First immunotherapeutic CAR-T cells against the immune checkpoint protein HLA-G.

BACKGROUND: CAR-T cells immunotherapy is a breakthrough in the treatment of hematological malignancies such as acute lymphoblastic leukemia (ALL) and B-cell malignancies. However, CAR-T therapies face major hurdles such as the lack of tumor-specific antigen (TSA), and immunosuppressive tumor microenvironment sometimes caused by the tumorous expression of immune checkpoints (ICPs) such as HLA-G. Indeed, HLA-G is remarkable because it is both a potent ICP and a TSA. HLA-G tumor expression causes immune escape by impairing innate and adaptive immune responses and by inducing a suppressive microenvironment. Yet, to date, no immunotherapy targets it. METHODS: We have developed two anti-HLA-G third-generation CARs based on new anti-HLA-G monoclonal antibodies. RESULTS: Anti-HLA-G CAR-T cells were specific for immunosuppressive HLA-G isoforms. HLA-G-activated CAR-T cells polarized toward T helper 1, and became cytotoxic against HLA-G+ tumor cells. In vivo, anti-HLA-G CAR-T cells were able to control and eliminate HLA-G+ tumor cells. The interaction of tumor-HLA-G with interleukin (IL)T2-expressing T cells is known to result in effector T cell functional inhibition, but anti-HLA-G CAR-T cells were insensitive to this inhibition and still exerted their function even when expressing ILT2. Lastly, we show that anti-HLA-G CAR-T cells differentiated into long-term memory effector cells, and seemed not to lose function even after repeated stimulation by HLA-G-expressing tumor cells. CONCLUSION: We report for the first time that HLA-G, which is both a TSA and an ICP, constitutes a valid target for CAR-T cell therapy to specifically target and eliminate both tumor cells and HLA-G+ suppressive cells.

HLA-G Neo-Expression on Tumors.

HLA-G is known to modulate the immune system activity in tissues where physiological immune-tolerance is necessary (i.e., maternal-fetal interface, thymus, and cornea). However, the frequent neo-expression of HLA-G in many cancer types has been previously and extensively described and is correlated with a bad prognosis. Despite being an MHC class I molecule, HLA-G is highly present in tumor context and shows unique characteristics of tissue restriction of a Tumor Associated Antigen (TAA), and potent immunosuppressive activity of an Immune CheckPoint (ICP). Consequently, HLA-G appears to be an excellent molecular target for immunotherapy. Although the relevance of HLA-G in cancer incidence and development has been proven in numerous tumors, its neo-expression pattern is still difficult to determine. Indeed, the estimation of HLA-G's actual expression in tumor tissue is limited, particularly concerning the presence and percentage of the new non-canonical isoforms, for which detection antibodies are scarce or inexistent. Here, we summarize the current knowledge about HLA-G neo-expression and implication in various tumor types, pointing out the need for the development of new tools to analyze in-depth the HLA-G neo-expression patterns, opening the way for the generation of new monoclonal antibodies and cell-based immunotherapies.

CD28/4-1BB CD123 CAR T cells in blastic plasmacytoid dendritic cell neoplasm.

Blastic plasmacytoid dendritic cell neoplasm (BPDCN) is associated with a remarkably poor prognosis and with no treatment consensus. The identification of relevant therapeutic targets is challenging. Here, we investigated the immune functions, antileukemia efficacy and safety of CD28/4-1BB CAR T cells targeting CD123 the interleukin (IL)-3 receptor alpha chain which is overexpressed on BPDCN. We demonstrated that both retroviral and lentiviral engineering CD28/4-1BB CD123 CAR T cells exhibit effector functions against BPDCN cells through CD123 antigen recognition and that they efficiently kill BPDCN cell lines and BPDCN-derived PDX cells. In vivo, CD28/4-1BB CD123 CAR T-cell therapy displayed strong efficacy by promoting a decrease of BPDCN blast burden. Furthermore we showed that T cells from BPDCN patient transduced with CD28/4-1BB CD123 CAR successfully eliminate autologous BPDCN blasts in vitro. Finally, we demonstrated in humanized mouse models that these effector CAR T cells exert low or no cytotoxicity against various subsets of normal cells with low CD123 expression, indicating a potentially low on-target/off-tumor toxicity effect. Collectively, our data support the further evaluation for clinical assessment of CD28/4-1BB CD123 CAR T cells in BPDCN neoplasm.

