Peptide-specific Cytotoxic T Lymphocytes Research Articles

Phase I Trial of a B7-1 (CD80) Gene Modified Autologous Tumor Cell Vaccine in Combination With Systemic Interleukin-2 in Patients With Metastatic Renal Cell Carcinoma

A reason that the immune system may fail to reject tumors is that T cells encounter tumor antigen derived peptides on the surface of tumor cells in a tolerizing rather than activating context since tumor cells do not express T cell co-stimulatory molecules such as B7-1 (CD80). In preclinical models over expression of B7-1 on the surface of tumor cells has been shown to activate T cells which kill tumor cells. We conducted a phase I clinical trial testing this approach in patients with metastatic renal cell carcinoma. Resected tumors from 15 patients were disaggregated and adapted to tissue culture, transduced with the B7-1 gene and injected subcutaneously as a vaccine. The dose of the vaccine was escalated in 3 separate cohorts of patients, and systemic interleukin-2 (IL-2) was administered as an adjuvant designed to enhance the proliferation of the vaccine activated T cells. Of the 15 patients 9 had measurable disease, 2 had a partial response and 2 had stable disease. Perivascular T cell infiltrates at autologous tumor delayed type hypersensitivity skin test sites developed in 3 of the 4 patients with stable disease or partial response. Although the patients experienced the usual and expected toxicity from the IL-2, there was no significant toxicity observed with the vaccine. The B7-1 gene modified autologous tumor cell vaccine is safe and can be combined with systemic IL-2 with acceptable toxicity. Immunological and clinical responses were observed in some of the patients. A phase II trial is reasonable to determine the efficacy of this approach.

A simple culture protocol to detect peptide-specific cytotoxic T lymphocyte precursors in the circulation.

The detection and monitoring of peptide-specific cytotoxic T lymphocyte (CTL) precursors is essential for successful peptide-based immunotherapy against cancers. In contrast to the development of effective methods of detecting antigen-specific CTL, such as ELISpot and HLA-class I tetramer assay, stimulation with peptide-pulsed antigen-presenting cells (APC) has for some time been conventionally employed to induce peptide-specific CTL from peripheral blood mononuclear cells (PBMC). This culture protocol, however, needs a substantial number of PBMC to test the reactivity against a panel of peptides. In the present study, we established a simple culture protocol which has no need of additional APC. Addition of a corresponding peptide every 3 days was found to induce not only Epstein-Barr virus (EBV)-specific CTL from healthy donors, but also tumor antigen-derived peptide-specific CTL from cancer patients. A 10-ml blood sample was almost sufficient to test the presence of CTL precursors against 20 different peptides in triplicate assays. Overall, this culture protocol can be useful in detecting and monitoring peptide-specific CTL precursors in the circulation in peptide-based immunotherapy against cancer.

ALK as a novel lymphoma-associated tumor antigen: identification of 2 HLA-A2.1–restricted CD8+ T-cell epitopes

AbstractOncogenic anaplastic lymphoma kinase (ALK) fusion proteins (NPM/ALK and associated variants) are expressed in about 60% of anaplastic large cell lymphomas (ALCLs) but are absent in normal tissues. In this study, we investigated whether ALK, which is expressed at high levels in lymphoma cells, could be a target for antigen-specific cell-mediated immunotherapy. A panel of ALK-derived peptides was tested for their binding affinity to HLA-A*0201 molecules. Binding peptides were assessed for their capacity to elicit a specific immune response mediated by cytotoxic T lymphocytes (CTLs) both in vivo, in HLA-A*0201 transgenic mice, and in vitro in the peripheral blood lymphocytes (PBLs) from healthy donors. Two HLA-A*0201–restricted CTL epitopes, p280-89 (SLAMLDLLHV) and p375-86 (GVLLWEIFSL), both located in the ALK kinase domain were identified. The p280-89– and p375-86–induced peptide-specific CTL lines were able to specifically release interferon-γ (IFN-γ) on stimulation with ALK peptide-pulsed autologous Epstein-Barr virus–transformed B cells (LCLs) or T2 cells. Anti-ALK CTLs lysed HLA-matched ALCL and neuroblastoma cell lines endogenously expressing ALK proteins. CTL activity was inhibited by anti-HLA-A2 monoclonal antibody CR11.351, consistent with a class I–restricted mechanism of cytotoxicity. These results show the existence of functional anti-ALK CTL precursors within the peripheral T-cell repertoire of healthy donors, clearly indicating ALK as a tumor antigen and ALK-derived peptides, p280-89 and p375-86, as suitable epitopes for the development of vaccination strategies.

