Peptide-pulsed T2 Cells Research Articles

Humoral immune responses against tumor‐associated antigen OVA66 originally defined by serological analysis of recombinant cDNA expression libraries and its potentiality in cellular immunity

Immunotherapy for cancer relies on the identification of tumor antigens and efficacy of antitumor immune responses. Serological analysis of recombinant cDNA libraries (SEREX), which is based on the spontaneous humoral responses against potential tumor antigens, has provided a novel strategy for searching novel tumor-associated candidates. Through SEREX analysis, we have identified 24 distinct gene clones by immunoscreening of a cDNA library derived from an ovarian cancer patient. Among these genes, a novel gene, OVA66, was found to be expressed significantly higher in carcinoma samples from cancer patients than in normal controls. Comparing humoral responses to OVA66 between tumor patients and healthy donors, it has been shown that the IgG level against OVA66 was significantly elevated in the serum of cancer patients from different histological types of cancer. To determine whether SEREX-defined OVA66 can trigger promising cytotoxic T lymphocyte (CTL) responses, human leukocyte antigen (HLA)-A*0201-restricted T-cell epitopes were predicted through a computational algorithm. Of four predicted peptides, p306-314 (L235) possesses the ability to induce efficient peripheral blood lymphocyte (PBL)-derived CTL responses capable of specifically recognizing peptide-pulsed T2 cells and lysing carcinoma cell lines expressing both HLA-A2 and OVA66 as determined by cytotoxicity and enzyme-linked immunospot assay (ELISPOT). Taken together, our results demonstrate that the SEREX-defined tumor-associated antigen OVA66 can elicit humoral immunity and may also serve as a potential candidate for T-cell-based immunotherapy for cancer.

Identification of an HLA-A*0201-Restricted T-Cell Epitope on the MPT51 Protein, a Major Secreted Protein Derived fromMycobacterium tuberculosis, by MPT51 Overlapping Peptide Screening

CD8+ T cells play a pivotal role in protection against Mycobacterium tuberculosis infection. We identified a novel HLA-A*0201-restricted CD8+ T-cell epitope on a dominant secreted antigen of M. tuberculosis, MPT51, in HLA-A*0201 transgenic HHD mice. HHD mice were immunized with plasmid DNA encoding MPT51 with gene gun bombardment, and gamma interferon (IFN-gamma) production by the immune splenocytes was analyzed. In response to overlapping synthetic peptides covering the mature MPT51 sequence, the splenocytes were stimulated to produce IFN-gamma by only one peptide, p51-70. Three-color flow cytometric analysis of intracellular IFN-gamma and cell surface CD4 and CD8 staining revealed that the MPT51 p51-70 peptide contains an immunodominant CD8+ T-cell epitope. Further analysis using computer algorithms permitted identification of a bona fide T-cell epitope, p53-62. A major histocompatibility complex class I stabilization assay using T2 cells confirmed that this epitope binds to HLA-A*0201. The T cells were capable of lysing MPT51 p53-62 peptide-pulsed T2 cells. In addition, MPT51 p53-62-specific memory CD8+ T cells were found in tuberculin skin test-positive HLA-A*0201+ healthy individuals. Use of this HLA-A*0201-restricted CD8+ T-cell epitope for analysis of the role of MPT51-specific T cells in M. tuberculosis infection and for design of vaccines against tuberculosis is feasible.

Identification of CS1 Peptides for Induction of Antigen-Specific CTLs in Multiple Myeloma.

