Specific and randomly derived immunoactive peptide mimotopes of mycobacterial antigens

The mycobacterial cell surface contains complex nonprotein antigens that are highly immunoactive in nature. However, these antigens are chemically heterogeneous and structurally complex, thereby limiting their applications. To identify their peptide mimotopes, phage-displayed peptide libraries Ph.D.-7 and Ph.D.-12 were panned on either defined template, monoclonal antibody (MAb) CS-35 against lipoarabinomannan (LAM), or a polyclonal rabbit immune serum reactive against whole cells of Mycobacterium bovis BCG. Panning on anti-LAM MAb CS-35 yielded two confirmed mimotopes of LAM, a 7-mer and a 12-mer, whereas panning on polyclonal serum yielded a large repertoire of mimotopes reactive against sera from BCG-immunized rabbits, one of which turned out to have the same sequence as the 7-mer LAM mimotope. The dissociation constant of the interaction between MAb CS-35 and a synthetic peptide corresponding to the 7-mer LAM mimotope was determined to be 7.55 microM. Dot blot assays were performed with peptides corresponding to the two LAM mimotopes to evaluate their diagnostic potential. Both peptides gave discernibly higher signals with a panel of tuberculosis (TB) patient sera than with sera from healthy controls. The peptides were also found to stimulate the release of tumor necrosis factor alpha and interleukin-12 cytokines in the J774A.1 cell line and primary bone marrow-derived macrophages, indicating that they may have immunomodulatory potential. The present study demonstrates that peptide mimotopes of known and unknown mycobacterial antigens could be isolated by using subtractive phage display techniques and that these peptides could have potential applications in areas such as TB diagnostics and immunotherapy.     

Anti-tumor immune responses and tumor regression induced with mimotopes of a tumor-associated T cell epitope

Mimotopes provide an alternative to natural T cell epitopes for cancer immune therapy, as they can recruit and stimulate T cell repertoires that deviate from the repertoires engaged with the tumor and exposed to disease-related immune suppression. Here, mimotopes of a shared tumor-associated T cell epitope in cutaneous lymphoma were tested for their capacities to induce clinical and immunological responses in cancer patients. The mimotope sequences had been determined by a combinatorial peptide library approach without knowledge of the corresponding natural tumor-associated antigen. Vaccination with these mimotopes together with helper T cell-inducing antigens led to complete tumor remission in the two patients tested. After each booster vaccination, enhanced frequencies of mimotope-specific CD8+ T cells were detected in the peripheral blood of the patients, and the CTL proved to be cytotoxic and tumoricidal when tested in vitro. These data provide a first indication of clinical efficacy of mimotopes in cancer patients.     

Evaluating strategies to enhance the anti-tumor immune response to a carbohydrate mimetic peptide vaccine

Carbohydrate mimetic peptides of tumor associated carbohydrate antigens (TACA) are T-cell-dependent antigens and, therefore, immunization with these surrogates is predicted to overcome the low immunogenicity of carbohydrate antigens. Consistent with this hypothesis, we show that among the potential immune cells involved, peptide immunization led to an increase in T-cell populations. While peptide mimetics may also function as TLR binding ligands, we did not observe evidence of involvement of NK cells. Examining tumor challenged animals, we observed that peptide immunization and not tumor cells rendered IL-12 responsiveness to T-cells, as T-cells from peptide-immunized mice produced IFN-gamma upon stimulation with IL-12. Cyclophosphamide administration enhanced the anti-tumor efficacy of the vaccine, which was achieved by enhancing T-cell responses with no effect on NK cell population. Prophylactic immunization of mice with a DNA construct encoding carbohydrate mimetic peptides indicated a specific role for the mimotope vaccine in anti-tumor immune responses. These data suggest a role for both CD4(+) and CD8(+) T-cells induced by mimotopes of TACA in protective immunity against tumor cells.     

