Immunogenicity of the carcinoembryonic antigen derived peptide 694 in HLA-A2 healthy donors and colorectal carcinoma patients

Carcinoembryonic antigen (CEACAM5) is commonly overexpressed in human colon cancer. Several antigenic peptides recognized by cytolytic CD8+ T-cells have been identified and used in colon cancer phase-I vaccination clinical trials. The HLA-A*0201-binding CEA_694–702 peptide was recently isolated from acid eluted MHC-I associated peptides from a human colon tumor cell line. However, the immunogenicity of this peptide in humans remains unknown. We found that the peptide CEA_694–702 binds weakly to HLA-A*0201 molecules and is ineffective at inducing specific CD8+ T-cell responses in healthy donors. Immunogenic-altered peptide ligands with increased affinity for HLA-A*0201 were identified. Importantly, the elicited cytolytic T lymphocyte (CTL) lines and clones cross-reacted with the wild-type CEA_694–702 peptide. Tumor cells expressing CEA were recognized in a peptide and HLA-A*0201 restricted fashion, but high-CEA expression levels appear to be required for CTL recognition. Finally, CEA-specific T-cell precursors could be readily expanded by in vitro stimulation of peripheral blood mononuclear cell (PBMC) from colon cancer patients with altered CEA peptide. However, the CEA-specific CD8+ T-cell clones derived from cancer patients revealed low-functional avidity and impaired tumor-cell recognition. Together, using T-cells to demonstrate the processing and presentation of the peptide CEA694-702, we were able to corroborate its presentation by tumor cells. However, the low avidity of the specific CTLs generated from cancer patients as well as the high-antigen expression levels required for CTL recognition pose serious concerns for the use of CEA694-702 in cancer immunotherapy.     

STEAP, a prostate tumor antigen, is a target of human CD8+ T cells

STEAP is a recently identified protein shown to be particularly overexpressed in prostate cancer and also present in numerous human cancer cell lines from prostate, pancreas, colon, breast, testicular, cervical, bladder and ovarian carcinoma, acute lymphocytic leukemia and Ewing sarcoma. This expression profile renders STEAP an appealing candidate for broad cancer immunotherapy. In order to investigate if STEAP is a tumor antigen that can be targeted by specific CD8⁺ T cells, we identified two high affinity HLA-A*0201 restricted peptides (STEAP_86–94 and STEAP_262–270). These peptides were immunogenic in vivo in HLA-A*0201 transgenic HHD mice. Peptide specific murine CD8 T cells recognized COS-7 cells co-transfected with HHD (HLA-A*0201) and STEAP cDNA constructs and also HLA-A*0201⁺ STEAP⁺ human tumor cells. Furthermore, STEAP_86–94 and STEAP_262–270 stimulated specific CD8⁺ T cells from HLA-A*0201⁺ healthy donors, and these peptide specific CD8⁺ T cells recognized STEAP positive human tumor cells in an HLA-A*0201-restricted manner. Importantly, STEAP_86–94-specific T cells were detected and reactive in the peripheral blood mononuclear cells in NSCLC and prostate cancer patients ex vivo. These results show that STEAP can be a target of anti-tumor CD8⁺ T cells and that STEAP peptides can be used for a broad-spectrum-tumor immunotherapy.     

An altered peptide ligand for naïve cytotoxic T lymphocyte epitope of TRP-2<Subscript>(180–188)</Subscript> enhanced immunogenicity

Tyrosinase-related protein-2 (TRP-2) is a non-mutated melanocyte differentiation antigen. The TRP-2-recognizing CD8⁺ T cells can evoke immune responses to melanoma in both humans and mice. Developing epitopes with amino acid replacements in their sequences might improve the low immunogenicity against this ‘self’ tumor antigen. We designed altered peptide ligands (APLs) of TRP-2_(180–188) (SVYDFFVWL) with preferred primary and auxiliary HLA-A*0201 molecule anchor residue replacement. These APLs were screened for MHC-affinity by affinity prediction plots and molecular dynamics simulation, and analyzed in vitro for stability and binding-affinity to molecular HLA-A*0201. We also investigated the CTLs activities induced by TRP-2 wild-type epitope and the APLs both in vitro in human PBMCs and HLA-A2.1/K^b transgenic mice. The results indicate that TRP-2 2M analog simultaneously had stronger binding-affinity and a lower dissociation rate to HLA-A*0201, than wild-type peptide. In addition, the analog 2M was superior to other APLs and wild-type epitope in terms of immunological efficacy ex vivo as measured by the ELISPOT assays of IFN-γ and granzyme B. These results demonstrate that TRP-2 2M is an agonist epitope that can induce anti-tumor immunity superior to its wild-type epitope, and has potential application in peptide-mediated immunotherapy.     

