肿瘤疫苗及其应用研究

随着肿瘤免疫学、疫苗新技术及分子医学等学科的不断发展，肿瘤疫苗研究与应用倍受关注。目前已有多种形式肿瘤疫苗进行了动物实验和临床研究，并取得了可喜成果。本文就近年来肿瘤疫苗的制备方法、作用机制及临床应用现状进行综述。     

白血病瘤苗对荷瘤小鼠细胞免疫功能影响的研究

目的：探讨白血病细胞疫苗主动免疫治疗在血液恶性肿瘤中的作用。方法：建立红白血病荷瘤小鼠,用自制的3种不同的白血病细胞瘤苗进行主动免疫治疗,用MTT比色法测其治疗2周和4周的Mφ和CTL的杀伤活性,并与对照组相比较。结果：（1）随着肿瘤细胞在小鼠体内的增长,小鼠的细胞免疫功能受到严重抑制,特异性细胞免疫功能的抑制滞后于非特异性细胞免疫功能。（2）灭活肿瘤细胞＋IFA＋细胞因子（rGM-CSF rIL-2 rIL-6)的肿瘤疫苗在提高荷瘤小鼠的细胞免疫功能方面,尤其是特异性细胞免疫功能方面优于灭活肿瘤细胞＋IFA肿瘤疫苗,而仅含灭活肿瘤细胞的疫苗作用不明显,结论：灭活肿瘤细胞＋IFA＋细胞因子（rGM-CSF rIL-2 rIL-6)的肿瘤疫苗在血液恶性肿瘤的特异性主动免疫治疗中很有潜力。     

肿瘤树突状细胞疫苗治疗的临床研究进展

树突状细胞(Dendritic cell,DC)作为功能最强的抗原递呈细胞,具有摄取、加工抗原、以MGC-Ⅰ、Ⅱ类肽结合物的形式递呈抗原的能力,在天然免疫和获得性免疫中都发挥着极其重要的作用.     

以树突状细胞为基础的肿瘤疫苗研究进展

树突状细胞作为一种目前已知的功能最强的抗原提呈细胞，可以体内外向Ｔ细胞提呈抗原并诱发ＣＴＬ反应。９０年代开始，ＤＣ在肿瘤免疫治疗中的作用逐渐受到重视。近年来，在ＤＣ的基础和临床应用研究方面取得了一些突破性的进展。     

肿瘤疫苗研究的现状

随着分子免疫学、分子生物学及基因工程技术的迅速发展,肿瘤疫苗的研究取得了令人鼓舞的成果.目前,肿瘤疫苗类型主要有以下几种.     

肿瘤生物治疗进展（三）——肿瘤疫苗

受疫苗在感染性疾病治疗中的影响，肿瘤疫苗在本世纪初开始使用于临床。有所不同的是：前者一般用于预防．而后者往往是治疗的目的。两种疫苗均是使用减毒的全细胞、细胞壁、特异性抗原或非致病性的活微生物来刺激病人的免疫系统。用肿瘤疫苗进行主动免疫治疗的目的是：克服因肿瘤产物造成的免疫抑制状态；刺激特异性免疫来攻击肿瘤细胞；增强肿瘤相关抗原(TAA)的免疫原性。     