Induction of IgG antibodies against the GD2 carbohydrate tumor antigen by vaccination with peptide mimotopes

The disialoganglioside GD2, a carbohydrate antigen, is expressed on all tumors of neuroectodermal origin, including melanoma, neuroblastoma, sarcoma and small cell lung cancer. Due to its specific expression on tumor surfaces, GD2 is an attractive target for immunotherapies. The mouse/human chimeric anti-GD2 mAb ch14.18 is already applied in melanoma and neuroblastoma trials as a passive immunotherapy. To establish an active immunotherapy alternative, we aimed to replace the poorly immunogenic ganglioside with immunogenic peptides. Previously, we used the ch14.18 antibody to select GD2 peptide mimics from a phage display library. In the present study, two mimics of the ch14.18 epitope were coupled to keyhole limpet hemocyanin and used for immunizing BALB/c mice. Induction of a specific humoral immune response towards the original antigen GD2, both purified and expressed on neuroblastoma and melanoma cells, could be demonstrated in ELISA, Western blot, and immunofluorohistochemistry. As the elicited antibodies were of the IgG isotype, the mimotope conjugates were capable of recruiting T cell help and inducing memory phenomena. In conclusion, we show that an epitope of the carbohydrate antigen GD2 can successfully be translated into immunogenic peptide mimotopes. Our immunization experiments indicate that GD2 mimotopes are suitable for active immunotherapy of GD2-expressing tumors.     

Identification of tumor-associated MHC class I ligands by a novel T cell-independent approach

Specific immunotherapy of cancer utilizes tumor-directed cytotoxic T lymphocytes (CTL) that lyse tumor cells presenting MHC class I-associated peptides derived from tumor-associated proteins. Many tumor-associated gene products are known, but corresponding T cell epitopes are only known for relatively few of these. The most commonly used approaches to identify such antigens require pre-existing CTL lines or clones. By using a CTL-independent high performance liquid chromatography mass spectrometry (HPLC MS)-based approach we identified HLA-A2-presented peptides from carcinoembryonic antigen and wild-type p53 with a copy number as low as eight molecules per cell. Potential epitopes were predicted from the sequences of known tumor antigens and the corresponding synthetic peptides were analyzed by nanocapillary HPLC MS. In parallel, peptides were extracted from fresh, solid tumor tissue or tumor cell lines and analyzed in the same way. Upon co-elution of a natural peptide with a predicted peptide of the same mass, the peptide sequence was confirmed by on-line tandem MS. This approach allows rapid screening of large numbers of tumor-associated gene products for naturally processed peptides presented by different MHC class I molecules as a prerequisite for efficient epitope identification and rapid transfer to therapeutic vaccine trials.     

Antigenicity and immunogenicity of Melan-A/MART-1 derived peptides as targets for tumor reactive CTL in human melanoma

Summary: Some cancer patients mount spontaneous T‐ and B‐cell responses against their tumor cells. Autologous tumor reactive CD8 cytolytic T lymphocyte (CTL) and CD4 T‐cell clones as well as antibodies from these patients have been used for the identification of genes encoding the target antigens. This knowledge opened the way for new approaches to the immunotherapy of cancer. In this review, we describe the characterization of the structure‐function properties of the melanocyte/melanoma tumor antigen Melan‐A/MART‐1, the assessment of the T‐cell repertoire available against this antigen in healthy individuals, and the analysis of naturally acquired and/or vaccine‐induced CTL responses to this antigen in patients with metastatic melanoma.     

MG7 mimotope-based DNA vaccination for gastric cancer

Gastric cancer is still one of the leading causes of cancer-related death worldwide. Prevention and treatment of gastric cancer through vaccination has been difficult owing to lack of a specific target and poor immunity. A number of vaccination strategies have been used to augment immune responses against gastric cancer and some progress has been made. In a series of studies, the authors have focused on gastric cancer vaccination approaches based on MG7 mimotopes, which are mimicry epitopes selected from phage-displayed oligopeptide libraries with a gastric cancer cell-specific monoclonal antibody, MG7-Ab. Strategies employed in these studies include viral or plasmid vectors in combination with carrier sequence or unmethylated CpG with synthetic peptides in nanoemulsion. The results demonstrated that MG7 mimotopes could effectively and specifically induce both cellular and humoral immune reactions and in vivo antitumor responses. In particular, a four-MG7 mimotope DNA vaccine was found to elicit much stronger antitumor immune responses in mice compared with its single-mimotope counterpart. These encouraging findings might pave the way for the development of novel MG7 antigen-based vaccination approaches for human gastric cancer. The review also discusses other immune-enhancing vaccination strategies for gastric cancer.     