Cytotoxic T lymphocyte epitopes from human heparanase can elicit a potent anti-tumor immune response in mice

Heparanase is expressed in almost all advanced tumors, and therefore it may serve as a potential target for tumor therapy. Our previous study has shown that heparanase can serve as a potential universal tumor-associated antigen (TAA) for the immunotherapy of advanced tumors. Further study demonstrated that the HLA-A*0201-restricted Cytotoxic T lymphocytes (CTL) epitopes Hpa525 (PAFSYSFFV), Hpa277 (KMLKSFLKA) and Hpa405 (WLSLLFKKL) from human heparanase could induce a potent anti-tumor immune response in vitro. The present study was designed to investigate whether the above peptides could induce immune responses in mice. Our results demonstrated that the effectors from heparanase peptide-immunized mice could effectively lyse various tumor cells that were heparanase positive and HLA-A*0201 matched. We also found that these peptide-specific CTLs did not lyse autologous lymphocytes that had low heparanase activity. Further study revealed that Hpa525, Hpa277, and Hpa405 peptides increased the frequency of IFN-γ-producing T cells as compared to a negative peptide. These results suggest that Hpa525, Hpa277, and Hpa405 peptides are novel HLA-A*0201-restricted CTL epitopes capable of inducing heparanase-specific CTLs in mice. Because heparanase is expressed in most advanced malignant tumors, Hpa525, Hpa277, and Hpa405 peptide-based vaccines may be useful for the immunotherapy of patients with advanced tumors.     

Identification of a novel HLA-A2-restricted cytotoxic T lymphocyte epitope from cancer-testis antigen PLAC1 in breast cancer

Identification of cytotoxic T lymphocyte (CTL) epitopes from tumor antigens is essential for the development of peptide vaccines against tumor immunotherapy. Among all the tumor antigens, the caner-testis (CT) antigens are the most widely studied and promising targets. PLAC1 (placenta-specific 1, CT92) was considered as a novel member of caner-testis antigen, which expressed in a wide range of human malignancies, most frequently in breast cancer. In this study, three native peptides and their analogues derived from PLAC1 were predicted by T cell epitope prediction programs including SYFPEITHI, BIMAS and NetCTL 1.2. Binding affinity and stability assays in T2 cells showed that two native peptides, p28 and p31, and their analogues (p28-1Y9?V, p31-1Y2L) had more potent binding activity towards HLA-A*0201 molecule. In ELISPOT assay, the CTLs induced by these four peptides could release IFN-γ. The CTLs induced by these four peptides from the peripheral blood mononuclear cells (PBMCs) of HLA-A*02⁺ healthy donor could lyse MCF-7 breast cancer cells (HLA-A*0201⁺, PLAC1⁺) in vitro. When immunized in HLA-A2.1/K^b transgenic mice, the peptide p28 could induce the most potent peptide-specific CTLs among these peptides. Therefore, our results indicated that the peptide p28 (VLCSIDWFM) could serve as a novel candidate epitope for the development of peptide vaccines against PLAC1-positive breast cancer.     

Generation of human tumor-specific CTLs in HLA-A2.1–transgenic mice using unfractionated peptides from eluates of human primary breast and ovarian tumors

