Carcinoembryonic Antigen Peptide Research Articles

A pilot trial of vaccination with Carcinoembryonic antigen and Her2/neu peptides in advanced colorectal cancer.

Checkpoint-blockade therapy (CBT) is approved for select colorectal cancer (CRC) patents, but additional immunotherapeutic options are needed. We hypothesized that vaccination with carcinoembryonic antigen (CEA) and Her2/neu (Her2) peptides would be immunogenic and well tolerated by participants with advanced CRC. A pilot clinical trial (NCT00091286) was conducted in HLA-A2+ or -A3+ Stage IIIC-IV CRC patients. Participants were vaccinated weekly with CEA and Her2 peptides plus tetanus peptide and GM-CSF emulsified in Montanide ISA-51 adjuvant for 3 weeks. Adverse events (AEs) were recorded per NIH Common Terminology Criteria for Adverse Events version 3. Immunogenicity was evaluated by interferon-gamma ELISpot assay of in vitro sensitized peripheral blood mononuclear cells and lymphocytes from the sentinel immunized node. Eleven participants were enrolled and treated; one was retrospectively found to be ineligible due to HLA type. All 11 participants were included in AEs and survival analyses, and the 10 eligible participants were evaluated for immunogenicity. All participants reported AEs: 82% were Grade 1-2, most commonly fatigue or injection site reactions. Two participants (18%) experienced treatment-related dose-limiting Grade 3 AEs; both were self-limiting. Immune responses to Her2 or CEA peptides were detected in 70% of participants. Median overall survival (OS) was 16 months; among those enrolled with no evidence of disease (n=3), median OS was not reached after 10 years of follow-up. These data demonstrate that vaccination with CEA or Her2 peptides is well tolerated and immunogenic. Further study is warranted to assess potential clinical benefits of vaccination in advanced CRC either alone or in combination with CBT.

Serum tumour markers as a diagnostic and prognostic tool in Libyan breast cancer

Results from studies on efficacy of carcinoembryonic antigen (CEA), carbohydrate antigen 15.3 (CA 15.3) and thymidine kinase (TK1) as diagnostic and prognostic tools for primary breast cancer (BC) have presented conflicting results, and usefulness of these markers for clinical use in BC remains unclear. The aim of this study is to evaluate potential of concentration of the sera CEA, CA15.3 and TK1 peptides' use as markers in the diagnosis and prognosis of breast lesions of Libyan patients. Serum tumour markers were studied in 20 healthy subjects, 30 patient with benign lesion diseases and 50 patients with histologically confirmed BC diagnosed at the National Cancer Institute (NCI), Misurata, Libya during the period 2005-2009. The concentrations of the BC patients' cutoff points used for diagnostic and prognostic sensitivity were 8.82 ng/ml, 35.57 U/ml and 32.57 U/mg/protein for CEA, CA15.3 and TK1, respectively. Increased CEA (>8.82 ng/ml), CA 15.3 (>35.57 U/ml) and TK1 (>32.57 U/mg/protein) concentrations were found in 62 %, 70 % and 78 % of the BC patients, respectively. For all three tumour markers, increased concentrations correlated increased tumour size and nodal involvement. Significantly higher serum TK1 levels were found in patients with advanced disease (p < 0.0001) and TK1 levels also correlated with disease-specific survival (DSS, p < 0.07). The combined data set of the three markers' data from three markers increased the diagnostic sensitivity to 90 %. The serum marker analysis for CEA, CA 15.3, and S-TK1 concentrations is shown to be a useful tool for identification of malignant cases in our BC population and for the prognostic evaluation of patients with primary BC. Increased concentrations of the markers were also observed to be higher in patients with advanced tumours and indicative of the development of distant metastasis.

Recent Advances in Active Specific Cancer Vaccine Treatment for Colorectal Cancer

Cloning techniques to identify genes and peptides of tumor-associated antigens have created new possibilities for the immunotherapy of patients with advanced cancer. Here, we review recent clinical trials of specific cancer vaccines, mainly HLA-restricted peptides, and epitope-encoding vectors for advanced colorectal cancer (CRC). Many researchers initially focused on carcinoembryonic antigen (CEA) as an immunologic target antigen that is overexpressed on virtually all CRCs. A recombinant vaccine containing the CEA gene and dendritic cells (DCs) loaded with CEA peptide was administered to patients with CEA-elevated CRC. Although CEA-specific responses were detected, the clinical responses were limited. Recently, new types of clinical trials--namely, a personalized protocol to take into account the immunological diversity of cytotoxic T cell responses among patients and a novel cancer-testis antigen protocol that uses multiple peptides derived from genes identified by the cDNA array method--have been introduced. The personalized protocol seemed to be better than the classical (non-personalized) protocol in terms of clinical response and survival. Novel cancer-testis antigen protocols that use multiple CRC-derived peptides were recently conducted in patients with advanced CRC. The preliminary study yielded promising results regarding specific T cell responses to peptides and survival benefits. In this review, we summarize these results and discuss future perspectives.

