Substitution analog peptide derived from HER-2 can efficiently induce HER-2-specific, HLA-A24 restricted CTLs

In order to broaden the possibility for anti-HER-2/neu (HER-2) immune targeting, it is important to identify HLA-A24 restricted peptide epitopes derived from HER-2, since HLA-A24 is one of the most common alleles in Japanese and Asian people. In the present study, we have screened HER-2-derived, HLA-A24 binding peptides for cytotoxic T lymphocyte (CTL) epitopes. A panel of HER-2-derived peptides with HLA-A24 binding motifs and the corresponding analogs designed to enhance HLA-A24 binding affinity were selected. Identification of HER-2-reactive and HLA-A24 restricted CTL epitopes were performed by a reverse immunology approach. To induce HER-2-reactive and HLA-A24 restricted CTLs, PBMCs from healthy donors were repeatedly stimulated with monocytes-derived, mature DCs pulsed with HER-2 peptide. Subsequent peptide-induced T cells were tested for the specificity by enzyme linked immunospot, cytotoxicity and tetramer assays. CTL clones were then obtained from the CTL lines by limiting dilution. Of the peptides containing HLA-A24 binding motifs, 16 peptides (nine mers) including wild type peptides (IC₅₀<1,000 nM) and substituted analog peptides (IC₅₀<50 nM) were selected for the present study. Our studies show that an analog peptide, HER-2(905AA), derived from HER-2(905) could efficiently induce HER-2-reactive and HLA-A24 restricted CTLs. The reactivity of the HER-2(905AA)-induced CTL (CTL905AA) was confirmed by different CTL assays. The CTL905AA clones also were able to lyse HER-2(+), HLA-A24(+) tumor cells and cytotoxicity could be significantly reduced in cold target inhibition assays using cold targets pulsed with the HER-2(905) wild type peptide as well as the inducing HER-2(905AA) analog peptide. A newly identified HER-2(905) peptide epitope is naturally processed and presented as a CTL epitope on HER-2 overexpressing tumor cells, and an MHC anchor-substituted analog, HER-2(905AA), can efficiently induce HER-2-specific, HLA-A24 restricted CTLs.     

Hierarchy among multiple H-2b-restricted cytotoxic T-lymphocyte epitopes within simian virus 40 T antigen.

Simian virus 40 large tumor (T) antigen contains three H-2Db-restricted (I, II/III, and V) and one H-2Kb-restricted (IV) cytotoxic T lymphocyte (CTL) epitopes. We demonstrate that a hierarchy exists among these CTL epitopes, since vigorous CTL responses against epitopes I, II/III, and IV are detected following immunization of H-2b mice with syngeneic, T-antigen-expressing cells. By contrast, a weak CTL response against the H-2Db-restricted epitope V was detected only following immunization of H-2b mice with epitope loss variant B6/K-3,1,4 cells, which have lost expression of CTL epitopes I, II/III, and IV. Limiting-dilution analysis confirmed that the lack of epitope V-specific CTL activity in bulk culture splenocytes correlated with inefficient expansion and priming of epitope V-specific CTL precursors in vivo. We examined whether defined genetic alterations of T antigen might improve processing and presentation of epitope V to the epitope V-specific CTL clone Y-5 in vitro and/or overcome the recessive nature of epitope V in vivo. Deletion of the H-2Db-restricted epitopes I and II/III from T antigen did not increase target cell lysis by epitope V-specific CTL clones in vitro. The amino acid sequence SMIKNLEYM, which species an optimized H-2Db binding motif and was found to induce CTL in H-2b mice, did not further reduce epitope V presentation in vitro when inserted within T antigen. Epitope V-containing T-antigen derivatives which retained epitopes I and II/III or epitope IV did not induce epitope V-specific CTL in vivo: T-antigen derivatives in which epitope V replaced epitope I failed to induce epitope V-specific CTL. Recognition of epitope V-H-2Db complexes by multiple independently derived epitope V-specific CTL clones was rapidly and dramatically reduced by incubation of target cells in the presence of brefeldin A compared with the recognition of the other T-antigen CTL epitopes by epitope specific CTL, suggesting that the epitope V-H-2Db complexes either are labile or are present at the cell surface at reduced levels. Our results suggest that processing and presentation of epitope V is not dramatically altered (reduced) by the presence of immunodominant CTL epitopes in T antigen and that the immunorecessive nature of epitope V is not determined by amino acids which flank its native location within simian virus 40 T antigen.     

