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The benefits of CAR-T therapy could be expanded to the treatment of solid tumors through the use of derived autologous αβ T cell, but clinical trials of CAR-T therapy for patients with solid tumors have so far been disappointing. CAR-T therapy also faces hurdles due to the time and cost intensive preparation of CAR-T cell products derived from patients as such CAR-T cells are often poor in quality and low in quantity. These inadequacies may be mitigated through the use of third-party donor derived CAR-T cell products which have a potent anti-tumor function but a constrained GVHD property. Vγ9Vδ2 TCR have been shown to exhibit potent antitumor activity but not alloreactivity. Therefore, in this study, CAR-T cells were prepared from Vγ9Vδ2 T (CAR-γδ T) cells which were expanded by using a novel prodrug PTA. CAR-γδ T cells suppressed tumor growth in an antigen specific manner but only during a limited time window. Provision of GITR co-stimulation enhanced anti-tumor function of CAR-γδ T cells. Our present results indicate that, while further optimization of CAR-γδ T cells is necessary, the present results demonstrate that Vγ9Vδ2 T cells are potential source of ‘off-the-shelf’ CAR-T cell products for successful allogeneic adoptive immunotherapy.

CAR-T therapy has achieved considerable treatment success in hematologic tumors by using patient derived autologous αβ T cells. There is an impetus to broaden the applicability of this approach by using a third-party donor derived CAR-T cell product which has a potent anti-tumor function but a constrained GVHD property. In this study, CAR-T cells were prepared from Vγ9Vδ2 T cells expanded by using a novel prodrug PTA and their anti-tumor functions were assessed in conjunction with persistency, localization, and phenotype. γδ T cells were successfully transduced with a CAR specific to CEA with signaling domains of CD3ζ and CD28 (CEA.CAR-γδ T cells), and exhibited potent tumor killing function in vitro. In a xenograft mouse model, CEA.CAR-γδ T cells suppressed CEA+ tumor growth though a limited time window. CEA.CAR-γδ T cells persisted and accumulated in the tumor even after tumor progression, however, ex vivo analysis revealed that those recovered at different time points from PBMC, spleen and tumors gradually lost tumor reactivity as assessed by IFN-γ production. Provision of GITR co-stimulation enhanced anti-tumor function of CEA.CAR-γδ T cells, the result of which imposes additional measurers to be adopted in CAR-γδ T cells for an allogeneic adoptive immunotherapy.

Simple Summary Pancreatic ductal adenocarcinoma is one of the most lethal malignancies, and there are vast unmet medical needs. In this study, we hypothesized that chimeric antigen receptor engineered T cell (CAR-T) targeting carcinoembryonic antigen (CEA) would be effective in the treatment of pancreatic ductal adenocarcinoma. In vivo experiments in a more clinically similar environment were considered necessary; we examined the antitumor effects of adoptive anti-CEA-CAR-T, using orthotopic xenograft mouse models of pancreatic ductal adenocarcinoma. As result, the therapeutic effect of anti-CEA-CAR-T therapy was related to the CEA expression level. Furthermore, the retrospective analysis of pathological findings from pancreatic ductal adenocarcinoma patients showed a correlation between the intensity of CEA immunostaining and tumor heterogeneity. These findings show that anti-CEA-CAR-T therapy can be useful for pancreatic ductal adenocarcinoma; furthermore, the pathological findings of CEA can be clinically used as biomarkers to select cases for anti-CEA-CAR-T therapy. Chimeric antigen receptor engineered T cell (CAR-T) therapy has high therapeutic efficacy against blood cancers, but it has not shown satisfactory results in solid tumors. Therefore, we examined the therapeutic effect of CAR-T therapy targeting carcinoembryonic antigen (CEA) in pancreatic adenocarcinoma (PDAC). CEA expression levels on the cell membranes of various PDAC cell lines were evaluated using flow cytometry and the cells were divided into high, medium, and low expression groups. The relationship between CEA expression level and the antitumor effect of anti-CEA-CAR-T was evaluated using a functional assay for various PDAC cell lines; a significant correlation was observed between CEA expression level and the antitumor effect. We created orthotopic PDAC xenograft mouse models and injected with anti-CEA-CAR-T; only the cell line with high CEA expression exhibited a significant therapeutic effect. Thus, the therapeutic effect of CAR-T therapy was related to the target antigen expression level, and the further retrospective analysis of pathological findings from PDAC patients showed a correlation between the intensity of CEA immunostaining and tumor heterogeneity. Therefore, CEA expression levels in biopsies or surgical specimens can be clinically used as biomarkers to select PDAC patients for anti-CAR-T therapy.