Analysis of HLA‐A24‐restricted peptides of carcinoembryonic antigen using a novel structure‐based peptide‐HLA docking algorithm

Carcinoembryonic antigen (CEA) is a very common tumor marker because many types of solid cancer usually produce a variety of CEA and a highly sensitive measuring kit has been developed. However, immunological responses associated with CEA have not been fully characterized, and specifically a weak immunogenicity of CEA protein as a tumor antigen is reported in human leukocyte antigen (HLA)-A24-restricted CEA peptide-based cancer immunotherapy. These observations demonstrated that immunogenic and potent HLA-A24-restricted CTL epitope peptides derived from CEA protein are seemingly difficult to predict using a conventional bioinformatics approach based on primary amino acid sequence. In the present study, we developed an in silico docking simulation assay system of binding affinity between HLA-A24 protein and A24-restricted peptides using two software packages, AutoDock and MODELLER, and a crystal structure of HLA-A24 protein obtained from the Protein Data Bank. We compared the current assay system with HLA-peptide binding predictions of the bioinformatics and molecular analysis section (BIMAS) in terms of the prediction capability using MHC stabilization and peptide-stimulated CTL induction assays for CEA and other HLA-A24 peptides. The MHC stabilization score was inversely correlated with the affinity calculated in the docking simulation alone (r = -0.589, P = 0.015), not with BIMAS score or the IFN-γ production index. On the other hand, BIMAS was not significantly correlated with any other parameters. These results suggested that our in silico assay system has potential advantages in efficiency of epitope prediction over BIMAS and ease of use for bioinformaticians.

DNA vaccine expressing repeated carcinoembryonic antigen (CEA) 625–667 induces strong immunity in mice

The efficacy of immunization with DNA plasmids for single truncated carcinoembryonic antigen (CEA) peptide or triple repeated CEA peptides in mice was evaluated. A DNA fragment the truncated CEA gene (nucleotide 625–667) encoding two helper T lymphocyte (HTL) epitopes was amplified by PCR and cloned for generating recombinant plasmids for single CEA 625–667 (pcDNA-CEA 625–667) or triple CEA 625–667 (pcDNA-triCEA 625–667), respectively. Subsequently, groups of BALB/c female mice were intramuscularly injected with pcDNA-CEA 625–667, pcDNA-triCEA 625–667 or control pcDNA3.0 vector, respectively. Ten days after the last immunization, the frequency of splenic CD4 + and CD8 + T cells in the mice was determined by flow cytometry. The antigen-specific proliferation of splenic T cells and cytokine production ex vivo were analyzed by 3H-TdR uptake and cytokine ELISA, respectively. The levels of serum antibodies against CEA in the mice were detected by Western blot and ELISA. Although immunization with plasmid for the CEA 625–667 peptide(s) did not alter the frequency of CD4 + and CD8 + T cells in mice, vaccination with plasmid for CEA peptide induced strong antigen-specific T cell proliferation, particularly for the plasmid encoding the triple-repeated CEA peptides, accompanied by significantly elevated levels of IFN-γ secreted by T cells from the mice immunized with triple-repeated peptides. Furthermore, immunization with the plasmid for CEA peptide stimulated higher levels of antigen-specific antibody responses in mice. Vaccination with the plasmid for the triple repeated CEA peptides induced stronger Th1 responses. Our findings may be useful for the development of effective DNA vaccine for the immunotherapy of cancer.

Identification of a broad coverage HLA-DR degenerate epitope pool derived from carcinoembryonic antigen