Identification of NY-ESO-1 peptide analogues capable of improved stimulation of tumor-reactive CTL

Expression of NY-ESO-1 in a high proportion of different human tumors makes this protein a very attractive vaccine target. NY-ESO-1 peptides, recognized by HLA-A2-restricted CTL, have recently been described. However, it remains unclear how efficiently tumors generate these epitopes, and whether peptide analogues can be used for optimal expansion and activation of NY-ESO-1-specific HLA-A2-restricted CTL. By generating unique CTL clones, we demonstrate that NY-ESO-1-positive tumor cells are efficiently killed by HLA-A2-restricted CTL specific for the peptide epitope NY-ESO-1 157-165. Presentation of this epitope is not affected by the presence or absence of the proteasome subunits low molecular proteins 2 and 7 and is not blocked by proteasome inhibitors, while it is impaired in the TAP-deficient cell line LBL 721.174. NY-ESO-1 157-165 peptide analogues were compared for their antigenicity and immunogenicity using PBL from melanoma patients. Three peptides, containing the carboxyl-terminal cysteine substituted for either valine, isoleucine, or leucine, were recognized at least 100 times more efficiently than the wild-type peptide by specific CTL. Peptide analogues were capable of stimulating the expansion of NY-ESO-1-specific CTL from PBL of melanoma patients much more efficiently than wild-type peptide. These findings define the processing requirements for the generation of the NY-ESO-1 157-165 epitope. Identification of highly antigenic NY-ESO-1 peptide analogues may be important for the development of vaccines capable of expanding NY-ESO-1-specific CTL in cancer patients.     

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.     

Ii-Key/HER-2/<Emphasis Type="Italic">neu</Emphasis>(776-90) hybrid peptides induce more effective immunological responses over the native peptide in lymphocyte cultures from patients with HER-2/<Emphasis Type="Italic">neu</Emphasis>+ tumors

We have demonstrated that coupling an immunoregulatory segment of the MHC class II-associated invariant chain (Ii), the Ii-Key peptide, to a promiscuous MHC class II epitope significantly enhances its presentation to CD4+ T cells. Here, a series of homologous Ii-Key/HER-2/neu(776-790) hybrid peptides, varying systematically in the length of the epitope(s)-containing segment, are significantly more potent than the native peptide in assays using T cells from patients with various types of tumors overexpressing HER-2/neu. In particular, priming normal donor and patient PBMCs with Ii-Key hybrid peptides enhances recognition of the native peptide either pulsed onto autologous dendritic cells (DCs) or naturally presented by IFN-gamma-treated autologous tumor cells. Moreover, patient-derived CD4+ T cells primed with the hybrid peptides provide a significantly stronger helper effect to autologous CD8+ T cells specific for the HER-2/neu(435-443) CTL epitope, as illustrated by either IFN-gamma ELISPOT assays or specific autologous tumor cell lysis. Hybrid peptide-specific CD4+ T cells strongly enhanced the antitumor efficacy of HER-2/neu(435-443) peptide-specific CTL in the therapy of xenografted SCID mice inoculated with HER-2/neu overexpressing human tumor cell lines. Our data indicate that the promiscuously presented vaccine peptide HER-2/neu(776-790) is amenable to Ii-Key-enhancing effects and supports the therapeutic potential of vaccinating patients with HER-2/neu+ tumors with such Ii-Key/HER-2/neu(776-790) hybrid peptides.     

Identification of a novel immunogenic HLA-A*0201-binding epitope of HER-2/neu with potent antitumor properties

