ALBERT

Description: Key Points The efficacy and safety of a novel redirected T-cell–based adoptive immunotherapy targeting hTERT for patients with adult T-cell leukemia. hTERT-specific T-cell receptor gene-transduced CD8+ T cells lyse ATL cells, but not normal cells, both in vitro and in vivo.

Description: Aurora kinase A (AURKA) is overexpressed in leukemias. Previously, we demonstrated that AURKA-specific CD8+ T cells specifically and selectively lysed leukemia cells, indicating that AURKA is an excellent target for immunotherapy. In this study, we examined the feasibility of adoptive therapy using redirected T cells expressing an HLA-A*0201–restricted AURKA207-215-specific T-cell receptor (TCR). Retrovirally transduced T cells recognized relevant peptide-pulsed but not control target cells. Furthermore, TCR-redirected CD8+ T cells lysed AURKA-overexpressing human leukemic cells in an HLA-A*0201–restricted manner, but did not kill HLA-A*0201+ normal cells, including hematopoietic progenitors. In addition, AURKA207-215-specific TCR-transduced CD4+ T cells displayed target-responsive Th1 cytokine production. Finally, AURKA207-215-specific TCR-transduced CD8+ T cells displayed antileukemia efficacy in a xenograft mouse model. Collectively, these data demonstrate the feasibility of redirected T cell–based AURKA-specific immunotherapy for the treatment of human leukemia.

Description: Abstract 4290 Background & Purpose: Recently we have identified a novel HLA-A*0201-restricted antigenic 9mer epitope (aa207-215: YLILEYAPL) derived from Aurora-A kinase (Aurora-A) which is capable of generating anti-leukemia cytotoxic T-lymphocytes (CTL).(Blood, 2009) To improve the feasibility of Aurora-A targeting cellular immunotherapy against leukemia, we have established a Aurora A207-215-specific CTL clone, and have obtained the full-length T-cell receptor (TCR) α/β genes for TCR gene transfer. In this study, using human leukemia cell lines and patients' leukemia cells, we examined the anti-leukemia reactivity of engineered T-cells with Aurora-A-specific TCR gene transfer. Methods: Full-length of an HLA-A*0201-restricted and Aurora-A207-215-specific TCR α/β genes (Vα3/J20/Cα, Vβ10.3(12)/J1.1/Cβ1, respectively) were cloned into bicistronic GaLV-pseudotyped retroviral vector. Using Retronectin (Takara Bio. Japan)-coated plates, Aurora-A-specific TCR α/β genes were inserted into lymphocytes. Whether this vector was capable of generating a functional Aurora-A207-215-specific TCR heterodimer was examined using Jurkat/MA cells (kindly gifted from Prof. Erik Hooijberg, Netherlands). The epitope-specific and leukemia specific cytotoxicity and IFN-γ production of gene-modified normal CD8+ and CD4+ T-cells were examined by 51Cr-releasing assay and ELISA. HLA-A*0201-restriction of engineered T-cell responses was examined by inhibition assay with antibodies, and HLA-A*0201 transduced human leukemia cell line: MEG01 which abundantly expresses Aurora-A. In vivo anti-leukemia effect of gene-modified CD8+T-cells was examined using NOD/SCID/γcnull (NSG) mice. Eventually, the on-target adverse effect of these Aurora-A-specific TCR-gene transferred CD8+T-cells against autologous hematopoietic progenitor cells was examined using cord blood CD34+ cells. Results: The Aurora-A specific-TCR expressing retroviral vector was capable of generating a functional TCR in Jurkat/MA cells which could produce luciferase in response to Aurora-A peptide on C1R-A2 cells in a dose dependent manner. Aurora-A-specific TCR-transduced CD8+ T-cells produced IFN-γ and exerted cytotoxicity against Aurora-A peptide-loaded C1R-A2 cells in an HLA-A*0201 restricted fashion. These engineered CD8+ T-cells also killed HLA-A*0201+ leukemia cell line and patient leukemia cells, but not HLA-A*0201+ normal PBMC and normal mitotic PHA-stimulated lymphoblasts. The anti-leukemia effect of These engineered CD8+ T cells was significantly abrogated by the anti-HLA-class I monoclonal antibody (MoAb), but not by anti-HLA-DR-MoAb. These engineered CD8+ T-cells killed HLA-A*0201-transduced MEG01 cells which were abundantly expressing Aurora-A, but not parent HLA-A*0201-negative MEG01 cells. Aurora-A-specific TCR gene transduced CD4+ T-cells produced IFN-γ in response to the epitope recognition, which was also in an HLA-A*0201-restricted fashion. Furthermore, Aurora-A-specific TCR-transduced CD8+ T cells did not damage the viability of autologous cord blood CD34+ cells in vitro. Finally, These engineered CD8+ T-cells successfully inhibited the engraftment and growth of inoculated leukemia cell line cells in the NOD/SCIDγ/cnull mice. Background: In this study, Aurora-A kinase-specific TCR gene transferred T-cells successfully recognized the target epitope and exerted the target-specific cytotoxicity. Additionally these engineered CD8+ T-cells exerted anti-leukemia effect both in vitro and in vivo. While those these transfectants did not damage autologous hematopoietic progenitor cells in vitro. Collectively, the novel anti-leukemia adoptive therapy using Aurora-A-specific TCR-gene transferred T-cells appears promising, and further investigations are warranted for the clinical application. Disclosures: No relevant conflicts of interest to declare.