CD4 T cells are important for anti-tumor immune responses. Aside from their role in the activation of CD8 T cells, CD4 T cells also mediate anti-tumor immune responses by recruiting innate immune effectors into the tumor microenvironment. Thus, the search for strategies to boost CD4 T cell immunity is an active area of research. Our goal in this study was to identify HLA-DR epitopes of carcinoembryonic antigen (CEA), a commonly over-expressed tumor antigen. HLA-DR epitopes of CEA were identified using the epitope prediction program, PIC (predicted IC(50)) and tested using in vitro HLA-DR binding assays. Following CEA epitope confirmation, IFN-gamma ELIspot assays were used to detect existing immunity against the HLA-DR epitope panel of CEA in breast and ovarian cancer patients. In vitro generated peptide-specific CD4 T cells were used to determine whether the epitopes are naturally processed from CEA protein. Forty-three epitopes of CEA were predicted, 15 of which had high binding affinity for 8 or more common HLA-DR molecules. A degenerate pool of four, HLA-DR restricted 15 amino acid epitopes (CEA.24, CEA.176/354, CEA.488, and CEA.653) consisting of two novel epitopes (CEA.24 and CEA.488) was identified against which 40% of breast and ovarian cancer patients had pre-existent T cell immunity. All four epitopes are naturally processed by antigen-presenting cells. Hardy-Weinberg analysis showed that the pool is useful in approximately 94% of patients. Patients with breast or ovarian cancer demonstrate pre-existent immune responses to the tumor antigen CEA. The degenerate pool of CEA peptides may be useful for augmenting CD4 T cell immunity.

Characterization of Genetically Modified T-Cell Receptors that Recognize the CEA:691-699 Peptide in the Context of HLA-A2.1 on Human Colorectal Cancer Cells

Carcinoembryonic antigen (CEA) is a tumor-associated protein expressed on a variety of adenocarcinomas. To develop an immunotherapy for patients with cancers that overexpress CEA, we isolated and genetically modified a T-cell receptors (TCRs) that specifically bound a CEA peptide on human cancer cells. HLA-A2.1 transgenic mice were immunized with CEA:691-699. A CEA-reactive TCR was isolated from splenocytes of these mice and was genetically introduced into human peripheral blood lymphocytes via RNA electroporation or retroviral transduction. Amino acid substitutions were introduced throughout the complementarity determining regions (CDR1, CDR2, and CDR3) of both TCR alpha and beta chains to improve recognition of CEA. Murine lymphocytes bearing the CEA-reactive TCR specifically recognized peptide-loaded T2 cells and HLA-A2.1(+) CEA(+) human colon cancer cells. Both CD8(+) and CD4(+) human lymphocytes expressing the murine TCR specifically recognized peptide-loaded T2 cells. However, only gene-modified CD8(+) lymphocytes specifically recognized HLA-A2.1(+) CEA(+) colon cancer cell lines, and tumor cell recognition was weak and variable. We identified two substitutions in the CDR3 of the alpha chain that significantly influenced tumor cell recognition by human peripheral blood lymphocytes. One substitution, T for S at position 112 (S112T), enhanced tumor cell recognition by CD8(+) lymphocytes, and a second dually substituted receptor (S112T L110F) enhanced tumor cell recognition by CD4(+) T cells. The modified CEA-reactive TCRs are good candidates for future gene therapy clinical trials and show the power of selected amino acid substitutions in the antigen-binding regions of the TCR to enhance desired reactivities.

A trial of vaccination with the carcinoembryonic antigen (CEA) peptide CAP 1–6D with montanide ISA 51 adjuvant or granulocyte- macrophage colony stimulating factor (GM-CSF) in HLA-A2+ patients with CEA producing adenocarcinomas of gastrointestinal (GI) track

13513 Advances in drug therapy have improved the survival of patients with GI tumors but most patients with metastatic disease are not cured. Immunotherapy is an additional modality of treatment. In this trial, the CEA peptide, CAP 1–6D, was combined with either GM-CSF or montanide ISA 51 as adjuvants and used to vaccinate patients with CEA producing GI tumors. End points were development of peptide responsive immunity, toxicity, and anti-tumor effects. To be eligible, patients were required to have a histologic diagnosis of adenocaricnoma of GI tract origin with a life expectancy of at least 6 months and to have either metastatic disease or to have completed standard adjuvant therapy after resection. Patients were required to have a CEA producing tumor, be HLA-A2 positive, have a performance status of 0–1, test negative for HIV, hepatitis B and C, and have adequate bone marrow, liver and kidney function. Treatment consisted of 1 mg of CAP 1–6D peptide combined with either 250 ug of GM-CSF given intradermally, or 2 ml of Montanide ISA 51 given subcutaneously every 3 weeks for 6 treatments. Blood was drawn for ELISpot assay at each visit. Nine (out of a planned 10) patients, 8 with colorectal cancer and 1 with pancreatic cancer, were entered before the peptide was deemed expired and withdrawn from clinical use by the NCI. Of five colorectal patients with metastatic disease at baseline 4 progressed and 1 had stable disease. The pancreatic cancer patient was treated adjuvantly but progressed during therapy. Three colorectal cancer patients treated adjuvantly were free of disease at baseline and at the completion of therapy. Treatments were well tolerated. ELISpot data on 5 patients showed that 4 patients developed peptide responsive immunity. Responsive patients included those receiving either GM-CSF or Montanide ISA 51 and both adjuvantly treated and progressive metastatic colorectal patients and the progressed pancreatic cancer patient. This vaccine regimen can be safely given to cancer patients and can produce peptide responsive immunity in the ELISpot assay but this does not correlate with clinical anti-tumor effect. No significant financial relationships to disclose.

