Peptide-specific CTL Research Articles

Phase I clinical study of a peptide vaccination for hepatitis C virus‐infected patients with different human leukocyte antigen‐class I‐A alleles

Hepatitis C virus (HCV) infection has a high risk of liver cirrhosis and hepatocellular carcinoma at later stages. We recently identified a peptide derived from the HCV core protein capable of inducing both cellular and humoral responses to nearly all HCV-positive patients in Japan with different human leukocyte antigen (HLA)-class I-A alleles. To assess the safety and immune responses to this novel peptide, we conducted a phase I dose-escalation study of the vaccination for 26 HCV-positive patients who were either non-responders to the interferon-based therapy (n = 23) or refused it (n = 3). The regimen was well tolerated, with no severe vaccine-related toxicity. Twenty-five and 22 patients completed the first and second cycle vaccination (6 and 12 vaccine injections), respectively. After a series of six vaccine injections, peptide-specific CTL activity was augmented in peripheral blood mononuclear cells from 15 of 25 patient samples, with an expected optimal dose of 1 mg peptide. After 12 vaccine injections, peptide-specific IgG production was augmented in plasma from the majority of patients (15 of 22 patients) tested, but not in a dose-dependent fashion. There were two HCV RNA responders with >1 log declines. Among patients whose pre-vaccination levels of alanine aminotransferase and alpha feto-protein exceeded the normal ranges, a <30% decrease was found in 7 of 24 and three of six patients, respectively. Because of its tolerability and higher rate of immune boosting, this protocol is recommended for a phase II study to investigate its clinical efficacy.

Analysis of the frequency and function of antigen specific CTL in different courses' patients with condyloma acuminata

Objective To explore the frequency and function of antigen-specific T cells at different courses of condyloma acuminate(CA)by means of the sHLA-A2/HPV-6 E7 tetramer constructed from HLA-A * 0201 and its restricted peptide HPV-6 E7(22-30).Methods Peripheral blood lymphecytes (PBL)were collected from the HLA-A2 positive individuals without clinical manifestations and those CA cases at different courses.The T2 (HLA-A2+)cells born with HPV-6 E7 were used as the priming cells to co-cultivate with the PBL taken from different courses of the patients to generate the peptide-specific CTL through long term co-cultivation.The HLA-A2/HPV-6 E7 tetramer was labeled with PE in vitro and the proliferated specific CTLs after cultivation were stained with fluorescence by making use of sHLA-A2/HPV-6 E7 tetramer and FITC-CD8 McAb.And the killing activities of these specific CTLs were tested by LDH assay.Results The frequency of specific CTLs in control group was (0.320±0.076)%.The frequencies of specific CTLs in recurrent CA and during remission were ( 1.100±0.064)%and ( 1.115±0.071)%,respectively,both of which were of no significant difference (t=0.34,P=0.74)and higher than that of the control.The killing activities in recurrent CA and during remission were (51.18±0.48)%and (59.33±1.71)%,respectively.The significant difference existed betwcen these two groups.Conclusion The tetramer can be applied directly to visualize antigen-specific CTLs efficiently and become the critical approach in the assessment of T cell immune responses in CA patients.The difference is not obvious in the frequencies of specific CTLs at different courses of CA.But the difference is prominent in the CTLs killing frequency at different courses of CA.The CTLs can be highly activated and eliminate the HPV in remission CA,so the disease can be cured. Key words: Human papillomavirus; E7 antigen; Condyloma acuminata; Tetramer; Cytotoxin T lymphocyte

Dendritic Cells Require STAT-1 Phosphorylated at Its Transactivating Domain for the Induction of Peptide-Specific CTL