Listeriolysin O-liposome-mediated cytosolic delivery of macromolecule antigen in vivo: enhancement of antigen-specific cytotoxic T lymphocyte frequency, activity, and tumor protection

Cytotoxic T lymphocytes (CTLs) are primed by peptide antigens that are endogenously processed in the cytosol and presented in the context of major histocompatibility complex I (MHC I) molecules of antigen-presenting cells (APCs). Exogenous soluble protein antigens do not gain efficient entry into the cytosol of APCs, and therefore requires a special cytosolic delivery method. We have developed such a delivery strategy adopting the well-elucidated cytosol-invading listerial endosomal escape mechanism, and report here an efficient delivery of exogenous whole protein antigen into the cytosol in a mouse model. Co-encapsulation of listeriolysin O (LLO) inside liposome (LLO-liposome) was required for delivery of ovalbumin (OVA) into the cytosol of APCs in primary cultures. LLO-liposome-mediated OVA immunization in mice engendered significantly higher OVA-specific CTL activity and increased antigenic peptide-specific CTL precursor (CTLp) frequency as compared to non-LLO-liposome or soluble OVA immunizations. Interferon-γ (IFN-γ) production upon specific stimulation by MHC I-restricted peptide was also significantly stronger by the inclusion of LLO in the liposomes. Rerouting of antigen into the cytosol by LLO-liposomes, however, did not reduce the extent of anti-OVA antibody responses. Moreover, LLO-liposome-antigen vaccination was robust in conferring protection to mice from lethal challenges with antigen-expressing tumor cells. Our study demonstrates a novel delivery system for efficient introduction of exogenous protein into the cytosol in vivo, priming cellular immune responses, which are protective in nature.

Functional detection of epithelial cell adhesion molecule specific cytotoxic T lymphocytes in patients with lung cancer, colorectal cancer and in healthy donors

Epithelial cell adhesion molecule (Ep-CAM) derived antigenic peptides have been identified that can be recognized by cytotoxic T lymphocytes (CTL) in a major histocompatibility complex (MHC) class I restricted fashion. Thus, altered expression of Ep-CAM in a variety of human tumors might render a potential target for T cell mediated therapy. We have examined, whether the novel HLA-A*0201 restricted peptide ILYENNVIT (184–192) corresponding to Ep-CAM and one heteroclitic modified variant peptide previously demonstrated to be immunogenic in the human system can elicit antigen specific CTL responses in HLA-A2 positive patients with history of Ep-CAM expressing cancer of lung and colon. Specific CTL recognition of T2 target cells pulsed with the native peptide as well as of the lung cancer cell line A549 indicates that an appropriate T cell repertoire can be expanded from peripheral blood from patients in clinical remission and with advanced cancer. Despite an overall low frequency, peptide specific precursor CTLs could be readily expanded from peripheral blood from 6/8 patients that were diagnosed previously with Ep-CAM expressing lung cancer and 4/8 control individuals (2/5 healthy donors and 2/3 colon cancer patients). CTLs from three of five lung cancer patients tested also lyzed the HLA-A2 + and Ep-CAM expressing lung cancer cell line A549. We did not detect an increased frequency of pCTLs after peripheral blood monocytes (PBMCs) were stimulated with the heteroclitic compound peptide. The results of our study indicate that Ep-CAM specific precursor CTL can be expanded in vitro and a specific T cell response against this epitope can be elicited in patients at various stages of lung cancer.

Protective Effects on A2KbTransgenic Mice That Were Immunized with Hepatitis B Virus X Antigen Peptides by the Activation of CD8+T Cells; XEP-3 Specific CTL Responses in thein vitroCulture