Description: Abstract 2059 Background & Purpose: Redirected T-cell based adoptive therapy using cancer antigen-specific T-cell receptor (TCR) gene transfer has proven promise, however its clinical efficacy still remains unsatisfactory. The less accumulation in number of infused redirected T cells at the local tumor site is one of the causes. In order to accumulate those tumor-responsive T cells inside tumor microenvironment, chemokine produced by tumor cells and/or tumor associated cells is an attractive target. In this study, we examined the feasibility of CC chemokine receptor 2 (CCR2) gene transfer into T cells beforehand redirected toward WT1 in order to enhance the anti-cancer reactivity, both in vitro and in vivo. Methods: HLA-A*24:02-restricted and WT1235–243-specific TCR-a/b genes were introduced into normal CD8+ T cells using our novel retroviral TCR-gene expression vector encompassing silencers for endogenous TCRs (WT1-si-TCR vector). mRNA expression of total 11 chemokines expressed by 10 human lung cancer cell lines was examined using QRT-PCR, then CCL2 (variably produced in 7 out of 10 examined cell lines) and the small lung cancer cell line, LK79 which abundantly produced CCL2 was chosen for the proof of concept. Cloned CCR2 gene was retrovirally introduced into Jurkat cells, Jurkat/MA cells and normal CD8+ T cells similarly redirected beforehand using WT1-siTCR vector. Introduced CCR2 was validated using flow cytometer and transwell experiments. Cytotoxicity was examined using standard 51chromium release assay. Cooperative functionality composed of CCL2-directinality and WT1-specific antitumor cytotoxicity mediated by those double gene transfectants was examined in vitro; values of LDH released from destroyed LK79 cells in the bottom well by migrated double gene transduced CD8+ T cells from the upper well were measured. Antitumor reactivity in vivo was assessed using xenograft mouse model using luciferase-transduced LK79 cells (LK79-luc). Direct effect of CCL2 ligation on WT1-TCR signaling in double gene transfectant was assessed using luciferase assay with double gene transduced TCR− Jurkat/MA cell line, which stably expresses hCD8a and NFAT-luciferase reporter genes (Jurkat/MA/CD8a/luc; kindly provided by Prof. Erik Hooijberg, Netherlands). Results: CCL2 sensitivity was successfully introduced by CCR2 gene transfer; CCR2 transduced Jurkat successfully directed toward CCL2 producing cell line, LK79 cells. CCR2 gene transduction did not impede WT1-specific cytotoxicity mediated by CD8+ T cells beforehand redirected using WT1-siTCR gene transfer, rather double gene transduction cooperatively endowed those transfectants with CCL2 sensitivity and WT1-specific cytotoxicity against LK79 cells. Furthermore, in vivo assay using xenograft mouse model, growth of subcutaneously inoculated LK79-luc cells was more efficiently suppressed by those double gene transduced CD8+ T cells than WT1-siTCR single gene trnasfectants, in particular, immediately after adoptive transfer. Finally, CCL2 synergistically enhanced the magnitude of cognate peptide evoked WT1-specific TCR signaling in a dose dependent manner. Even without WT1 peptide ligation, such TCR signaling was similarly evoked by CCL2 to some extent. Conclusion: In this study, our results demonstrate that forced expression of CCR2 on CTL beforehand redirected toward WT1 enhanced its anti-cancer reactivity both in vitro and in vivo. This in vivo enhancement of antitumor reactivity may be caused by increased number of effector cells and enhanced WT1-TCR signaling generated both by CCL2 in the local tumor microenvironment. Although further studies are warranted, CCR2 gene transfer into redirected WT1-specific tumor-reactive CTL may be feasible for the treatment of human cancers. Disclosures: No relevant conflicts of interest to declare.