Phosphorylation of transcription factor STAT-1 on Y701 regulates subcellular localization whereas phosphorylation of the transactivating domain at S727 enhances transcriptional activity. In this study, we investigate the impact of STAT-1 and the importance of transactivating domain phosphorylation on the induction of peptide-specific CTL in presence of the TLR9-dependent immune adjuvant IC31. STAT-1 deficiency completely abolished CTL induction upon immunization, which was strongly reduced in animals carrying the mutation of the S727 phospho-acceptor site. A comparable reduction of CTL was found in mice lacking the type I IFN (IFN-I) receptor, whereas IFN-gamma-deficient mice behaved like wild-type controls. This finding suggests that S727-phosphorylated STAT-1 supports IFN-I-dependent induction of CTL. In adoptive transfer experiments, IFN-I- and S727-phosphorylated STAT-1 were critical for the activation and function of dendritic cells. Mice with a T cell-specific IFN-I receptor ablation did not show impaired CTL responses. Unlike the situation observed for CTL development S727-phosphorylated STAT-1 restrained proliferation of naive CD8(+) T cells both in vitro and following transfer into Rag-deficient mice. In summary, our data reveal a dual role of S727-phosphorylated STAT-1 for dendritic cell maturation as a prerequisite for the induction of CTL activity and for T cell autonomous control of activation-induced or homeostatic proliferation.

Characterization of cytomegalovirus pp65-HLA-A24 peptide-specific CTL lines from metastatic melanoma patients

Because of advances in immunological technology for detecting a very small number of blood CTL cells, clinicians have been able to monitor cellular immunity against CMV and evaluate the status of CMV infections in highly advanced cancer patients or transplant recipients. Our previous study using healthy volunteer PBLs revealed a significant increase in CMV HLA-A24 tetramer+ CTLs after stimulation in vitro with autologous DCs. However, the efficiency of CMV A24 peptide-specific CTL expansion in highly advanced cancer patients has yet to be studied in detail. In the present study, we tried to characterize and expand HLA-A*2402 CMVpp65 peptide (QYDPVAALF)-specific tetramer+ CTLs from HLA-A*2402+ metastatic melanoma patients, and eventually demonstrated that expansion efficiency was closely related to both post-stimulation CMV tetramer frequency and anti-CMV IgG titer. This is a novel finding regarding in vitro CMVpp65-A24 peptide-specific CTL expansion based on metastatic cancer patient-derived PBLs. Interestingly, the current results using metastatic melanoma PBLs showed a much higher frequency of CMVpp65-A24 tetramer+ CTLs and expansion efficiency than in healthy volunteers. Finally, we were successful in cloning CMVpp65 HLA-A24 peptide-specific TCR cDNAs from in vitro expanded CTL lines derived from melanoma patients. Additionally, CMVpp65 HLA-A24 peptide-specific TCR cDNA was transduced into naive T cells from patients and functionally reconstructed. The results showed that cloned CMV-specific TCR genes were efficient in reconstituting specific anti-CMV activity and might be good tools for adoptive immunotherapy against CMV infections.

Impaired Mast Cell-Driven Immune Responses in Mice Lacking the Transcription Factor NFATc2

The three calcium-dependent factors NFATc1, c2, and c3 are expressed in cells of the immune system and play pivotal roles in modulating cellular activation. With regard to NFATc2, it was reported that NFATc2-deficient mice display increased immune responses in several models for infection and allergy in vivo. This led to the assumption that NFATc2 is involved in the maintenance of immune homeostasis. Using the synthetic TLR7 agonist imiquimod as an adjuvant in epicutaneous peptide immunization, we observed that both the inflammatory reaction and the peptide-specific CTL response are severely impaired in NFATc2-deficient mice. Detailed analyses revealed that early production of proinflammatory cytokines, lymph node hypertrophy, and migration of Langerhans cells are strongly reduced in NFATc2-deficient animals. With the aid of mast cell-deficient mice and reconstitution experiments using mast cells derived from either NFATc2-deficient mice or wild-type controls, we were able to show that NFATc2 expressed in mast cells is critical for the initiation of inflammation, migration of Langerhans cells, and the development of full-blown CTL responses following epicutaneous immunization. Thus, NFATc2 is an important factor controlling mast cell accessory function at the interface of innate and adaptive immunity.