Background: Viral antigens presented on the cell surface in association with MHC class I molecules are recognized by CD8+ T cells. MHC restricted peptides are important in eliciting cellular immune responses. As peptide antigens have a weak immunigenicity, pH-sensitive liposomes were used for peptide delivery to induce effective cytotoxic T lymphocyte (CTL) responses. In the previous study, as the HBx peptides could induce specific CTLs in vitro, we tested whether the HLA-A2/K transgenic mice that were immunized by HBx-derived peptides could be protected from a viral challenge. Methods: HBx-peptides encapsulated by pH-sensitive liposomes were prepared. A2K transgenic mice were immunized i.m. on days one and seven with the indicated concentrations of liposome-encapsulated peptides. Three weeks later, mice were infected with 1×10pfu/head of recombinant vaccinia virus (rVV)-HBx via i.p. administration. The ovaries were extracted from the mice, and the presence of rVV-HBx in the ovaries was analyzed using human TK 143B cells. IFN-γsecretion by these cells was directly assessed using a peptide-pulsed target cell stimulation assay with either peptide-pulsed antigen presenting cells (APCs), concanavalin A (2μg/ml), or a vehicle. To generate peptide-specific CTLs, splenocytes obtained from the immunized mice were stimulated with 20μg/ml of each peptide and restimulated with peptide-pulsed APC four times. The cytotoxic activity of the CTLs was assessed by standard Cr-release assay and intracellular IFN-γassay. Results: Immunization of these peptides as a mixture in pH-sensitive liposomes to transgenic mice induced a good protective effect from a viral challenge by inducing the peptide-specific CD8+ T cells. Mice immunized with 50μg /head were much better protected against viral challenge compared to those immunized with 5μg/head, whereas the mice immunized with empty liposomes were not protected at all. After in vitro CTL culture by peptide stimulation, however, specific cytotoxicity was much higher in the CTLs from mice immunized with 5μg/head than 50μg/head 42 Yu Kyeong Hwang, et al. group. Increase of the number of cells that intracellular IFN-γsecreting cell among CD8+ T cells showed similar result. Conclusion: Mice immunized with XEPs within pH-sensitive liposome were protected against viral challenge. The protective effect depended on the amount of antigen used during immunization. XEP-3-specific CTLs could be induced by peptide stimulation in vitro from splenocytes obtained from immunized mice. The cytotoxic effect of CTLs was measured by Cr-release assay and the percentage of accumulated intracellular IFN-γ secreting cells after in vitro restimulation was measured by flow cytometric analysis. The result of Cr-release cytotoxicity test was well correlated with that of the flow cytometric analysis. Viral protection was effective in immunized group of 50μg/head, while in the in vitro restimulation, it showed more spectific response in 5μg/head group. (Immune Network 2002;2(1): 41-48)

Detection of peptide-specific cytotoxic T-lymphocyte precursors used for specific immunotherapy of pancreatic cancer.

The prognosis of pancreatic cancer is extremely poor with a 5-year survival of approximately 3%. Thus, the development of new treatment modalities, including a specific immunotherapy, is required. Our study investigated whether cytotoxic T-lymphocyte (CTL) precursors reacting to peptides with vaccine candidates (13 peptides for HLA-A2+ or -A24+ patients, respectively) were detectable in the prevaccination peripheral blood mononuclear cells (PBMCs) of 15 pancreatic cancer patients. Peptide-specific CTL precursors were detectable in the majority (11 of 15, 73%) of patients, with a mean positive number of 1.5 peptides (ranging from 0-5 peptides) per patient. Positive peptide profiles varied among patients. These results may provide a scientific basis for a new kind of cancer immunotherapy, namely, a CTL precursor-oriented peptide vaccine, for pancreatic cancer patients.

Identification of the HLA-A24 peptide epitope within cytomegalovirus protein pp65 recognized by CMV-specific cytotoxic T lymphocytes.

Among cytomegalovirus (CMV) tegument proteins, phosphoprotein 65 (pp65) has been identified as the important target antigen of the cytotoxic T lymphocyte (CTL) response against the virus. We synthesized seven CMV-pp65-derived peptides carrying an HLA-A24-binding motif, and investigated the ability of these peptides to induce CMV-specific CTL. We identified one nonamer peptide (pp65113-121; VYALPLKML) able to bind HLA-A24 and induce CTL responses in vitro in peripheral blood mononuclear cells (PBMC) from CMV-seropositive individuals. The peptide-specific CTLs generated were capable of recognizing pp65 expressed on CMV-infected fibroblasts as well as pp65113-121 peptide bound to the surface of C1R-A*2402 cells in an HLA-A24-restricted manner. The pp65113-121 peptide thus might be considered a synthetic peptide vaccine in HLA-A24-positive individuals.

Generation of cytotoxic T lymphocytes by MHC class I ligands fused to heat shock cognate protein 70.