HER-2/neu oncoprotein is overexpressed in a variety of human tumors and is associated with aggressive disease. Immunogenic HER-2/neu CTL epitopes have been used as vaccines for the treatment of HER-2/neu positive malignancies with limited success. By applying prediction algorithms for MHC class I ligands and proteosomal cleavages, in this study, we describe the identification of HER-2/neu decamer LIAHNQVRQV spanning residues 85-94 (HER-2(10(85))). HER-2(10(85)) proved to bind with high affinity to HLA-A2.1 and was stable for 4 h in an off-kinetics assay. This peptide was immunogenic in HLA-A2.1 transgenic (HHD) mice inducing peptide-specific CTL, which responded to tumor cell lines of various origin coexpressing human HER-2/neu and HLA-A2.1. This demonstrates that HER-2(10(85)) is naturally processed from endogenous HER-2/neu. Five of sixteen HER-2/neu+ HLA-A2.1+ breast cancer patients analyzed had HER-2(10(85))-reactive T cells ranging from 0.35-0.70% of CD8+ T cells. Depletion of T regulatory cells from PBMC enabled the rapid expansion of HLA-A2.1/HER-2(10(85))pentamer+/CD8+ cells (PENT+/CD8+), whereas significantly lower numbers of CTL could be generated from unfractionated PBMC. HER-2(10(85))-specific human CTL recognized the HER-2/neu+ HLA-A2.1+ tumor cell line SKBR3.A2, as determined by IFN-gamma intracellular staining and in the high sensitivity CD107alpha degranulation assay. Finally, HER-2(10(85)) significantly prolonged the survival of HHD mice inoculated with the transplantable ALC.A2.1.HER tumor both in prophylactic and therapeutic settings. These data demonstrate that HER-2(10(85)) is an immunogenic peptide, capable of eliciting CD8-mediated responses in vitro and in vivo, providing the platform for further exploitation of HER-2(10(85)) as a possible target for anticancer immunotherapy.     

Trastuzumab Mediated T-Cell Response against HER-2/Neu Overexpressing Esophageal Adenocarcinoma Depends on Intact Antigen Processing Machinery

Background

Esophageal adenocarcinoma (EAC) is a highly aggressive disease with poor prognosis, which frequently exhibits HER-2 gene amplification. Trastuzumab, the humanized antibody against HER-2, has potent growth inhibitory effects on HER-2 overexpressing cancers. One effect of trastuzumab is that it causes HER-2 receptor internalization and degradation, enhancing presentation of HER-2 epitopes on MHC-Class I molecules. This enhances the ability of HER-2 specific cytotoxic T lymphocytes (CTLs) to recognize and kill cancer cells. Novel strategies targeting the HER-2 receptor either directly by trastuzumab and/or indirectly by inducing a CTL response against HER-2 epitopes with, for instance, DC immunotherapy and consequently combining these strategies might prove to be very effective.

Methodology/Principal Findings

In this study we report that trastuzumab has potent growth inhibitory effects on two HER-2 overexpressing EAC cell lines OE33 and OE19. However, we found that trastuzumab and HER-2 specific CTLs act synergistically in inducing tumor lysis in OE33 but not in OE19. We discovered that in OE19 this deficient response is due to a down-regulation of the Transporter Associated with Antigen Processing-2 (TAP-2). TAP-2 is an important member of the Antigen Processing Machinery (APM), and is one of the essential elements for loading antigens on MHC class I molecules. Importantly, we demonstrated that by inducing re-expression of TAP-2 in OE19 with INF-γ treatment or by incubating the cells with INF-γ producing CTLs, the specific anti HER-2 CTL tumor lysis response and synergistic effect with trastuzumab can be restored.

Conclusion

An inefficient response of HER-2 overexpressing EAC to trastuzumab and/or DC immunotherapy can be due to a down-regulated TAP-2 expression and thus a deficient APM. Future studies combining trastuzumab with IFN-γ and/or immune-therapies inducing potent anti HER-2 CTL responses could lead to an effective combinatorial strategy for successful treatment of HER-2 overexpressing but APM defective cancers.     

Fusion to Listeriolysin O and delivery by Listeria monocytogenes enhances the immunogenicity of HER-2/neu and reveals subdominant epitopes in the FVB/N mouse