HER-2/neu oncoprotein is overexpressed in a variety of human tumors and is associated with malignant transformation and aggressive disease. Due to its overexpression in tumor cells and because it has been shown to be immunogenic, this protein represents an excellent target for T-cell immunotherapy. Peptide extracts derived from primary HLA-A*0201-positive (+) HER-2/neu⁺ human tumors by acid elution (acid cell extracts (ACEs)) were tested for their capacity to elicit in HLA-A*0201 transgenic mice, cytotoxic T lymphocytes (CTLs) lysing HLA-A*0201⁺ HER-2/neu⁺ tumor cells. Injections of ACE in transgenic mice induced CTLs capable of specifically lysing HER-2/neu⁺ tumor cell lines (also including the original HER-2/neu⁺ primary tumor cells from which the ACEs were derived) in an HLA-A*0201–restricted fashion. Adoptive transfer of ACE-induced CTLs was sufficient to significantly prolong survival of SCID mice inoculated with HLA-A*0201⁺ HER-2/neu⁺ human tumor cell lines. Cytotoxicity of such ACE-induced CTL lines was directed, at least as detected herein, also against the HER-2/neu peptides HER-2 (9₃₆₉) and HER-2 (9₄₃₅) demonstrating the immunodominance of these epitopes. HER-2 peptide–specific CTLs generated in the HLA-A*0201–transgenic mice, upon peptide immunization, lysed in vitro HER-2/neu⁺ human tumor cell lines in an HLA-A*0201–restricted manner and, when adoptively transferred, conferred sufficient protection in SCID mice inoculated with the same human tumor cell lines as above. However, CTLs induced by ACEs displayed enhanced efficacy in the therapy of xenografted SCID mice compared with the HER-2 peptide–specific CTLs (i.e., HER-2 [9₃₆₉] or HER-2 [9₄₃₅]). Even by administering mixtures of CTLs specific for each of these peptides, the prolongation of survival achieved was still inferior compared with that obtained with ACE-induced CTLs. This suggested that additional epitopes may contribute to the immunogenicity of such tumor-derived ACEs. Thus, immunization with ACEs from HER-2/neu⁺ primary tumor cells appears to be an effective approach to generate multiple and potent CTL-mediated immune responses against HER-2/neu⁺ tumors expressing the appropriate HLA allele(s). By screening ACE-induced CTL lines with synthetic peptides encompassing the HER-2/neu sequence, it is feasible to identify immunodominant epitopes which may be used in mixtures as vaccines with enhanced efficacy in both the prevention and therapy of HER-2/neu⁺ malignancies.This work was supported by grants from the Regional Operational Program Attika (No. 20, MIS code 59605GR) to M.P., and from the GSRT Program (No. PENED 01ED55) to C.N.B.     

Identification and modification of an HLA-A*0201-restricted cytotoxic T lymphocyte epitope from Ran antigen

Ran is considered to be a promising target for tumor-specific immunotherapy because its protein is exclusively expressed in tumor tissues, though its mRNA can be expressed in most normal tissues. In our study, we obtained four candidate wild-type epitopes designated Ran1, Ran2, Ran3, and Ran4, derived from the Ran antigen with the highest predicted affinity with MHC-I, indicated by affinity prediction plots and molecular dynamics simulation. However, in vitro affinity assays of these epitopes showed only a moderate affinity with MHC-I. Thus, we designed altered peptide ligands (APLs) derived from Ran wild-type epitopes with preferred primary and auxiliary HLA-A*0201 molecule anchor residue replacement. Of the eight tested peptides, the 1Y analog had the strongest binding-affinity and lowest-dissociation rate to HLA-A*0201. Additionally, we investigated the CTLs activities induced by Ran wild-type peptides and the APLs in human PBMCs and in HLA-A*0201/K^b transgenic mice. Ran1 1Y was superior to other APLs and wild-type peptides in eliciting epitope-specific CTL immune responses both in vitro and in vivo. In summary, a wild-type epitope of the tumor-specific antigen Ran, expressed broadly in many tumors, was identified and designated Ran1. An APL of Ran1, Ran1 1Y, was further designed and verified in vitro and in vivo and found to elicit a stronger Ran-specific CTL response, indicating a potential anti-tumor application in the future.     

