have demonstrated that HER-2/neu-gene-modified DC vaccine is more potent than DNA vaccine in both protective and preventive animal tumor models. Therefore, DCs genetically engineered to express tumor antigens such as HER-2/neu represent a new direction in DC vaccine of breast cancer.     

Therapeutic HER2/Neu DNA vaccine inhibits mouse tumor naturally overexpressing endogenous neu.

The therapeutic efficacy of HER2/c-erbB-2/neu DNA immunization on mouse tumor cells expressing exogenous human or rat p185neu but not on mouse tumor cells naturally expressing mouse p185neu has been demonstrated. We investigated the feasibility of using N-terminal rat neu DNA immunization on mouse tumor overexpressing endogenous p185neu and enhancing the therapeutic efficacy of this vaccine by fusion to various cytokine genes, including interleukin-2 (IL-2), interleukin-4 (IL-4), or granulocyte-macrophage colony-stimulating factor. In a therapeutic model, N'-neu-IL-2 DNA vaccine was significantly better than N'-neu DNA vaccine, and N'-neu DNA vaccine was significantly better than control DNA or N'-neu-IL-4 DNA vaccine. The therapeutic efficacy of DNA vaccines was correlated with tumor infiltration of CD8+ T cells. Depletion of CD8+ T cells completely abolished the therapeutic effects of N'-neu-IL-2 DNA vaccine and N'-neu DNA vaccine. Depletion of CD4+ T cells after tumor implantation had no influence on N'-neu-IL-2 DNA vaccine, but enhanced the therapeutic efficacy of N'-neu DNA vaccine. Our results demonstrate that rat N'-neu DNA vaccine has a therapeutic effect on established tumor through the CD8+ T-cell-dependent pathway. Depletion of CD4+ T cells or fusion to the IL-2 gene can thus further enhance the therapeutic effects of N'-neu DNA immunization on mouse tumor expressing endogenous p185neu.     

Enhanced immunity by NeuEDhsp70 DNA vaccine Is needed to combat an aggressive spontaneous metastatic breast cancer.

The rapid growth and ruthless metastasis of tumors demand effective broad immune responses. Dendritic cells (DCs) are critical in developing tumor vaccines. Recent investigations have been focused on modifying tumor antigens to target DCs to induce immune responses efficiently in vivo. In this study, human hsp70 was fused to the extracellular domain of rat Her2/Neu (NeuEDhsp70) for enhancing anti-tumor immunity in aggressive breast tumor models. NeuEDhsp70-conditioned DCs produced significant IL-12p40 and effectively presented NeuED antigens to T cells in vitro. NeuEDhsp70 DNA vaccine induced enhanced Neu-specific antibody and IFN-gamma-producing cellular immune responses in vivo. Although NeuEDhsp70 and NeuED DNA vaccines elicited comparable therapeutic anti-tumor immunity in an aggressive primary breast tumor model, NeuEDhsp70 DNA vaccine significantly increased survival and reduced metastasis in an aggressive spontaneous metastatic breast tumor model. Results from animal experiments with depletion of immune cells or with deficiency of CD40 molecules indicate that T cells and CD40 molecules are critical in the anti-tumor immunity induced by NeuEDhsp70 DNA vaccine. These observations suggest that NeuEDhsp70 DNA vaccine is a promising reagent to induce potent anti-tumor immunity to an aggressive spontaneous metastatic breast tumor.     

Induction of effective therapeutic antitumor immunity by direct in vivo administration of lentiviral vectors

Ex vivo lentivirally transduced dendritic cells (DC) have been described to induce CD8+ and CD4+ T-cell responses against various tumor-associated antigens (TAAs) in vitro and in vivo. We report here that direct administration of ovalbumin (OVA) encoding lentiviral vectors caused in vivo transduction of cells that were found in draining lymph nodes (LNs) and induced potent anti-OVA cytotoxic T cells similar to those elicited by ex vivo transduced DC. The cytotoxic T-lymphocyte (CTL) response following direct injection of lentiviral vectors was highly effective in eliminating target cells in vivo up to 30 days after immunization and was efficiently recalled after a boost immunization. Injection of lentiviral vectors furthermore activated OVA-specific CD4+ T cells and this CD4 help was shown to be necessary for an adequate primary and memory CTL response. When tested in therapeutic tumor experiments with OVA+ melanoma cells, direct administration of lentiviral vectors slowed down tumor growth to a comparable extent with the highest dose of ex vivo transduced DC. Taken together, these data indicate that direct in vivo administration of lentiviral vectors encoding TAAs has strong potential for anticancer vaccination.     