Heat shock protein-peptide complex in the treatment of glioblastoma

Vaccination immunotherapies offer the promise of long-term tumor control, and preclinical trials have found promising results. Active immunotherapy uses the adaptive immune response to specifically kill tumor cells. Tumor-specific antigens are processed by antigen-presenting cells and recognized by specific effector lymphocytes. However, basic vaccination strategies with tumor lysates have been unsuccessful in inducing antiglioma immunity in clinical trials. Gliomas are known to modulate the activity of antigen-presenting cells to reduce antitumor immune activity. Recently, tumor-derived heat shock proteins have been found to more effectively activate the immune response. Widely expressed, heat shock proteins are thought to present protein peptide fragments in a format conducive to processing by antigen-presenting cells. As a part of the protein synthesis machinery, peptides complexed with heat shock proteins are effectively representative of antigens expressed by the cell; these peptides convey the specificity of this vaccination strategy. The heat shock protein-peptide vaccine is one of many promising immunotherapeutic strategies being evaluated in clinical trials. These can be broadly classified as active, passive and adoptive, each with advantages and disadvantages. Here, we compare and contrast heat shock protein-peptide vaccines with other immunotherapies and describe the outcomes of clinical trials to date.     

Mimotopes identify conformational epitopes on parvalbumin, the major fish allergen

Parvalbumin, the major fish allergen, is recognized by allergen-specific IgE of more than 90% of all fish-allergic patients. A detailed knowledge of allergenic structures is crucial for developing a vaccine inducing blocking antibodies specifically directed towards the IgE binding epitopes. In the present study we aimed to use the phage display technique to generate mimotopes, which mimic epitopes on parvalbumin. Parvalbumin-specific IgE was purified from sera of fish-allergic patients and used for screening of a constrained decamer phage library. After four rounds of biopanning using parvalbumin-specific IgE, five phage clones were selected which were specifically recognized by parvalbumin-specific IgE as well as IgG. DNA sequencing and peptide alignment revealed a high degree of sequence similarities between the mimotopes. Interestingly, on the surface of natural parvalbumin three regions could be defined by computational mimotope matching. In accordance, previously defined allergenic peptides of cod parvalbumin highlighted areas in close proximity or overlapping with the mimotope matching sites. From the presented data we conclude that our approach identified conformational epitopes of parvalbumin relevant for IgE and IgG binding. We suggest that these mimotopes are suitable candidates for an epitope-specific immunotherapy of fish-allergic patients.     

T-cell immunity against tumors,a delicate balancing act involving dendritic cells

T-cell immunity occurs naturally against tumors induced by viruses and other causes. In the latter case self antigens are increasingly found to be targets of tumor associated CTL. In all categories of tumors the T cell response usually falls short of the maximally possible response. This situation calls for vaccination, primarily in situations of low tumor burden and adoptive transfer with tumor specific T cells in case of higher tumor burden. We recently observed that the outcome of immunization with vaccines containing tumor virus CTL epitopes strongly depends on mode of epitope delivery. Surprisingly, vaccination with MHC class I binding peptides cause CTL tolerance associated with enhanced tumor outgrowth rather than immunity. Such specific CTL tolerance can be induced by a single injection of 1 μg of peptide in adjuvant. However, in vivo presentation of the same peptides on dendritic cells or in viral vector (adenovirus) causes strong antitumor protection. Thus tumors may escape from immune attack by specific tolerance induction. Tumor specificity of autoreactive CTL can be achieved by T cells directed against tumor associated self antigens of limited tissue distribution. Alternatively useful CTL can be directed against strongly overexpressed self antigens, as illustrated in our lab by the successful eradication of tumors overexpressing wild type p53 tumor suppressor protein, by the adoptive transfer of a wild p53-specific CTL clone. Apparently the low expression of p53 in many tissues does not cause the CTL clone to inflict tissue damage, while the p53 overexpressing tumor cells are specifically targeted and eradicated. Recently we showed that CD40 signalling can replace CD4+ T-cells in priming of helper dependent tumor-specific CD8+ responses. Blockade of CD40L results in profound inhibition of CTL priming that is overcome by CD40 signalling. CD40 signalling converted CTL tolerization by a tolerogenic peptide into strong CTL priming. Moreover, supplementation of an already protective tumor-specific peptide vaccine with a CD40 activating antibody endows this vaccine with therapeutic CTL activity in tumor-bearing mice. The CD40–CD40L pair acts as a switch determining CTL priming or tolerance. This supports the clinical use of CD40 stimulating agents as components of anticancer vaccines.     