Immunization with gp96 and heat shock cognate protein 70 (hsc70) purified with in vivo bound naturally occurring peptides or bound to synthetic peptides by in vitro reconstitution has been shown to induce peptide-specific cytotoxic T lymphocytes (CTL). In addition, mycobacterial heat shock protein 70 covalently fused to ovalbumin (OVA)-derived fragments has been shown to generate MHC class I-restricted CTL responses. Here, we genetically fused five different CTL epitopes, including peptides derived from Plasmodium yoelii circumsporozoite protein, tumor antigens, HY antigen and OVA, to either the N- or C-terminus of murine hsc70 and expressed the resulting proteins in Escherichia coli. Vaccination with all five fusion proteins induced peptide-specific CTL, indicating that no cognate flanking regions of CTL epitopes are necessary for the immune response. The point of injection was crucial for CTL induction. CD4(+) T cells were not required for the priming of CD8(+) T cells and vaccination with bone marrow-derived dendritic cells pulsed with hsc70 fusion proteins also elicited CTL responses. Furthermore, by using deletion mutants of hsc70, we identified amino acid residues 280-385 of hsc70 as the region most critical for inducing the CTL response.

Immune responses to tumour antigens: implications for antigen specific immunotherapy of cancer.

Tumour associated antigens recognised by cellular or humoral effectors of the immune system are potential targets for antigen specific cancer immunotherapy. Different categories of cancer antigens have been identified that induce cytotoxic T lymphocyte (CTL) responses in vitro and in vivo, namely: (1) "cancer testis" (CT) antigens, expressed in different tumours and normal testis, (2) melanocyte differentiation antigens, (3) point mutations of normal genes, (4) self antigens that are overexpressed in malignant tissues, and (5) viral antigens. Clinical studies with peptides and proteins derived from these antigens have been initiated to study the efficacy of inducing specific CTL responses in vivo. Immunological and clinical parameters for the assessment of antigen specific immune responses have been defined-delayed type hypersensitivity (DTH), CTL, autoimmmune, and tumour regression responses. Specific DTH and CTL responses and tumour regression have been observed after the intradermal administration of tumour associated peptides alone. Peptide specific immune reactions were enhanced after using granulocyte macrophage stimulating factor (GM-CSF) as a systemic adjuvant by increasing the frequency of dermal antigen presenting Langerhans cells. Complete tumour regression has been observed in the context of measurable peptide specific CTL. However, in single cases with disease progression after an initial tumour response, either a loss of single antigens targeted by CTL or of the presenting major histocompatibility complex (MHC) class I allele was detected, pointing towards immunisation induced immune escape. Cytokines to modulate antigen and MHC class I expression in vivo are being evaluated to prevent immunoselection. Recently, a new CT antigen, NY-ESO-1, has been identified on the basis of spontaneous antibody responses to tumour associated antigens. NY-ESO-1 appears to be one of the most immunogenic antigens known to date, with spontaneous immune responses observed in 50% of patients with NY-ESO-1 expressing cancers. Clinical studies have been initiated to evaluate the immunogenicity of different NY-ESO-1 constructs to induce both humoral and cellular immune responses in vivo.

Immunotherapy of melanoma.

Over the past decade, tumour immunology has progressed enormously, as a result of the application of a range of new technologies. Screening of tumour gene expression libraries using cancer patients' sera and cancer-specific T lymphocytes has defined a large number of tumour-specific antigenic proteins. The repertoire of known tumour antigens is rapidly expanding as a consequence of these advances. In addition, over the last few years the analysis of human T-cell responses specific for several tumour antigens in cancer patients has been made possible by the use of novel staining reagents (i.e. tetramers). This has allowed the characterization and monitoring of specific tumour responses, and has provided an opportunity to greatly accelerate the development of new anticancer vaccines. Recent results have demonstrated that the immune system can detect the presence of malignant cells in cancer patients, and have raised hopes for the use of antigenic cancer proteins as vaccines to induce tumour-specific cell-mediated immunity. Tumour immunology is now on solid technical and conceptual footing. However, the challenge remains to apply to patients the results obtained in the laboratories.

The peptide-specific alloreactive human T cell repertoire varies largely between individuals and is not extended in HLA-A*0205--anti-HLA-A*0201 pairings.