Despite progress in the treatment of multiple myeloma (MM), the disease remains incurable. Therefore novel therapeutic approaches are required to achieve better outcome in patients. Development of peptide-based immunotherapy against tumor specific or associated antigens offers an attractive approach that may lead to tumor-targeted cellular therapy in MM patients. Recently, CS1 (CD2 subset 1, CRACC, SLAMF7), a cell surface glycoprotein of the CD2 family, was found to be highly expressed by the tumor cells of the majority of MM patients but not other normal tissues. Importantly, a novel anti-CS1 HuLuc63 mAb induced significant anti-myeloma killing in vitro and in vivo (Abstract #950059). In addition, CS1 may play a role in MM cell survival (Abstract #953659). In this study, we asked whether CS1 is a suitable myeloma-associated antigen for cellular therapy in MM. We wanted to identify potential immunogenic peptides derived from CS1 and tested whether these peptides evoke MM-specific cytotoxic T lymphocytes (CTLs). First, we predicted the potential peptide sequences of CS1 that could bind to HLA-A*0201 molecule using three databases (RANKPEP, BIMAS and NetMHC). A total of four peptides were selected and synthesized according to the high binding scores among all the databases. Next, peptide-T2 cells binding assay confirmed these peptides with high binding affinity to HLA-A*0201 molecule (Fluorescence index: 2.19, 3.28, 3.44 and 3.49). To generate the peptide-specific CTLs, HLA-A*0201-positive normal human CD8+ T lymphocytes were stimulated with autologuous dendritic cells (DCs) or T2 cells pulsed with candidate CS1-peptides. The CTL cell lines were established after several rounds restimulation. We have evaluated the immunogenicity of the expanded CTLs by the stimulation with peptide-pulsed or unpulsed T2 cells. Our data demonstrated that one peptide-induced CTLs (CS1-CTLs) possessed high antigen-specific immune responses with the HLA-A*0201-restriction relative to a certain level of immune responses displayed by another three peptide-induced CTLs. We further evaluated the proliferative activity of CS1-CTLs by 3H-TdR incorporation assay. Upon stimulation by peptide-pulsed T2 cells, the cell proliferation of CS1-specific CTLs increased approximately 55-fold, compared with unstimulated cells (pulsed vs. unpulsed: 22894 vs. 423 cpm). The activation of CS1-CTLs was monitored by the expression of CD25 (IL-2Rα chain) on peptide-stimulated CS1-CTLs (%CD25+ cells: pulsed vs. unpulsed: 98% vs. 13%). CS1-CTLs significantly secreted 14-fold higher levels of IFN- γ in response to peptide-pulsed T2 cells (pulsed vs. unpulsed: 615 vs. 46 pg/ml). Importantly, the expanded CS1-CTLs generated high cytotoxicity (93%) in response to peptide pulsed T2 cells. Furthermore, more than 30% of killing activity by CS1-CTLs against HLA-A*0201+CS1+ MCCAR cells was observed at the effector: target ratio of 20:1. In contrast, these CS1-CLTs did not kill HLA-A*0201+/CS1− U266 and HLA-A*0201−/CS1+ MM1S target cells. These results support CS1 as an antigenic target for the induction of peptide-specific CTLs against MM cells. Further functional studies are underway to confirm potential CS1-specific cellular therapy to treat MM.

DKK1-Specific Cytotoxic T Lymphocytes Are Potent Effector Cells for the Treatment of Multiple Myeloma in SCID-hu Model.

Dickkopf-1 (DKK1), a secreted protein and Wnt signaling pathway inhibitor, is highly expressed by the tumor cells of most multiple myeloma (MM) patients and contributes to osteolytic bone disease by inhibiting the differentiation of osteoblasts. DKK1 has been targeted as a myeloma-associated antigen by immunotherapeutical approaches based on DKK1-directed monoclonal antibodies and DKK1-specific cytolytic T lymphocytes (CTLs). The present study was conducted to develop a treatment strategy through adoptive transfer of DKK1-specific CTLs in a SCID-hu system. Using DKK1 peptide-pulsed dendritic cells (DCs), we successfully generated DKK1-specific CTL lines and clones from HLA-A2+ MM patients in vitro. These CTLs not only had cytolytic activity against DKK1 peptide-pulsed T2 cells, but also significantly lysed autologous myeloma tumor cells, HLA-A2+ and DKK1+ MM cell lines U266 and IM-9, and primary tumor cells in vitro. No killing was observed against HLA-A2+ normal lymphocytes, including B cells and HLA-A2+ DKK1- XG1 or HLA-A2- myeloma cell lines or primary myeloma cells from patients. To determine the in vivo antitumor activity, SCID-hu mice were engrafted with primary MM cells expressing high levels of DKK1 from patients and treated with DKK1-specific CTLs after one month of tumor injection. Control mice were treated with naïve CD8+ T cells or PBS alone. All mice were examined by X-ray, tumor burden was measured according to levels of circulating human IgG, and survival rates were determined. All mice established human myeloma within one month after tumor inoculation, at which time the level of circulating human IgG was greater than 600 ng/ml in mouse serum. After another 3 weeks, the level of circulating human IgG was greater than 2,500 ng/ml in the serum of all control mice, while only 40% mice treated with DKK1-specific CTLs had high levels of circulating human IgG. X-ray examination showed that osteolytic bone disease and a large tumor burden were found in all control mice, while the established tumor was eradicated in 60% mice treated with DKK1-specific CTLs. Results of immunofluorescence studies showed that only tumor-reactive DKK1-specific CD8+ T cells were able to infiltrate into the tumor and mediate apoptosis in tumor cells. All control mice succumbed to myeloma within one month of establishment of the myeloma, while 9 out of 15 mice treated with DKK1-specific CTLs were macroscopically disease-free at the end of the experiment. These results suggest that DKK1-specific CTLs are able to eradicate established patient-derived primary myeloma in the host and that adoptive transfer of DKK1-specific CTLs may be used for myeloma therapy.