Immunotherapy, phase I study of immunotherapy with carcinoembryonic antigen peptide -pulsed, autologous human cultured dendritic cells in patients with advanced non-small cell lung cancer

12517 Background: To study toxicity tolerance and dose-effect relationship of carcinoembryuonic antigen peptide-pulsed dendritic cells in patients with advanced lung cancer. Methods: cells preparations enriched for autologous DCs were generated from the patients plastic adherent peripheral blood mononuclear cells in media supplemented with granulocyte macrophage colony -stimulating factor and interleukin-4. 37C0, 5%CO2 culture for 7–10. The DCs were loaded with carcinoembryonic antigen(CEA) in day 7, Groups of three to six patients received four weekly or biweekly i.v. infusions of the CAP-1-loaded DC in escalating dose levels of 1 × 106, 1 × 107, and 1 × 108 cells/dose. Patients with lung cancer received iv injections DCs. There were no toxicities directly referable to the treatments. Results: Total 22 patients with lung cancer received DCs immunotherapy. DCs infusion were 2.5×106-9.6×107,means 15.03×106. The early clinical trials suggest that vaccination with CEA vaccines is safe, producing few side-effects, and can lead to CEA-specific immunity. Conclusions: We conclude that it is feasible and safe to generate and administer large numbers of DCs loaded with CEA peptide to patients with lung cancer. No significant financial relationships to disclose.

Analyses of Recombinant Vaccinia and Fowlpox Vaccine Vectors Expressing Transgenes for Two Human Tumor Antigens and Three Human Costimulatory Molecules

The poor immunogenicity of tumor antigens and the antigenic heterogeneity of tumors call for vaccine strategies to enhance T-cell responses to multiple antigens. Two antigens expressed noncoordinately on most human carcinomas are carcinoembryonic antigen (CEA) and MUC-1. We report here the construction and characterization of two viral vector vaccines to address these issues. The two viral vectors analyzed are the replication-competent recombinant vaccinia virus (rV-) and the avipox vector, fowlpox (rF-), which is replication incompetent in mammalian cells. Each vector encodes the transgenes for three human costimulatory molecules (B7-1, ICAM-1, and LFA-3, designated TRICOM) and the CEA and MUC-1 transgenes (which also contain agonist epitopes). The vectors are designated rV-CEA/MUC/TRICOM and rF-CEA/MUC/TRICOM. Each of the vectors is shown to be capable of faithfully expressing all five transgenes in human dendritic cells (DC). DCs infected with either vector are shown to activate both CEA- and MUC-1-specific T-cell lines to the same level as DCs infected with CEA-TRICOM or MUC-1-TRICOM vectors. Thus, no evidence of antigenic competition between CEA and MUC-1 was observed. Human DCs infected with rV-CEA/MUC/TRICOM or rF-CEA/MUC/TRICOM are also shown to be capable of generating both MUC-1- and CEA-specific T-cell lines; these T-cell lines are in turn shown to be capable of lysing targets pulsed with MUC-1 or CEA peptides as well as human tumor cells endogenously expressing MUC-1 and/or CEA. These studies provide the rationale for the clinical evaluation of these multigene vectors in patients with a range of carcinomas expressing MUC-1 and/or CEA.

Integrating Bench With Bedside: The Role of Vaccine Therapy in the Treatment of Solid Tumors