Inhibition of iNKT Cells by the HLA-G-ILT2 Checkpoint and Poor Stimulation by HLA-G-Expressing Tolerogenic DC.

Invariant Natural Killer T (iNKT) cells are a small and distinct population of T cells crucial in immunomodulation. After activation by alpha-GalactosylCeramide (αGC), an exogenic glycolipid antigen, iNKT cells can rapidly release cytokines to enhance specific anti-tumor activity. Several human clinical trials on iNKT cell-based anti-cancer are ongoing, however results are not as striking as in murine models. Given that iNKT-based immunotherapies are dependent mainly on antigen-presenting cells (APC), a human tolerogenic molecule with no murine homolog, such as Human Leucocyte Antigen G (HLA-G), could contribute to this discrepancy. HLA-G is a well-known immune checkpoint molecule involved in fetal-maternal tolerance and in tumor immune escape. HLA-G exerts its immunomodulatory functions through the interaction with immune inhibitory receptors such as ILT2, differentially expressed on immune cell subsets. We hypothesized that HLA-G might inhibit iNKT function directly or by inducing tolerogenic APC leading to iNKT cell anergy, which could impact the results of current clinical trials. Using an ILT2-transduced murine iNKT cell line and human iNKT cells, we demonstrate that iNKT cells are sensitive to HLA-G, which inhibits their cytokine secretion. Furthermore, human HLA-G+ dendritic cells, called DC-10, failed at inducing iNKT cell activation compared to their autologous HLA-G‒ DCs counterparts. Our data show for the first time that the HLA-G/ILT2 ICP is involved in iNKT cell function modulation.

A First-in-Human Phase I Study of INVAC-1, an Optimized Human Telomerase DNA Vaccine in Patients with Advanced Solid Tumors.

PURPOSE: Human telomerase reverse transcriptase (hTERT) is highly expressed in >85% of human tumors and is thus considered as a good tumor-associated antigen candidate for vaccine development. We conducted a phase I study to investigate the safety, tolerability, clinical response, and immunogenicity of INVAC-1, a DNA plasmid encoding a modified hTERT protein in patients with relapsed or refractory solid tumors. PATIENTS AND METHODS: INVAC-1 was either administered by intradermal route followed by electroporation or by Tropis, a needle-free injection system. Safety and tolerability were monitored by clinical and laboratory assessments. Progression-free survival and overall survival were reported using Kaplan-Meier survival analysis. Immunogenicity was studied by ELISpot, Luminex, and Flow Cytometry. RESULTS: Twenty-six patients were treated with INVAC-1 administered at three dose levels (100, 400, and 800 µg). Vaccination was well tolerated and no dose-limiting toxicity was reported. One treatment-related grade 3 SAE was reported. Fifty-eight percent of patients experienced disease stabilization. PFS was 2.7 months, median OS was 15 months, and 1-year survival was reached for 65% of patients. INVAC-1 vaccination stimulated specific anti-hTERT CD4 T-cell response as well as cytotoxic CD8 T-cell response. No evidence of peripheral vaccine-induced immunosuppression was observed. CONCLUSIONS: INVAC-1 vaccination was safe, well tolerated, and immunogenic when administered intradermally at the three tested doses in patients with relapsed or refractory cancers. Disease stabilization was observed for the majority of patients (58%) during the treatment period and beyond.See related commentary by Slingluff Jr, p. 529.

A Modified mRNA Vaccine Targeting Immunodominant NS Epitopes Protects Against Dengue Virus Infection in HLA Class I Transgenic Mice.