Five overlapping fragments of rat HER-2/neu have been expressed in recombinant Listeria monocytogenes. Each fragment of HER-2/neu is secreted as a fusion protein with a truncated, nonhemolytic form of listeriolysin O (LLO). Lm-LLO-EC1, Lm-LLO-EC2, and Lm-LLO-EC3 overlap the extracellular domain of HER-2/neu, whereas Lm-LLO-IC1 and Lm-LLO-IC2 span the intracellular domain. All five strains controlled the growth of established NT-2 tumors, a rat HER-2/neu-expressing tumor line derived from a spontaneously arising mammary tumor in a FVB/N HER-2/neu-transgenic mouse. The antitumor effect of each of these vaccine constructs was abrogated by the in vivo depletion of CD8(+) T cells, although only one known epitope has been defined previously and is present in Lm-LLO-EC2. Anti-HER-2/neu CTL responses were generated by each of the rLm vaccine constructs. With the use of a panel of 3T3 cell lines expressing overlapping fragments of HER-2/neu, regions of HER-2/neu with potential CD8(+) T cell epitopes have been defined. DNA vaccines expressing either a fragment or full-length HER-2/neu were constructed in LLO-fused and non-LLO-fused forms. CTL analysis of the DNA vaccines revealed a broadening in the regions of HER-2/neu recognizable as targets when the target Ag is fused to LLO. These studies show the efficacy of L. monocytogenes-based HER-2/neu vaccines in a murine model of breast cancer and also that the immunogenicity of self-Ags can be increased by fusion to LLO and delivery by L. monocytogenes revealing subdominant epitopes.     

Liposome-based systems for anti-tumor vaccination: influence of lipopeptide adjuvants

We have developed liposome-based synthetic constructs incorporating peptide epitope(s) (ErbB2 p63-67 CTL which is overexpressed in many tumors and/or HA 307-319 T-helper) and lipopeptide adjuvants (Pam3CysSerSer, Pam3CysAlaGly) in order to elicit an anti-tumor immune response. The epitopes, derivatized with a linker containing a cysteine residue, were conjugated on preformed vesicles (dia. approximately 100 nm) containing lipopeptides functionalized with thiol reactive groups (maleimide or bromoacetyl). The therapeutic efficacy of these constructs was evaluated on a Balb/c mice tumor model inoculated with syngenic murine renal carcinoma (Renca) cells expressing human ErbB2 (Her2/neu) receptor. A successful therapeutic vaccination was obtained which was antigen specific. Furthermore, it appeared that the nature of the polar head group of the lipopeptide adjuvant and also its type of functionalization influence the efficacy of the construct. In our study, the best results were obtained with formulations containing a Pam3CSS anchor in association with the CTL and Th epitopes. Considering these promising results studies are in progress with a new generation of liposomes that incorporate a neutral lipid--lacking adjuvant properties--that serves as anchor of the peptide epitopes and new adjuvants synthesized in our laboratory, which are screened for their antitumour activity in a therapeutic setting.     

Improved immunogenicity of an immunodominant epitope of the HER-2/neu protooncogene by alterations of MHC contact residues

The HER-2/neu (HER-2) oncogene is expressed in normal epithelial surfaces at low levels and overexpressed in several types of tumors. The low immunogenicity against this self tumor Ag can be improved by developing epitopes with amino acid replacements in their sequences. In this study, three HER-2/neu.369 (HER-2.369) analogue peptides, produced by modifying both anchor positions by introducing L, V, or T at position 2 and V at the C terminus, were analyzed for their capacity to induce CTLs in vitro from human PBMC and in vivo in HLA-A2.1/Kb transgenic mice. One of the analogues (HER-2.369 V2V9) sensitized target cells for HER-2-specific recognition by human CTLs and induced specific CTLs in vitro at 100-fold lower concentrations than the HER-2.369 wild-type epitope. These CTLs were also able to recognize the wild-type epitope and HER-2-expressing tumors in an MHC-restricted manner. Furthermore, a 100-fold lower amount of the HER-2.369 V2V9 analogue compared with the wild-type epitope was required to induce CTLs in HLA-A2.1/Kb transgenic mice. However, the V2V9 analogue demonstrated only marginally better binding to the MHC class I A2 allele compared with wild type. To establish thermodynamic parameters, we developed radiolabeled F3*Y analogues from both the HER-2.369 epitope and the V2V9 analogue. Our results indicate that the high biological activity of the HER-2.369 V2V9 epitope is associated with a slower dissociation kinetic profile, resulting in an epitope with greater HLA-A2 stability.     