The treatment of solid tumors continues to be one of the major challenges facing both patients and the oncology community. An improved understanding of how the immune system recognizes and eradicates tumor cells has led to an intense interest in therapeutic vaccine development, representing one of the most successful applications of bench-to-bedside translational research. Extensive “proof of principle” evidence from the bench has used murine models to demonstrate that active immunization can mediate effective tumor treatment. In 1991, a vaccine strategy employing recombinant vaccinia virus engineered to express carcinoembryonic antigen (CEA) was reported. Initial animal experiments documented the ability of vaccinia-CEA to induce humoral and cellular immunity by targeting a commonly expressed solid tumor antigen in a highly immunogenic vaccine—a result since confirmed in transgenic mice and in vitro experiments with human T cells. The first clinical trial with this vaccine was published in 1996, and numerous other studies, largely phase I in format, were conducted throughout the last 8 years, demonstrating the safety and feasibility of various poxvirus vaccines in patients with advanced solid tumors. While these studies were not designed or powered to determine clinical effectiveness, intriguing anecdotal evidence suggested that vaccination induced CEA-specific T cells, and even objective clinical responses, in some patients. The completion of these early-phase clinical trials provided important safety data and guided additional bench experiments aimed at improving the therapeutic effectiveness of CEA-based poxvirus vaccines. For example, careful analysis of in vitro T-cell responses from vaccinated patients led to the identification of HLA-restricted T-cell epitopes and later demonstrated that poxvirus vaccines could break tolerance against these epitopes in patients. This also allowed Schlom et al to identify a modified CEA agonist peptide that was more efficient in inducing T-cell responses against the native CEA peptide and CEAexpressing tumor cells. Another problem identified from early clinical trials was that repeated administration of vaccinia virus induced strong neutralizing antibodies that prohibited subsequent boosting of immune responses against the tumor antigen. Animal studies utilizing a prime-boost approach, wherein vaccinia virus is used once for priming followed by booster vaccinations with nonreplicating poxvirus vectors such as fowlpox virus, were shown to be superior to using either vector alone. This was later confirmed in a clinical trial. Additional vaccine improvements were provided by the coexpression of powerful costimulatory molecules in the viral genome, designed to augment the activation of T cells after engagement of the T-cell receptor. Preclinical experiments in mice and with human T cells in vitro resulted in selecting a triad of costimulatory molecules (B7-1, ICAM-1, and LFA-3) that significantly enhanced immune responses of the poxvirus vaccines, and augmented tumor regression in animals. Other studies evaluated the benefits of adjuvant cytokines such as granulocyte-macrophage colony-stimulating factor (GM-CSF) and interleukin-2 (IL-2) for promoting CEA-specific immunity in clinical trials after a benefit was seen in murine models. In this issue of the Journal of Clinical Oncology, Marshall et al, combine many of these methods for improving poxvirus vaccination into a single vaccine strategy for patients with advanced CEA-expressing solid tumors. They utilized a vaccinia virus expressing CEA encoding the agonist epitope, along with three costimulatory molecules (B7-1, ICAM-1, and LFA-3) for priming an immune response, followed by booster vaccinations with a fowlpox virus expressing the same transgenes. In addition, they treated some cohorts with GM-CSF administered near the vaccine site to promote local recruitment of dendritic cells. They also evaluated a split dose of the booster vaccine in an JOURNAL OF CLINICAL ONCOLOGY E D I T O R I A L VOLUME 23 NUMBER 4 FEBRUARY 1 2005

A novel ELISPOT assay to enhance detection of antigen-specific T cells employing antigen-presenting cells expressing vector-driven human B7-1

The ELISPOT assay is used to detect T-cell responses in patients enrolled in vaccine clinical trials; it is a relatively sensitive assay that can be performed without in vitro stimulation (IVS) of PBMC. However, this assay may be limited in some cases because of a weak signal 1 (as is the case for tumor-associated antigens [TAA]), or by a limited number of PBMC available from patients. The objective of this study is to enhance the sensitivity of the ELISPOT by increasing the level of signal 2, in this case, B7-1 expression on antigen-presenting cells (APC), allowing for a more sensitive detection of antigen-specific T-cell precursors. C1RA2 cells were used as APC and were uninfected, or infected with either recombinant avipox (fowlpox)-B7-1 (rF-B7-1) or control fowlpox wild-type (FP-WT) vector. The expressions of B7-1, MHC Class II, as well as HLA-A2 on the infected cells were analyzed by flow cytometry. A nearly threefold increase in B7-1 mean fluorescence intensity (MFI) occurred in the rF-B7-1-infected C1RA2 cells with no changes in MHC Class II or HLA-A2 expression. Various PBMC/APC ratios were used to further analyze the use of rF-B7-1-infected C1RA2 cells as APC in the ELISPOT assay. Fewer APC were required to activate PBMC when C1RA2 infected with rF-B7-1 were used as APC. Furthermore, using a PBMC/APC ratio of 1:1, similar detection was achieved using fewer PBMC. In addition, we demonstrated that the reactivity can be blocked by adding anti-B7-1 antibody. We performed the assay using APC on five normal healthy donors. All five donors showed substantial increases in PF to the Flu matrix peptide (Flu peptide) using the rF-B7-1-infected C1RA2 cells. Finally, we evaluated five cancer patients who received a carcinoembryonic antigen (CEA) vaccine-based therapy. Increases in CEA peptide precursors were noted in all five patients using the B7-1-infected APC. In conclusion, we have demonstrated the ability to enhance the sensitivity of the ELISPOT assay through infection of C1RA2 with rF-B7-1.