Alloreactive T cells recognize framework or peptide-dependent determinants on foreign MHC molecules. Among the peptide-dependent alloreactive T cells a significant proportion is specific for one particular peptide presented by the allo-MHC molecule as antigen-specific T cells would do. Such alloreactive, peptide-specific T cells are referred to as 'allorestricted'. High-avidity HLA-A*02 allorestricted cytotoxic T lymphocyte (CTL) clones specific for peptide libraries can be generated from HLA-A*02(-) donors. We made use of this technique to study the role of closely related self-HLA molecules on shaping of the alloreactive T cell repertoire. Peripheral blood lymphocytes from HLA-A*0205 individuals were stimulated by HLA-A*0201 targets pulsed with an HLA-A*0201 peptide library. We did not observe a bias towards peptide-specific CTL in the HLA-A*0201-directed alloreactive repertoire of HLA-A*0205 donors as compared to HLA-A*02(-) donors. Comparison of the alloreactive T cell response between two donors having similar HLA haplotypes demonstrated that the allorestricted T cell repertoire is largely different between individuals.

Synthetic Peptides Induce a Cytotoxic Response against Human Papillomavirus Type-18

Objectives. Over 90% of cervical carcinomas express human papillomavirus (HPV) E6 and E7 proteins. These unique antigens are ideal targets for the development of cytotoxic T-lymphocytes (CTL) for antitumor immunotherapy. In this study we identify peptides from HPV-18 E6 and E7 proteins that bind to HLA class I molecules. We further show that these peptides are able to induce peptide-specific CTL from an HLA-A2-positive (+) peripheral blood donor in vitro.Methods. A computer-assisted algorithm was devised to identify peptides from HPV-18 E6 and E7 proteins that bind to HLA-A2 molecules. Peptides that were predicted to bind were synthesized and their binding activity was determined. HLA-A2+ irradiated stimulator cells pulsed with HPV-18 peptides were incubated with HLA-A2+ peripheral blood mononuclear cells. Cytotoxicity assays were performed to assess specific cell lysis.Results. Of 295 possible sequences, the computer-assisted algorithm predicted 10 peptides that would have a high probability of binding to HLA-A2. The 4 strongest binding peptides were analyzed for their ability to induce cytotoxic cells against HPV-18 peptide-pulsed targets. Two of the peptides induced significant lysis.Conclusions. There are limited data on peptide-based immunotherapy for HPV-18+ tumors. The combination of our computer-assisted algorithm and binding assay permits rapid selection of potential CTL epitopes. We identified two peptides that were able to induce peptide-specific lysis. These two epitopes are candidates for a peptide-based vaccine against HPV-18+ tumors. The model described has broad applications and can be used in the development of immunotherapy for other types of cancers.

Identification of hepatitis B virus-specific CTL epitopes presented by HLA-A*2402, the most common HLA class I allele in East Asia

Background/Aims : The aim of this study was to identify and characterize hepatitis B virus (HBV)-specific cytotoxic T lymphocytes (CTL) epitopes presented by human leukocyte antigen (HLA)-A*2402, most common HLA class I allele in East Asia. Methods : HLA-A*2402-restricted CTL epitopes were identified by reverse immunogenetics. Immunogenecity of these epitopes was investigated using peripheral blood mononuclear cell (PBMC) from HLA-A24 + patients with acute hepatitis B. Results : An HLA-A*2402 stabilization assay demonstrated that 36 of 63 HBV peptides carrying HLA-A*2402 anchor residues have high- and medium-HLA-A*2402 binding affinity. Two (C117–125 and P756–764) of the 36 peptides induced peptide-specific CTLs. CTL clones and lines specific for these peptides killed HBV recombinant vaccinia virus-infected target cells expressing HLA-A*2402, indicating that these two peptides are CTL epitopes presented by HLA-A*2402. These two peptides were able to induce specific CTLs in 7 and 11 of 12 HLA-A24 + patients with acute hepatitis B, respectively. Conclusions : We identified two immunodominant CTL epitopes restricted by HLA-A*2402. Because HLA-A*2402 is the most common allele in East Asia, a region in which there are approximately 200 million HBV carriers, these epitopes will be useful for analysis of CTL responses in patients from East Asia.

Generation of hepatitis C virus-specific cytotoxic T lymphocytes from healthy individuals with peptide-pulsed dendritic cells.