Dengue virus (DENV) induces strong T and B cell responses upon infection. Hence, it is difficult to determine the contribution of cell-mediated immunity alone in the long lasting protection against DENV infection and disease. Numerous CD4+ and CD8+ T cell epitopes have been identified, mainly in the non-structural proteins of DENV. Taking into account the immunogenicity and peptide sequence conservation among the different DENV serotypes, a minimal DENV antigen, called DENV1-NS, has been designed. This antigen is enriched in conserved and highly antigenic epitopes located in the NS3, NS4B, and NS5 regions of DENV1. To evaluate the ability of the DENV1-NS poly-epitope to express the antigenic peptides in the context of different HLA class I molecules, we established its in vivo immunogenicity by measuring, after DNA immunization and electroporation, the activation of DENV-specific CD8 T cells in transgenic mice expressing the human HLA-A*0201, -A*2402, -B*0702, and -B*3502 class I alleles. We then engineered a lipid nanoparticle (LNP) encapsulated modified mRNA vaccine encoding DENV1-NS and tested immunogenicity and protection in these human HLA class I transgenic mice, after transient blockade of the interferon (IFN) type I receptor. Significant protection was observed, after two injections of the mRNA vaccine. Collectively, these data strongly support the development of T cell-based vaccines targeting immunodominant T cell epitopes that generate potent virus-specific T cell responses conferring immunity against DENV infection.

A DNA telomerase vaccine for canine cancer immunotherapy.

Telomerase reverse transcriptase (TERT) is highly expressed in more than 90% of canine cancer cells and low to absent in normal cells. Given that immune tolerance to telomerase is easily broken both naturally and experimentally, telomerase is an attractive tumor associated antigen for cancer immunotherapy. Indeed, therapeutic trials using human telomerase peptides have been performed. We have developed an immunogenic yet catalytically inactive human telomerase DNA construct that is in clinical trials with patients presenting solid tumors. Paralleling this human construct, we have developed a canine telomerase DNA vaccine, called pDUV5. When administered intradermally to mice combined with electrogene transfer, pDUV5 induced canine TERT specific cytotoxic T-cells as measured by IFN-γ ELISpot assay. Intradermal vaccination of healthy dogs with 400 µg of pDUV5 generated strong, broad and long lasting TERT specific cellular immune responses. In vitro immunization with cTERT peptides revealed the maintenance of cTERT specific T-cells in PBMCs from tumor bearing dogs showing that this repertoire was not depleted. This study highlights the potential of pDUV5 as a cancer vaccine and supports its evaluation for the treatment of spontaneous canine tumors.

Strong antigen-specific T-cell immunity induced by a recombinant human TERT measles virus vaccine and amplified by a DNA/viral vector prime boost in IFNAR/CD46 mice.

Cancer immunotherapy is seeing an increasing focus on vaccination with tumor-associated antigens (TAAs). Human telomerase (hTERT) is a TAA expressed by most tumors to overcome telomere shortening. Tolerance to hTERT can be easily broken both naturally and experimentally and hTERT DNA vaccine candidates have been introduced in clinical trials. DNA prime/boost strategies have been widely developed to immunize efficiently against infectious diseases. We explored the use of a recombinant measles virus (MV) hTERT vector to boost DNA priming as recombinant live attenuated measles virus has an impressive safety and efficacy record. Here, we show that a MV-TERT vector can rapidly and strongly boost DNA hTERT priming in MV susceptible IFNAR/CD46 mouse models. The cellular immune responses were Th1 polarized. No humoral responses were elicited. The 4 kb hTERT transgene did not impact MV replication or induction of cell-mediated responses. These findings validate the MV-TERT vector to boost cell-mediated responses following DNA priming in humans.

Anticancer DNA vaccine based on human telomerase reverse transcriptase generates a strong and specific T cell immune response.