Self-Reactive CTL Reveal a Strong Focus of the CD8+ T cell Receptor Repertoire on Self MHC (46.12)

Abstract Mouse CD8+ T cells can be activated to lyse targets expressing self class I molecules more potently than allogeneic targets. Self-reactive CTL express IL-17 and can be enriched following intracellular staining. Such CTL lyse targets in an MHC restricted fashion. This recognition is independent of the bound peptides, as demonstrated by cold-target inhibition studies. Activation of OT-1 transgenic T cells using traditional APC results in peptide specific CTL recognition, stimulation of the same T cells induces peptide independent, yet Kb-restricted recognition, irrespective of whether RAG sufficient or deficient mice were used. Self-reactive CTL efficiently lyse targets expressing wild type Kb or Kb-mutants with amino acid substitutions on the β-strands of the antigen binding groove, but not mutants with substitutions on the α1 and α2 helices, suggesting self MHC recognition is mediated by interactions of the CDR1 and CDR2 regions of the TCR with the α-helices of the class I molecules, and not by bound peptides. The ability of CTL to react in an MHC restricted, yet peptide independent, manner raises important questions about the nature of TCR-MHC interactions. We propose a novel "pre-positive selection" check point in thymic development and in peripheral T cell activation in recruitment of TCR-MHC complexes at the immunological synapse during T cell activation and homeostatic recognition.

A Head-to-Head Comparison between Cyclophosphamide and Ontak in Reducing the Influence of Tregs on Self (Tumor) Peptide-Specific CTL Responses

Introduction: Regulatory (suppressor) T cells limit immune responses including those directed against tumor-associated, self-antigens. Several approaches have been taken to reduce the numbers and/or function of these Tregs during vaccination against tumor-associated antigens. However, the immunomodulatory drugs used in these protocols have never been rigorously compared head-to-head for their ability to down-regulate Treg subsets and to up-regulate self peptide-specific responses. To this end FoxP3 promoter-GFP knock-in mice were used to track GFP+ Tregs following exposure to cyclophosphamide (CTX) or Ontak (Denileukin Diftitox/DAB289IL-2), two FDA-approved therapeutics currently being tested for their ability to boost anti-cancer vaccine responses. Transgenic mice expressing both this reporter construct and human HLA-A2 antigen were used to generate HLA-A2-restricted, CTL responses against a self-peptide derived from murine Blimp-1, a plasma cell-associated transcription factor representing a putative vaccine for plasma cell dyscrasias. The effects of CTX and Ontak on these responses also were determined.Method: To track Tregs and to determine an optimal point for self-peptide immunization, FoxP3 promoter-GFP knock-in mice were pre-bled and injected i.p. with PBS, CTX (2mg once) or Ontak (5ug on days 1, 3, 15 and 18). Mice were bled periodically for up to 41 days and assessed for expression of FoxP3(GFP)+/CD25+ and FoxP3(GFP)+/CD25− Tregs and other T cell subsets by flow cytometry. To determine the functional consequence of these treatments, “quadruple” transgenic/knock-out mice (mouse MHC-I−/−, human HLA-A2 and β2 microgloblin transgenic, FoxP3 promoter-GFP knock-in) were treated with CTX or Ontak, mock immunized or immunized with murine Blimp-1 peptide at day 12 (CTX treatment) or day 23 (Ontak treatment), and boosted twice at 14 day intervals. Mice were sacrificed 14 days thereafter, spleen, lymph node and bone marrow assayed for Treg and other T cell subsets, and splenic cells re-challenged in vitro with Blimp-1 peptide. Cultures were evaluated 7 days later for CTL activity in a standard 51Cr-release killing assays using Blimp-1 or control peptide-pulsed HLA-A2+ human B lymphoma cells as targets.Results: CTX dramatically decreased the absolute number of blood FoxP3+/CD25+ and FoxP3+/CD25− Treg cells by day 3 (32 ± 2% and 18 ±3% of starting numbers on day 0 respectively) but had less of an effect on “responder” CD4+/FoxP3− T cells (64 ± 4% at lowest on day3, p<0.001) or CD8+ T cells (68 ± 6% at lowest on day 3, p<0.001). FoxP3+ cells did not fully recover over a 40-day period while CD8+ T cells recovered by day 14. Ontak preferentially decreased FoxP3+/CD25+ Treg numbers (30 ± 4% of starting population) and had no effect on FoxP3+/CD25− Tregs (96 ± 15% of starting numbers at day 5). FoxP3+/CD25+ Tregs did not completely recover at any point evaluated. Ontak modestly reduced the number of “responder” CD4+/FoxP3− cells (64 ± 5% at day 5) but had little effect on CD8+ T cells (85 ± 7% of starting numbers at day 5). CD4+ and CD8+ T cells recovered by days 23 and 15 respectively after Ontak treatment. In some experiments, Ontak induced an increase in CD8+ cells above baseline levels 23–28 days after treatment. Despite specific killing of FoxP3+/CD25+ Tregs and a minimal effect on CD8+ T cells following Ontak treatment, Blimp-1 peptide-specific CTL responses were augmented only after CTX treatment.Conclusion: CTX and Ontak influence different sub-populations of Tregs potentially impacting self (tumor) peptide-induced CTL responses differently. CTX appears more effective in augmenting Blimp-1-specific CTL responses in this study, possibly because of its ability to eliminate both CD25+ and CD25− Tregs, results suggest that CD25− Tregs play an important role in limiting self antigen-specific responses. In addition the data suggest that a combination of CTX and Ontak may be optimal in generating immune responses to peptides for immunotherapy of B cell malignancies and, perhaps, for other cancers.