Melanomas with concordant loss of multiple melanocytic differentiation proteins: immune escape that may be overcome by targeting unique or undefined antigens

Melanoma-reactive HLA-A*0201-restricted cytotoxic T lymphocyte (CTL) lines generated in vitro lyse autologous and HLA-matched allogeneic melanoma cells and recognize multiple shared peptide antigens from tyrosinase, MART-1, and Pmel17/gp100. However, a subset of melanomas fail to be lysed by these T cells. In the present report, four different HLA-A*0201⁺ melanoma cell lines not lysed by melanoma-reactive allogeneic CTL have been evaluated in detail. All four are deficient in expression of the melanocytic differentiation proteins (MDP) tyrosinase, Pmel17/gp100, gp75/trp-1, and MART-1/Melan-A. This concordant loss of multiple MDP explains their resistance to lysis by melanoma-reactive allogeneic CTL and confirms that a subset of melanomas may be resistant to tumor vaccines directed against multiple MDP-derived epitopes. All four melanoma lines expressed normal levels of HLA-A*0201, and all were susceptible to lysis by xenoreactive-peptide-dependent HLA-A*0201-specific CTL clones, indicating that none had identifiable defects in antigen-processing pathways. Despite the lack of shared MDP-derived antigens, one of these MDP-negative melanomas, DM331, stimulated an effective autologous CTL response in vitro, which was restricted to autologous tumor reactivity. MHC-associated peptides isolated by immunoaffinity chromatography from HLA-A1 and HLA-A2 molecules of DM331 tumor cells included at least three peptide epitopes recognized by DM331 CTL and restricted by HLA-A1 or by HLA-A*0201. Recognition of these CTL epitopes cannot be explained by defined, shared melanoma antigens; instead, unique or undefined antigens must be responsible for the autologous-cell-specific anti-melanoma response. These findings suggest that immunotherapy directed against shared melanoma antigens should be supplemented with immunotherapy directed against unique antigens or other undefined antigens, especially in patients whose tumors do not express MDP. Received: 31 October 1997 / Accepted: 4 August 1999     

Screening and identification of severe acute respiratory syndrome-associated coronavirus-specific CTL epitopes

Severe acute respiratory syndrome (SARS) is a highly contagious and life-threatening disease that emerged in China in November 2002. A novel SARS-associated coronavirus was identified as its principal etiologic agent; however, the immunopathogenesis of SARS and the role of special CTLs in virus clearance are still largely uncharacterized. In this study, potential HLA-A*0201-restricted spike (S) and nucleocapsid protein-derived peptides were selected from an online database and screened for potential CTL epitopes by in vitro refolding and T2 cell-stabilization assays. The antigenicity of nine peptides which could refold with HLA-A*0201 molecules was assessed with an IFN-gamma ELISPOT assay to determine the capacity to stimulate CTLs from PBMCs of HLA-A2(+) SARS-recovered donors. A novel HLA-A*0201-restricted decameric epitope P15 (S411-420, KLPDDFMGCV) derived from the S protein was identified and found to localize within the angiotensin-converting enzyme 2 receptor-binding region of the S1 domain. P15 could significantly enhance the expression of HLA-A*0201 molecules on the T2 cell surface, stimulate IFN-gamma-producing CTLs from the PBMCs of former SARS patients, and induce specific CTLs from P15-immunized HLA-A2.1 transgenic mice in vivo. Furthermore, significant P15-specific CTLs were induced from HLA-A2.1-transgenic mice immunized by a DNA vaccine encoding the S protein; suggesting that P15 was a naturally processed epitope. Thus, P15 may be a novel SARS-associated coronavirus-specific CTL epitope and a potential target for characterization of virus control mechanisms and evaluation of candidate SARS vaccines.     

Identification and characterization of a HER-2/neu epitope as a potential target for cancer immunotherapy

Our aim is to develop peptide vaccines that stimulate tumor antigen-specific T-lymphocyte responses against frequently detected cancers. We describe herein a novel HLA-A*0201-restricted epitope, encompassing amino acids 828–836 (residues QIAKGMSYL), which is naturally presented by various HER-2/neu ⁺ tumor cell lines. HER-2/neu(828-836), [HER-2(9₈₂₈)], possesses two anchor residues and stabilized HLA-A*0201 on T2 cells in a concentration-dependent Class I binding assay. This peptide was stable for 3.5 h in an off-kinetic assay. HER-2(9₈₂₈) was found to be immunogenic in HLA-A*0201 transgenic (HHD) mice inducing peptide-specific and functionally potent CTL and long-lasting anti-tumor immunity. Most important, using HLA-A*0201 pentamer analysis we could detect increased ex vivo frequencies of CD8⁺ T-lymphocytes specifically recognizing HER-2(9₈₂₈) in 8 out of 20 HLA-A*0201⁺ HER-2/neu ⁺ breast cancer patients. Moreover, HER-2(9₈₂₈)-specific human CTL recognized the tumor cell line SKOV3.A2 as well as the primary RS.A2.1.DR1 tumor cell line both expressing HER-2/neu and HLA-A*0201. Finally, therapeutic vaccination with HER-2(9₈₂₈) in HHD mice was proven effective against established transplantable ALC.A2.1.HER tumors, inducing complete tumor regression in 50% of mice. Our data encourage further exploitation of HER-2(9₈₂₈) as a promising candidate for peptide-based cancer vaccines.     