Human telomerase reverse transcriptase (hTERT) is overexpressed in more than 85% of human cancers regardless of their cellular origin. As immunological tolerance to hTERT can be overcome not only spontaneously but also by vaccination, it represents a relevant universal tumor associated antigen (TAA). Indeed, hTERT specific cytotoxic T lymphocyte (CTL) precursors are present within the peripheral T-cell repertoire. Consequently, hTERT vaccine represents an attractive candidate for antitumor immunotherapy. Here, an optimized DNA plasmid encoding an inactivated form of hTERT, named INVAC-1, was designed in order to trigger cellular immunity against tumors. Intradermal injection of INVAC-1 followed by electrogene transfer (EGT) in a variety of mouse models elicited broad hTERT specific cellular immune responses including high CD4+ Th1 effector and memory CD8+ T­cells. Furthermore, therapeutic INVAC­1 immunization in a HLA-A2 spontaneous and aggressive mouse sarcoma model slows tumor growth and increases survival rate of 50% of tumor-bearing mice. These results emphasize that INVAC-1 based immunotherapy represents a relevant cancer vaccine candidate.

Optimization of a gene electrotransfer procedure for efficient intradermal immunization with an hTERT-based DNA vaccine in mice.

DNA vaccination consists in administering an antigen-encoding plasmid in order to trigger a specific immune response. This specific vaccine strategy is of particular interest to fight against various infectious diseases and cancer. Gene electrotransfer is the most efficient and safest non-viral gene transfer procedure and specific electrical parameters have been developed for several target tissues. Here, a gene electrotransfer protocol into the skin has been optimized in mice for efficient intradermal immunization against the well-known telomerase tumor antigen. First, the luciferase reporter gene was used to evaluate gene electrotransfer efficiency into the skin as a function of the electrical parameters and electrodes, either non-invasive or invasive. In a second time, these parameters were tested for their potency to generate specific cellular CD8 immune responses against telomerase epitopes. These CD8 T-cells were fully functional as they secreted IFNγ and were endowed with specific cytotoxic activity towards target cells. This simple and optimized procedure for efficient gene electrotransfer into the skin using the telomerase antigen is to be used in cancer patients for the phase 1 clinical evaluation of a therapeutic cancer DNA vaccine called INVAC-1.

Universal cancer peptide-based therapeutic vaccine breaks tolerance against telomerase and eradicates established tumor.

PURPOSE: To evaluate CD4(+) helper functions and antitumor effect of promiscuous universal cancer peptides (UCP) derived from telomerase reverse transcriptase (TERT). EXPERIMENTAL DESIGN: To evaluate the widespread immunogenicity of UCPs in humans, spontaneous T-cell responses against UCPs were measured in various types of cancers using T-cell proliferation and ELISPOT assays. The humanized HLA-DRB1*0101/HLA-A*0201 transgenic mice were used to study the CD4(+) helper effects of UCPs on antitumor CTL responses. UCP-based antitumor therapeutic vaccine was evaluated using HLA-A*0201-positive B16 melanoma that express TERT. RESULTS: The presence of a high number of UCP-specific CD4(+) T cells was found in the blood of patients with various types of cancer. These UCP-specific T cells mainly produce IFN-γ and TNF-α. In HLA transgenic mice, UCP vaccinations induced high avidity CD4(+) T(H)1 cells and activated dendritic cells that produced interleukin-12. UCP-based vaccination breaks self-tolerance against TERT and enhances primary and memory CTL responses. Furthermore, the use of UCP strongly improves the efficacy of therapeutic vaccination against established B16-HLA-A*0201 melanoma and promotes tumor infiltration by TERT-specific CD8(+) T cells. CONCLUSIONS: Our results showed that UCP-based vaccinations strongly stimulate antitumor immune responses and could be used to design efficient immunotherapies in multiple types of cancers.

Analysis of spontaneous tumor-specific CD4 T-cell immunity in lung cancer using promiscuous HLA-DR telomerase-derived epitopes: potential synergistic effect with chemotherapy response.