Critical components of a DNA fusion vaccine able to induce protective cytotoxic T cells against a single epitope of a tumor antigen

DNA vaccines can activate immunity against tumor Ags expressed as MHC class I-associated peptides. However, priming of CD8(+) CTL against weak tumor Ags may require adjuvant molecules. We have used a pathogen-derived sequence from tetanus toxin (fragment C (FrC)) fused to tumor Ag sequences to promote Ab and CD4(+) T cell responses. For induction of CD8(+) T cell responses, the FrC sequence has been engineered to remove potentially competitive MHC class I-binding epitopes and to improve presentation of tumor epitopes. The colon carcinoma CT26 expresses an endogenous retroviral gene product, gp70, containing a known H2-L(d)-restricted epitope (AH1). A DNA vaccine encoding gp70 alone was a poor inducer of CTL, and performance was not significantly improved by fusion of full-length FrC. However, use of a minimized domain of FrC, with the AH1 sequence fused to the 3' position, led to rapid induction of high levels of CTL. IFN-gamma-producing epitope-specific CTL were detectable ex vivo and these killed CT26 targets in vitro. The single epitope vaccine was more effective than GM-CSF-transfected CT26 tumor cells in inducing an AH1-specific CTL response and equally effective in providing protection against tumor challenge. Levels of AH1-specific CTL in vivo were increased following injection of tumor cells, and CTL expanded in vitro were able to kill CT26 cells in tumor bearers. Pre-existing immunity to tetanus toxoid had no effect on the induction of AH1-specific CTL. These data demonstrate the power of epitope-specific CTL against tumor cells and illustrate a strategy for priming immunity via a dual component DNA vaccine.     

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.     

Liposome-based Systems for Anti-tumor Vaccination: Influence of Lipopeptide Adjuvants

We have developed liposome-based synthetic constructs incorporating peptide epitope(s) (ErbB2 p63-67 CTL which is overexpressed in many tumors and/or HA 307-319 T-helper) and lipopeptide adjuvants (Pam3CysSerSer, Pam3CysAlaGly) in order to elicit an anti-tumor immune response. The epitopes, derivatized with a linker containing a cysteine residue, were conjugated on preformed vesicles (dia. ～ 100 nm) containing lipopeptides functionalized with thiol reactive groups (maleimide or bromoacetyl). The therapeutic efficacy of these constructs was evaluated on a Balb/c mice tumor model inoculated with syngenic murine renal carcinoma (Renca) cells expressing human ErbB2 (Her2/neu) receptor. A successful therapeutic vaccination was obtained which was antigen specific. Furthermore, it appeared that the nature of the polar head group of the lipopeptide adjuvant and also its type of functionalization influence the efficacy of the construct. In our study, the best results were obtained with formulations containing a Pam₃CSS anchor in association with the CTL and Th epitopes. Considering these promising results studies are in progress with a new generation of liposomes that incorporate a neutral lipid – lacking adjuvant properties – that serves as anchor of the peptide epitopes and new adjuvants synthesized in our laboratory, which are screened for their antitumour activity in a therapeutic setting.     

Re-evaluating the generation of a "proteasome-independent" MHC class I-restricted CD8 T cell epitope

The proteasome is primarily responsible for the generation of MHC class I-restricted CTL epitopes. However, some epitopes, such as NP(147-155) of the influenza nucleoprotein (NP), are presented efficiently in the presence of proteasome inhibitors. The pathways used to generate such apparently "proteasome-independent" epitopes remain poorly defined. We have examined the generation of NP(147-155) and a second proteasome-dependent NP epitope, NP(50-57), using cells adapted to growth in the presence of proteasome inhibitors and also through protease overexpression. We observed that: 1) Ag processing and presentation proceeds in proteasome-inhibitor adapted cells but may become more dependent, at least in part, on nonproteasomal protease(s), 2) tripeptidyl peptidase II does not substitute for the proteasome in the generation of NP(147-155), 3) overexpression of leucine aminopeptidase, thymet oligopeptidase, puromycin-sensitive aminopeptidase, and bleomycin hydrolase, has little impact on the processing and presentation of NP(50-57) or NP(147-155), and 4) proteasome-inhibitor treatment altered the specificity of substrate cleavage by the proteasome using cell-free digests favoring NP(147-155) epitope preservation. Based on these results, we propose a central role for the proteasome in epitope generation even in the presence of proteasome inhibitors, although such inhibitors will likely alter cleavage patterns and may increase the dependence of the processing pathway on postproteasomal enzymes.     

The carboxyl-terminal 120-residue polypeptide of infectious bronchitis virus nucleocapsid induces cytotoxic T lymphocytes and protects chickens from acute infection.