肿瘤疫苗的研究现状

肿瘤在体内的排斥/控制主要依赖于肿瘤抗原特异性T淋巴细胞.T淋巴细胞通过TCR与肿瘤细胞表面表达的HLA-抗原肽复合物的相互作用来识别其靶分子.与特异性HLA-抗原肽复合物有价值的相互作用诱导了淋巴细胞克隆的扩增以及一系列的免疫反应.目前,从理论上已经提出了以整个肿瘤细胞,或纯化的肿瘤抗原作为疫苗(包括整个蛋白质分子和抗原肽及纯化的DNA分子)来提高免疫系统对肿瘤的识别和排斥.本文从免疫系统对肿瘤细胞的识别、疫苗诱导的体内抗肿瘤免疫的假设机制、疫苗接种所诱导的抗肿瘤反应以及临床结果几个方面,对当前肿瘤疫苗的研究现状作一简要综述,并对肿瘤疫苗研究中尚待解决的问题作了简单的归纳.     

Antigen discovery for the development of cancer immunotherapy

Central to successful cancer immunotherapy is effective T cell antitumor immunity. Multiple targeted immunotherapies engineered to invigorate T cell-driven antitumor immunity rely on identifying the repertoire of T cell antigens expressed on the tumor cell surface. Mass spectrometry-based survey of such antigens (“immunopeptidomics”) combined with other omics platforms and computational algorithms has been instrumental in identifying and quantifying tumor-derived T cell antigens. In this review, we discuss the types of tumor antigens that have emerged for targeted cancer immunotherapy and the immunopeptidomics methods that are central in MHC peptide identification and quantification. We provide an overview of the strength and limitations of mass spectrometry-driven approaches and how they have been integrated with other technologies to discover targetable T cell antigens for cancer immunotherapy. We highlight some of the emerging cancer immunotherapies that successfully capitalized on immunopeptidomics, their challenges, and mass spectrometry-based strategies that can support their development.     

Clonal diversity of the T-cell population responding to a dominant HLA-A2 epitope of HER-2/neu after active immunization in an ovarian cancer patient

Natural antigen processing and presentation of antigen is thought to be important for the generation of a broad functional repertoire of antigen-specific T cells. In this study, the T-cell repertoire to an immunodominant human leukocyte antigen A2 (HLA-A2) binding peptide epitope of HER-2/neu, p369-377, was examined in a patient following immunization with a peptide-based vaccine consisting of helper peptides encompassing HLA-A2 peptide epitopes. The responding T-cell repertoire generated was both phenotypically and functionally diverse. A total of 21 p369-377 clones were generated from this patient. With the exception of two clones, all clones were CD3(+). Sixteen of the clones were CD8(+)/CD4(-). Five of the clones were CD4(+)/CD8(-), despite being generated with an HLA-A2 binding peptide. Nineteen of 21 of clones expressed the alpha beta-T-cell receptor (TCR). The remaining two clones expressed the gamma delta T-cell response (TCR). Selected alpha beta-TCR clones, both CD8(+) and CD4(+), could lyse HLA-A2 transfected HER2 overexpressing tumor cells and p369-377-loaded B-lymphoblastic cell line. In addition to their lytic capabilities these clones could be induced to produce interferon-gamma (IFN-gamma) specifically in response to p369-377 peptide stimulation. The 2 gamma delta-TCR clones expressed CD8 and lysed HLA-A2(+) HER-2/neu(+) tumor cells, but not HLA-A2(-) HER-2/neu(+) tumor cells. One of gamma delta-TCR clones also released IFN-gamma directly in response to p369-377 stimulation. These results suggest that a tumor antigen TCR, directed against a specific epitope, can be markedly polyclonal at multiple levels including CD4/CD8 and TCR.     

