Ikuei Nukaya

Identification of HLA-A24 epitope peptides of carcinoembryonic antigen which induce tumor-reactive cytotoxic T lymphocyte

Carcinoembryonic antigen (CEA), which is expressed in several cancer types, is a potential target for specific immunotherapy. HLA-A24 is the most frequent allele among Japanese and is also frequently present in Asians and Caucasians. We tested CEA-encoded HLA-A24 binding peptides for their capacity to elicit anti-tumor cytotoxic T lymphocytes (CTL) in vitro. For this purpose, we used CD8+ T lymphocytes from peripheral blood mononuclear cells (PBMC) of a healthy donor and autologous peptide-pulsed dendritic cells as antigen-presenting cells. This approach enabled us to identify 2 peptides, QYSWFVNGTF and TYACFVSNL, which were capable of eliciting CTL lines that lysed tumor cells expressing HLA-A24 and CEA. The cytotoxicity to tumor cells by the CTL lines was antigen-specific since it was inhibited by peptide-pulsed cold target cells as well as by anti-class I major histocompatibility complex (MHC) and anti-CD3 monoclonal antibodies (MAbs). The antigen specificity of the 2 CTL lines was examined using several tumor cell lines of various origins and for their peptide-dose responses. The identification of these novel CEA epitopes for CTL offers the opportunity to design and develop epitope-based immunotherapeutic approaches for treating HLA-A24+ patients with tumors that express CEA.

Immunotherapy of solid cancer using dendritic cells pulsed with the HLA-A24-restricted peptide of carcinoembryonic antigen.

Abstract. Carcinoembryonic antigen (CEA), an oncofetal glycoprotein overexpressed in most gastrointestinal and lung cancers, is a candidate molecule for cancer immunotherapy. Recently, a CEA-derived 9-mer peptide, CEA652 (TYACFVSNL), has been identified as the epitope of cytotoxic T lymphocytes restricted with human leukocyte antigen (HLA)-A24, which is present in 60% of the Japanese population and in some Caucasians. The authors performed a clinical study of a vaccine using autologous dendritic cells (DCs) pulsed with CEA652 and adjuvant cytokines, natural human interferon alpha (nhuIFN-α), and natural human tumor necrosis factor alpha (nhuTNF-α), for the treatment of patients with CEA-expressing advanced metastatic malignancies. Ten HLA-A24 patients with advanced digestive tract or lung cancer were enrolled in the study to assess toxicity, tolerability and immune responses to the vaccine. DCs were generated from plastic adherent monocytes of granulocyte colony-stimulating factor (G-CSF)-mobilized peripheral blood mononuclear cells (PBMCs) in the presence of granulocyte/macrophage colony-stimulating factor (GM-CSF) and interleukin 4 (IL-4). Generated DCs showing an immature phenotype were loaded with CEA652 and injected into patients intradermally and subcutaneously with 50% of the dose administered by each route every 2 weeks for a total of ten vaccinations. The total dose of administered DCs ranged from 2.7×107 cells to 1.6×108 cells. Adjuvant cytokines, i.e., 1×106 U/body of nhuIFN-α and nhuTNF-α, were administered to patients twice a week during the vaccination period. No severe toxicity directly attributable to the treatment was observed, and the vaccine was well tolerated. In the delayed-type hypersensitivity (DTH) skin test, two patients showed a positive skin response to peptide-pulsed DCs after vaccination, although none of the patients tested positive prior to vaccination. In the two patients who showed a positive skin response disease remained stable for 6 and 9 months respectively. These results suggest that active immunization using DCs pulsed with CEA652 peptide in combination with the administration of adjuvant cytokines is a safe and feasible treatment procedure.