Mcl-1 and anticancer vaccination: identification of an HLA-A2-restricted epitope

The remarkable clinical benefit of donor-lymphocyte infusions as well as the finding that human T cells can destroy chemotherapy-resistant cell lines from chronic myeloid leukemia and multiple myeloma, have prompted the development of immunotherapeutic strategies against hematological cancers.1 Among these approaches, active specific immunization or vaccination is emerging as a valuable tool to boost the adaptive immune system against malignant cells. In this regard, interesting clinical responses have been described in the initial vaccination trials against hematological cancers.2 Due to the phenomenon of 'immune escape', strategies are being developed that conceptually focus on minimizing the risk of immune escape by specifically targeting proteins that are important for the function, survival and growth of cancer cells. To this end, antiapoptotic molecules that enhance the survival of cancer cells and facilitate their escape from cytotoxic therapies represent prime candidates. Indeed, most malignancies are characterized by defects in apoptotic signaling cascades, for example, an overexpression of the Bcl-2 family (for example Bcl-2, Bcl-X(L) or Mcl-1), and the inhibitor of apoptosis proteins (IAPs; for example survivin). Spontaneous cellular immune responses against these proteins have been identified as frequent features in cancer patients. However, whereas several survivin-based vaccination trials are currently ongoing,3 the potency of the antiapoptotic members of the Bcl-2 family as vaccination targets still needs to be addressed. In this regard, clinical vaccination trials in patients suffering from multiple myeloma and acute myeloid leukemia (AML) are on its way at the University Hospital Herlev, Denmark, including the Bcl-2 family member Mcl-1. Mcl-1 plays an important role in cancer as it has been directly linked to the resistance to conventional forms of therapies and poor prognosis.4 Recently, we demonstrated that cancer patients of different origin host spontaneous T-cell responses specifically against Mcl-1-derived peptides presented in the context of the HLA-A1 and -A3 antigen.5, 6, 7 However, the Mcl-1-based clinical vaccination trials await the identification of an HLA-A2-restricted epitope from Mcl-1 as HLA-A2 is the most frequent HLA-allele in the Caucasian population expressed by 50%. In the present study, the amino-acid sequence of the Mcl-1 protein was screened for the most probable HLA-A2 nona-peptide epitopes, using a cytotoxic T lymphocytes (CTL) epitope prediction algorithm available at http://www.cbs.dtu.dk/services/NetCTL/.8 The algorithm integrates prediction of peptide MHC class-I binding, proteasomal C-terminal cleavage and TAP-transport efficiency. Based on the prediction, nine Mcl-1-deduced peptides were synthesized: Mcl-1(13), NLYCGGAGL; Mcl-1(118), AIMSPEEEL; Mcl-1(139), AVLPLLELV; Mcl-1(159), SLPSTPPPA; Mcl-1(245), SLSRVMIHV; Mcl-1(250), MIHVFSDGV; Mcl-1(266), TLISFGAFV; Mcl-1(330), NVLLAFAGV; Mcl-1(333), LAFAGVAGV. Using the ELISPOT IFN- secretion assay, we examined peripheral blood lymphocytes (PBL) from six renal carcinoma patients for the presence of specific T-cell responses against the Mcl-1-derived peptides. This method has previously been highly effective for identifying tumor-specific CTL in cancer patients.9 In one of the patients, we were able to detect a strong T-cell response against the Mcl-1(139), AVLPLLELV. Subsequently, we stimulated PBL in vitro from this patient with Mcl-1(139) pulsed autologous dendritic cells. After four in vitro stimulations, CTL-clones were established by limiting dilution in 96-well plates using peptide-pulsed, irradiated peripheral blood mononuclear cells as feeder cells in the presence of 120 U ml-1 IL-2. After a short expansion step, the specificity of the growing clones was analyzed in standard 51Cr-release assays. To this end, either with or without T2-cells loaded with Mcl-1(139) or an irrelevant peptide (HIV-1 pol 476–484) served as targets. A number of T-cell clones only killed T2 cells pulsed with Mcl-1(139). One of these T-cell clones was selected for further expansion. This clone effectively lysed peptide-pulsed T2 cells at an effector/target (ET) ratio at 1:1 (Figure 1). Moreover, we tested the ability of Mcl-1-specific CTL to lyse human AML-blasts enriched directly ex vivo from the bone marrow of an AML patient. Consequently, we depleted T cells (CD3+) and B cells (CD19+) from the bone marrow of an HLA-A2+ AML patient. The highly enriched AML-blasts (CD3-, CD19-) were used as target cells in a standard 51Cr-release assay. As shown in Figure 1 the Mcl-1-specific T-cell clone efficiently lysed the enriched leukemia cells in a HLA-dependent manner. Inhibition with either the HLA-specific antibody W6/3210 or cold target cells (peptide-pulsed T2 cells) completely abrogated killing. In contrast, the addition of T2 cells without peptide only showed a very limited dilution effect.