Description: Key Points T cells expressing a CAR consisting of scFv #213 targeting WT1 peptide/HLA-A*2402 complex killed HLA-A*2402+ WT1+ tumor cell lines. The therapeutic efficacy of #213 scFv CAR-T cells was shown to be enhanced by DC vaccine in a murine xenograft model.

Description: Adoptive T-cell therapy for malignancies using redirected T cells genetically engineered by tumor antigen-specific T-cell receptor (TCR) gene transfer is associated with mispairing between introduced and endogenous TCR chains with unknown specificity. Therefore, deterioration of antitumor reactivity and serious autoimmune reactivity are major concerns. To address this problem, we have recently established a novel retroviral vector system encoding siRNAs for endogenous TCR genes (siTCR vector). In this study, to test the clinical application of siTCR gene therapy for human leukemia, we examined in detail the efficacy and safety of WT1-siTCR–transduced T cells. Compared with conventional WT1-TCR (WT1-coTCR) gene-transduced T cells, these cells showed significant enhancement of antileukemia reactivity resulting from stronger expression of the introduced WT1-specific TCR with inhibition of endogenous TCRs. Notably, WT1-siTCR gene-transduced T cells were remarkably expandable after repetitive stimulation with WT1 peptide in vitro, without any deterioration of antigen specificity. WT1-siTCR gene–transduced T cells from leukemia patients successfully lysed autologous leukemia cells, but not normal hematopoietic progenitor cells. In a mouse xenograft model, adoptively transferred WT1-siTCR gene-transduced T cells exerted distinct antileukemia efficacy but did not inhibit human hematopoiesis. Our results suggest that gene-immunotherapy for leukemia using this WT1-siTCR system holds considerable promise.