Identification of a WT1 protein‐derived peptide, WT1187, as a HLA‐A*0206‐restricted, WT1‐specific CTL epitope

The Wilms' tumor gene WT1 is overexpressed in various kinds of hematopoietic malignancies as well as solid cancers, and this protein has been demonstrated to be an attractive target antigen for cancer immunotherapy. WT1-specific CTL epitopes with a restriction of HLA-A 2402 or HLA-A 0201 have been already identified. In the present study it has been demonstrated that a 9-mer WT1-derived WT1(187) peptide, which had already been shown to elicit a WT1-specific CTL response with a restriction of HLA-A 0201, can also elicit a CTL response with a restriction of HLA-A 0206. In all three different HLA-A 0206(+) healthy donors examined, WT1(187) peptide-specific CTL could be generated from peripheral blood mononuclear cells, and the CTL showed cytotoxic activity that depended on dual expression of WT1 and HLA-A 0206 molecules. The present study describes the first identification of a HLA-A 0206-restricted, WT1-specific CTL epitope. The present results should help to broaden the application of WT1 peptide-based immunotherapy from only HLA-A 0201-positive to HLA-A 0206-positive cancer patients as well.

DNA fusion gene vaccination mobilizes effective anti‐leukemic cytotoxic T lymphocytes from a tolerized repertoire

The majority of known human tumor-associated antigens derive from non-mutated self proteins. T cell tolerance, essential to prevent autoimmunity, must therefore be cautiously circumvented to generate cytotoxic T cell responses against these targets. Our strategy uses DNA fusion vaccines to activate high levels of peptide-specific CTL. Key foreign sequences from tetanus toxin activate tolerance-breaking CD4(+) T cell help. Candidate MHC class I-binding tumor peptide sequences are fused to the C terminus for optimal processing and presentation. To model performance against a leukemia-associated antigen in a tolerized setting, we constructed a fusion vaccine encoding an immunodominant CTL epitope derived from Friend murine leukemia virus gag protein (FMuLV(gag)) and vaccinated tolerant FMuLV(gag)-transgenic (gag-Tg) mice. Vaccination with the construct induced epitope-specific IFN-gamma-producing CD8(+) T cells in normal and gag-Tg mice. The frequency and avidity of activated cells were reduced in gag-Tg mice, and no autoimmune injury resulted. However, these CD8(+) T cells did exhibit gag-specific cytotoxicity in vitro and in vivo. Also, epitope-specific CTL killed FBL-3 leukemia cells expressing endogenous FMuLV(gag) antigen and protected against leukemia challenge in vivo. These results demonstrate a simple strategy to engage anti-microbial T cell help to activate epitope-specific polyclonal CD8(+) T cell responses from a residual tolerized repertoire.

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.