CD8 T cell responses to myelin oligodendrocyte glycoprotein-derived peptides in humanized HLA-A*0201-transgenic mice

Multiple sclerosis (MS) is a demyelinating inflammatory disease of the CNS. Though originally believed to be CD4-mediated, additional immune effector mechanisms, including myelin-specific CD8(+) T cells, are now proposed to participate in the pathophysiology of MS. To study the immunologic and encephalitogenic behavior of HLA-A*0201-binding myelin-derived epitopes in vivo, we used a humanized HLA-A*0201-transgenic mouse model. Eight HLA-A*0201-binding peptides derived from myelin oligodendrocyte glycoprotein (MOG), an immunodominant myelin self-Ag, were identified in silico. After establishing their relative affinity for HLA-A*0201 and their capacity to form stable complexes with HLA-A*0201 in vitro, their immunological characteristics were studied in HLA-A*0201-transgenic mice. Five MOG peptides, which bound stably to HLA-A*0201 exhibited strong immunogenicity by inducing a sizeable MOG-specific HLA-A*0201-restricted CD8(+) T cell response in vivo. Of these five candidate epitopes, four were processed by MOG-transfected RMA target cells and two peptides proved immunodominant in vivo in response to a plasmid-encoding native full-length MOG. One of the immunodominant MOG peptides (MOG(181)) generated a cytotoxic CD8(+) T cell response able to aggravate CD4(+)-mediated EAE. Therefore, this detailed in vivo characterization provides a hierarchy of candidate epitopes for MOG-specific CD8(+) T cell responses in HLA-A*0201 MS patients identifying the encephalitogenic MOG(181) epitope as a primary candidate.     

Assessment of immunogenicity of human Melan-A peptide analogues in HLA-A*0201/Kb transgenic mice

Previous studies have shown that substitution of single amino acid residues in human Melan-A immunodominant peptides Melan-A27-35 and Melan-A26-35 greatly improved their binding and the stability of peptide/HLA-A*0201 complexes. In particular, one Melan-A peptide analogue was more efficient in the generation of Melan-A peptide-specific and melanoma-reactive CTL than its parental peptide in vitro from human PBL. In this study, we analyzed the in vivo immunogenicity of Melan-A natural peptides and their analogues in HLA-A*0201/Kb transgenic mice. We found that two human Melan-A natural peptides, Melan-A26-35 and Melan-A27-35, were relatively weak immunogens, whereas several Melan-A peptide analogues were potent immunogens for in vivo CTL priming. In addition, induced Melan-A peptide-specific mouse CTL cross-recognized natural Melan-A peptides and their analogues. More interestingly, these mouse CTL were also able to lyse human melanoma cell lines in vitro in a HLA-A*0201-restricted, Melan-A-specific manner. Our results indicate that the HLA-A*0201/Kb transgenic mouse is a useful animal model to perform preclinical testing of potential cancer vaccines, and that Melan-A peptide analogues are attractive candidates for melanoma immunotherapy.     