替莫唑胺缓释微球局部植入治疗大鼠脑胶质瘤的疗效

目的：研究替莫唑胺缓释微球（temozolomide/PLGA microsphere, TMMS）局部植入对大鼠C6胶质瘤的治疗效果。方法：将C6大鼠胶质瘤细胞接种于鼠脑左侧尾状核,制备大鼠脑胶质瘤模型。分别用替莫唑胺（temozolomide, TM）口服及TMMS肿瘤局部植入治疗,观察大鼠的一般情况、生存期、肿瘤体积大小、病理学变化;免疫组织化学染色检测胶质瘤组织中增殖细胞核抗原（proliferadion cell nuclear antigen, PCNA）蛋白的表达;TUNEL法检测胶质瘤组织细胞的凋亡。结果：TMMS治疗大鼠的生存期较假手术组、空载体组、替莫唑胺口服组明显延长\[（31.2±6.21）d vs （20.7±4.83）、（19.2±6.23）、（24.7±6.31）d;P<0.05或P<0.01\]。MRI检查显示经TMMS治疗后脑内瘤灶体积较假手术组、空载体组、替莫唑胺口服组明显缩小\[（28.8±6.41）mm3 vs （56.4±6.92）、（58.2±5.36）、（46.7±7.28）mm3;P<0.05或P<0.01\];TMMS治疗后肿瘤细胞PCNA表达率较假手术组、空载体组、替莫唑胺口服组显著降低\[（20.2±4.33）% vs （63.2±5.91）%、（62.1±7.88）%、（41.7±6.71）%;P<001\],细胞凋亡率也明显增高\[（32.31±317）% vs （8.63±1.52）%、（9.25±2.31）%、（16.14±3.42）%;P<0.01\]。结论：TMMS局部植入治疗大鼠脑胶质瘤能显著抑制脑胶质瘤细胞增殖、诱导肿瘤细胞凋亡、延长大鼠生存期,具有潜在的临床应用价值。     

RNA转染树突状细胞肿瘤疫苗的研究进展

树突状细胞(DC)是体内功能最强的专职抗原递呈细胞,能激发并维持机体的初始免疫反应。通过不同方式将肿瘤抗原信息负载DC制备肿瘤疫苗是肿瘤免疫治疗的一个重要方面。而通过RNA转染DC制备核酸肿瘤疫苗是最近发展起来的新型制备疫苗的方法。现对RNA-DC 疫苗的研究进展作一综述。     

RNA转染树突状细胞肿瘤疫苗的研究进展

树突状细胞（DC）是体内功能最强的专职抗原递呈细胞,能激发并维持机体的初始免疫反应。通过不同方式将肿瘤抗原信息负载DC制备肿瘤疫苗是肿瘤免疫治疗的一个重要方面。而通过RNA转染DC制备核酸肿瘤疫苗是最近发展起来的新型制备疫苗的方法。现对RNA—DC疫苗的研究进展作一综述。     

肿瘤疫苗研究的回顾与展望

190 2年利用肿瘤疫苗 (tumor vaccine,简称瘤苗 )对癌症患者进行免疫治疗的尝试标志着肿瘤免疫学的开端。近一个世纪来有关瘤苗的研究始终是围绕着肿瘤的抗原性及机体的抗肿瘤免疫应答这个肿瘤免疫学的核心问题展开的。在世纪之交对肿瘤疫苗的研究、应用与开发作一回顾与展望可能是有益的。1　百年历史回顾1 .1　早期阶段 (上世纪初～中叶 ) :盲目尝试在基础免疫学 (P.Ehrlich,E.Metchinikoff等 )特别是抗感染免疫治疗成就 (von Behring对白喉的抗毒素血清治疗获首届诺贝尔生理学及医学奖 ,1 90 1 )的启发与推动下 ,1 90 2年 Leyden和 Bl…     

Heat shock protein 105 peptide vaccine could induce antitumor immune reactions in a phase I clinical trial

Heat shock protein 105 (HSP105) is overexpressed in many cancers, including colorectal cancer (CRC) and esophageal cancer (EC). We carried out a phase I clinical trial of HLA‐A24‐ and HLA‐A2‐restricted HSP105 peptide vaccines in patients with CRC or EC. In this additional study of the trial, we examined the immunological efficacy of the novel vaccine. Thirty patients with advanced CRC or EC underwent HSP105 peptide vaccination. Immunological responses were evaluated by ex vivo and in vitro γ‐interferon enzyme‐linked immunospot assays and their correlation with patients’ prognosis was analyzed. The HSP105 peptide vaccines induced peptide‐specific CTLs in 15 of 30 patients. Among HLA‐A24 patients (n = 15), 7 showed induction of CTLs only ex vivo, whereas among HLA‐A2 patients (n = 15), 4 showed the induction ex vivo and 6 in vitro. Heat shock protein 105‐specific CTL induction correlated with suppression of cancer progression and was revealed as a potential predictive biomarker for progression‐free survival (P = .008; hazard ratio = 3.03; 95% confidence interval, 1.34‐6.85) and overall survival (P = .025; hazard ratio = 2.72; 95% confidence interval, 1.13‐6.52). Production of cytokines by HSP105 peptide‐specific CTLs was observed at the injection sites (skin) and tumor tissues, suggesting that HSP105‐specific CTLs not only accumulated at vaccination sites but also infiltrated tumors. Furthermore, we established 2 HSP105 peptide‐specific CTL clones, which showed HSP105‐specific cytokine secretion and cytotoxicity. Our results suggest that the HSP105 peptide vaccine could induce immunological effects in cancer patients and improve their prognosis.     