Induction of anti-tumor cytotoxic T cell responses through PLGA-nanoparticle mediated antigen delivery

Nanotechnology-based antigen delivery has been developing as a vaccine strategy due to its dose-sparing and prolonged antigen presentation features. In the current study, we examined the feasibility of nanoparticle (NP)-mediated delivery of antigenic peptides to efficiently induce cytotoxic T lymphocyte responses against tumor-associated self-antigens in C57BL/6 mouse models. The biodegradable poly(D,L-lactide-co-glycolide) nanoparticle (PLGA-NP) carrying murine melanoma antigenic peptides, hgp100(25-33) and TRP2(180-188), were prepared by double emulsion method. Efficient uptake of PLGA-NP by murine dendritic cells was shown in vitro and in vivo, using NP labeled with the fluorescent dye DiD. Intradermal injection of peptide-loaded PLGA-NP into mice induced antigen-specific T cell responses more strongly than the peptides mixed with Freund's adjuvant. More importantly, vaccination with PLGA-NP carrying both TRP2(180-188) and a toll-like receptor 4 agonist, monophosphoryl lipid A, significantly delayed growth of subcutaneously inoculated B16 melanoma cells in a prophylactic setting. Furthermore, the anti-tumor activity of NP-mediated peptide vaccination was significantly augmented by combined treatment with interferon-γ, which might prevent tumor escape through up-regulation of MHC class I expression on tumor cells. Our findings demonstrate the feasibility of NP-mediated antigen delivery for cancer immunotherapy, in particular when immune escape mechanisms of tumor cells are blocked simultaneously.     

应用噬菌体随机肽库技术筛选SARS-CoV S蛋白模拟表位

目的筛选SARS病毒(Severe acutere spiratorysyndromeas sociated coronavirus,SARSCoV)结构蛋白S(Spike)特异性的模拟表位,为抗SARS疫苗研究提供基础。方法以抗SARS病毒S蛋白的单克隆抗体为固相筛选分子,对人工合成的噬菌体随机12肽库进行5轮“吸附洗脱扩增”的筛选,随机挑选30个克隆,经噬菌体酶联免疫吸附(ELISA)法和交叉反应实验鉴定阳性克隆,再进行DNA序列分析和竞争抑制结合实验,以确定SARS病毒S蛋白的模拟表位。结果经噬菌体富集后,从随机挑选的30个克隆中得到了29个编码8种12肽的阳性克隆,8条肽与S蛋白的320～350氨基酸序列有较高同源性,确定YF、W、E和K氨基酸残基为模拟表位的骨架结构。结论用噬菌体12肽库成功筛选到了SARS病毒S蛋白的模拟表位,为基于S蛋白的肽疫苗研制提供了基础。     

Hepatitis B surface antigen presentation and HLA-DRB1*- lessons from twins and peptide binding studies

The aim of this study was to investigate the underlying mechanisms of the genetic association between certain HLA-DRB1* alleles and the immune response to HBsAg vaccination. Therefore, HBsAg peptide binding to HLA-DR molecules was measured in vitro by peptide binding ELISAs. Additionally, HBsAg-specific T cell reaction and cytokine profile of immune response were analysed ex vivo in ELISPOT assays and DR-restriction of T-cell proliferative responses was investigated with HBsAg specific T cell clones. In addition, we compared HBsAg specific T cell responses of 24 monozygotic and 3 dizygotic twin pairs after HBsAg vaccination. Our results showed that the peptide binding assays did not reflect antigen presentation in vivo. DR alleles associated with vaccination failure like DRB1*0301 and 0701 efficiently presented HBsAg peptides. In 11 of 24 investigated monozygotic twin pairs we observed pronounced differences in the recognition of HBsAg peptides. This study indicates that HLA-DR associations with HBsAg vaccination response are not caused by differences in peptide binding or by a shift in the Th1/Th2 profile. Our findings strongly argue for differences in the T cell recognition of peptide/MHC complexes as the critical event in T cell responsiveness to HBsAg.     