Clinical response in Japanese metastatic melanoma patients treated with peptide cocktail-pulsed dendritic cells

BackgroundMetastatic, chemotherapy-resistant melanoma is an intractable cancer with a very poor prognosis. As to immunotherapy targeting metastatic melanoma, HLA-A2+ patients were mainly enrolled in the study in Western countries. However, HLA-A24+ melanoma patients-oriented immunotherapy has not been fully investigated. In the present study, we investigated the effect of dendritic cell (DC)-based immunotherapy on metastatic melanoma patients with HLA-A2 or A24 genotype.MethodsNine cases of metastatic melanoma were enrolled into a phase I study of monocyte-derived dendritic cell (DC)-based immunotherapy. HLA-genotype analysis revealed 4 cases of HLA-A*0201, 1 of A*0206 and 4 of A*2402. Enriched monocytes were obtained using OptiPrep™ from leukapheresis products, and then incubated with GM-CSF and IL-4 in a closed serum-free system. After pulsing with a cocktail of 5 melanoma-associated synthetic peptides (gp100, tyrosinase, MAGE-2, MAGE-3 and MART-1 or MAGE-1) restricted to HLA-A2 or A24 and KLH, cells were cryopreserved until used. Finally, thawed DCs were washed and injected subcutaneously (s.c.) into the inguinal region in a dose-escalation manner.ResultsThe mean percentage of DCs rated as lin-HLA-DR+ in melanoma patients was 46.4 ± 15.6 %. Most of DCs expressed high level of co-stimulatory molecules and type1 phenotype (CD11c+HLA-DR+), while a moderate number of mature DCs with CD83 and CCR7 positive were contained in DC products. DC injections were well tolerated except for transient liver dysfunction (elevation of transaminases, Grade I-II). All 6 evaluable cases except for early PD showed positive immunological responses to more than 2 melanoma peptides in an ELISPOT assay. Two representative responders demonstrated strong HLA-class I protein expression in the tumor and very high scores of ELISPOT that might correlate to the regression of metastatic tumors. Clinical response through DC injections was as follows : 1CR, 1 PR, 1SD and 6 PD. All 59 DC injections in the phase I study were tolerable in terms of safety, however, the maximal tolerable dose of DCs was not determined.ConclusionsThese results suggested that peptide cocktail-treated DC-based immunotherapy had the potential for utilizing as one of therapeutic tools against metastatic melanoma in Japan.

Adoptive Transfer of MAGE-A4 T-cell Receptor Gene-Transduced Lymphocytes in Patients with Recurrent Esophageal Cancer

Purpose: Preparative lymphodepletion, the temporal ablation of the immune system, has been reported to promote persistence of transferred cells along with increased rates of tumor regression in patients treated with adoptive T-cell therapy. However, it remains unclear whether lymphodepletion is indispensable for immunotherapy with T-cell receptor (TCR) gene–engineered T cells. Experimental Design: We conducted a first-in-man clinical trial of TCR gene-transduced T-cell transfer in patients with recurrent MAGE-A4–expressing esophageal cancer. The patients were given sequential MAGE-A4 peptide vaccinations. The regimen included neither lymphocyte-depleting conditioning nor administration of IL2. Ten patients, divided into 3 dose cohorts, received T-cell transfer. Results: TCR-transduced cells were detected in the peripheral blood for 1 month at levels proportional to the dose administered, and in 5 patients they persisted for more than 5 months. The persisting cells maintained ex vivo antigen-specific tumor reactivity. Despite the long persistence of the transferred T cells, 7 patients exhibited tumor progression within 2 months after the treatment. Three patients who had minimal tumor lesions at baseline survived for more than 27 months. Conclusions: These results suggest that TCR-engineered T cells created by relatively short-duration in vitro culture of polyclonal lymphocytes in peripheral blood retained the capacity to survive in a host. The discordance between T-cell survival and tumor regression suggests that multiple mechanisms underlie the benefits of preparative lymphodepletion in adoptive T-cell therapy. Clin Cancer Res; 21(10); 2268–77. ©2015 AACR.

T-cell receptor gene therapy targeting melanoma-associated antigen-A4 inhibits human tumor growth in non-obese diabetic/SCID/γcnull mice