Induction of protective and therapeutic antitumor immunity by a DNA vaccine with a glioma antigen, SOX6

We previously reported identifying SOX6 as a glioma antigen by serological screening using a testis cDNA library. Its preferential expression and frequent IgG responses in glioma patients indicate that SOX6 may be a useful target for immunotherapy. To examine whether cytotoxic T-lymphocyte (CTL) responses specific for SOX6 to destroy glioma can be generated in vivo, we treated glioma-bearing mice by vaccination with a plasmid DNA encoding murine full-length SOX6 protein. Following SOX6-DNA vaccination, CTLs specific for SOX6-expressing glioma cells were induced, while normal autologous-cells that had restrictedly expressed SOX6 during embryogenesis were not destroyed. Furthermore, DNA vaccination with SOX6 exerted protective and therapeutic antitumor responses in the glioma-bearing mice. This antitumor activity was abrogated by the depletion of CD4 positive T cells and/or CD8 positive T cells. These results suggest that the SOX6 protein has multiple CTL and helper epitopes to induce antitumor activity and the effectiveness of SOX6-DNA vaccine for the prevention and treatment of glioma.     

Polyomavirus-driven Merkel cell carcinoma: Prospects for therapeutic vaccine development

Great strides have been made in cancer immunotherapy including the breakthrough successes of anti-PD-(L)1 checkpoint inhibitors. In Merkel cell carcinoma (MCC), a rare and aggressive skin cancer, PD-(L)1 blockade is highly effective. Yet, ~50% of patients either do not respond to therapy or develop PD-(L)1 refractory disease and, thus, do not experience long-term benefit. For these patients, additional or combination therapies are needed to augment immune responses that target and eliminate cancer cells. Therapeutic vaccines targeting tumor-associated antigens, mutated self-antigens, or immunogenic viral oncoproteins are currently being developed to augment T-cell responses. Approximately 80% of MCC cases in the United States are driven by the ongoing expression of viral T-antigen (T-Ag) oncoproteins from genomically integrated Merkel cell polyomavirus (MCPyV). Since T-Ag elicits specific B- and T-cell immune responses in most persons with virus-positive MCC (VP-MCC), and ongoing T-Ag expression is required to drive VP-MCC cell proliferation, therapeutic vaccination with T-Ag is a rational potential component of immunotherapy. Failure of the endogenous T-cell response to clear VP-MCC (allowing clinically evident tumors to arise) implies that therapeutic vaccination will need to be potent anśd synergize with other mechanisms to enhance T-cell activity against tumor cells. Here, we review the relevant underlying biology of VP-MCC, potentially applicable therapeutic vaccine platforms, and antigen delivery formats. We also describe early successes in the field of therapeutic cancer vaccines and address several clinical scenarios in which VP-MCC patients could potentially benefit from a therapeutic vaccine.     

Recombinant modified vaccinia Ankara primes functionally activated CTL specific for a melanoma tumor antigen epitope in melanoma patients with a high risk of disease recurrence

Recombinant plasmid DNA and attenuated poxviruses are under development as cancer and infectious disease vaccines. We present the results of a phase I clinical trial of recombinant plasmid DNA and modified vaccinia Ankara (MVA), both encoding 7 melanoma tumor antigen cytotoxic T lymphocyte (CTL) epitopes. HLA-A*0201-positive patients with surgically treated melanoma received either a "prime-boost" DNA/MVA or a homologous MVA-only regimen. Ex vivo tetramer analysis, performed at multiple time points, provided detailed kinetics of vaccine-driven CTL responses specific for the high-affinity melan-A(26-35) analogue epitope. Melan-A26-35-specific CTL were generated in 2/6 patients who received DNA/MVA (detectable only after the first MVA injection) and 4/7 patients who received MVA only. Ex vivo ELISPOT analysis and in vitro proliferation assays confirmed the effector function of these CTL. Responses were seen in smallpox-vaccinated as well as vaccinia-naive patients, as defined by anti-vaccinia antibody responses demonstrated by ELISA assay. The observations that 1) CTL responses were generated to only 1 of the recombinant epitopes and 2) that the magnitude of these responses (0.029-0.19% CD8(+) T cells) was below the levels usually seen in acute viral infections suggest that to ensure high numbers of CTL specific for multiple recombinant epitopes, a deeper understanding of the interplay between CTL responses specific for the viral vector and recombinant epitopes is required.     