Cytotoxic T-lymphocyte responses to human papillomavirus type 16 E5 and E7 proteins and HLA-A*0201-restricted T-cell peptides in cervical cancer patients

Previously, we found that human papillomavirus type 16 (HPV-16) E5 protein is a tumor rejection antigen and can induce cytotoxic T-lymphocyte (CTL) activity. Therefore, in this study, human leukocyte antigen A*0201 (HLA-A*0201)-restricted human CTL epitopes of HPV-16 E5 protein were identified using a bioinformatics approach, and the abilities of these predicted peptides to induce an immune response in HLA-A*0201 transgenic mice were confirmed by assaying E5-specific CTLs and in vitro-generated CTLs from normal peripheral blood T lymphocytes of HLA-A2-positive human donors. Second, the CTL responses to HLA-A*0201 CTL epitopes (E5 63-71 and E7 11-20) were examined in HPV-16-infected patients with HLA-A2. Third, the effect of HLA-A-type alleles on CTL activities in response to the entire E5 and E7 proteins was examined in cervical cancer patients. E5 and E7 peptides (but not the whole proteins) stimulated E5- and E7-specific CTL recall responses in HPV-16- and HLA-A2-positive cervical cancer patients, and HPV-16 E5 and E7 proteins stimulated na?ve T cells in HPV-16-negative cervical cancer patients with HLA-A11 and -A24 haplotypes. In summary, this is the first demonstration that E5 63-71 is an HLA-A*0201-restricted T-cell epitope of HPV-16 E5.     

Adoptive T cell immunotherapy of human uveal melanoma targeting gp100

HLA-A*0201-restricted CTL against human gp100 were isolated from HLA-A*0201/K(b) (A2/K(b))-transgenic mice immunized with recombinant canarypox virus (ALVAC-gp100). These CTL strongly responded to the gp100(154-162) epitope, in the context of both the chimeric A2/K(b) and the wild-type HLA-A*0201- molecule, and efficiently lysed human HLA-A*0201(+), gp100(+) melanoma cells in vitro. The capacity of the CTL to eradicate these tumors in vivo was analyzed in A2/K(b)-transgenic transgenic mice that had received a tumorigenic dose of human uveal melanoma cells in the anterior chamber of the eye. This immune-privileged site offered the unique opportunity to graft xenogeneic tumors into immunocompetent A2/K(b)-transgenic mice, a host in which they otherwise would not grow. Importantly, systemic (i.v.) administration of the A2/K(b)-transgenic gp100(154-162)-specific CTL resulted in rapid elimination of the intraocular uveal melanomas, indicating that anti-tumor CTL are capable of homing to the eye and exerting their tumoricidal effector function. Flow cytometry analysis of ocular cell suspensions with HLA-A*0201-gp100(154-162) tetrameric complexes confirmed the homing of adoptively transferred CTL. Therefore, the immune-privileged state of the eye permitted the outgrowth of xenogeneic uveal melanoma cells, but did not protect these tumors against adoptive immunotherapy with highly potent anti-tumor CTL. These data constitute the first direct indication that immunotherapy of human uveal melanoma may be feasible.     

Generation of CTL recognizing an HLA-A*0201-restricted epitope shared by MAGE-A1, -A2, -A3, -A4, -A6, -A10, and -A12 tumor antigens: implication in a broad-spectrum tumor immunotherapy

MAGE-A1, -A2, -A3, -A4, -A6, -A10, and -A12 are expressed in a significant proportion of primary and metastatic tumors of various histological types and are targets of tumor Ag-specific CTL. Individual MAGE-A expression varies from one tumor type to the other but, overall, the large majority of tumors expresses at least one MAGE-A Ag. Therefore, targeting epitopes shared by all MAGE-A Ags would be of interest in immunotherapy against a broad spectrum of cancers. In the present study, we describe a heteroclitic MAGE-A peptide (p248V9) that induces CTL in vivo in HLA-A*0201 transgenic HHD mice and in vitro in healthy donors. These CTL are able to recognize two low HLA-A*0201 affinity peptides differing at their C-terminal position and derived from MAGE-A2, -A3, -A4, -A6, -A10, and -A12 (p248G9) and MAGE-A1 (p248D9). Interestingly, p248V9-specific CTL respond to endogenous MAGE-A1, -A2, -A3, -A4, -A6, -A10, and -A12 in an HLA-A*0201-restricted manner and recognize human HLA-A*0201(+)MAGE-A(+) tumor cells of various histological origin. Therefore, this heteroclitic peptide may be considered as a potent candidate for a broad-spectrum tumor vaccination.     

