DNA疫苗诱导的小鼠抗肿瘤特异性细胞免疫和体液免疫

目的:检测以双表达质粒pIRES为载体构建的带有全序列癌胚抗原(CEA)基因和IL-2基因的核苷酸疫苗对机体特异性抗肿瘤免疫反应的激活效果。方法:利用分子生物学技术,将CEA基因片段和IL-2基因片段连接于真核双表达质粒pIRES中,用肌内注射方法接种核酸疫苗;检测疫苗在小鼠肌肉组织中的表达情况及其对小鼠脾细胞CEA特异性细胞免疫和体液免疫反应的激活效果。结果:小鼠经肌内注射质粒后,免疫组化证实该核酸疫苗可在体内有效表达CEA分子;免疫检测显示注射后小鼠血清中出现抗CEA抗体,脾细胞IL-4分泌增强,特异性淋巴细胞增值反应明显并伴有自然杀伤细胞NK活性显著增高。结论:实验所构建的核酸疫苗pIRES-CEA,pIRES-IL-2,pIRES-IL-2等可在小鼠体内高效表达并表现出良好的免疫原性。     

Human autoantibodies to carcinoembryonic antigen (CEA) induced by a vaccinia-CEA vaccine.

Carcinoembryonic antigen (CEA) is a well-characterized oncofetal glycoprotein whose overexpression by human adenocarcinomas has been a target for cancer immunotherapy. Limited information is available regarding the ability of patients to mount an antibody response to this self-antigen following vaccination. Recombinant vaccinia viruses encoding full-length or internally deleted cDNAs for human CEA were used to vaccinate 32 patients with CEA-expressing adenocarcinomas, predominantly of colorectal origin. CEA-specific autoantibodies were induced by vaccination in 7 of 32 patients. None of the patients had CEA antibodies detected before vaccination. CEA specificity of the antibodies initially identified by ELISA was confirmed by competitive inhibition analysis as well as recognition of recombinant CEA produced in baculovirus-infected insect cell cultures and human cell cultures by Western blot. The CEA autoantibodies were predominantly IgG1, with a minority of patients also demonstrating IgM autoantibodies. CEA antibodies were of low titer and low avidity, based on competitive inhibition assays. These autoantibodies did not affect clinical serum CEA protein quantitation. Furthermore, elevated serum CEA levels commonly encountered in patients with advanced adenocarcinoma did not hinder detection of low avidity polyclonal CEA antibodies. CEA antibodies such as those induced in these pilot trials are projected to have modest antitumor activity. Thus, additional Phase I/II trials of recombinant vaccinia-CEA with alternative prime-boost approaches and/or augmentation strategies are warranted in an effort to enhance the frequency and avidity of CEA-specific autoantibodies and cytolytic T cells before Phase III trials.     

癌胚抗原与γ-干扰素核酸疫苗对机体特异性细胞免疫的激活作用

目的检测以双表达质粒pIRES为载体构建的带有全序列癌胚抗原(carcinoembry-onic antigens,CEA)基因和γ-干扰素(interfer-on-γ,IFN-γ)基因的核苷酸疫苗对机体特异性抗肿瘤免疫反应的激活效果。方法利用分子生物学技术,将CEA基因片段和IFN-γ基因片段连接于真核双表达质粒pIRES中,用肌肉注射方法接种核酸疫苗;检测疫苗在小鼠肌肉组织中的表达及其对小鼠脾细胞CEA特异性细胞免疫反应的激活效果。结果小鼠经肌肉注射质粒后,核酸疫苗可在体内有效表达CEA和IFN-γ分子;小鼠特异性淋巴细胞增殖反应明显增高,并且伴有自然杀伤细胞(natural killercell,NK细胞)活性显著增高,F=15289·67,P<0·001。结论实验所构建的核酸疫苗pIRES-CEA、pIRES-CEA-IFN-γ和pIRES-CEA-IFN-γ等可在小鼠体内高效表达,并表现出良好的细胞免疫原性。     