Immunization with tumor-derived ER chaperone grp170 elicits tumor-specific CD8+ T-cell responses and reduces pulmonary metastatic disease

Glucose-regulated protein (grp) 170 is a molecular chaperone localized in endoplasmic reticulum (ER), which has been demonstrated to interact with the peptides translocated by transporter associated with antigen presentation (TAP). In our study, we have evaluated the therapeutic efficacy of tumor-derived grp170 against the highly metastatic and poorly immunogenic murine melanoma B16F10. Immunization of mice with grp170 preparations from autologous tumor significantly delayed progression of the primary cancer and reduced established pulmonary metastases, which was associated with the prolonged survival of metastases-bearing mice. However, grp170 from normal liver or antigenically distinct tumor failed to exhibit therapeutic effect. In addition, tumor-derived grp170 elicited a potent cytotoxic T-lymphocyte response specific for B16F10 tumor, which correlates with in vivo protective effects. Adoptive transfer of splenocytes obtained from B16F10-grp170-primed animals remarkably suppressed pulmonary metastases. Depletion of either CD4(+) or CD8(+) T cells in priming phase significantly abrogated the tumor immunity induced by the B16F10-grp170. However, the vaccine activity was intact when CD4(+), not CD8(+), T cells were depleted in effector phase. It suggests that CD4(+) T helper cells play an important role in the generation of effective antitumor response, whereas CD8(+) T cells are predominantly involved in direct killing of tumor cells. These observations have strong clinical implications for using tumor-derived grp170 as a therapeutic vaccine against metastatic diseases.     

Administration route-dependent vaccine efficiency of murine dendritic cells pulsed with antigens

Dendritic cells (DCs) loaded with tumour antigens have been successfully used to induce protective tumour immunity in murine models and human trials. However, it is still unclear which DC administration route elicits a superior therapeutic effect. Herein, we investigated the vaccine efficiency of DC2.4 cells, a murine dendritic cell line, pulsed with ovalbumin (OVA) in the murine E.G7-OVA tumour model after immunization via various routes. After a single vaccination using 1 x 10(6)OVA-pulsed DC2.4 cells, tumour was completely rejected in the intradermally (i.d.; three of four mice), subcutaneously (s.c.; three of four mice), and intraperitoneally (i.p.; one of four mice) immunized groups. Double vaccinations enhanced the anti-tumour effect in all groups except the intravenous (i.v.) group, which failed to achieve complete rejection. The anti-tumour efficacy of each immunization route was correlated with the OVA-specific cytotoxic T lymphocyte (CTL) activity evaluated on day 7 post-vaccination. Furthermore, the accumulation of DC2.4 cells in the regional lymph nodes was detected only in the i.d.-and s.c.-injected groups. These results demonstrate that the administration route of antigen-loaded DCs affects the migration of DCs to lymphoid tissues and the magnitude of antigen-specific CTL response. Furthermore, the immunization route affects vaccine efficiency.     

Efficacy of the NCCV Cocktail‐1 vaccine for refractory pediatric solid tumors: A phase I clinical trial