A multicenter phase I/II study of gemcitabine and personalized peptide vaccination combination therapy for metastatic pancreatic cancer patients

4633 Background: The present multicenter phase I/II study was conducted to confirm the efficacy and toxicity of immunochemotherapy for metastatic pancreatic cancer. Methods: HLA-A2 or A24 positive patients with histologically or cytologically proven pancreatic adenocarcinoma with at least one measurable metastatic lesion were eligible for the study. Other eligibility criteria included: age of 20–80 years, ECOG performance status (PS) of 0 or 1, and adequate organ function. Gemcitabine was given intravenously at a dose of 1,000 mg/m2 over 30 min once a week for three weeks, followed by 1 week of rest, and three or four peptides that had been positive for pre-existing peptide-specific immune responses in the circulation (personalized peptide vaccine) were injected subcutaneously in the femoral area once a week. The objective response rate was assessed according to RECIST. Results: Twenty patients from 2 institutions were enrolled in this study. The median age was 63 years (range: 41–80 years). The PS was 0 in 10 patients (50%) and 1 in 10 patients (50%). The efficacy and toxicity were analyzed in 20 patients who received at least one course (8 weeks) of immunochemotherapy. Although no complete response was seen, a partial response was achieved in 5 patients, resulting in an overall response rate of 25%. Eleven patients (55%) had stable disease. The median overall survival was 8.5 months with a 1-year survival rate of 33%. The major grade 3–4 toxicities using CTCAE criteria were neutropenia (25%), anemia (20%), thrombocytopenia (20%) and anorexia (5%). All 17 dermatologic reactions at the vaccination site were scored as grade 1 or 2. No treatment-related deaths occurred during the study. Augmentation of peptide-specific CTL responses in the post-vaccination peripheral blood mononuclear cells was observed in 72%, while increased titer of peptide-specific IgG antibodies was observed in the post-vaccination plasma in 78%. Conclusions: This study could be recommended for further stages of clinical trials because of safety, boosting of immune responses, and potential clinical benefits. Author Disclosure Employment or Leadership Consultant or Advisory Role Stock Ownership Honoraria Research Expert Testimony Other Remuneration GreenPeptide GreenPeptide GreenPeptide Grant-in-Aid for Scientific Research from Research Center of Innovative Cancer Therapy of the 21st Century COE Program for Medical Science of Japan, Grant-in-Aid for Scientific Research from the Ministry of Education, Culture, Sports, Science and Technology of Japan

Phase I/II study of individualized peptide vaccines for HLA-A2/A24 positive patients with hormone-refractory prostate cancer

5155 Background: Due to a lack of standard effective treatment strategies for hormone-refractory prostate cancer (HRPC), the development of new therapeutic vaccine strategies remains of considerable interest. To evaluate both the toxicicity and capability to induce immune and clinical responses, a phase I/II study of MHC class-I restricted peptide vaccination was carried out in patients with HRPC. Methods: 23 HRPC patients positive for human leukocyte antigen (HLA)-A2 and/or -A24 were enrolled in this study. We prepared 32 kinds of 9mer peptides as HLA-A2 vaccines and 25 kinds of 9mer peptides as HLA-A24 vaccines. To select vaccine candidates among the 57 different kinds of peptides, we measured both peptide-specific CTL precursors and peptide-specific IgG in the patients’ pre-vaccination PBMCs and sera respectively. Based on the results of pre-vaccination CTL and IgG responses, positive peptides (up to 4 kinds) were individually selected for each patient and patients were vaccinated subcutaneously with e...

Clinical and immunological evaluation of anti-apoptosis protein, survivin-derived peptide vaccine in phase I clinical study for patients with advanced or recurrent breast cancer

BackgroundWe previously reported that survivin-2B, a splicing variant of survivin, was expressed in various types of tumors and that survivin-2B peptide might serve as a potent immunogenic cancer vaccine. The objective of this study was to examine the toxicity of and to clinically and immunologically evaluate survivin-2B peptide in a phase I clinical study for patients with advanced or recurrent breast cancer.MethodsWe set up two protocols. In the first protocol, 10 patients were vaccinated with escalating doses (0.1–1.0 mg) of survivin-2B peptide alone 4 times every 2 weeks. In the second protocol, 4 patients were vaccinated with the peptide at a dose of 1.0 mg mixed with IFA 4 times every 2 weeks.ResultsIn the first protocol, no adverse events were observed during or after vaccination. In the second protocol, two patients had induration at the injection site. One patient had general malaise (grade 1), and another had general malaise (grade 1) and fever (grade 1). Peptide vaccination was well tolerated in all patients. In the first protocol, tumor marker levels increased in 8 patients, slightly decreased in 1 patient and were within the normal range during this clinical trial in 1 patient. With regard to tumor size, two patients were considered to have stable disease (SD). Immunologically, in 3 of the 10 patients (30%), an increase of the peptide-specific CTL frequency was detected. In the second protocol, an increase of the peptide-specific CTL frequency was detected in all 4 patients (100%), although there were no significant beneficial clinical responses. ELISPOT assay showed peptide-specific IFN-γ responses in 2 patients in whom the peptide-specific CTL frequency in tetramer staining also was increased in both protocols.ConclusionThis phase I clinical study revealed that survivin-2B peptide vaccination was well tolerated. The vaccination with survivin-2B peptide mixed with IFA increased the frequency of peptide-specific CTL more effectively than vaccination with the peptide alone, although neither vaccination could induce efficient clinical responses. Considering the above, the addition of another effectual adjuvant such as a cytokine, heat shock protein, etc. to the vaccination with survivin-2B peptide mixed with IFA might induce improved immunological and clinical responses.