Treatment of colon and breast carcinoma cells with 5-fluorouracil enhances expression of carcinoembryonic antigen and susceptibility to HLA-A(*)02.01 restricted, CEA-peptide-specific cytotoxic T cells in vitro.

Cancer vaccines directed against tumor associate antigen (TAA) have produced encouraging results in preclinical models but not in cancer patients. A major limitation of this strategy is the relative degree of tolerance to these antigens and the low and heterogeneous tumor cell expression of TAA and major histocompatibility complex (MHC). Previous studies have shown that 5-fluorouracil (5-FU) can upregulate the expression of membrane-associated carcino-embryonic antigen (CEA), and MHC molecules in colon and breast carcinoma cell lines. We have investigated whether this drug can also enhance their sensitivity to the lytic effects of CEA-peptide specific Cytotoxic T cell lymphocytes (CTL). The CEA peptide-specific CTLs generated in our laboratory from normal HLA-A(*)02.01(+) donor PBMCs, were able to kill HLA-A(*)02.01(+)/CEA(+) breast (MCF-7-T103) and colon (HLA-A(*)02.01 gene-transfected HT-29 and C22.20) carcinoma cells in HLA-A(*)02.01 restricted manner. The treatment of target cells with 5-FU, enhanced their CEA expression and susceptibility to CTL-mediated lysis. Cold competition assays confirmed these results, thus supporting the hypothesis that immune target cell lysis and 5-FU mediated enhancement were dependent on CEA peptide presentation by cancer cells. 5-FU treatment of functionally "mature" CTL after in vitro expansion, did not reduce their cytolytic activity against MT-2 target cells but, when the anti-metabolite was added during the immune-sensitization phase, CTL generation was significantly inhibited. These results provide a rationale for investigating a possible new role of 5-FU as an immuno targeting amplifier agent in breast and colorectal cancer patients immunized with CEA-directed cancer vaccines.

Induction of CEA-specific Cytotoxic T Lymphocytes by Murine Dendritic Cells Expressing CEA

Background: Carcinoembryonic antigen (CEA) is well-known soluble tumor marker frequently detectable in peripheral blood of carcinoma patients and considered as good target for antigen-specific immunotherapy. In this study, we used a replication-deficient adenovirus containing CEA to study CTL induction in vitro after adenovirus-mediated gene transfer into DC. Methods: DC were obtained from mouse bone marrow and cultured with IL-4 and GM-CSF. For measuring CTL activity, splenocytes were harvested from the mice, which were immunized with DC that had been infected AdV-CEA or pulsed with CEA peptide. Untreated DC was used as a control. Splenocytes were re-stimulated in vitro with DC pulsed with CEA peptide for 7 days and CTL activity with CEA peptide-pulsed EL-4 cells were assessed in a standard Cr-release assay. The frequencies of antigen-specific cytokine-secreting T cell were determined with mIFN-γ ELISPOT. Results: DC infected with recombinant adenovirus expressing CEA induced CEA-specific CTL responses in vivo. Splenocyte induced from mice immunized with AdV-CEA-infected DC increase in the number of IFN-γ secreting T cells compared with those from mice immunized with CEA peptide-pulsed DC. Conclusion: These results suggested that DC infected with recombinant adenovirus has advantages over other forms of vaccination and could provide an alternative approach vaccination therapies. (Immune Network 2003;3(4):295-301)

Preparation and characterization of anti-anti-idiotypic monoclonal antibody ("Ab1-like Ab3") in relation to carcinoembryonic antigen.