Pediatric refractory solid tumors are aggressive malignant diseases, resulting in an extremely poor prognosis. KOC1, FOXM1, and KIF20A are cancer antigens that could be ideal targets for anticancer immunotherapy against pediatric refractory solid tumors with positive expression for these antigens. This nonrandomized, open‐label, phase I clinical trial evaluated the safety and efficacy of the NCCV Cocktail‐1 vaccine, which is a cocktail of cancer peptides derived from KOC1, FOXM1, and KIF20A, in patients with pediatric refractory solid tumors. Twelve patients with refractory pediatric solid tumors underwent NCCV Cocktail‐1 vaccination weekly by intradermal injections. The primary endpoint was the safety of the NCCV Cocktail‐1 vaccination, and the secondary endpoints were the immune response, as measured by interferon‐r enzyme‐linked immunospot assay, and the clinical outcomes including tumor response and progression‐free survival. The NCCV Cocktail‐1 vaccine was well tolerated. The clinical response of this trial showed that 4 patients had stable disease after 8 weeks and 2 patients maintained remission for >11 months. In 4, 8, and 5 patients, the NCCV Cocktail‐1 vaccine induced the sufficient number of peptide‐specific CTLs for KOC1, FOXM1, and KIF20A, respectively. Patients with high peptide‐specific CTL frequencies for KOC1, FOXM1, and KIF20A had better progression‐free survival than those with low frequencies. The findings of this clinical trial showed that the NCCV Cocktail‐1 vaccine could be a novel therapeutic strategy, with adequate effects against pediatric refractory solid tumors. Future large‐scale trials should evaluate the efficacy of the NCCV Cocktail‐1 vaccination.     

Protective antitumor immunity induced by tumor cell lysates conjugated with diphtheria toxin and adjuvant epitope in mouse breast tumor models

Cancer cell vaccine-based immunotherapy has received increasing interest in many clinical trials involving patients with breast cancer. Combining with appropriate adjuvants can enhance the weak immunogenic properties of tumor cell lysates (TCL). In this study, diphtheria toxin (DT) and two tandem repeats of mycobacterial heat shock protein 70 (mHSP70) fragment 407-426 (M2) were conjugated to TCL with glutaraldehyde, and the constructed cancer cell vaccine was named DT-TCL-M2. Subcutaneous injection of DT-TCL-M2in mice effectively elicited tumor-specific polyclonal immune responses, including humoral and cellular immune responses. High levels of antibodies against TCL were detected in the serum of immunized mice with ELISA and verified with Western blot analyses. The splenocytes from immunized mice showed potent cytotoxicity on Ehrlich ascites carcinoma cells. Moreover, the protective antitumor immunity induced by DT-TCL-M2 inhibited tumor growth in a mouse breast tumor model. DT-TCL-M2 also attenuated tumor-induced angiogenesis and slowed tumor growth in a mouse intradermal tumor model. These findings demonstrate that TCL conjugated with appropriate adjuvants induced effective antitumor immunity in vivo. Improvements in potency could further make cancer cell vaccines a useful and safe method for preventing cancer recurrence after resection.     

A tumor lysate is an effective vaccine antigen for the stimulation of CD4+ T-cell function and subsequent induction of antitumor immunity mediated by CD8+ T cells

To develop a potent cancer vaccine, it is important to study how to prepare highly immunogenic antigens and to identify the most appropriate adjuvants for the antigens. Here we show that a tumor lysate works as an effective antigen to prime CD4⁺ T-cell help when baculovirus is employed as an adjuvant. When immunized intradermally with the combination (BLP) of baculovirus, a CT26 tumor lysate, and a cytotoxic T-cell epitope peptide before a tumor challenge, 60% of mice rejected tumors. In contrast, all mice vaccinated with baculovirus plus a tumor lysate (BL) developed tumors. In addition, flow cytometry showed that tumor-specific, interferon γ-producing CD8⁺ cytotoxic T lymphocytes (CTLs) were robustly activated by intradermal immunization with BLP. When BLP was administered therapeutically to tumor-bearing mice, antitumor efficacy was better compared to BL. The established tumor was completely eradicated in 50–60% of BLP-treated mice, and induction of tumor-specific CTLs was observed, suggesting that the antitumor efficacy of BLP is mediated by CD8⁺ T cells. Numerous CD4⁺ T cells infiltrated the tumors of BLP-treated mice, whereas the antitumor effect of BLP almost disappeared after removal of the tumor lysate from BLP or after depletion of BLP-immunized mice of CD4⁺ T cells. Thus, the combination of a peptide, lysate, and baculovirus provides stronger antitumor immunity than does a peptide plus baculovirus or a lysate plus baculovirus; effectiveness of BLP is determined by functioning of CD4⁺ T cells stimulated with a tumor lysate.