带免疫佐剂CEA基因疫苗的构建及其稳定同步表达的研究

背景与目的：基因疫苗是目前肿瘤免疫基因治疗研究的热点。目前癌胚抗原阳性肿瘤的治疗效果不佳,本研究拟利用质粒pVAX1构建带有免疫佐剂白细胞介素2同步表达的癌胚抗原(carcinoembryonic antigen,CEA)基因疫苗,探讨一种新的肿瘤生物治疗方法。方法：利用RT-PCR从患者肿瘤组织中钓取CEA目的基因cDNA;运用分子克隆技术,通过内部核糖体进入位点(IRES),将CEA cDNA和hIL-2 cDNA连接于质粒pVAX1中,采用化学发光免疫法和ELISA双抗体夹心法分别检测CEA抗原和hIL-2因子的表达;同时用蛋白质分析软件分析和预测目的抗原的特性。结果：测序结果证实本实验所构建的重组质粒插入DNA序列无错配,质粒上所接入的CEAcDNA与GenBank公布的M29540和M17303的序列99．8％同源,经蛋白质分析软件预测,其抗原性、跨膜结构片段、信号肽位点和二、三级结构特征与同源癌胚抗原基本一致。hIL-2 cDNA与母本序列100％同源。检测结果说明该重组质粒在体外CHO细胞株中可同步表达CEA和hIL-2分子。结论：从肿瘤组织中成功地获取了CEA cDNA,所构建的pVCEA2重组质粒能在体外细胞中同步表达CEA和IL-2蛋白分子,这为进一步体内实验研究基因治疗CEA阳性肿瘤提供了新的可能途径。     

Natural antibodies against the carcinoembryonic antigen (CEA) and a related antigen, NCA, in human sera

Natural antibodies directed against CEA and a related antigen, NCA, have been demonstrated in all normal and pathological sera using an EIA, although they have never been detected by RIA. These antibodies are not anti-blood group antibodies, as their titer was decreased only slightly by absorption with blood group substances. The study of their reactivity with deglycosylated antigens demonstrated that they were directed against peptidic epitopes. Antibodies against NCA or CEA, purified using specific immunosorbents, cross-reacted with all the antigens of the "CEA family" but not or only weakly with unrelated antigens.     

Dendritic cells pulsed with an anti-idiotype antibody mimicking carcinoembryonic antigen (CEA) can reverse immunological tolerance to CEA and induce antitumor immunity in CEA transgenic mice

In this report, we have studied the immunogenicity of the nominal antigen, carcinoembryonic antigen (CEA), and that of an anti-idiotype antibody, 3H1, which mimics CEA and can be used as a surrogate for CEA. We have demonstrated that immunization of CEA transgenic mice with bone marrow-derived mature dendritic cells (DC) loaded with anti-idiotype 3H1 or CEA could reverse CEA unresponsiveness and result in the induction of CEA-specific immune responses and the rejection of CEA-transfected MC-38 colon carcinoma cells, C15. Immunized mice splenocytes proliferated in an antigen-specific manner by a mechanism dependent on the functions of CD4, MHC II, B7-2, CD40, CD28, and CD25. However, immune splenic lymphocytes isolated from 3H1-DC-vaccinated mice when stimulated in vitro with 3H1 or CEA secreted significantly higher levels of Th1 cytokines than did CEA-DC vaccinated mice. DC vaccination also induced antigen-specific effector CD8+ T cells capable of expressing interleukin-2, IFN-gamma, and tumor necrosis factor (TNF)-alpha and displayed cytotoxic activity against C15 cells in an MHC class I-restricted manner. 3H1-DC vaccination resulted in augmented CTL responses and the elevated expression of CD69, CD25, and CD28 on CD8(+) CTLs. The immune responses developed in 3H1-DC-immunized mice resulted in rejection of C15 tumor cells in nearly 100% of experimental mice, whereas only 40% of experimental mice immunized with CEA-DC were protected from C15 tumor growth. These findings suggest that under the experimental conditions used, 3H1-DC vaccination was better than CEA-DC vaccination in breaking immune tolerance to CEA and inducing protective antitumor immune responses in this murine model transgenic for human CEA.     

Durable carcinoembryonic antigen (CEA)-specific humoral and cellular immune responses in colorectal carcinoma patients vaccinated with recombinant CEA and granulocyte/macrophage colony-stimulating factor.