Specific cytotoxic T-lymphocyte (CTL) responses to nucleocapsid of infectious bronchitis virus (IBV) were identified by using target cells infected with a Semliki Forest virus (SFV) vector. Effector cells for CTL assays were collected from chickens infected with the Gray strain of IBV or inoculated with a DNA plasmid encoding nucleocapsid proteins. IBV-specific CTL epitopes were mapped within the carboxyl-terminal 120 amino acids of the nucleocapsid protein. CTL lysis of target cells infected with SFV encoding nucleocapsid was major histocompatibility complex restricted and mediated by CD8+ T cells. In addition, splenic T cells collected from chickens inoculated in the breast muscle with a DNA plasmid encoding this CTL epitope(s) recognized target cells infected with wild-type virus or an SFV vector encoding nucleocapsid proteins. CTL activity of splenic T cells collected from chicks immunized with a DNA plasmid encoding CTL epitopes was cross-reactive, in that lysis of target cells infected with serologically distinct strains of IBV was dose responsive in a manner similar to that for lysis of target cells infected with the homologous strain of IBV. Furthermore, chickens immunized with a DNA plasmid encoding a CTL epitope(s) were protected from acute viral infection.     

Immunogenic HER-2/neu peptides as tumor vaccines

During the last decade, a large number of tumor-associated antigens (TAA) have been identified, which can be recognized by T cells. This has led to renewed interest in the use of active immunization as a modality for the treatment of cancer. HER-2/neu is a 185-KDa receptor-like glycoprotein that is overexpressed by a variety of tumors including breast, ovarian, lung, prostate and colorectal carcinomata. Several immunogenic HER-2/neu peptides recognized by cytotoxic T lymphocytes (CTL) or helper T lymphocytes (TH) have been identified thus far. Patients with HER-2/neu over-expressing cancers exhibit increased frequencies of peripheral blood T cells recognizing immunogenic HER-2/neu peptides. Various protocols for generating T cell-mediated immune responses specific for HER-2/neu peptides have been examined in pre-clinical models or in clinical trials. Vaccination studies in animals utilizing HER-2/neu peptides have been successful in eliminating tumor growth. In humans, however, although immunological responses have been detected against the peptides used for vaccination, no clinical responses have been described. Because HER-2/neu is a self-antigen, functional immune responses against it may be limited through tolerance mechanisms. Therefore, it would be interesting to determine whether abrogation of tolerance to HER-2/neu using appropriate adjuvants and/or peptide analogs may lead to the development of immune responses to HER-2/neu epitopes that can be of relevance to cancer immunotherapy. Vaccine preparations containing mixtures of HER-2/neu peptides and peptide from other tumor-related antigens might also enhance efficacy of therapeutic vaccination. This article is a symposium paper from the conference “Progress in Vaccination against Cancer 2004 (PIVAC 4)”, held in Freudenstadt-Lauterbad, Black Forest, Germany, on 22–25 September 2004     

HER-2/neu peptide specificity in the recognition of HLA-A2 by natural killer cells

Although natural killer (NK) cells have been described as non-MHC-restricted, new evidence suggests that NK activity can be either up- or down-regulated after interaction with the peptide–MHC-class-I complex expressed on target cells. However, the epitope(s) recognized by NK cells have remained ill-defined. We investigated NK cell recognition of synthetic peptides representing a portion of a self-protein encoded by the HER-2/neu (HER-2) proto-oncogene and presented by HLA-A2. HER-2 nonapeptides C85, E89, and E75 were found partially to protect T2 targets from lysis by freshly isolated and interleukin-2(IL-2)-activated NK cells (either HLA-A2⁺ or A2⁻). This inhibition was not solely due to changes in the level of HLA-A2 expression or conformation of serological HLA-A2 epitopes. Using single-amino-acid variants at position 1 (P1) of two HER-2 peptides, we observed that protection of targets was dependent on the sequence and the side-chain. These results suggest similarities in the mechanism of target recognition by NK and T cells. This information may be important for understanding the mechanisms of tumor escape from immunosurveillance and could help explain the aggressiveness of HER-2-overexpressing tumor cells. Received: 16 March 1999 / Accepted: 3 June 1999     

HER-2/neu mediated down-regulation of MHC class I antigen processing prevents CTL-mediated tumor recognition upon DNA vaccination in HLA-A2 transgenic mice