IgE peptide-specific CTL inhibit IgE production: A transient IgE suppression model in wild-type and HLA-A2.1 transgenic mice

Effect of IgE peptide-specific CTL on IgE antibody production was studied in mouse models. CTL elicited in B6.A2Kb tg mice against a human IgE peptide nonamer, pWV, lysed human IgE-secreting U266 myeloma cells and inhibit IgE production by these cells. U266 transfected with mouse A2Kb transgene (U266-A2Kb) were optimally lysed by these CTL, because the α3 domain of A2Kb interacts well with the CD8 co-receptors. The CTL generated were more effective in inhibiting IgE production by U266-A2Kb cells than lysing these cells. IgE production by and progression of U266 myeloma were suppressed in B6.A2Kb tg mice rendered tolerant to these cells and vaccinated with pWV along with CpG. We also studied the CTL response elicited in wild-type mice by a mouse nonameric IgE peptide, PI-1, along with CpG. This treatment caused a transient suppression of the IgE response in mice previously sensitized to an antigen. In mice treated with this regimen repeatedly, the IgE response was fully recovered 20 days after each treatment. Notably, while IgE peptide/CpG-treated mice remained unresponsive to antigen challenge in vivo, antigen-specific IgE production can be elicited by antigen in cultured splenocytes from these mice. Moreover, IgE peptide/CpG also inhibited an on-going IgE response, including IgE production by bone marrow cells. Taken together, these observations indicate that a CTL-based IgE peptide vaccine targeting IgE-secreting B/plasma cells may be safely employed as a therapeutic approach for suppressing IgE production.

In Vivo Eradication of Human Clonigenic Acute Lymphoblastic Leukemic Cells Expressing the Wilms Tumor Protein, WT-1, by HLA Restricted WT-1 Specific T-Cells in NOD/SCID Mice Monitored by Bioluminescent Imaging.