PURPOSE: Previous studies have indicated that carcinoembryonic antigen (CEA) might be a suitable immunotherapeutic target in colorectal carcinoma (CRC). The aim of the present study was to analyze the immunological and clinical effects of vaccination with CEA together with the adjuvant granulocyte/macrophage colony-stimulating factor (GM-CSF). EXPERIMENTAL DESIGN: Twenty-four resected CRC patients without macroscopic disease were immunized seven times with recombinant CEA at four different dose levels over a 12-month period. Half of the patients received GM-CSF (80 microg/day for 4 consecutive days) at each immunization. Patients were monitored immunologically for 36 months and clinically for 76 months. T-cell response was evaluated by a [(3)H]thymidine incorporation assay, and IgG response was determined by ELISA. RESULTS: Minor local side effects were common. All 12 patients (100%) in the GM-CSF group developed a CEA-specific T-cell as well as an IgG response. The corresponding figures in the CEA alone group were 9 of 12 (75%) and 8 of 12 (66%), respectively. GM-CSF significantly augmented the amplitude of the T-cell response and the IgG titers. No dose-response relationship was noted. The immune responses at 12 months persisted 24 months after the last vaccination. Anti-CEA IgG titers were associated with increased survival (P < 0.05), whereas standard prognostic factors had no relationship, with the exception of serum CEA value. CONCLUSIONS: Vaccination with recombinant CEA and GM-CSF appears to be a nontoxic regimen inducing potent and durable antigen-specific IgG and T-cell response. The results of this study justify more extensive trials with recombinant CEA protein for immunotherapy of CRC.     

Fully human IgG and IgM antibodies directed against the carcinoembryonic antigen (CEA) Gold 4 epitope and designed for radioimmunotherapy (RIT) of colorectal cancers

Background  

Human monoclonal antibodies (MAbs) are needed for colon cancer radioimmunotherapy (RIT) to allow for repeated injections. Carcinoembryonic antigen (CEA) being the reference antigen for immunotargeting of these tumors, we developed human anti-CEA MAbs.     

Establishment and characterization of a carcinoembryonic antigen (CEA)-producing cell line from a human carcinoma of the exocrine pancreas

A human carcinoembryonic antigen (CEA)-producing cell line, T3M-4, has been established from explant cultures of a primary human pancreatic exocrine adenocarcinoma transplanted into nude mice. The tumor had metastasized in the patient. The tumor obtained from metastatic lymph nodes was the initial source for implantation in athymic nude mice. In the primary culture, host fibroblasts were eliminated by the use of the antiserum raised against nude mouse cells. T3M-4 cells have been continuously propagated in vitro during the past 26 months. The cells grew in a monolayered sheet with about 31 hours of population doubling time. The cells exhibited epithelial morphologic features resembling the structure of the original tumor, and they showed tumor takes when inoculated into athymic nude mice. Xenografts established from the cell line have retained a similar histology to the original tumor on serial transplantation. Chromosomal analysis revealed the cell line to be a human aneuploid one with a hyperdiploid mode. T3M-4 cells possess the characteristic function of CEA secretion in vitro in culture and in vivo in nude mice bearing the tumors produced by inoculation with the cultured cells. In view of these characteristics, T3M-4 cell line represents a new human pancreatic exocrine adenocarcinoma cell line that produces CEA.     

LL-37 enhances adaptive antitumor immune response in a murine model when genetically fused with M-CSFR (J6-1) DNA vaccine

DNA vaccine against M-CSFR(J6-1) (macrophage colony-stimulating factor receptor cloned from the J6-1 leukemic cell line) has shown both protective and therapeutic effects. In this study, to explore the adjuvant effects of LL-37 to M-CSFR(J6-1) DNA vaccines, we constructed genetically fused vaccines encoding M-CSFR(J6-1) and LL-37(pF). After immunizing BALB/c mice, specific humoral and cellular immune responses were detected. Compared with pR (encoding the extracellular region of M-CSFR(J6-1)), pF was more effective in inducing humoral and cytotoxic immune response, prolonging survival of mice challenged with SP2/0-CSFR(J6-1) tumor cells, and inducing IFN-gamma and IL-4 release by splenocytes. In this study, we also constructed pLL37 (encoding the mature LL-37) and coadministrated pLL37 and pR to see whether the genetic fusion was necessary. We found that compared with pR alone, pLL37+pR could not prolong survival of mice challenged with SP2/0-CSFR(J6-1) tumor cells. Our results suggest that when genetically fused with M-CSFR(J6-1), LL-37 could enhance adaptive immune response against M-CSFR(J6-1) in a murine model challenged with tumor cells bearing M-CSFR(J6-1).     