To study DNA vaccination directed against human HER-2 in the HHD mouse Tg strain, we created a novel HER-2-expressing syngeneic tumor transplantation model. We found that a DNA vaccine encoding the full length HER-2 DNA protected HHD mice from HER-2⁺ tumor challenge by a CTL independent mechanism. A more efficient approach to induce HLA-A2 restricted CTLs, through immunization with a multi-epitope DNA vaccine expressing the HLA-A2 restricted HER-2 369–377, 435–443 and 689–697 epitopes, resulted in high numbers of peptide specific T cells but failed to induce tumor protection. Subsequently we discovered that HER-2 transfected tumor cells down-regulated MHC class I antigen expression and exhibited a series of defects in the antigen processing pathway which impaired the capacity to produce and display MHC class I peptide-ligands to specific CTLs. Our data demonstrate that HER-2 transfection is associated with defects in the MHC class I presentation pathway, which may be the underlying mechanism behind the inability of CTLs to recognize tumors in this HLA-A2 transgenic model. As defective MHC class I presentation may be a common characteristic of HER-2 expressing tumors, vaccines targeting HER-2 should aim at inducing an integrated immune response where also CD4⁺ T cells and antibodies are important components. S. Vertuani and C. Triulzi contributed equally to this work.     

Defining MHC class II T helper epitopes for WT1 tumor antigen

The product of Wilms‘ tumor gene 1 (WT1) is overexpressed in diverse human tumors, including leukemia, lung and breast cancer, and is often recognized by antibodies in the sera of patients with leukemia. Since WT1 encodes MHC class I-restricted peptides recognized by cytotoxic T lymphocytes (CTL), WT1 has been considered as a promising tumor-associated antigen (TAA) for developing anticancer immunotherapy. In order to carry out an effective peptide-based cancer immunotherapy, MHC class II-restricted epitope peptides that elicit anti-tumor CD4⁺ helper T lymphocytes (HTL) will be needed. In this study, we analyzed HTL responses against WT1 antigen using HTL lines elicited by in vitro immunization of human lymphocytes with synthetic peptides predicted to serve as HTL epitopes derived from the sequence of WT1. Two peptides, WT1_124–138 and WT1_247–261, were shown to induce peptide-specific HTL, which were restricted by frequently expressed HLA class II alleles. Here, we also demonstrate that both peptides-reactive HTL lines were capable of recognizing naturally processed antigens presented by dendritic cells pulsed with tumor lysates or directly by WT1+ tumor cells that express MHC class II molecules. Interestingly, the two WT1 HTL epitopes described here are closely situated to known MHC class I-restricted CTL epitopes, raising the possibility of stimulating CTL and HTL responses using a relatively small synthetic peptide vaccine. Because HTL responses to TAA are known to be important for promoting long-lasting anti-tumor CTL responses, the newly described WT1 T-helper epitopes could provide a useful tool for designing powerful vaccines against WT1-expressing tumors.     

Identification of new HER2/neu-derived peptide epitopes that can elicit specific CTL against autologous and allogeneic carcinomas and melanomas.

Twenty-two new HLA-A2.1-binding peptides derived from the protooncogene HER2/neu were identified and analyzed for their capacity to elicit peptide and tumor-specific CTL responses. We used peptide-pulsed autologous DC from the ascites of patients with ovarian carcinomas to induce CTL. Of the 22 tested new HER2/neu-derived epitopes that could bind HLA-A2 with high (IC50 < 50 nM) or intermediate (50 nM < IC50 < 500 nM) affinity, we report the recognition by CTL of at least four novel epitopes, including HER2(9435), HER2(9665), HER2(9689), and HER2(10952), and confirm that of the known HER2 (9369) epitope. These epitopes were able to elicit CTL that specifically killed peptide-sensitized target cells and, most importantly, a HER2/neu-transfected cell line and the autologous tumor cells. We also confirm that HER2/neu is overexpressed in several melanoma lines, and as a new finding, report that some of these lines are sensitive to CTL induced by the HER2 (9369), HER2(9435), and HER2(9689) epitopes. Finally, CTL clones specific for HER2 (9369), HER2(9435), and HER2(9689) epitopes were isolated from tumor-specific CTL lines, further demonstrating the immunodominance of these epitopes. These findings broaden the potential application of HER2/neu-based immunotherapy.