In order to analyze the epitope structure of carcinoembryonic antigen (CEA) and the idiotype network system, seven anti-idiotypic monoclonal antibodies (anti-Id MoAbs) were generated from a BALB/c mouse immunized with anti-CEA MoAb P1-356, which recognized a synthetic peptide P1 of CEA, domain I. These MoAbs were divided into four groups. The anti-Id MoAbs specifically reacted with MoAb P1-356, but not with any MoAb, and inhibited the binding of MoAb P1-356 to CEA, indicating that all of these anti-Id MoAbs recognized private idiotopes at the combining sites of MoAb P1-356. Polyclonal anti-anti-idiotypic antiserum (Ab3) generated with anti-Id MoAbs M315 (Ab2), blocked the binding of MoAb P1-356 (Ab1) to CEA and reacted with CEA(Ag) and synthetic peptide P1. The analysis of serological assays suggested that it contains "Ab1-like Ab3." Therefore, we prepared anti-anti-Id MoAbs using anti-Id MoAb M315. Among 13 candidates, anti-anti-Id MoAb 11B2 was selected, because it competed with MoAb P1-356 (Ab1) binding to CEA. A direct binding assay using MoAb11B2 showed that it reacted with purified CEA and with CEA synthetic peptide P1. In addition, MoAb 11B2 (Ab3) reacted with both CEA-producing cultured cells and colonic cancerous tissues in immunostaining. These results indicate that anti-anti-Id MoAb 11B2 is "Ab1-like Ab3." Therefore, it is suggested that anti-Id MoAb M315 bears an "internal image" of the MoAb P1-356-defined epitope on CEA.

Strategies for cancer therapy using carcinoembryonic antigen vaccines.

Advances in molecular biology and immunology have renewed interest in the development of vaccines for the treatment or prevention of cancer. Research over the past 10 years has focused on the identification of suitable tumour antigens to use as targets for a variety of vaccine strategies. Carcinoembryonic antigen (CEA) was one of the first tumour antigens described, and is commonly expressed by a wide range of adenocarcinomas. Recent studies have identified several human-leukocyte-antigen-restricted epitopes (short peptides) within the CEA protein that can be recognised by human T lymphocytes (T cells). Although CEA-expressing tumour cells are generally weakly recognised by the immune system, several new strategies have been used to enhance immune responses against CEA. This includes using antibodies directed against CEA; inserting the CEA gene into recombinant viruses and bacteria as viral and bacterial vaccines; pulsing the CEA protein, peptides, DNA or RNA onto dendritic cells (specialised antigen-presenting cells); and combining CEA vaccines with cytokines or co-stimulatory molecules to increase vaccine effectiveness. Other factors that might be important in establishing systemic immunity against CEA are the dose, route, timing, and choice of vector and adjuvants for vaccine administration. Further research in understanding the fundamental processes involved in tumour-cell recognition by the immune system, better animal models, and improved clinical trial designs will help to define the full potential of CEA as a target for cancer vaccine development.

Carcinoembryonic Antigen Peptide-Pulsed Dendritic Cells in Patients with Metastatic Cancer

Because of high mortality rate of metastatic cancers and significant toxicity of chemotherapy and radiotherapy, new treatment approaches such as immunotherapy warrant evaluation. The basis for modern approaches to immunotherapy is the observation that many tumors possess antigens that can be recognized by antigen-specific T cells leading to tumor rejection. The recognition of tumor-associated antigens is generally MHC class I-restricted1 and has been localized to eight to ten amino acid peptide fragments. These fragments bind in the groove of the MHC complex where they are presented to T lymphocytes that express receptors specific for the particular peptide. Among the described tumor antigens is carcinoembryonic antigen (CEA), expressed on most gastrointestinal adenocarcinomas, 50% of breast cancers, and 70% of non–small-cell lung cancers.2 The immunogenicity of CEA in man has been established in studies where patients have been immunized with recombinant, human CEA along with GM-CSF3, vaccinia vectors containing the gene for CEA4, and anti-idiotype specific for CEA.5 Peptide fragments of CEA have been found to be epitopes for T-cell lines generated from patients previously vaccinated with vaccinia-CEA. The most potent peptide in this study, designated CAP-1, is an HLA-A2 restricted CTL epitope.4 Because naive precursors of cytotoxic T cells specific for tumor antigens are present in very low numbers, methods for stimulating the T cells are necessary. Approaches have included viral vectors, administration of antigen with adjuvants and gene-transduced tumor cells. A more recent development has been the emerging understanding of the critical role that antigen-presenting cells, such as dendritic cells (DC), play in inducing T and B-cell responses in vivo. Numerous studies have demonstrated that DC loaded with tumor antigen can induce T-cell responses in vitro and in vivo murine models without significant toxicity. We and others have developed methods for generating a sufficient number of DC in vitro for use in immunotherapy studies.6-9 Furthermore, we have demonstrated that these DC loaded with CEA and other peptides are capable of inducing CEA specific T-cell responses in vitro.10 Therefore, we hypothesize that DC loaded with tumor-associated peptides such as the CEA peptide CAP-1 will be safe and induce CAP-1 specific Tcell responses in vivo in humans with metastatic cancers. Our objectives are: a) to determine the safety and dose-limiting toxicity of intravenous and intradermal injections of autologous, DC pulsed with an HLA-A2 restricted CEA peptide; b) to evaluate cellular immune response to the CEA peptide.