Differential immune responses mediated by adenovirus- and lentivirus-transduced DCs in a HER-2/neu overexpressing tumor model

Recent investigations have demonstrated that adenoviral and lentiviral vectors encoding HER-2 can be utilized in cancer immunotherapy. However, it is not known whether both viral systems elicit a similar immune response. Here, we compare the immune response in mice induced by dendritic cells (DCs) infected with either recombinant adenovirus or lentivirus encoding rat HER-2 (rHER-2). Both vaccine types yielded similar control of tumor growth, but we found clear differences in their immune responses 10 days after DC immunization. Adenovirus rHER-2-transduced DCs elicited locally and systemically high frequencies of CD4+ and CD8+ T cells, while lentivirus rHER-2-transduced DCs predominantly led to CD4+ T-cell infiltration at the tumor site. Splenocytes from mice immunized with lentivirus rHER-2-transduced DCs secreted higher levels of interferon (IFN)-γ, mainly by CD4+ T cells, following stimulation by RM-1-mHER-2 tumors. In contrast, the adenovirus vaccinated group exhibited CD4+ and CD8+ T cells that both contributed to IFN-γ production. Besides an established cellular immune response, the rHER-2/DC vaccine elicited a significant humoral response that was highest in the adenovirus group. DC subsets and regulatory T cells in the spleen were also differentially modulated in the two vaccine systems. Finally, adoptive transfer of splenocytes from both groups of immunized mice strongly inhibited in vivo tumor growth. Our results suggest that not only the target antigen but also the virus system may determine the nature and magnitude of antitumor immunity by DC vaccination.     

Adenovirus-transduced dendritic cells stimulate cellular immunity to melanoma via a CD4(+) T cell-dependent mechanism

We previously showed that genetic immunization of C57BL/6 mice with recombinant adenovirus encoding human TRP2 (Ad-hTRP2) was able to circumvent tolerance and induce cellular and humoral immune responses to murine TRP2 associated with protection against metastatic growth of B16 melanoma. In the present study we compared delivery of Ad-hTRP2 with cultured dendritic cells (DC) and direct injections of Ad-hTRP2. We show that application of Ad-hTRP2 with cultured DC enhanced protective immunity to B16 melanoma cells. Most importantly, delivery of recombinant adenovirus with DC alters the character of the immune response resulting in preferential stimulation of strong cellular immunity in the absence of significant humoral immunity to the encoded antigen. Adoptive transfer of lymphocytes from mice immunized with Ad-hTRP2-transduced DC confirmed that cellular components of the immune response were responsible for rejection of B16 melanoma. The protective efficacy of Ad-hTRP2-transduced DC clearly depended on the presence of CD4(+) T helper cells. Furthermore, AD-hTRP2-transduced DC, but not direct injection of Ad-hTRP2, were effective in the presence of neutralizing anti-adenoviral antibodies. These preclinical studies demonstrate the superiority of melanoma vaccines consisting of cultured DC transduced with recombinant adenoviruses encoding melanoma antigens.     

Induction of therapeutic antitumor immunity by in vivo administration of a lentiviral vaccine

Direct in vivo administration of a lentiviral vaccine has been shown to transduce dendritic cells (DCs) in order to induce antigen-specific CD8+ T cell responses, but the efficacy of antitumor immunity has not been reported. In this study we tested whether direct in vivo administration of a lentiviral vaccine can induce selfantigen- based therapeutic antitumor immunity in murine tumor models. Lentiviral vector (LV) transduced DCs efficiently in vitro and was able to transduce DCs in vivo. LV-transduced DCs effectively presented antigens to T cells. Compared with a naked DNA tyrosinase-related protein-2 (TRP2)-heat shock protein-70 (hsp70) vaccine, the TRP2-specific interferon-gamma-producing CD8+ T cell response was augmented by direct in vivo administration of an LV-TRP2hsp70 vaccine, which induced significant therapeutic antitumor immunity in subcutaneous B16 and subcutaneous GL-26 models. Moreover, in vivo administration of an LV-NeuEDhsp70 vaccine induced significant therapeutic antitumor immunity against spontaneous breast tumors in a BALB/c- Neu transgenic model. Our observations indicate that direct in vivo administration of a lentiviral vaccine not only enhances antigen-specific CD8+ T cell responses, but also generates significant therapeutic antitumor activities.     