Induction of immune responses and safety profiles in rhesus macaques immunized with a DNA vaccine expressing human prostate specific antigen

Prostate specific antigen (PSA) is a widely used marker for prostate cancer, which is secreted by normal prostate cells at low levels, but is produced more substantially by cancer cells. We have previously reported on the use of a DNA vaccine construct that encodes for human PSA gene to elicit host immune responses against cells producing PSA. DNA immunization strategy delivers DNA constructs encoding for a specific immunogen into the host, who becomes the in vivo protein source for the production of antigen. This antigen then is the focus of the resulting immune response. In this study, we examine the induction of immune responses and safety profiles in rhesus macaques immunized with DNA-based PSA vaccine. We observed induction of PSA-specific humoral response as well as positive PSA-specific lymphoproliferative (LPA) response in the vaccinated macaques. We also observed that the stimulated T cells from the PSA-immunized rhesus macaques produced higher levels of Th1 type cytokine IFN-gamma than the control vector immunized animals. On the other hand, DNA immunization did not result in any adverse effects in the immunized macaques, as indicated by complete blood counts, leukocyte differentials and hepatic and renal chemistries. The macaques appeared healthy, without any physical signs of toxicity throughout the observation period. In addition, we did not observe any adverse effect on the vaccination site. The apparent safety and immunogenecity of DNA immunization in this study suggest that further evaluation of this vaccination strategy is warranted.     

Streptococcal preparation OK-432 promotes the capacity of dendritic cells (DCs) to prime carcinoembryonic antigen (CEA)-specific cytotoxic T lymphocyte responses induced with genetically modified DCs that express CEA

Cancer immunotherapy using dendritic cells (DCs) adenovirally transduced with the whole tumor-associated antigen (TAA) gene is an effective approach. Streptococcal preparation OK-432 is useful for stimulating DCs in terms of maturation. In this study, we established carcinoembryonic antigen (CEA)-specific cytotoxic T lymphocytes (CTLs) using in vitro stimulation with adenovirally modified human DCs that express CEA. We investigated whether OK-432 stimulation could be more effective in inducing CEA-specific CTLs compared with other typical stimuli. DCs adenovirally transduced with the CEA gene were cultured under various conditions with tumor necrosis factor (TNF)-alpha, lipopolysaccharide (LPS), or OK-432. A cytotoxicity assay using peripheral blood mononuclear cell (PBMC)-derived CTLs was performed in a 4 h-51Cr release assay. OK-432 stimulated immature DCs to acquire a mature phenotype and to produce significant amounts of T-helper 1 cytokines. In all groups (immature DCs, TNF-alpha/DCs, LPS/DCs, OK-432/DCs), CEA-specific CTLs were generated. OK-432-stimulated DCs (HLA-A24) induced the most potent cytotoxic activity against CEA-expressing targets (A24) but not against controls. OK-432/DCs were able to induce markedly potent CTLs specific to target cells pulsed with CEA652 peptide (HLA-A24-restricted peptide), although others failed to induce potent CTLs. In conclusion, the CTL induction protocol using adenovirally modified DCs that express CEA after maturation with OK-432 showed a potent antitumor activity against CEA-expressing target cells, and is therefore promising for clinical applications as a cancer vaccine therapy.     

Cell mediated cytotoxicity of human colon carcinoma cells by a monoclonal antibody (R4) recognizing the carcinoembryonic antigen (CEA) and CEA-related molecules