Induction of Cytotoxic T Lymphocytes Specific to Malignant Glioma Using T2 Cells Pulsed with HLA-A2-restricted Interleukin-13 Receptor α2 Peptide <italic>in vitro</italic>

Interleukin-13 receptor alpha2 (IL-13Ralpha2) is a glioma-restricted cell-surface epitope not otherwise detected within the central nervous system. The present study is a report of a novel approach of targeting malignant glioma with IL-13Ralpha2-specific cytotoxic T lymphocyte (CTL) induced from the peripheral blood mononuclear cells of healthy donors by multiple stimulations with human leukocyte antigen (HLA)-A2-restricted IL-13Ralpha2(345-353) peptide-pulsed T2 cells. The induced CTL showed specific lysis against T2 cells pulsed with the peptide and HLA-A2+ glioma cells expressing IL-13R2(345-353), while HLA-A2 glioma cell lines that express IL-13Ralpha2(345-353) could not be recognized by CTL. The peptide-specific activity was inhibited by anti-HLA class I monoclonal antibody. These results suggest that the induced CTL specific for IL-13Ralpha2(345-353) peptide could be a potential target of specific immunotherapy for HLA-A2 patients with malignant glioma.

Efficient tumor cell lysis mediated by a Bcl-X(L) specific T cell clone isolated from a breast cancer patient.

Based on the detection of spontaneous immune responses in cancer patients with cancer of different origin, Bcl-X(L) was recently described as a highly interesting tumor antigen recognized by CD8 positive cytotoxic T lymphocytes. To further characterize Bcl-X(L) as a tumor antigen we isolated and expanded Bcl-X(L) specific T cells from the peripheral blood of a breast cancer patient hosting a strong Bcl-X(L) specific T cell response. We describe that HLA-A2 restricted Bcl-X(L) specific T cell clones very efficiently lyse peptide pulsed T2 cells. Furthermore, tumor cell lines of different origin, i.e., breast cancer, colon cancer, and melanoma, are efficiently lysed in an HLA-dependent manner. Finally, ex vivo-isolated leukemia cells, but not non-malignant B and T cells are killed by Bcl-X(L) specific T cells. Our data underline Bcl-X(L) as an universal tumor antigen widely applicable in specific anticancer immunotherapy.

Functional capacity of Mcl-1-specific cytotoxic T-cells

Recently, using the interferon-gamma (IFN-) ELISPOT we demonstrated that both hematopoetic and solid cancer patients host spontaneous T-cell responses against Mcl-1-derived peptides.1, 2 Thus, strong and frequent cytotoxic T lymphocytes (CTL) responses against Mcl-1 were detected in chronic lymphocytic leukemia (CLL) patients, melanoma patients, pancreatic cancer patients and breast cancer patients, whereas no responses could be detected in healthy individuals. We have identified several Mcl-1-derived peptide epitopes, for example, the HLA-A3-restricted peptide Mcl-195–103 (RLLFFAPTR). The IFN- ELISPOT assay is one of the most useful techniques for immunological monitoring of CTL responses and has gained increased application as a measure of specific T-cell activation. However, it does not assess cell-mediated cytotoxicity directly as IFN- secretion is not limited to cytolytic cells. Thus, although it has been shown that IFN- ELISPOT reactivity in most cases correlates with the capacity to exhibit cytotoxic function, the formal proof for this notion can only be obtained directly. Subsequently, to further characterize Mcl-1-specific T cells we isolated and cloned such cells from PBL from a breast cancer patient hosting a strong Mcl-1-specific T-cell response. Thus, lymphocytes were stained with anti-CD8 antibodies and HLA-A3/Mcl-195–103 pentamers (Proimmune, Oxford, UK) and analyzed by fluorescence-activated cell sorting (FACS). Double-positive cells were sorted as single cells into 96-well plates containing a mixture of irradiated PBL from three healthy donors and IL-2. After in vitro expansion the functional capacity of the Mcl-195–103-specific clones was analyzed in standard 51Cr release assays. To this end, either T2-A3 cells loaded with Mcl-195–103 or an irrelevant peptide served as targets. A number of clones only killed T2 cells pulsed with Mcl-195–103. One of these clones was selected for expansion. This clone effectively lysed peptide-pulsed T2-cells at an effector:target ratio of 1:1 (Figure 1a).