PURPOSE: To investigate the presence and impact of spontaneous telomerase-specific CD4 T-cell responses in cancer patients. EXPERIMENTAL DESIGN: A multistep approach was used to design novel pan-HLA-DR-restricted peptides from telomerase. T-cell clones isolated from cancer patients were used to characterize the polarization of telomerase-specific CD4 response. The presence of spontaneous CD4 T-cell response against telomerase was monitored in 84 metastatic non-small cell lung cancer (NSCLC) patients before first-line chemotherapy (CT) using IFN-γ ELISPOT assay. Then we analyzed the impact of the pretherapeutic telomerase-specific CD4 T immunity on clinical outcome in patients according to their respective response to CT. RESULTS: We described four novel telomerase-derived CD4 epitopes referred as universal cancer peptides (UCP) that effectively bind to most commonly found human MHC class II alleles. UCP-specific CD4 T-cell repertoire is present in human and UCP-specific CD4 T-cell clones generated from cancer patients exhibited high avidity and are Th1 polarized. Significant frequency (38%) of naturally occurring UCP-specific T-cell responses were detected before CT in advanced NSCLC but not in healthy volunteers. This response was shown to significantly increase overall survival (OS) of patients responding to CT (Median OS: 53 vs. 40 weeks, P = 0.034). CONCLUSIONS: These results show for the first time a potential synergistic effect of telomerase-specific CD4 T-cell response with CT response in NSCLC and underline the potential role of tumor-specific CD4 T-cell response on the efficiency of conventional anticancer therapy.

Immunogenicity of a recombinant lentiviral vector carrying human telomerase tumor antigen in HLA-B*0702 transgenic mice.

Over expression of telomerase represents a hallmark of cancer cells and the induction of T cell immunity against this universal tumor antigen have gained promising interest for anticancer immunotherapy. In this study we evaluated a recombinant lentiviral vector expressing the human telomerase reverse transcriptase (lv-hTERT) vaccination in the humanized HLA-B*0702 transgenic (HLA-B7 Tg) mice. A single lv-hTERT vector immunization induces potent and broad HLA-B7-restricted CTL responses against hTERT. Unlike conventional hTERT peptide or DNA immunization, the lv-hTERT vector triggers high and sustained IFN-gamma producing CD8(+) T cell responses in HLA-B7 Tg mice. The avidity and in vivo cytotoxicity of CD8(+) T cells were stronger in lv-hTERT vector-immunized mice than in hTERT peptide or DNA vaccinated groups. The study also showed that the use of prime-boost vaccination drastically improved the magnitude and strength of lentivector-primed CD8(+) T cells. Our data indicated that lentiviral delivery of hTERT is suitable for enhancing cellular immunity against hTERT and offers a promising alternative for telomerase-based cancer vaccine.

Targeting human telomerase reverse transcriptase with recombinant lentivector is highly effective to stimulate antitumor CD8 T-cell immunity in vivo.

The success of active immunotherapy is based on the vaccine's ability to overcome immune tolerance through recalibrating the immune system so that it is able to recognize tumor antigens as foreign rather than self. In this study, we used a lentiviral vector system to target human telomerase reverse transcriptase (lv-hTERT), a widely expressed tumor antigen. Immunization of HLA-A*0201 transgenic HHD mice with recombinant lv-hTERT vector induces potent and diversified cytotoxic T lymphocyte responses that recognize in vitro murine tumor cells, which overexpress telomerase. Compared with peptide-based vaccinations, the lv-hTERT vector triggers better and more sustained CD8(+) T-cell response against self/TERT epitope in vivo. The study found that the additional use of a heterologous boosted vaccination drastically improves self/TERT-specific CD8 responses in lv-hTERT primed mice. Both primary and long-lasting self/TERT-specific CD8(+) T-cell responses induced with Iv-hTERT vector required the presence of CD4 T cells in vivo. This lv-hTERT-based active immunotherapy efficiently inhibits the growth of telomerase expressing tumors (B16/HLA-A2.1 murine melanoma) in HHD mice. These data show that targeting hTERT with lentivector is highly effective in stimulating a broad range of CD8 T-cell immunity that can be exploited for cancer immunotherapy.

Design of a HIV-1-derived HLA-B07.02-restricted polyepitope construct.