Description: Abstract 4169 Background and Purpose Currently adult T-cell leukemia (ATL) is one of the most chemotherapy-resistant T cell malignancies and the prognosis of ATL patients still remains very poor. Although several novel treatment options, for example anti-chemokine receptor 4 (CCR4) monoclonal antibody, look promise, development of other novel treatment options still remains warranted. Currently activated status of human telomerase reverse transcriptase (hTERT) in ATL cells has been underscored. Thus, in this study, in order to develop a novel T cell-based immunotherapy for the treatment of ATL, we investigated the feasibility of redirected T-cell using hTERT-specific T-cell receptor (TCR) gene transfer. Methods Approval for this study was obtained from the Institutional Review Board of Ehime University Hospital. HLA-A*24:02-restricted and hTERT461–469epitope (residues: VYGFVRACL)-specific TCR -a/b genes (Tajima K et al. Int J Cancer 2004) cloned from our established hTERT-specific CTL clone were inserted into a novel GaLV-pseudotyped retroviral vector encoding built-in siRNAs for constant regions of endogenous TCR a /b genes (hTERT-siTCR vector). hTERT-siTCR vector was transfected into normal CD8+ T cells in RetroNectin (Takara Bio Inc.) coated-plates and these redirected CD8+ T cells (hTERT-siTCR CD8) were used as effector cells. HLA-A*24:02+ or A*24:02− ATL cell lines, HTLV-1 infected T-cell lines and freshly isolated ATL cells from patients were examined as target cells. All patients gave written informed consents in accordance with the declaration of Helsinki. Normal HLA-A*24:02+CD4+ T cells andHLA-A*24:02+cord blood CD34+ mononuclear cells (CB-CD34+) were similarly obtained from healthy volunteers. Expression of hTERT mRNA and hTERT protein in ATL cell lines, freshly isolated ATL cells, and HTLV-1 infected T cells were evaluated by quantitative real time PCR (QRT-PCR) using DCt method and Western blotting. Antileukemia reactivity mediated by hTERT-siTCR CD8 against ATL cells were examined by standard 51chromium release assay and flow-based CD107a assay. For the safety assessment, effector cells-mediated cytotoxicity against CB-CD34+ cells as normal hematopoietic progenitors was similarly examined. Peripheral blood mononuclear cells (PBMCs) from ATL patients were obtained on admission and in stable disease or remission after receiving treatments, and hTERT461–469 epitope responsive CD8+ T cells were detected using hTERT461–469peptide/ HLA-A*2402 tetramer or ELISPOT assay. Results QRT-PCR revealed that both freshly isolated ATL cells and ATL cell lines markedly overexpressed hTERT mRNA, while that in normal CD4+ cells and CB-CD34+ cells was less than undetectable. hTERT-siTCR CD8 cells were more than 40% positive for hTERT/HLA-A*2402 tetramer and successfully displayed HLA-A*2402-restricted and hTERT461–469-specific cytocidal effect against target-loaded C1R-A24 cells. Additionally, hTERT-siTCR CD8 cells successfully discriminated A*2402+ ATL cell lines (ATN-1, TL-Su) from A*2402− ones (TLO-m1, HUT-102); those all cell lines similarly overexpressed hTERT. Furthermore, those effector cells successfully killed freshly isolated HLA-A*2402+ ATL cells, but not A*2402− ones. Finally, hTERT461–469-specific cytotoxic T-lymphocyte precursors were detected at variable levels in PBMCs obtained from HLA-A*2402+ ATL patients, which shows hTERT overexpressed by ATL cells may be naturally processed in vivo. Conclusion Although much further invetigations are warranted, in our preliminary study, we have shown for the first time that hTERT could be a potential therapeutic target of redirected T cell-based immunotherapy for the treatment of ATL. We are now in detail examining efficacy and safety of those redirected T cells using xenograft mouse model. Disclosures: No relevant conflicts of interest to declare.