Adoptive cell therapy with lymphocytes that have been genetically engineered to express tumor‐reactive T‐cell receptors (TCR) is a promising approach for cancer immunotherapy. We have been exploring the development of TCR gene therapy targeting cancer/testis antigens, including melanoma‐associated antigen (MAGE) family antigens, that are ideal targets for adoptive T‐cell therapy. The efficacy of TCR gene therapy targeting MAGE family antigens, however, has not yet been evaluated in vivo. Here, we demonstrate the in vivo antitumor activity in immunodeficient non‐obese diabetic/SCID/γcnull (NOG) mice of human lymphocytes genetically engineered to express TCR specific for the MAGE‐A4 antigen. Polyclonal T cells derived from human peripheral blood mononuclear cells were transduced with the αβ TCR genes specific for MAGE‐A4, then adoptively transferred into NOG mice inoculated with MAGE‐A4 expressing human tumor cell lines. The transferred T cells maintained their effector function in vivo, infiltrated into tumors, and inhibited tumor growth in an antigen‐specific manner. The combination of adoptive cell therapy with antigen peptide vaccination enhanced antitumor activity, with improved multifunctionality of the transferred cells. These data suggest that TCR gene therapy with MAGE‐A4‐specific TCR is a promising strategy to treat patients with MAGE‐A4‐expressing tumors; in addition, the acquisition of multifunctionality in vivo is an important factor to predict the quality of the T‐cell response during adoptive therapy with human lymphocytes. (Cancer Sci 2012; 103: 17–25)

An Efficient Large-Scale Retroviral Transduction Method Involving Preloading the Vector into a RetroNectin-Coated Bag with Low-Temperature Shaking

In retroviral vector-mediated gene transfer, transduction efficiency can be hampered by inhibitory molecules derived from the culture fluid of virus producer cell lines. To remove these inhibitory molecules to enable better gene transduction, we had previously developed a transduction method using a fibronectin fragment-coated vessel (i.e., the RetroNectin-bound virus transduction method). In the present study, we developed a method that combined RetroNectin-bound virus transduction with low-temperature shaking and applied this method in manufacturing autologous retroviral-engineered T cells for adoptive transfer gene therapy in a large-scale closed system. Retroviral vector was preloaded into a RetroNectin-coated bag and incubated at 4°C for 16 h on a reciprocating shaker at 50 rounds per minute. After the supernatant was removed, activated T cells were added to the bag. The bag transduction method has the advantage of increasing transduction efficiency, as simply flipping over the bag during gene transduction facilitates more efficient utilization of the retroviral vector adsorbed on the top and bottom surfaces of the bag. Finally, we performed validation runs of endoribonuclease MazF-modified CD4+ T cell manufacturing for HIV-1 gene therapy and T cell receptor-modified T cell manufacturing for MAGE-A4 antigen-expressing cancer gene therapy and achieved over 200-fold (≥1010) and 100-fold (≥5×109) expansion, respectively. In conclusion, we demonstrated that the large-scale closed transduction system is highly efficient for retroviral vector-based T cell manufacturing for adoptive transfer gene therapy, and this technology is expected to be amenable to automation and improve current clinical gene therapy protocols.

Optimization of lentiviral vector transduction into peripheral blood mononuclear cells in combination with the fibronectin fragment CH-296 stimulation

Large scale T-cell expansion and efficient gene transduction are required for adoptive T-cell gene therapy. Based on our previous observations, human peripheral blood mononuclear cells (PBMCs) can be expanded efficiently while conserving a naïve phenotype by stimulating with both recombinant human fibronectin fragment (CH-296) and anti-CD3 monoclonal antibodies. In this article, we explored the possibility of using this co-stimulation method to generate engineered T cells using lentiviral vector. Human PBMCs were stimulated with anti-CD3 together with immobilized CH-296 or anti-CD28 antibody as well as anti-CD3/anti-CD28 conjugated beads and transduced with lentiviral vector simultaneously. Co-stimulation with CH-296 gave superior transduction efficiency than with anti-CD28. Next, PBMCs were stimulated and transduced with anti-CD3/CH-296 or with anti-CD3/CD28 beads. T-cell expansion, gene transfer efficiencies and immunophenotypes were analysed. Stimulation with anti-CD3/CH-296 resulted in more than 10-times higher cell expansion and higher gene transfer efficiency with conservation of the naïve phenotype compared with anti-CD3/CD28 stimulation method. Thus, lentiviral transduction with anti-CD3/CH-296 co-stimulation is an efficient way to generate large numbers of genetically modified T cells and may be suitable for many gene therapy protocols that use adoptive T-cell transfer therapy.