Hyperthermia Enhances CTL Cross-Priming

Dendritic cells (DCs) loaded with killed allogeneic melanoma cells can cross-prime naive CD8(+) T cells to differentiate into melanoma-specific CTLs in 3-wk cultures. In this study we show that DCs loaded with killed melanoma cells that were heated to 42 degrees C before killing are more efficient in cross-priming of naive CD8(+) T cells than DCs loaded with unheated killed melanoma cells. The enhanced cross-priming was demonstrated by several parameters: 1) induction of naive CD8(+) T cell differentiation in 2-wk cultures, 2) enhanced killing of melanoma peptide-pulsed T2 cells, 3) enhanced killing of HLA-A*0201(+) melanoma cells in a standard 4-h chromium release assay, and 4) enhanced capacity to prevent tumor growth in vitro in a tumor regression assay. Two mechanisms might explain the hyperthermia-induced enhanced cross-priming. First, heat-treated melanoma cells expressed increased levels of 70-kDa heat shock protein (HSP70), and enhanced cross-priming could be reproduced by overexpression of HSP70 in melanoma cells transduced with HSP70 encoding lentiviral vector. Second, hyperthermia resulted in the increased transcription of several tumor Ag-associated Ags, including MAGE-B3, -B4, -A8, and -A10. Thus, heat treatment of tumor cells permits enhanced cross-priming, possibly via up-regulation of both HSPs and tumor Ag expression.

A new way to generate cytolytic tumor-specific T cells: electroporation of RNA coding for a T cell receptor into T lymphocytes

Effective T cell receptor (TCR) transfer until now required stable retroviral transduction. However, retroviral transduction poses the threat of irreversible genetic manipulation of autologous cells. We, therefore, used optimized RNA transfection for transient manipulation. The transfection efficiency, using EGFP RNA, was >90%. The electroporation of primary T cells, isolated from blood, with TCR-coding RNA resulted in functional cytotoxic T lymphocytes (CTLs) (>60% killing at an effector to target ratio of 20:1) with the same HLA-A2/gp100-specificity as the parental CTL clone. The TCR-transfected T cells specifically recognized peptide-pulsed T2 cells, or dendritic cells electroporated with gp100-coding RNA, in an IFNgamma-secretion assay and retained this ability, even after cryopreservation, over 3 days. Most importantly, we show here for the first time that the electroporated T cells also displayed cytotoxicity, and specifically lysed peptide-loaded T2 cells and HLA-A2+/gp100+ melanoma cells over a period of at least 72 h. Peptide-titration studies showed that the lytic efficiency of the RNA-transfected T cells was similar to that of retrovirally transduced T cells, and approximated that of the parental CTL clone. Functional TCR transfer by RNA electroporation is now possible without the disadvantages of retroviral transduction, and forms a new strategy for the immunotherapy of cancer.

DKK-1 Is a Widely Expressed, Potent Tumor-Associated Antigen in Multiple Myeloma Recognized by Cytotoxic T Lymphocytes.

Idiotype proteins have been used for immunotherapy in multiple myeloma (MM); and the results thus far have been disappointing. Therefore, the identification and use of novel and more potent tumor-associated antigens are urgently needed to improve the efficacy of the treatment. Dickkopf-1 (DKK1), a secreted protein and Wnt signaling pathway inhibitor, is highly expressed by the tumor cells of most myeloma patients and may be responsible for suppressed osteoblast formation. Based on these data and the fact that DKK1 is not expressed in normal tissues except placenta, prostate, and mesenchymal stem cells, we hypothesized that DKK1 is a novel tumor-associated antigen in MM. In this study we examined the capacity of DKK1 to trigger the induction of antimyeloma cytotoxic T lymphocyte (CTL) responses. Using DKK-1 peptide-pulsed dendritic cells (DCs), we successfully generated DKK-1 peptide (DKK120-29 and DKK166-74V)-specific CTL lines and clones from HLA-A2+ MM patients and healthy individuals. In our experiments, mature DCs obtained from cultures of blood monocytes were pulsed with HLA-A2-restricted DKK1 peptides. Autologous T cells were then stimulated weekly with these DCs, and cytotoxicity was examined against DKK1 peptide-pulsed T2 cells, myeloma cell lines, and primary myeloma cells isolated from patients. After 4 cycles of in vitro stimulation and subsequent T cell cloning and expansion, specific CD8+ CTL lines and clones were obtained. These CTLs not only had cytolytic activity against DKK1 peptide-pulsed T2 cells, but also significantly lysed HLA-A2+ MM cell line U266 and primary tumor cells in vitro. No killing was observed against HLA-A2+ normal lymphocytes including B cells or HLA-A2− myeloma cell lines or primary myeloma cells from patients. The CTL response could be inhibited by anti-HLA-A2 antibody indicating that the response was indeed restricted by HLA-A2 molecules. IFN-γ, but not IL-4, was secreted by the T cells indicating that the effector cells were type-1 CD8+ T cells. Further functional studies are underway. Thus, these results demonstrate that DKK1-specific CTLs are able to lyse myeloma tumor cells including primary myeloma cells from patients, and identify DKK1 as a potentially important antigenic target for antimyeloma immunotherapeutic strategies