Vaccination against Chlamydial Genital Tract Infection after Immunization with Dendritic Cells Pulsed Ex Vivo with Nonviable Chlamydiae

Chlamydia trachomatis, an obligate intracellular bacterial pathogen of mucosal surfaces, is a major cause of preventable blindness and sexually transmitted diseases for which vaccines are badly needed. Despite considerable effort, antichlamydial vaccines have proven to be elusive using conventional immunization strategies. We report the use of murine bone marrow–derived dendritic cells (DC) pulsed ex vivo with killed chlamydiae as a novel approach to vaccination against chlamydial infection. Our results show that DC efficiently phagocytose chlamydiae, secrete IL-12 p40, and present chlamydial antigen(s) to infection sensitized CD4⁺ T cells. Mice immunized intravenously with chlamydial-pulsed DC produce protective immunity against chlamydial infection of the female genital tract equal to that obtained after infection with live organisms. Immunized mice shed ∼3 logs fewer infectious chlamydiae and are protected from genital tract inflammatory and obstructive disease. Protective immunity is correlated with a chlamydial-specific Th1-biased response that closely mimics the immune response produced after chlamydial infection. Thus, ex vivo antigen-pulsed DC represent a powerful tool for the study of protective immunity to chlamydial mucosal infection and for the identification of chlamydial protective antigens through reconstitution experiments. Moreover, these findings might impact the design of vaccine strategies against other medically important sexually transmitted diseases for which vaccines are sought but which have proven difficult to develop.     

Development of cell-based tuberculosis vaccines: genetically modified dendritic cell vaccine is a much more potent activator of CD4 and CD8 T cells than peptide- or protein-loaded counterparts.

Genetically modified dendritic cell (DC)-based vaccines have not been explored for immunization against tuberculosis. A gene-modified DC vaccine expressing Mycobacterium tuberculosis (M.tb) antigen 85A (Ag85A) was developed by using a recombinant replication-deficient adenoviral gene transfer vector (AdAg85A). AdAg85A-transduced DC vaccine (AdAg85/DC) expressed higher levels of IL-12 and was much more immunogenic than Ag85 protein-loaded (pro/DC) or CD4/CD8 T cell peptide-loaded (pep/DC) DC vaccines. Compared to pro/DC or pep/DC, AdAg85/DC elicited a remarkably higher level of ex vivo IFN-gamma production by CD4 and CD8 T cells at weeks 2, 6, and 12 postimmunization, which was coupled with higher frequencies of antigen-specific T cells. By an in vivo CD8 or CD4 T cell cytotoxicity (CTL) assay, AdAg85/DC was shown to provoke much higher and more sustained levels of CD8 and CD4 CTL activity up to 12 weeks postimmunization. Intramuscular (im) AdAg85/DC immunization was more potent than the iv route of AdAg85/DC immunization. Such stronger immunogenicity of im AdAg85/DC vaccination was corroborated with better protection from M.tb challenge. Our results thus suggest that genetically modified DC-based TB vaccine is superior to subunit DC vaccines and has the potential for therapeutic applications.     

HER-2/neu胞外配体第2结构域致敏的DC与CIK共培养对高表达HER-2/neu乳腺癌细胞杀伤作用的研究

目的探讨HER-2/neu胞外配体第2结构域(RLD2)作为肿瘤疫苗在抗乳腺癌免疫中的作用。方法纯化出目的蛋白HER-2/neu胞外配体第2结构域,同时选取10例经免疫组化证实为HER-2/neu阳性的乳腺癌,用RLD2分别负荷这10名患者外周血来源的树突状细胞(DC),再诱导抗原特异的细胞因子诱导的杀伤细胞(CIK),将DC与C IK共培养,研究其对HER-2/neu阳性、阴性肿瘤细胞株和自体乳腺癌细胞杀伤活性的作用。结果RLD2诱导的特异性CIK对HER-2/neu阳性肿瘤细胞SKBR-3以及自体乳腺癌细胞的杀伤活性,较之单独CIK组更高,差异有统计学意义(P<0.01);RLD2诱导的特异性C IK对HER-2/neu阴性细胞MDA-435的杀伤活性与单独C IK组之间差异无统计学意义(P>0.05)。结论负载RLD2的DCs诱导的C IK对HER-2/neu阳性肿瘤细胞以及自体乳腺癌肿瘤有特异性杀伤能力,RLD2可能成为新型肿瘤疫苗应用于临床乳腺癌免疫治疗。     