Donor lymphocytes expressing the herpes simplex virus thymidine kinase suicide gene: detailed immunological function following add-back after haplo-identical transplantation

BACKGROUND AIMS Haplo-identical hematopoietic stem cell transplantation (HSCT) with add-back of donor lymphocytes expressing the herpes simplex virus thymidine kinase suicide gene (TK cells) is one of the most widely applied promising new gene therapy approaches. However, the immunological status of added-back TK cells after HSCT has yet to be well characterized. METHODS We investigated TK cells through the use of flow cytometry, T-cell receptor (TCR) Vβ repertoire spectratyping and linear amplification-mediated polymerase chain reaction followed by insertion site analysis in a patient enrolled in our clinical trial. RESULTS A comparison of onset with remission of acute graft-versus-host disease confirmed that TK cells were predominantly eliminated and that proliferative CD8(+) non-TK cells were also depleted in response to ganciclovir administration. The TCR Vβ-chain repertoire of both TK cells and non-TK cells markedly changed after administration of ganciclovir, and, whereas the TCR repertoire of non-TK cells returned to a normal spectratype long after transplantation, that of TK cells remained skewed. With the long-term prophylactic administration of acyclovir, TK cells oligoclonally expanded and the frequency of spliced variants of TK cells increased. Known cancer-associated genes were not evident near the oligoclonally expanded herpes simplex virus (HSV)-TK insertion sites. CONCLUSIONS We demonstrate obvious differences in immunological status between TK cells and non-TK cells. In addition, we speculate that long-term prophylactic administration of acyclovir increases the risk of oligoclonal expansion of spliced forms of TK cells.

Stimulation through very late antigen-4 and -5 improves the multifunctionality and memory formation of CD8⁺ T cells.

T cells express multiple integrin molecules. The significance of signaling through these molecules on acquisition of T‐cell effector functions and memory formation capacity remains largely unknown. Moreover, the impact of stimulation through these signals on the generation of T cells for adoptive immunotherapy has not been elucidated. In this study, using a recombinant fragment of fibronectin, CH‐296, we demonstrated that stimulation via very late Ag (VLA)‐4 and VLA‐5 in human and BALB/c mouse CD8+ T cells, in combination with TCR stimulation, enhances effector multifunctionality and in vivo memory formation. Using TCR‐transgenic mouse‐derived CD8+ T cells expressing TCR specific for the syngeneic CMS5 fibrosarcoma‐derived tumor Ag, we showed that stimulation by CH‐296 improved the ability of tumor‐specific CD8+ T cells to inhibit CMS5 tumor growth when adoptively transferred into hosts with progressing tumors. Improved antitumor effects were associated with decreased infiltration of Foxp3+CD4+ Treg cells in tumors. These results suggest that stimulation via VLA‐4 and VLA‐5 modulates the qualities of effector T cells and could potentially increase the efficacy of adoptive therapy against cancer.

In vivo persistence of adoptively transferred TCR gene-transduced lymphocytes with anti-tumor reactivity in patients with MAGE-A4 expressing esophageal cancer

The application of adoptive immunotherapy with tumor-specific T cells has been limited because of the short life span of the transferred T cells unless the host has been manipulated. Engineering the antigen receptor gene in patients’ lymphocytes is one promising strategy to create antigen-specific lymphocytes without senescent phenotypes. The strategy provides an opportunity to broaden the types of cancer to be treated. However, this concept has not been tested in the epithelial cancer patients. We completed a phase I clinical trial of TCR gene therapy targeting MAGE-A4 to treat esophageal cancer patients without lympho-depleting pre-conditioning. The trial was designed as a cell-dose escalation consisting of three cohorts, 2x10E8, 1x10E9 and 5x10E9 cells/patient. The treatment was tolerable with no adverse events associated with transferred cells. In all ten patients of the 3 cell-doses, the transferred lymphocytes were detected in their peripheral blood in a dose-dependent manner during the first 14 days. In 4 patients, the infused cells have been persisting more than 5 months after the transfer. The T cell clones were established from the transferred lymphocytes that were harvested more than 100 days after the transfer. These clones sustained the reactivity to the antigen-expressing tumor cells. Three patients showed SD or long tumor free status. These results suggest that this approach may extend the availability of adoptive T cell therapy for epithelial cancer patients by providing tumor-reactive and long surviving lymphocytes reducing the risk of intensive pre-treatments.