Differential CTLs specific for prostate-specific antigen in healthy donors and patients with prostate cancer

Induction of CTL responses specific for prostate-specific antigen (PSA)-derived peptides in healthy individuals and patients with prostate cancer (PC) was investigated. Eight PSA-derived peptides that have the potential to bind HLA-A2 molecules were examined. Peripheral blood lymphocytes isolated from HLA-A2-positive volunteers were expanded using autologous mature, PSA-derived peptide-pulsed dendritic cells. The expansion of IFN-gamma-secreting CD8+ T cells specific for three of the eight PSA-derived peptides (PSA-2(108-117), PSA-4(141-150) and PSA-6(146-154)) was detected in healthy individuals, but not in patients with PC. Using HLA-A2/peptide tetramers, the PSA-specific CD8+ T cells were detectable at low frequency both in healthy individuals and patients with PC. Using flow cytometric cytotoxicity assays, the expanded effectors from healthy individuals were able to kill the PSA-expressing epithelial cell line LNCaP and the peptide-pulsed T2 cells in a MHC class I-restricted manner without involving NK activity. However, such killing by effectors expanded from prostatectomized patients involved a complete or a significant NK activity. Specific recognition of PSA-derived peptides in healthy individuals may occur by an adaptive CTL immune response, while such recognition in PC patients may additionally or alternatively be mediated by an innate NK immune response. In conclusion, our work indicates that the PSA-specific CD8+ T cells exist in both healthy individuals and PC patients, but they have impaired function in patients as they failed to release IFN-gamma and to kill targets without involving NK activity.

Identification and characterization of epitopes of the receptor for hyaluronic acid–mediated motility (RHAMM/CD168) recognized by CD8+ T cells of HLA-A2–positive patients with acute myeloid leukemia

AbstractThe receptor for hyaluronic acid–mediated motility (RHAMM/CD16881) has been described as a leukemia-associated antigen. To define T-cell epitopes of RHAMM/CD168 toward specific immunotherapies for acute myeloid leukemia (AML), 10 potential HLA-A2–binding RHAMM/CD168 peptides (R1 to R10) were synthesized based on computer algorithms and screened by enzyme-linked immunospot (ELISPOT) analysis using CD8+ T cells isolated from peripheral blood (PB) of patients with AML and healthy donors. We found that CD8+ cells from 7 of 13 (54%) patients with AML presensitized with peptides R3 (ILSLELMKL) or R5 (SLEENIVIL) specifically recognized T2 cells pulsed with R3 (39%) or R5 (15%) peptide. In contrast, only 4 of 21 (19%) healthy volunteers had CD8+ cells reactive with R3- or R5-pulsed T2 cells after presensitization. The presence of R3 peptide–specific effector T cells in the peripheral blood of patients with AML could be confirmed by staining as HLA-A2/R3 peptide tetramer+ CCR7-CD45RA+ cells. In chromium-51 release assays, peptide-primed CD8+ T cells from patients with AML were able to lyse RHAMM/CD168 peptide–pulsed T2 cells, AML blasts, and dendritic cells generated thereof (AML DCs). Transfection of COS7 cells with RHAMM/CD168 cDNA revealed that peptides R3 and R5 are naturally processed epitopes of RHAMM/CD168 that are presented in an HLA-A2–restricted manner. In summary, RHAMM/CD168 is a promising target for immunotherapies in patients with AML, and we have therefore initiated a clinical vaccination trial with R3 peptide. Because RHAMM/CD168 is also expressed in various other hematologic malignancies and solid tumors, vaccines targeting this antigen may have even wider application.

Human CTL Epitopes Prostatic Acid Phosphatase-3 and Six-Transmembrane Epithelial Antigen of Prostate-3 as Candidates for Prostate Cancer Immunotherapy