In hepatitis C virus (HCV) infection, cytotoxic T lymphocytes (CTL) are involved in liver inflammation and contribute to the reduction of viral load. Antibodies for HCV-CTL precursor frequencies (CTLpf) are relatively low in chronic hepatitis C, and this may be related to the poor CTL response in vivo. The aim of this study was to assess the efficacy of dendritic cells (DC) as antigen-presenting cells in CTL generation from low CTLpf. To confirm the rationale of using DC to prime naive T cells, five HCV-uninfected individuals were enrolled in the study. We obtained DC by maturation from peripheral progenitors under stimulation with granulocyte-macrophage colony-stimulating factor (GM-CSF), interleukin (IL)-4 and IL-1alpha. Autologous T cells were cultured with DC or concanavalin-A-induced blasts loaded with four HCV-derived peptides bearing human leukocyte antigen (HLA)-A*0201 or -A24 motifs for 28 days under IL-7 and IL-2 stimulation. The lytic activity against peptide-pulsed targets was assessed by using a [51Cr]-releasing assay. The DC strongly expressed HLA class I, II, B7-1 and B7-2, but not phenotypic markers of T-, B-, natural killer (NK)-cells or monocytes. The CD8-positive, HLA-class I-restricted and HCV peptide-specific CTL were generated with DC from HLA-A antigen-matched subjects, whereas no CTL activity was detected with concavalin (Con-A) blasts. We were thus able to generate HCV specific CTL from naive precursors with peptide-pulsed DC. This DC-based system can be used to generate CTL of desired antigen specificity, even from a source with low CTLpf.

Analysis of MAGE-3 derived synthetic peptide as a human lung cancer antigen recognized by cytotoxic T lymphocytes.

The human MAGE-3 gene was originally discovered in melanoma cells that encode tumor antigens, and has been reported to be expressed in various types of tumors, including lung cancer, but not in normal tissues other than testis or placenta. Our aim in this study was to clarify whether HLA-A2 restricted MAGE-3 peptide (FLWGPRALV) could be a lung cancer antigen recognized by cytotoxic T lymphocytes (CTL). MAGE-3-derived peptide-specific CTL were induced from the peripheral blood mononuclear cells (PBMC) of HLA-A0201-positive healthy donors and the regional lymph node lymphocytes (RLNL) of HLA-A2-positive patients with lung cancer by multiple stimulations with peptide-pulsed HLA-A0201-positive antigen-presenting cells. Lymphocytes stimulated with MAGE-3 peptide exhibited specific lysis of Epstein-Barr virus-transformed B cells (EBV-B) pulsed with MAGE-3 peptide, but not with control peptide derived from influenza matrix protein, erbB-2, or wild type p53. Specific activity for MAGE-3-presenting targets was found after the second stimulation, and increased depending on the number of stimulations. The peptide-specific activity was inhibited by the addition of monoclonal antibodies against MHC class I and HLA-A2. Such CTL also recognized tumor cell lines expressing both HLA-A2 and MAGE-3 in an MHC class I-restricted manner, but did not recognize tumor cell lines that did not express HLA-A2 or MAGE-3. These results suggested the MAGE-3 peptide could be a potential target of specific immunotherapy for HLA-A2 patients with lung cancer.

Identification of human antitumor cytotoxic T lymphocytes epitopes of recoverin, a cancer-associated retinopathy antigen, possibly related with a better prognosis in a paraneoplastic syndrome

Cancer-associated retinopathy (CAR) is a rare paraneoplastic syndrome, and the recoverin-specific autoantibody is suggested to contribute to the pathogenesis of retinopathy, including apoptosis of retinal cells. Because it is known that CAR+ cancer patients have a preferable prognosis, we hypothesized that aberrantly expressed recoverin in cancer cells can become a target of cytotoxic T lymphocytes (CTL). Here we tested nine recoverin-derived HLA-A24-binding peptides for their capacity to elicit antitumor CTL. We observed recoverin-specific CTL responses in two HLA-A24+ CAR+ cancer patients. In addition, the CTL responses were obtained from three of ten CAR– cancer patients and two of six healthy individuals. The CTL precursor frequency of CAR+ cancer patients and that of CAR– cancer patients was higher than that of healthy individuals. Of nine recoverin peptides, R49 (QFQSIYAKF), R49.2 (QFQSIYAKFF), and R64 (AYAQHVFRSF) were discovered to induce the peptide-specific CTL. Taken together, our present data suggest that peripheral activation of recoverin-specific antitumor CTL is likely to contribute to the preferable prognosis of CAR+ cancer patients. Moreover, in cases other than CAR+ cancer patients, recoverin may offer the opportunity to design epitope-based immunotherapeutic approaches for treating HLA-A24+ cancer patients with a recoverin-expressing tumor.