WT-1 is expressed in 60–80% of acute leukemias, CML and high risk forms of MDS. Its expression has been hypothesized to be critical to the growth or survival of leukemic stem cells. Previously, alloreactive HLAA0201− T-cells recognizing a complex of WT-1 peptide and HLA A0201 were reported to prevent growth of leukemic HLA A0201+ CD34+ Ph+CML progenitor cells in NOD/ SCID mice (Transplantation, vol 75, No9, 2003). In this study, we have assessed the capacity of HLA-restricted, WT-1 peptide specific CTL (WT1-CTL) lacking alloreactivity to prevent the outgrowth of a human acute preB-lymphocytic leukemia (B-ALL)in NOD/SCID mice. This leukemia contained 65% of the blasts expressed WT-1 as determined by FACS analysis. For these studies the leukemic cells were transduced to express a luciferase reporter gene, permitting sequential monitoring of growth in vivo by bioluminescent imaging. WT-1 specific T-cells were generated from normal HLA A0201+ donor PBMC by in vitro sensitization with autologous dendritic cells loaded with the immunogenic HLA A0201 binding WT-1 peptide, RMFPNAPYL, and shown to be selectively cytotoxic against HLA A0201+WT-1+ leukemias and peptide loaded PHA blasts. T-cells from the same donor sensitized with autologous EBV BLCL and exhibiting HLA A0201 restricted EBV-specific cytotoxic activity served as controls. WT-1-CTL or EBV CTL were co-incubated in vitro with the WT-1+ HLA A0201+ BALL-LUC at a 4:1 effector target ratio for 7 hours at 37°C. Thereafter, separate groups of 5 NOD/SCID mice received intravenous infusions of cells from each of the co-cultures, at doses providing 12 × 106 WT1 CTL or EBVCTL and 3 × 106 BALL-LUC cells/mouse. A third group received 3×106 BALL-LUC alone. Leukemia growth was monitored at 2–3 day intervals from day 1–45 post infusion. In all 3 groups, BALL-LUC could be detected in the thorax by imaging at day 1. In mice treated with BALL-LUC alone or together with EBV-CTL, signal accumulation in the thorax increased steadily through 45 days of observation. By day 17, BALL-LUC were also detected throughout the head, abdomen and pelvis, and thereafter also increased until sacrifice at day 45. Autopsy confirmed presence of leukemic nodules in the lung and leukemic cells in blood, spleen and marrow as well as other organs. In contrast, in mice treated with WT1-CTL+ BALL LUC, signal intensity in lung decreased by day 4. In 4/5 of these mice, BALL-LUC could not be detected thereafter. In one mouse from this group, BALL-LUC were first detected in the head 31 days post infusion. At autopsy on day 45, this mouse had detectable BALL in the skull but in no other sites. WT-1 expression of residual leukemic cells is being analyzed. The other mice treated with WT-1 CTL had no detectable residual disease. These results suggest that clonogenic BALL cells express WT-1 and are susceptible to eradication in vivo by WT-1 peptide specific cytotoxic T-cells. The elimination of such clonogenic leukemic cells is sufficient to prevent subsequent development of leukemia.

80: High avidity cyclin E-derived peptide-specific CTL contribute to induction of remission after stem cell transplantation

80: High avidity cyclin E-derived peptide-specific CTL contribute to induction of remission after stem cell transplantation

CDK2-Specific CTLs Can Be Generated In Vivo from Donor Derived T Cells in HLA-A24+ Patients with Leukemia in Remission after Allogeneic Stem Cell Transplantation.

Aberrantly expressed self-antigens in leukemic cells serve as leukemia-associated-antigens (LAAs) of leukemia reactive T cells. Although such self-antigen-derived LAAs potentially induce high avidity CTLs in patients with leukemia, these CTLs usually do not persist due to apoptosis upon encountering leukemic cells. In allogeneic stem cell transplant (allo-SCT) recipients with leukemia, residual leukemic cell may sensitize donor-derived T cells by LAA in vivo and induce high avidity CTLs specific to leukemic cells. Cyclin-dependent kinase 2 (CDK2) is a cell cycle regulator protein that is aberrantly expressed in AML, MDS, ALL and MCL cells. We previously reported that CDK2-derived nonamer peptides (CDK2158: TYTHEVVTL, CDK2167: WYRQPEILL, CDK2178: KYYSTAVDI) avidly bound to HLA-A24 molecule to elicit each peptide-specific CTL from peripheral blood mononuclear cells (PBMCs) of healthy individuals (Blood. 106 (11): a3103. 2005). When we generated CDK2158-specific CD8+ T cells from an HLA identical sibling donor of a patient with AML by stimulating donor PBMCs with CDK2158-coated HLA-A24-transfected T2, CDK2158-specific CD8+ T cells preferentially killed the recipient AML cells which aberrantly expressed CDK2 proteins. The percentages of specific lysis in the 51Cr-release assay at an E/T ratio of 40 were 36.8% for AML cells and 24.8% for autologous PBMCs. To determine if CDK2-specific CD8+ T cells are present in PBMCs from allo-SCT patients, we studied 14 patients possessing HLA-A24 [6 patients with AML (2 AML-M0, 3 AML-M2, 1 AML with multilinage dysplasia), 1 with MDS, 1 with CML, 2 with ALL, 2 with MCL and 2 with RCC] using CDK2158/A24 and CDK2178/A24 multimers. Cryopreserved PBMCs obtained before and 4–73 months after allo-SCT were assayed for multimer staining. The source of graft was unrelated BM in 4, related BM in 2, related PBMC in 3 and CB in 5 patients. All CB and 1 BM graft were HLA mismatched. Seven patients were in complete remission (CR) at the time of SCT while 7 were in non-CR. Ten patients remained in CR 4–79 months after SCT. Small populations (0.13–0.77 % of lymphocyte) of CDK2158 and CDK2178-specific CTL were detectable in 5 patients (3 with AML-M2, 1 with CML and 1 with ALL) 10–73 months after SCT. All of the 5 remained in CR 11–79 months after SCT. Only one of them had active cGvHD at sampling. On the other hand, among 7 patients who did not show an increase in the proportion of neither CDK2158 nor CDK2178-specific CTLs, 2 patients with AML-M0 relapsed 4 and 5 months after SCT, respectively. Both of them had active cGvHD at sampling. CDK2-specific CTLs were also undetectable in 2 patients with RCC after allo-SCT. These data provide evidence for the first time that CDK2-specific CTLs can be induced from donor-derived T cells without peptide immunization possibly due to in vivo sentitization of donor T cells by residual leukemic cells. Vaccination with CDK2-derived peptides after allo-SCT may therefore be useful in inducing CDK2-specific CTLs and thereby preventing relapse of leukemia. [Display omitted]