Specific immunotherapy of prostate cancer may be an alternative or be complementary to other approaches for treatment of recurrent or metastasized disease. This study aims at identifying and characterizing prostate cancer-associated peptides capable of eliciting specific CTL responses in vivo. Evaluation of peptide-induced CTL activity in vitro was done following immunization of HLA-A2 transgenic (HHD) mice. An in vivo tumor rejection was tested by adoptive transfer of HHD immune lymphocytes to nude mice bearing human tumors. To confirm the existence of peptide-specific CTL precursors in human, lymphocytes from healthy and prostate cancer individuals were stimulated in vitro in the presence of these peptides and CTL activities were assayed. Two novel immunogenic peptides derived from overexpressed prostate antigens, prostatic acid phosphatase (PAP) and six-transmembrane epithelial antigen of prostate (STEAP), were identified; these peptides were designated PAP-3 and STEAP-3. Peptide-specific CTLs lysed HLA-A2.1+ LNCaP cells and inhibited tumor growth on adoptive immunotherapy. Furthermore, peptide-primed human lymphocytes derived from healthy and prostate cancer individuals lysed peptide-pulsed T2 cells and HLA-A2.1+ LNCaP cells. Based on the results presented herein, PAP-3 and STEAP-3 are naturally processed CTL epitopes possessing anti-prostate cancer reactivity in vivo and therefore may constitute vaccine candidates to be investigated in clinical trials.

Identification of a new HLA-A*0201-restricted CD8+ T cell epitope from hepatocellular carcinoma-associated antigen HCA587

For the development of peptide-based cancer immunotherapies, we aimed to identify specific HLA-A*0201-restricted CTL epitopes in hepatocellular carcinoma (HCC) associated antigen HCA587, which has been identified as a member of the cancer/testis (CT) antigens highly expressed in HCC. We first combined the use of an HLA-A*0201/peptide binding algorithm and T2 binding assays with the induction of specific CD8(+) T cell lines from normal donors by in vitro priming with high-affinity peptides, then IFN-gamma release and cytotoxicity assays were employed to identify the specific HLA-A*0201 CD8(+) T cell epitope using peptide-loaded T2 cells or the HCA587 protein(+) HCC cell line HepG2. In the six candidate synthesized peptides, two peptides showed higher binding ability in T2 binding assays. No. 2 peptide, encompassing amino acid residues FLAKLNNTV (HCA587(317-325)), was able to activate a HCA587-specific CD8(+) T-cell response in human lymphocyte cultures from two normal donors and two HCC patients, and these HCA587-specific CD8(+) T cells recognized peptide-pulsed T2 cells as well as the HCA587 protein(+) HCC cell line HepG2 in IFN-gamma release and cytotoxicity assays. The results indicate that no. 2 peptide is a new HLA-A*0201-restricted CTL epitope capable of inducing HCA587-specific CTLs. Our data suggest that identification of this new HCA587/HLA-A*0201 peptide FLAKLNNTV may facilitate the design of peptide-based immunotherapies for the treatment of HCA587-bearing HCC patients.

Primary Human Lymphocytes Transduced with NY-ESO-1 Antigen-Specific TCR Genes Recognize and Kill Diverse Human Tumor Cell Lines

cDNAs encoding TCR alpha- and beta-chains specific for HLA-A2-restricted cancer-testis Ag NY-ESO-1 were cloned using a 5'RACE method from RNA isolated from a CTL generated by in vitro stimulation of PBMC with modified NY-ESO-1-specific peptide (p157-165, 9V). Functionality of the cloned TCR was confirmed by RNA electroporation of primary PBL. cDNA for these alpha- and beta-chains were used to construct a murine stem cell virus-based retroviral vector, and high titer packaging cell lines were generated. Gene transfer efficiency in primary T lymphocytes of up to 60% was obtained without selection using a method of precoating retroviral vectors onto culture plates. Both CD4(+) and CD8(+) T cells could be transduced at the same efficiency. High avidity Ag recognition was demonstrated by coculture of transduced lymphocytes with target cells pulsed with low levels of peptide (<20 pM). TCR-transduced CD4 T cells, when cocultured with NY-ESO-1 peptide pulsed T2 cells, could produce IFN-gamma, GM-CSF, IL-4, and IL-10, suggesting CD8-independent, HLA-A2-restricted TCR activation. The transduced lymphocytes could efficiently recognize and kill HLA-A2- and NY-ESO-1-positive melanoma cell lines in a 4-h (51)Cr release assay. Finally, transduced T cells could efficiently recognize NY-ESO-1-positive nonmelanoma tumor cell lines. These results strongly support the idea that redirection of normal T cell specificity by TCR gene transfer can have potential applications in tumor adoptive immunotherapy.