Description: Abstract 3153 Purpose: In antitumor adoptive immunotherapy, the utility of tumoricidal CD8+ T cells are mainly highlighted, while in tumor immunity, the importance of tumor-reactive CD4+ T cells is also well documented. However, because the number of well-characterized tumor-associated epitopes recognized by CD4+ T cells still remains small, application of tumor-reactive CD4+ T cells is limited. In order to circumvent this drawback, redirection of CD4+ T cells to well-characterized HLA class I-restricted CD8+ T-cell epitope seems promising. In this study, using an HLA class I-restricted and WT1-specific T-cell receptor (TCR) gene transfer, we, in detail, examined helper functions mediated by those gene-modified CD4+T cells in redirected T cell-based antileukemia adoptive immunotherapy. Methods: HLA-A*2402-restricted and WT1235–243-specific TCR α/β genes were inserted into our unique retroviral vector encoding shRNAs for endogenous TCRs (WT1-siTCR vector), and was employed for gene-modification both of CD4+ and CD8+ T cells to express WT1-specific TCR. (1) WT1 epitope-responsive cytokine production mediated by WT1-siTCR-transduced CD4+ T cells (WT1-siTCR/CD4) was measured using bead-based immunoassay and ELISA assay. (2) WT1 epitope-ligation induced co-stimulatory molecules by WT1-siTCR/CD4 was assessed using flow cytometry. (3) Impacts on WT1 epitope and leukemia-specific responses; cytocidal activity, proliferation and differentiation into memory T-cell phenotype, mediated by WT1-siTCR-transduced CD8+ T cells (WT1-siTCR/CD8) provided by concurrent WT1-siTCR/CD4 were assessed using 51Cr-release assay, CD107a/intracellular IFN-γ assay, CFSE dilution assay and flow cytometry. (4) WT1 epitope-ligation triggered chemokine production mediated by WT1-siTCR/CD4 was assessed using real-time PCR, then chemotaxis mediated by WT1-siTCR/CD8 in response to those chemokines was assessed using a transwell experiment. (5) In vivo tumor trafficking mediated by WT1-siTCR/CD4 was assessed using bioluminescence imaging assay. (6) Finally, WT1-siTCR/CD4-caused in vivo augmentation of antileukemia functionality mediated by WT1-siTCR/CD8 was assessed similarly using a xenografted mouse model. Results: WT1-siTCR/CD4 showed a terminal effector phenotype; positive for transcription factor T-bet, but negative for Bcl-6 or Foxp3. Upon recognition of WT1 epitope, WT1-siTCR/CD4 produced Th1, but not Th2 cytokines in the context of HLA-A*2402, which simultaneously required HLA class II molecules on target cells. WT1 epitope-ligation enhanced WT1-siTCR/CD4 to express cell-surface OX40. In the presence of WT1-siTCR/CD4, but not non-gene-modified CD4, effector functions mediated by WT1-siTCR/CD8 in response to WT1 epitope and leukemia cells, including cytocidal activity based on CD107a expression and IFN-γ production was enhanced. Such augmentation was mediated by humoral factors produced by WT1 epitope-ligated WT1-siTCR/CD4. Additionally, proliferation and differentiation into memory phenotype, notably CD45RA- CD62L+ central memory phenotype, mediated by WT1-siTCR/CD8 in response to both WT1 epitope and leukemia cells were also augmented, accompanied with increased expression of intracellular Bcl-2 and cell-surface IL-7R. Next, CCL3/4 produced by activated WT1-siTCR/CD4 triggered chemotaxis of WT1-siTCR/CD8 which express the corresponding receptor, CCR5. Using bioluminescence imaging, intravenously infused WT1-siTCR/CD4 successfully migrated towards leukemia cells inoculated in a NOG mouse. Finally, co-infused WT1-siTCR/CD4 successfully augmented immediate accumulation towards leukemia cells and antileukemia reactivity mediated by WT1-siTCR/CD8 in a xenografted mouse model. Conclusion: Using GMP grade WT1-siTCR vector, redirected CD4+ T cells to HLA class I-restricted WT1 epitope successfully recognized leukemia cells and augmented in vivo antileukemia functionality mediated by similarly redirected CD8+ T cells, encompassing tumor trafficking, cytocidal activity, proliferation and differentiation into memory cells. The latter seem to support the longevity of transferred antileukemia efficacy. Taking together, coinfusion of redirected CD4+ T cells to HLA class I-restricted WT1 epitope seems feasible and advantageous for the successful WT1-targeting redirected T cell-based immunotherapy against human leukemia. Disclosures: No relevant conflicts of interest to declare.

Description: Key Points WT1-specific TCR-redirected T-cell therapy for AML and MDS is safe, and the T cells persisted in vivo and trafficked to bone marrow. Transient decreases of leukemic cell in bone marrow were shown.