Preventive and therapeutic vaccination with PAP-3, a novel human prostate cancer peptide, inhibits carcinoma development in HLA transgenic mice

Conventional treatment of recurrent and metastasized prostate cancer (CaP) remains inadequate; this fact mandates development of alternative therapeutic modalities, such as specific active or passive immunotherapy. Previously, we reported the identification of a novel highly immunogenic HLA-A*0201-restricted Prostatic Acid Phosphatase-derived peptide (PAP-3) by a two-step in vivo screening in an HLA-transgenic (HHD) mouse system. In the present study we aimed at elucidating the efficiency of PAP-3-based vaccine upon active antitumor immunization. To this end we established preventive and therapeutic carcinoma models in HHD mice. The 3LL murine Lewis lung carcinoma clone D122 transduced to express HLA-A*0201 and PAP served as a platform for these models. The HLA-A*0201–PAP-3 complex specific recombinant single chain scFV-PAP-3 antibodies were generated and used to confirm an endogenous PAP processing resulting in PAP-3 presentation by HLA-A*0201. PAP-3 based vaccines significantly decreased tumor incidence in a preventive immunization setting. Therapeutic vaccination of HHD mice with PAP-3 led to rejection of early established tumors and to increase of mouse survival. These results strongly support a therapeutic relevance of the identified CTL epitope upon active antitumor immunization. The newly established carcinoma model presented herein might be a useful tool for cancer vaccine design and optimization.     

Identification of an HLA-A*0201-restricted CD8+ T-cell epitope encoded within Leptospiral immunoglobulin-like protein A

Leptospirosis is an important zoonosis in humans. Immunity against leptospiral infection was thought to be primarily humoral, and limited studies have addressed the role of CD8⁺ T cells. Leptospiral immunoglobulin-like protein A (LigA) is an important protective antigen of Leptospira and a potential target for Leptospira-specific cell-mediated immunity. In this study, twenty LigA-derived peptides were tested their binding affinity and stability for the HLA-A*0201 molecule. Peptides with high binding affinity and stability for HLA-A*0201 were then assessed their capacity to elicit specific cytotoxic T-lymphocyte (CTL) responses using cytotoxicity, ELISPOT assays for IFN-γ and HLA-A*0201-peptide tetramer assays. We identified a HLA-A*0201-restricted epitope, LigA_305–313 KLIVTPAAL in Leptospira LigA. CTLs specific for LigA_305–313 were elicited both in HLA-A2.1/K^b transgenic mice and in patients with a clinical and/or laboratory diagnosis of leptospirosis. Staining of the HLA-A*0201–LigA_305–313 tetramer revealed the presence of LigA_305–313-specific CTLs in peripheral blood mononuclear cells (PBMCs) sourced from five patients infected with three different serovars of Leptospira. In conclusion, we report the existence of specific cytotoxic CD8⁺ T cells in patients with leptospirosis and we suggest that the newly identified epitope, LigA_305–313, will be helpful in enhancing the understanding of the mechanism of immunity to leptospirosis.     

HER-2/neu and hTERT cryptic epitopes as novel targets for broad spectrum tumor immunotherapy

Tolerance to tumor-nonmutated self proteins represents a major obstacle for successful cancer immunotherapy. Since this tolerance primarily concerns dominant epitopes, we hypothesized that targeting cryptic epitopes that have a low affinity for HLA could be an efficient strategy to breach the tolerance to tumor Ags. Using the P1Y heteroclitic peptide approach, we identified low affinity cryptic HLA-A*0201-restricted epitopes derived from two widely expressed tumor Ags, HER-2/neu and hTERT. The P1Y variants of four HER-2/neu (neu(391), neu(402), neu(466), neu(650))- and two hTERT (hTERT(572) and hTERT(988))-derived low affinity peptides exhibited strong affinity for HLA-A*0201 and stimulated specific CTL from healthy donor PBMCs. These CTL specifically recognized HER-2/neu- and hTERT-expressing tumor cells of various histological origins. In vivo studies showed that HLA-A*0201 transgenic HHD mice vaccinated with the P1Y variant peptides generated CTL that specifically lysed Ag-expressing tumor cells, thus recognizing the cognate endogenous Ags. These results suggest that heteroclitic variants of low affinity, cryptic epitopes of widely expressed tumor Ags may serve as valid tools for tumor immunotherapy.