OBJECTIVE: To design a vaccine construct containing various but conserved HIV-1-derived epitopes and generating broad CD8 T cell responses. METHODS: HLA-B7 transgenic H-2KD KO transgenic mice were used to identify potential new HLA-B07.02-restricted HIV-1-derived epitopes. Immunological recognition of these epitopes was confirmed by IFN-gamma ELISpot assays with PBMCs from HLA-B*0702 HIV-1-infected individuals. For these peptides as well as others previously identified, the capacity to induce cross-reactive responses against their frequent allelic variants was evaluated in the mouse model. A set of epitopes inducing strong T cell responses against various and conserved regions of HIV-1 was selected. A DNA vaccine was designed to express them as a unique antigen with or without a three amino acid ARY extension flanking each epitope. The spectrum of CD8 T responses generated by polyepitope constructs was tested in HLA-B7 transgenic mice. RESULTS: Five new epitopes were identified in accessory and regulatory HIV-1 proteins. Twelve HLA-B07.02-restricted epitopes were selected on the basis of their structural conservation and cross-reactive immunogenicity. The ARY N-terminal extension flanking each epitope markedly increases their affinity for TAP and the use of this flanking extension in polyepitope vaccine has a sizable advantage to induce CD8 T cell cytotoxic responses in mice following DNA immunization. CONCLUSION: The HLA-B7 mouse model allows to rapidly identify various HIV-1-derived epitopes of vaccine interest. Grouped in a polyepitope construct designed to increase their processing, this vaccine may be suitable for inducing multiple and relevant HIV-1-specific CTL responses in humans.

Presentation of telomerase reverse transcriptase, a self-tumor antigen, is down-regulated by histone deacetylase inhibition.

Histone deacetylases (HDAC) modify the architecture of chromatin, leading to decreased gene expression, an effect that is reversed by HDAC inhibition. The balance between deacetylation and acetylation is central to many biological events including the regulation of cell proliferation and cancer but also the differentiation of immune T cells. The effects of HDAC inhibition on the interaction between antitumor effector T cells and tumor cells are not known. Here, we studied presentation of a universal self-tumor antigen, telomerase reverse transcriptase, in human tumor cells during HDAC inhibition. We found that HDAC inhibition with trichostatin A was associated with a decreased presentation and diminished killing of tumor cells by CTLs. Using gene array analysis, we found that HDAC inhibition resulted in a decrease of genes coding for proteasome catalytic proteins and for tapasin, an endoplasmic reticulum resident protein involved in the MHC class I pathway of endogenous antigen presentation. Our findings indicate that epigenetic changes in tumor cells decrease self-tumor antigen presentation and contribute to reduced recognition and killing of tumor cells by cytotoxic T lymphocytes. This mechanism could contribute to tumor escape from immune surveillance.

Immunogenicity and tolerance following HIV-1/HBV plant-based oral vaccine administration.

Transgenic tobacco plants expressing a HIV-1 polyepitope associated with hepatitis B (HBV) virus-like particles (VLPs) were previously described. It is demonstrated here that oral administration of these transgenic plants to humanized HSB mice to boost DNA-priming can elicit anti-HIV-1 specific CD8+ T cell activation detectable in mesenteric lymph nodes. Nevertheless, a significant regulatory T cell activation was induced in vivo by the vaccination protocols. The balance between tolerance and immunogenicity remains the main concern in the proof of concept of plant-based vaccine.

B subunit of Shiga toxin-based vaccines synergize with alpha-galactosylceramide to break tolerance against self antigen and elicit antiviral immunity.