A peptide mimotope of hepatitis C virus E2 protein is immunogenic in mice and block human anti‐HCV sera

Conformational B‐cell epitopes on the HCV E2 protein recognized by human antibodies were characterized by the use of a peptide mimotope named K1. K1 was identified by two HCV anti‐E2 monoclonal antibodies (mAbs) following selection and purification of phage clones containing a 15‐mer random peptide insert. Murine antisera to the mimotope K1 recognized the E2 protein. Five of eight human sera from patients who had cleared HCV recognized the K1 mimotope. Binding to E2 in four individuals with the capacity to block E2–CD81 interaction was inhibited by the mimotope K1. The results demonstrate that anti‐E2 antibodies in sera from patients who have cleared HCV infection are directed against a conformational B‐cell epitope on E2 that can be mimicked with linear synthetic peptides. These findings could have implications for vaccine design by employing linear mimotopes to direct B‐cell responses against those specific E2 epitopes that may correlate with immunity. J. Med. Virol. 82:1655–1665, 2010. 2010 Wiley‐Liss, Inc.     

针对MUC1黏蛋白DC疫苗的研究进展

树突状细胞(DC)作为抗原递呈细胞在激活肿瘤特异性免疫中发挥重要作用,DC疫苗为肿瘤免疫治疗提供了一种有效手段.MUC1是一种高分子量糖蛋白,属于粘蛋白家族成员,在多种上皮性肿瘤中异常表达,是肿瘤免疫治疗的理想靶抗原.本文综述了MUC1的生物学特征、DC对MUC1的递呈和以MUC1为靶点DC疫苗的抗肿瘤效果.     

Peptide competition at the level of MHC-binding sites using T cell clones from a rheumatoid arthritis patient.

The inhibition of antigen presentation in rheumatoid arthritis by blocking peptide binding to MHC at the antigen presenting cell (APC) level was investigated using various synthetic peptides derived from the 65 kDa mycobacterial protein. Human T cell clones from tuberculosis and rheumatoid arthritis patients were stimulated with peptides in the presence of irradiated APCs (autologous or DR homozygous EBV-B cell lines). Two peptides (residues 65-85 and 412-426) were found to be able to bind to the HLA-DR1 protein. Cross-competition was observed between these peptides when APCs were cultured with a suboptimal concentration of stimulator peptide in the presence of various concentrations of competitor peptides and T cell clones from tuberculosis patients as responder cells. These T cell clones responded not only to the peptides but also to the native protein. In other experiments, we used T cell clones from a rheumatoid arthritis patient to demonstrate the blocking of MHC-binding sites by adding the p412-426 in the recognition of DR1 restricted T cell clone specific to p65-85; MHC binding was not observed using a control peptide (residues 198-217). This approach has permitted the identification of MHC-specific blockers. Further experimentation is required to determine the particular amino acids involved in MHC binding. Our data support the idea that modulation of antigen presentation by peptide competition could be a useful tool for immunotherapy in autoimmune diseases.     

Decreased specific CD8+ T cell cross-reactivity of antigen recognition following vaccination with Melan-A peptide

The aim of T cell vaccines is the expansion of antigen-specific T cells able to confer immune protection against pathogens or tumors. Although increase in absolute cell numbers, effector functions and TCR repertoire of vaccine-induced T cells are often evaluated, their reactivity for the cognate antigen versus their cross-reactive potential is rarely considered. In fact, little information is available regarding the influence of vaccines on T cell fine specificity of antigen recognition despite the impact that this feature may have in protective immunity. To shed light on the cross-reactive potential of vaccine-induced cells, we analyzed the reactivity of CD8(+) T cells following vaccination of HLA-A2(+) melanoma patients with Melan-A peptide, incomplete Freund's adjuvant and CpG-oligodeoxynucleotide adjuvant, which was shown to induce strong expansion of Melan-A-reactive CD8(+) T cells in vivo. A collection of predicted Melan-A cross-reactive peptides, identified from a combinatorial peptide library, was used to probe functional antigen recognition of PBMC ex vivo and Melan-A-reactive CD8(+) T cell clones. While Melan-A-reactive CD8(+) T cells prior to vaccination are usually constituted of widely cross-reactive naive cells, we show that peptide vaccination resulted in expansion of memory T cells displaying a reactivity predominantly restricted to the antigen of interest. Importantly, these cells are tumor-reactive.