Vaccination with helper-dependent adenovirus enhances the generation of transgene-specific CTL

Recombinant adenoviral vectors (AdV) have been used experimentally as vaccines to present antigenic transgenes in vivo. However, administration of first-generation vectors (FG-AdV) is often limited by their induction of antiviral immunity. To address this limitation, helper-dependent vectors (HD-AdV) were developed that lack viral coding regions. While the administration of HD-AdV results in long-term gene expression in vivo, their utility as immunogens has never been examined. Direct vaccination with 10(8) blue-forming units (BFU) of HD-AdV injected into C57BL/6 mice lead to superior transgene-specific CTL and antibody responses when compared to the same amount of a FG-AdV. The antibody responses to viral antigens were high in response to both the vectors. As a mechanism to reduce viral exposure, dendritic cells (DC) were transduced with HD-AdV in vitro and then used as a cell-based vaccine. DC transduced with HD-AdV expressed higher levels of transgene-specific mRNA and up to 1200-fold higher levels of transgene protein than did DC transduced with a FG-AdV. In addition, HD-AdV-transduced DC stimulated superior transgene-specific CTL responses when administered in vivo, an effect that was further enhanced by maturing the DC with LPS prior to administration. In contrast to direct immunization with HD-AdV, vaccination with HD-AdV-transduced DC was associated with limited antibody responses against the AdV. We conclude that HD-AdV stimulates superior transgene-specific immune responses when compared to a FG-AdV, and that immunization with a DC-based vaccine maintains this efficacy while limiting antiviral reactivity.     

'Survival gene' Bcl-xl potentiates DNA-raised antitumor immunity

T-cell priming is strongly affected by the longevity of antigen-bearing dendritic cells (DCs), which are typically short-lived in lymphoid tissues. 'Survival gene' Bcl-xl is critical for the lifespan of DCs in vivo. Here, we showed that in vivo coadministration of Bcl-xl under control of the DC-specific promoter (CD11c-Bcl-xl) and TRP2hsp70 DNA prolonged T-cell stimulation by DCs and augmented TRP2-specific-IFN-gamma-producing CD8+ T-cell responses. Consistent with these findings, enhanced protection and significant therapeutic immunity to B16 melanoma was generated by this coimmunization strategy, which also augmented therapeutic immunity to GL-26 tumor. In this B16 melanoma model, results from animal experiments with depletion of immune cells indicate that CD8+ T cells and NK cells are important in the antitumor immunity induced by this coimmunization strategy. These observations suggest that 'survival gene' Bcl-xl potentiates the magnitude of antigen-specific-CD8+ T-cell responses and the efficacy of antitumor immunity induced by DNA vaccine, and is relevant for the design of in vivo targeted DC-based vaccine strategies to improve immunity against cancer.     

树突状细胞肿瘤疫苗联合免疫佐剂治疗小鼠乳腺癌的初步探讨

目的研究免疫佐剂绿脓杆菌菌毛是否可以提高树突状细胞肿瘤疫苗的治疗作用.方法使用EMT6乳腺癌株种植BALB/c小鼠建立肿瘤模型,分成4组,次日在小鼠皮下分别注射生理盐水、免疫佐剂、树突状细胞肿瘤疫苗和树突状细胞肿瘤疫苗＋免疫佐剂.定期测量肿瘤大小,21 d后处死小鼠称量肿瘤重量.结果注射树突状细胞肿瘤疫苗＋免疫佐剂组肿瘤生长得到了明显抑制,与空白对照组比较,P=0.001,与单纯应用树突状细胞肿瘤疫苗组比较,P=0.047.结论绿脓杆菌菌毛作为免疫佐剂,可以和树突状细胞肿瘤疫苗发生协同作用,提高对小鼠乳腺癌的治疗效果.     