The nontoxic B subunit of Shiga toxin (STxB) targets in vivo Ag to dendritic cells that preferentially express the glycolipid Gb(3) receptor. After administration of STxB chemically coupled to OVA (STxB-OVA) or E7, a polypeptide derived from HPV, in mice, we showed that the addition of alpha-galactosylceramide (alpha-GalCer) resulted in a dramatic improvement of the STxB Ag delivery system, as reflected by the more powerful and longer lasting CD8(+) T cell response observed even at very low dose of immunogen (50 ng). This synergy was not found with other adjuvants (CpG, poly(I:C), IFN-alpha) also known to promote dendritic cell maturation. With respect to the possible mechanism explaining this synergy, mice immunized with alpha-GalCer presented in vivo the OVA(257-264)/K(b) complex more significantly and for longer period than mice vaccinated with STxB alone or mixed with other adjuvants. To test whether this vaccine could break tolerance against self Ag, OVA transgenic mice were immunized with STxB-OVA alone or mixed with alpha-GalCer. Although no CTL induction was observed after immunization of OVA transgenic mice with STxB-OVA, tetramer assay clearly detected specific anti-OVA CD8(+) T cells in 8 of 11 mice immunized with STxB-OVA combined with alpha-GalCer. In addition, vaccination with STxB-OVA and alpha-GalCer conferred strong protection against a challenge with vaccinia virus encoding OVA with virus titers in the ovaries reduced by 5 log compared with nonimmunized mice. STxB combined with alpha-GalCer therefore appears as a promising vaccine strategy to more successfully establish protective CD8(+) T cell memory against intracellular pathogens and tumors.

Immunogenic HLA-B7-restricted peptides of hTRT.

Telomerase reverse transcriptase (TRT) is the first bona fide common tumor antigen. While several 9mer peptides of the human TRT have been identified for HLA-A2, little information exists on peptides for the remaining HLA types. Here, we used a multi-step approach to select and characterize a panel of HLA-B7 9mer peptides as candidate immunogens. In sequence, we used algorithm-based predictions, in vivo immunization of HLA-B7 transgenic (Tg) mice, in vitro immunization of human blood lymphocytes from two normal donors and two cancer patients, in vivo processing in HLA-B7 Tg mice and HLA-B7 supertype binding. We found a correlation between the in vivo immunogenicity and the actual HLA-B7 binding avidity of the seven predicted peptides. Furthermore, endogenous processing correlated with in vitro immunogenicity in human PBMC and HLA-B7 supertype binding. Peptide (1123)LPSDFKTIL(1131) (p1123) with the wider spectrum of supertype binding displayed the highest immunogenicity overall and was endogenously processed in several human lymphoblastoid cells. Since no single step of the screening/selection process could substitute for the whole approach, we conclude that the identification of MHC class I-restricted peptides for potential vaccination of cancer patients remains, by and large, an empirical process.

Immunogenic HLA-B*0702-restricted epitopes derived from human telomerase reverse transcriptase that elicit antitumor cytotoxic T-cell responses.

PURPOSE: The human telomerase reverse transcriptase (hTERT) is considered as a potential target for cancer immunotherapy because it is preferentially expressed in tumor cells. To increase the applicability of hTERT-based immunotherapy, we set out to identify CTL epitopes in hTERT restricted by HLA-B*0702 molecule, a common MHC class I allele. EXPERIMENTAL DESIGN: HLA-B*0702-restricted peptides from hTERT were selected by using a method of epitope prediction and tested for their immunogenicity in human (in vitro) and HLA-B*0702 transgenic mice (in vivo). RESULTS: All the six hTERT peptides that were predicted to bind to HLA-B*0702 molecule were found to induce primary human CTL responses in vitro. The peptide-specific CD8+ CTL lines were tested against various hTERT+ tumor cells. Although differences were observed according to the tumor origin, only three CTL lines specific for p277, p342, and p351 peptides exhibited cytotoxicity against tumor cells in a HLA-B*0702-restricted manner. In addition, this cytotoxicity was inhibited by the addition of peptide-loaded cold target cells and indicated that these epitopes are naturally processed and presented on the tumor cells. Further, in vivo studies using humanized HLA-B*0702 transgenic mice showed that all the candidate peptides were able to induce CTL responses after peptide immunization. Furthermore, vaccination with a plasmid DNA encoding full-length hTERT elicited peptide-specific CTL responses, indicating that these epitopes are efficiently processed in vivo. CONCLUSIONS: Together with previously reported hTERT epitopes, the identification of new CTL epitopes presented by HLA-B*0702 increases the applicability of hTERT-based immunotherapy to treating cancer.