Inhibition of Tumor Growth with Antiangiogenic Cancer Vaccine Using Epitope Peptides Derived from Human Vascular Endothelial Growth Factor Receptor 1

Antiangiogenic therapy is now considered to be one of promising approaches to treat various types of cancer. In this study, we examined the possibility of developing antiangiogenic cancer vaccine targeting vascular endothelial growth factor receptor 1 (VEGFR1) overexpressed on endothelial cells of newly formed vessels in the tumor. Epitope-candidate peptides were predicted from the amino acid sequence of VEGFR1 based on their theoretical binding affinities to the corresponding HLAs. The A2/Kb transgenic mice, which express the alpha1 and alpha2 domains of human HLA-A*0201, were immunized with the epitope candidates to examine their effects. We also examined whether these peptides could induce human CTLs specific to the target cells in vitro. The CTL responses in A2/Kb transgenic mice were induced with vaccination using identified epitope peptides restricted to HLA-A*0201. Peptide-specific CTL clones were also induced in vitro with these identified epitope peptides from peripheral blood mononuclear cells donated by healthy volunteers with HLA-A*0201. We established CTL clones in vitro from human peripheral blood mononuclear cells with HLA-A*2402 as well. These CTL clones were shown to have potent cytotoxicities in a HLA class I-restricted manner not only against peptide-pulsed target cells but also against target cells endogenously expressing VEGFR1. Furthermore, immunization of A2/Kb transgenic mice with identified epitope peptides restricted to HLA-A*0201 was associated with significant suppression of tumor-induced angiogenesis and tumor growth without showing apparent adverse effects. These results strongly suggest that VEGFR1 is a promising target for antiangiogenic cancer vaccine and warrants further clinical development of this strategy.

Ii-Key/HER-2/neu(776-790) hybrid peptides induce more effective immunological responses over the native peptide in lymphocyte cultures from patients with HER-2/neu+ 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.

CD1d-Restricted T Cells License B Cells to Generate Long-Lasting Cytotoxic Antitumor ImmunityIn vivo

Although resting B cells are known for being poorly immunogenic and for inducing T-cell tolerance, we have here attempted to test whether their immunogenicity could be enhanced by CD1d-restricted invariant T cells (iNKT) to a point where they could be used in cellular vaccines. We found that the addition of the iNKT ligand alpha-galactosylceramide (alphaGalCer) to peptide-loaded B cells overcame peptide-specific T-cell unresponsiveness and allowed for the generation of peptide-specific memory CTL immunity. This CTL was induced independently of CD4 T and natural killer cells but required iNKT and CD8 T cells. B cells directly primed CTL, and the alphaGalCer and the peptide must be presented on the same cell. Importantly, our B-cell-based vaccine is comparable in efficiency with dendritic cell-based vaccines, inducing similar CTL responses as well as providing an effective regimen for preventing and suppressing s.c. and metastatic tumors. Therefore, with the help of iNKT, peptide-pulsed B cells can establish long-lasting antitumor immunity and so show promise as the basis for an alternative cell-based vaccine.