突变的erbB-2基因在树突状细胞中的表达

树突状细胞属于白细胞系,其功能为捕捉抗原并将该抗原呈递给T淋巴细胞而引发免疫应答。本研究在包装细胞中克隆了一个突变的erbB-2基因,并将该基因导入到CD34~ 来源的树突状细胞中。经检测,该基因失去了致癌性,不能转化NIH3T3细胞,但能表达较高水平的突变蛋白,组化染色和流式细胞术都确证了这一点。经基因修饰的树突状细胞将在乳腺癌的免疫治疗中具有潜在的应用价值。     

AE37: a novel T-cell-eliciting vaccine for breast cancer

INTRODUCTION: Immunotherapy, including vaccines targeting the human EGFR2 (HER-2/neu) protein, is an active area of investigation in combatting breast cancer. Several vaccines are currently undergoing clinical trials, most of which are CD8(+) T-cell-eliciting vaccines. AE37 is a promising primarily CD4(+) T-cell-eliciting HER-2/neu breast cancer vaccine currently in clinical trials. AREAS COVERED: This article reviews preclinical investigations as well as findings from completed and ongoing Phase I and Phase II clinical trials of the AE37 vaccine. EXPERT OPINION: Clinical trials have shown the AE37 vaccine to be safe and capable of generating peptide-specific, durable immune responses. This has been shown in patients with any level of HER-2/neu expression. Early clinical findings suggest there may be benefit to AE37 vaccination in preventing breast cancer recurrence.     

Protective T cell immunity in mice following protein-TLR7/8 agonist-conjugate immunization requires aggregation, type I IFN, and multiple DC subsets

The success of a non-live vaccine requires improved formulation and adjuvant selection to generate robust T cell immunity following immunization. Here, using protein linked to a TLR7/8 agonist (conjugate vaccine), we investigated the functional properties of vaccine formulation, the cytokines, and the DC subsets required to induce protective multifunctional T cell immunity in vivo. The conjugate vaccine required aggregation of the protein to elicit potent Th1 CD4+ and CD8+ T cell responses. Remarkably, the conjugate vaccine, through aggregation of the protein and activation of TLR7 in vivo, led to an influx of migratory DCs to the LN and increased antigen uptake by several resident and migratory DC subsets, with the latter effect strongly influenced by vaccine-induced type I IFN. Ex vivo migratory CD8-DEC205+CD103-CD326- langerin-negative dermal DCs were as potent in cross-presenting antigen to naive CD8+ T cells as CD11c+CD8+ DCs. Moreover, these cells also influenced Th1 CD4+ T cell priming. In summary, we propose a model in which broad-based T cell-mediated responses upon vaccination can be maximized by codelivery of aggregated protein and TLR7/8 agonist, which together promote optimal antigen acquisition and presentation by multiple DC subsets in the context of critical proinflammatory cytokines.     

Induction of CD4(+) T cell-dependent antitumor immunity by TAT-mediated tumor antigen delivery into dendritic cells

Dendritic cell-based (DC-based) immunotherapy represents a promising approach to the prevention and treatment of many diseases, including cancer, but current strategies have met with only limited success in clinical and preclinical studies. Previous studies have demonstrated that a TAT peptide derived from the HIV TAT protein has the ability to transduce peptides or proteins into various cells. Here, we describe the use of TAT-mediated delivery of T cell peptides into DCs to prolong antigen presentation and enhance T cell responses. While immunization of mice with DCs pulsed with an antigenic peptide derived from the human TRP2 protein generated partial protective immunity against B16 tumor, immunization with DCs loaded with a TAT-TRP2 peptide resulted in complete protective immunity, as well as significant inhibition of lung metastases in a 3-day tumor model. Although both DC/TRP2 and DC/TAT-TRP2 immunization increased the number of TRP2-specific CD8(+) T cells detected by K(b)/TRP2 tetramers, T cell activity elicited by DC/TAT-TRP2 was three- to tenfold higher than that induced by DC/TRP2. Furthermore, both CD4(+) and CD8(+) T cells were required for antitumor immunity demonstrated by experiments with antibody depletion of subsets of T cells, as well as with various knockout mice. These results suggest that a TAT-mediated antigen delivery system may have important clinical applications for cancer therapy.