Identification of human antitumor cytotoxic T lymphocytes epitopes of recoverin, a cancer-associated retinopathy antigen, possibly related with a better prognosis in a paraneoplastic syndrome

Cancer-associated retinopathy (CAR) is a rare paraneoplastic syndrome, and the recoverin-specific autoantibody is suggested to contribute to the pathogenesis of retinopathy, including apoptosis of retinal cells. Because it is known that CAR+ cancer patients have a preferable prognosis, we hypothesized that aberrantly expressed recoverin in cancer cells can become a target of cytotoxic T lymphocytes (CTL). Here we tested nine recoverin-derived HLA-A24-binding peptides for their capacity to elicit antitumor CTL. We observed recoverin-specific CTL responses in two HLA-A24+ CAR+ cancer patients. In addition, the CTL responses were obtained from three of ten CAR– cancer patients and two of six healthy individuals. The CTL precursor frequency of CAR+ cancer patients and that of CAR– cancer patients was higher than that of healthy individuals. Of nine recoverin peptides, R49 (QFQSIYAKF), R49.2 (QFQSIYAKFF), and R64 (AYAQHVFRSF) were discovered to induce the peptide-specific CTL. Taken together, our present data suggest that peripheral activation of recoverin-specific antitumor CTL is likely to contribute to the preferable prognosis of CAR+ cancer patients. Moreover, in cases other than CAR+ cancer patients, recoverin may offer the opportunity to design epitope-based immunotherapeutic approaches for treating HLA-A24+ cancer patients with a recoverin-expressing tumor.

Identification of human antitumor cytotoxic T lymphocytes epitopes of recoverin, a cancer-associated retinopathy antigen, possibly related with a better prognosis in a paraneoplastic syndrome.

Cancer-associated retinopathy (CAR) is a rare paraneoplastic syndrome, and the recoverin-specific autoantibody is suggested to contribute to the pathogenesis of retinopathy, including apoptosis of retinal cells. Because it is known that CAR(+) cancer patients have a preferable prognosis, we hypothesized that aberrantly expressed recoverin in cancer cells can become a target of cytotoxic T lymphocytes (CTL). Here we tested nine recoverin-derived HLA-A24-binding peptides for their capacity to elicit antitumor CTL. We observed recoverin-specific CTL responses in two HLA-A24(+) CAR(+) cancer patients. In addition, the CTL responses were obtained from three of ten CAR(-) cancer patients and two of six healthy individuals. The CTL precursor frequency of CAR(+) cancer patients and that of CAR(-) cancer patients was higher than that of healthy individuals. Of nine recoverin peptides, R49 (QFQSIYAKF), R49.2 (QFQSIYAKFF), and R64 (AYAQHVFRSF) were discovered to induce the peptide-specific CTL. Taken together, our present data suggest that peripheral activation of recoverin-specific antitumor CTL is likely to contribute to the preferable prognosis of CAR(+) cancer patients. Moreover, in cases other than CAR(+) cancer patients, recoverin may offer the opportunity to design epitope-based immunotherapeutic approaches for treating HLA-A24(+) cancer patients with a recoverin-expressing tumor.

Antibody-conjugated MHC class I tetramers can target tumor cells for specific lysis by T lymphocytes.

To demonstrate that antibody-guided targeting of antigenic MHC class I-peptide tetramer on tumor cells can render them susceptible to lysis by relevant cytotoxic T lymphocytes (CTL), biotinylated HLA-A*0201/Flu matrix peptide complexes were tetramerized on streptavidin molecules previously coupled to Fab' fragments from monoclonal antibodies (mAb) specific for cell surface markers such as carcinoembryonic antigen (CEA), ErbB-2 or CD20. Flow cytometry analysis showed that coating of the HLA-A2-peptide complexes on the four HLA-A2-negative human cancer lines tested (including a CEA-positive colon carcinoma, an ErbB-2(+) breast carcinoma and two CD20(+) B lymphomas) was entirely dependent upon the specificity of the conjugated antibody fragments. More importantly, HLA-A2-restricted Flu matrix peptide-specific CTL were then found to lyse specifically and efficiently the MHC-coated target cells. These results open the way to the development of new immunotherapy strategies based on antibody targeting of MHC class I-peptide complexes.