We report a novel anti-CEA monoclonal antibody (MAb) designated R4, which mediates antibody-dependent cell-mediated cytotoxicity (ADCC) of human colon carcinoma cells and displays differential reactivity for human carcinomas versus the normal counterparts. R4 (IgG1) reacted with the cell surface of 6 colon carcinoma cell lines expressing CEA. Western blot analysis and epitope mapping using native and baculovirus recombinant CEA and non specific cross-reacting antigen (NCA) demonstrated that MAb R4 recognizes a proteinic epitope located on the 3' end of the domain I shared by CEA and NCA molecules. Immunohistochemical analysis demonstrated an intense staining of MAb R4 with the majority of the neoplastic tissues tested, including colon (13/13), stomach (2/2), breast (9/10), lung (7/10) and endometrial (2/4) carcinomas, whereas no reactivity with the correspondent normal tissues was observed. Using human PBLs from healthy donors as effector cells, we have shown that MAb R4 mediated antibody dependent-cell mediated cytotoxicity (ADCC) of human carcinoma cells LS-174T, CBS and WiDr. This activity was enhanced after PBLs activation with interleukin-2 (IL-2). The specificity of MAb R4 for an epitope shared by two tumor overexpressed antigens, CEA and NCA, resulting in an intense reactivity with neoplastic cells and the peculiar property to mediate ADCC, indicate that MAb R4 might be a novel powerful reagent for diagnostic and immunotherapy of carcinoma patients.     

Pilot study of a dual gene recombinant avipox vaccine containing both carcinoembryonic antigen (CEA) and B7.1 transgenes in patients with recurrent CEA-expressing adenocarcinomas.

Coordinated presentation of antigen and costimulatory molecules has been shown to result in the induction of an antigen-specific T-cell response rather than the development of anergy. This study evaluated the vaccine ALVAC-CEA B7.1, a canary pox virus that has been engineered to encode the gene for the tumor-associated antigen carcinoembryonic antigen (CEA) and B7.1, a T-cell costimulatory molecule. Patients with CEA-expressing tumors were immunized with 2.5 x 10(7) (n = 3), 1.0 x 10(8) (n = 6), and 4.5 x 10(8) (n = 30) plaque-forming units intradermally every other week for 8 weeks. Patients with stable or responding disease received monthly boost injections. Biopsies of vaccine sites were obtained 48 h after vaccination to evaluate leukocytic infiltration and CEA expression. Induction of CEA-specific T-cell precursors was assessed by an ELISPOT assay looking for the production of IFN-gamma. Therapy was well tolerated, without significant toxicity attributable to vaccine. All patients had evidence of leukocytic infiltration and CEA expression in vaccine biopsy sites. Six patients with elevated serum CEA values at baseline had declines in their levels lasting 4-12 weeks. These patients all had stable disease after four vaccinations. After four vaccinations, patients who were HLA-A-2-positive demonstrated increases in their CEA-specific T-cell precursor frequencies to a CEA-A2-binding peptide from baseline. The number of prior chemotherapy regimens was inversely correlated with the ability to generate a T-cell response. ALVAC-CEA B7.1 is safe in patients with advanced, recurrent adenocarcinomas that express CEA, and it is associated with the induction of a CEA-specific T-cell response.     

alpha-Fetoprotein (AFP), human chorionic gonadotropin (HCG), and carcinoembryonic antigen (CEA) demonstrated in the immature glands of mediastinal teratocarcinoma: a case report

A 21-year-old man with mediastinal teratocarcinoma showed high levels of serum tumor markers: alpha-fetoprotein (AFP), human chorionic gonadotropin (HCG), and carcinoembryonic antigen (CEA). After modified VAC chemotherapy (vincristine, dactinomycin, and cyclophosphamide), AFP and HCG levels in the serum were reduced significantly, while serum CEA increased gradually to the level of 6.5 ng/ml. Histopathologic study of the tumor obtained by surgical resection and autopsy revealed teratocarcinoma (a combination of embryonal carcinoma and teratoma). By the indirect immunoperoxidase technique, the antigens AFP, HCG, and CEA were stained in the immature glandular tissues of teratoma. By serial section of the tumor, the antigens tested were found in the same gland of the tumor. In addition, the tumor contained high levels of AFP (17353.8 ng/g.wt.), beta-HCG (beta-HCG) (125.05 ng/g.wt.), and CEA (11.37 ng/g.wt.).     