Boosting Vaccinations with Peptide-Pulsed CD34+ Progenitor-Derived Dendritic Cells Can Expand Long-Lived Melanoma Peptide-Specific CD8+ T Cells in Patients with Metastatic Melanoma

The immunogenicity of dendritic cell (DC)-based vaccines has been shown in patients with advanced cancer, but it has not yet been established whether the elicited cancer-specific immunity is durable and whether it can be maintained by boosting vaccinations. The authors showed earlier, in 18 HLA-A*0201 metastatic melanoma patients, that four vaccinations over 6 weeks with peptide-loaded CD34-DCs (the induction phase) expand in the blood melanoma-specific CD8+ T cells, as documented by melanoma peptide-specific IFN-gamma ELISPOT and cytotoxic T-lymphocyte (CTL) activity against melanoma cell lines. The authors show here that the melanoma peptide-specific CD8+ T-cell immunity is short-lived, but it could be reactivated in 7 of 11 patients who received four boosting vaccinations with peptide-loaded CD34-DCs. Expansion of recall memory CD8+ T cells was confirmed by tetramer binding and CTL activity against melanoma peptide-pulsed T2 cells. In two patients boosted over 15 months, induced melanoma peptide-specific recall memory CD8+ T cells lasted at least 6 months. Thus, boosting vaccination with peptide-loaded CD34-DCs can expand long-lived tumor peptide-specific immunity.

Expansion of melanoma-specific cytolytic CD8+ T cell precursors in patients with metastatic melanoma vaccinated with CD34+ progenitor-derived dendritic cells.

Cancer vaccines aim at inducing (a) tumor-specific effector T cells able to reduce/eliminate the tumor mass, and (b) long-lasting tumor-specific memory T cells able to control tumor relapse. We have shown earlier, in 18 human histocompatibility leukocyte antigen (HLA)-A*0201 patients with metastatic melanoma, that vaccination with peptide-loaded CD34–dendritic cells (DCs) leads to expansion of melanoma-specific interferon γ–producing CD8+ T cells in the blood. Here, we show in 9 out of 12 analyzed patients the expansion of cytolytic CD8+ T cell precursors specific for melanoma differentiation antigens. These precursors yield, upon single restimulation with melanoma peptide–pulsed DCs, cytotoxic T lymphocytes (CTLs) able to kill melanoma cells. Melanoma-specific CTLs can be grown in vitro and can be detected in three assays: (a) melanoma tetramer binding, (b) killing of melanoma peptide–pulsed T2 cells, and (c) killing of HLA-A*0201 melanoma cells. The cytolytic activity of expanded CTLs correlates with the frequency of melanoma tetramer binding CD8+ T cells. Thus, CD34-DC vaccines can expand melanoma-specific CTL precursors that can kill melanoma antigen–expressing targets. These results justify the design of larger follow-up studies to assess the immunological and clinical response to peptide-pulsed CD34-DC vaccines.

Efficient induction of cytotoxic T lymphocytes specific to hepatocellular carcinoma using HLA-A2-restricted MAGE-n peptide in vitro

MAGE-n is a new member of MAGE gene family and has been demonstrated closely associated with hepatocellular carcinoma (HCC). In this study, MAGE-n-derived peptide-specific cytotoxic T lymphocytes (CTL) were induced from the peripheral blood mononuclear cells of healthy donors by multiple stimulations with HLA-A2-restricted MAGE-n peptide-pulsed T2 cells. The induced CTLs exhibited specific lysis against T2 cells pulsed with the peptide and HLA-A2 + HCC cells expressing MAGE-n, while HLA-A2 + HCC cell lines that did not express MAGE-n could not be recognized by the CTLs. The peptide-specific activity was inhibited by anti-MHC class I monoclonal antibody. These results suggested the MAGE-n peptide could be a potential target of specific immunotherapy for HLA-A2 patients with HCC.

Immunological monitoring of patients with melanoma after peptide vaccination using soluble peptide/HLA-A2 dimer complexes.

To facilitate the immunologic monitoring of a peptide vaccine trial, a novel, empty dimeric HLA-A2 molecule (A2 dimer) that could be loaded with peptides was produced. The dimer comprises the extracellular domain of HLA-A2 noncovalently linked to a fusion protein consisting of human beta2-microglobulin joined to the human IgG1 Fc domain. Peptide-loaded dimer complexes were used to assess the function of peptide-specific T cells. HLA-A2 gp100 peptide dimers stimulated interferon (IFN)-gamma production by the gp100-specific TIL-1520 cell line. Gp100/A2 dimer stimulation in combination with intracellular cytokine staining was used to analyze peptide-specific T-cell responses in patients with melanoma after vaccination with the modified gp100: 209-2M peptide in adjuvant. Titration analysis of the amount of peptide-loaded dimer required to stimulate gp100-specific T cells was used to estimate the functional avidity of effector/memory CD8+ T lymphocytes. The number of peptide-specific T cells detected in the peripheral blood of vaccinated patients using this assay was comparable to the number determined by staining with fluoresceinated gp100: 209-2M HLA-A2 tetramers. IFN-gamma production by T cells was comparable after stimulation with peptide-pulsed dimers, T2 cells, or autologous dendritic cells. Peptide-loaded A2 dimers could also be used directly to stimulate T cells in the ELISPOT assay.