Induction of carcinoembryonic antigen (CEA)-specific cytotoxic T-lymphocyte responses in vitro using autologous dendritic cells loaded with CEA peptide or CEA RNA in patients with metastatic malignancies expressing CEA.

The application of dendritic cells (DC) to the active immunotherapy of cancer currently relies on the generation of potent DC capable of presenting tumor antigens such as carcinoembryonic antigen (CEA). It is unknown whether the T cells of patients with advanced malignancies can be reliably stimulated against tumor antigens by their autologous DC. In this study, starting with the peripheral blood mononuclear cells (PBMC) of patients with metastatic malignancies expressing CEA, autologous DCs were generated in vitro in serum-free media supplemented with GM-CSF and IL-4. The DCs from HLA A2 positive patients were loaded with the CEA peptide CAP-1 and the DCs from HLA A2 negative patients were depleted of bystander lymphocytes and loaded with mRNA encoding CEA. The DC preparations were tested to determine their phenotype and were used to stimulate autologous PBMC twice, separated by 10-14 days. The stimulated cells were then tested for their ability to lyse CEA-expressing target cells. We successfully generated an adequate number of DC for a clinical trial from all patients. The harvested DC preparations contained 49% DC and 87% DC if depleted of bystander lymphocytes. Phenotypic analysis showed the typical pattern of CD11c+ CD40+ CD86+ HLA-DR+ CD80(low) CD83(low) CD14(low). All preparations but one were able to stimulate CEA-specific cytotoxic T-lymphocyte (CTL) activity, suggesting that the majority of patients are not anergic to CEA and possess functional DC. The CTL activity was similar for the CEA peptide and CEA RNA-loaded DC.

In vitro induction of HLA-A2402-restricted and carcinoembryonic-antigen-specific cytotoxic T lymphocytes on fixed autologous peripheral blood cells.

HLA-A2402-restricted and carcinoembryonic-antigen(CEA)-specific cytotoxic T lymphocytes (CTL) were induced by culturing human peripheral blood mononuclear cells (PBMC) on formalin-fixed autologous adhesive PBMC that had been loaded with CEA-bound latex beads. The CTL killed the CEA-producing HLA-type matched cancer cells, but not the non-producers of CEA, at an effector/target ratio of 10 within 24 h. On the basis of available HLA-A24-binding peptides, we have also attempted to identify the epitope peptide recognized by the CTL. The peptide CEA652(9), TYACFVSNL, stimulated the CTL most strongly when pulsed on HLA-A2402-expressing target cells. The other nine peptides so far tested were also active, but less efficient in their effect on CTL. The CTL failed to kill target cells pulsed with the HLA-A2-binding CEA peptide, CAP-1. The CTL were also generated on the fixed adherent cells previously pulsed with the peptide CEA652(9). Cytotoxic activity of the CTL was inhibited by monoclonal antibodies against CD3, CD8, and MHC class I molecules. These results suggest that human autologous CTL will be inducible on the autologous fixed PBMC without use of the cultured target cancer cells if tumor antigenic protein is available.

Dendritic cells pulsed with CEA peptide induce CEA-specific CTL with restricted TCR repertoire.

The human carcinoembryonic antigen (CEA), which is expressed in several cancer types is a potential target for antigen-specific immunotherapy. In this study, we show that dendritic cells (DC) pulsed with an HLA class I restricted CEA cytotoxic T lymphocyte (CTL) peptide epitope can stimulate T cells to kill CEA peptide loaded T2 target cells as well as CEA expressing tumor lines in the presence of interleukin-7 (IL-7) in an HLA-restricted manner. This has been demonstrated for carcinoma patients as well as healthy donors. The DC-CEA + IL-7 stimulated cultures contained predominantly CD3+CD8+CD56- cells indicative of MHC class I restricted CTL. In addition, DC-CEA + IL-7 stimulated cells showed higher levels of CD69 expression compared with cells stimulated with IL-7 alone, implying an activated phenotype. When the T-cell receptor (TCR) from CTL cultures stimulated with DC-CEA + IL-7 was analyzed, an oligoclonal pattern of expression was found for certain V beta subfamilies compared with the polyclonal patterns shown by IL-7 or phytohemagglutinin stimulated T cells from the same donors. This TCR restriction appeared to be maintained and enhanced after additional rounds of restimulation with DC-CEA + IL-7. The association between cytotoxicity and TCR restriction suggests that TCR analysis may be useful as an in vitro indicator to monitor alterations in the T-cell population in response to antigen-specific immunotherapies.