Specifically targeted killing of carcinoembryonic antigen (CEA)-expressing cells by a retroviral vector displaying single-chain variable fragmented antibody to CEA and carrying the gene for inducible nitric oxide synthase

The generation of retroviral vectors that infect specific cell types through recognition of cell surface antigens is a promising and effective approach to targeted gene therapy of cancer. Carcinoembryonic antigen (CEA), a highly characterized, cell surface glycoprotein overexpressed by various tumor cells, provides a specific tool for tumor tissue-specific targeting by retroviral vectors. The conventional suicidal gene delivery systems need additional drugs other than their gene products. The inducible nitric oxide synthase (iNOS) gene product yields nitric oxide (NO), which directly induces autocytotoxicity and cytolysis of bystander cells. In the present study, we have developed a novel bifunctional Moloney murine leukemia virus-based recombinant retroviral vector that displays a chimeric envelope protein containing a single-chain variable fragmented (scFv) antibody to CEA and carries the iNOS gene in the genome. The resultant bifunctional retroviral vector showed a specific delivery of the iNOS gene to human CEA-expressing carcinoma cells, resulting in the direct and efficient killing of CEA-expressing carcinoma cells by induction of apoptosis. This is the first report of successful killing of CEA-expressing cells by specific targeting of the iNOS gene. This approach may offer a one-step procedure for effective gene therapy of CEA-expressing tumors.     

Immunogenicity of a p210(BCR-ABL) fusion domain candidate DNA vaccine targeted to dendritic cells by a recombinant adeno-associated virus vector in vitro

Chronic myelogenous leukemia (CML) is characterized by a t(9;22) translocation, which results in the expression of chimeric BCR-ABL fusion oncoproteins that are necessary for oncogenesis, unique to the leukemic clones, and represent enticing targets for immunotherapy. As a strategy for the immunotherapy of CML, we constructed a recombinant adeno-associated virus vector encoding the p210(BCR-ABL) b3a2 variant fusion region with flanking sequences (CWRBA) and used it to express the BCR-ABL fusion region within primary human dendritic cells (DCs), the most potent antigen-presenting cells currently known. Peripheral blood mononuclear cells from healthy donors were primed and restimulated in vitro with autologous DCs transduced with purified CWRBA, CWRAP (negative control), or pulsed with a peptide corresponding to the fusion domain (positive control). No specific responses were generated using DCs transduced with CWRAP. In contrast, CWRBA-transduced DCs primed autologous T cells in an antigen-specific, MHC-restricted fashion to levels comparable with the positive control. CWRBA-transduced DCs elicited both cytotoxic CD4+/Th1 and CD8+ responses, although the former were more readily detected in this system. Cytotoxicity against a tumor cell line endogenously expressing the p210(BCR-ABL) b3a2 variant fusion region was also demonstrable. In addition, HLA-DRB5(*)0101+DRA (DR2a) was identified as a new restriction element capable of presenting the b3a2 BCR-ABL fusion region epitope. Thus, the construct developed herein may serve as a candidate vaccine for gene-based antigen-specific immunotherapy of CML and may serve as a paradigm for the use of DCs transduced with recombinant adeno-associated virus vectors encoding multiepitope immunogens for vaccine development.     

Serum‐derived carcinoembryonic antigen (CEA) activates fibroblasts to induce a local re‐modeling of the extracellular matrix that favors the engraftment of CEA‐expressing tumor cells

Elevated levels of the carcinoembryonic antigen (CEA; CEACAM5) in the serum of colorectal cancer (CRC) patients represent a clinical biomarker that correlates with disease recurrence. However, a mechanistic role for soluble CEA (sCEA) in tumor progression and metastasis remains to be established. In our study, we report that sCEA acts as a paracrine factor, activating human fibroblasts by signaling through both the STAT3 and AKT1‐mTORC1 pathways, promoting their transition to a cancer‐associated fibroblast (CaF) phenotype. sCEA‐activated fibroblasts express and secrete higher levels of fibronectin, including cellular EDA⁺‐fibronectin (Fn‐EDA) that selectively promote the implantation and adherence of CEA‐expressing cancer cells. Immunohistochemical analyses of liver tissues derived from CRC patients with elevated levels of sCEA reveal that the expression of cellular Fn‐EDA co‐registers with CEA‐expressing liver metastases. Taken together, these findings indicate a direct role for sCEA as a human fibroblast activation factor, in priming target tissues for the engraftment of CEA‐expressing cancer cells, through the differentiation of tissue‐resident fibroblasts, resulting in a local change in composition of the extracellular matrix.