BackgroundCAR-T cell therapy has shown highly effective clinical results in several diseases, but further improvement is necessary to target a wider range of antigens and tumors. In particular, excessive activation of CAR-T cells leads to cell exhaustion and reduction of naive/memory T cells’ population, which are important for long-term immune response. Therefore, suppressing non-antigen-specific activity is necessary for CAR-T cell production. However, when targeting tumor-related antigens that are also expressed on T cells, CAR-T cells recognize the antigens on the T cells, resulting in fratricide, poor cell growth, differentiation, and exhaustion during cell production process.In this study, we investigated a method for producing CAR-T cells targeting CD38 antigen that is common to T cells and tumor cells. CD38 is a suitable target antigen for CAR-T cell therapy because it is highly expressed in lymphocyte malignant tumors including B-cell non-Hodgkin’s lymphoma and multiple myeloma. However, as it is also intermediately expressed in normal blood cells, unwanted activation of CAR-T cells may be caused.MethodsWe tried to suppress the expression of CD38 in CAR-T cells by co-expressing CD38 siRNAs, and prevent activation during cell production by modifying the signal domain of anti-CD38-CAR to the newly developed JAK/STAT-CAR. JAK/STAT-CAR contains the intracellular domain of the IL-2 receptor β chain and the STAT3 binding motif, which have been shown to improve the proliferation of CAR-T cells and suppresses differentiation compared to conventional second-generation CAR-T cells.For further improvement, CAR-T cells were prepared in the presence of the tyrosine kinase inhibitor Dasatinib to suppress activation during the cell manufacturing process.ResultsCD38 siRNA co-expressing CAR-T cells showed decreased expression of CD38 and exhaustion markers, and the further reduction of exhaustion marker expression was observed in JAK/STAT CAR-T cells. However, compared to CAR-T cells targeting other antigens, CD38-CAR-T cells tended to be more exhausted and differentiated. As Dasatinib treatment maintained a high proportion of naive/memory T cells and was able to suppress exhaustion, combination of these approaches (CD38 siRNA-expressing CD38-JAK/STAT CAR-T cells with Dasatinib treatment) showed long-term persistence of antitumor activity in in vitro re-challenge assay.ConclusionsCD38 siRNA co-expressing CD38-JAK/STAT CAR-T cells produced in the presence of a tyrosine kinase inhibitor are expected to be suppressed excessive activation and maintain long-term antigen-specific activity. This approach is also expected to be applied to other CAR-T cell therapies targeting tumor-related antigens expressed on T cells.

Target-specific genome editing using engineered nucleases has become widespread in various fields. Long gene knock-in and single-base substitutions can be performed by homologous recombination (HR), but the efficiency is usually very low. To improve the efficiency of knock-in with single-stranded oligo DNA nucleotides (ssODNs), we have investigated optimal design of ssODNs in terms of the blocking mutation, orientation, size, and length of homology arms to explore the optimal parameters of ssODN design using reporter systems for the detection of single-base substitutions. We have also investigated the difference in knock-in efficiency among the delivery forms and methods of Cas9 and sgRNA. The knock-in efficiencies for optimized ssODNs were much higher than those for ssODNs with no blocking mutation. We have also demonstrated that Cas9 protein/sgRNA ribonucleoprotein complexes (Cas9-RNPs) can dramatically reduce the re-cutting of the edited sites.

As potential pandemic vaccines, DNA/RNA vaccines, viral vector vaccines and protein-based vaccines have been rapidly developed to prevent pandemic spread worldwide. In this study, we designed plasmid DNA vaccine targeting the SARS-CoV-2 Spike glycoprotein (S protein) as pandemic vaccine, and the humoral, cellular, and functional immune responses were characterized to support proceeding to initial human clinical trials. After intramuscular injection of DNA vaccine encoding S protein with alum adjuvant (three times at 2-week intervals), the humoral immunoreaction, as assessed by anti-S protein or anti-receptor-binding domain (RBD) antibody titers, and the cellular immunoreaction, as assessed by antigen-induced IFNγ expression, were up-regulated. In IgG subclass analysis, IgG2b was induced as the main subclass. Based on these analyses, DNA vaccine with alum adjuvant preferentially induced Th1-type T cell polarization. We confirmed the neutralizing action of DNA vaccine-induced antibodies by a binding assay of RBD recombinant protein with angiotensin-converting enzyme 2 (ACE2), a receptor of SARSCoV-2, and pseudo-virus assay, TCID assay with live SARS-CoV-2. Further B cell epitope mapping analysis using a peptide array showed that most vaccine-induced antibodies recognized the S2 and RBD subunits. Finally, DNA vaccine protected hamsters form SARSCoV-2 infection. In conclusion, DNA vaccine targeting the spike glycoprotein of SARS-CoV-2 might be an effective and safe approach to combat the COVID-19 pandemic. Funding: This study was supported by Project Promoting Support for Drug Discovery grants (JP20nk0101602 and JP21nf0101623h102) from the Japan Agency for Medical Research and Development and Panasonic Co. (Japan). To fight against the worldwide COVID-19 pandemic, the development of an effective and safe The Department of Health Development and Medicine is an endowed department supported by Anges, Daicel, and FunPep. The Department of Clinical Gene Therapy is financially supported by Novartis, AnGes, Shionogi, Boeringher, Fancl, Saisei Mirai Clinics, Rohto and Funpep. Declaration of Interest: R.M. is a stockholder of FunPep and Anges. T.O. T.K. and Y.S. are employees of Anges. R.I, A.T, H.K, S.K, E.T, S.M, and H.T are employees of FunPep. R.M, H.T, and A.T. are FunPep stockholders. All other authors declare no competing interests. Ethical Approval: All experiments were approved by the Ethical Committee for Animal Experiments of the Osaka University Graduate School of Medicine.

Background Despite recent impressive successes in chimeric antigen receptor (CAR)-T cell therapy, there are still considerable clinical challenges. To improve T cell persistence and antitumor effect, which are critical for clinical responses, various efforts have been made to optimize the CAR design such as the inclusion of a costimulatory domain(s). It is known that non-specific activation of CAR-T cells is greatly influenced by the CAR design, and excessive T cell activation leads exhaustion of T cells and depletion of naive/memory subsets important for durable clinical responses. Thus, the CAR construct needs to be optimized so that transduced T cells persist and induce potent antigen-specific response with reduced non-specific activation. For optimal T cell activation and proliferation, three signals including TCR (signal 1), co-stimulatory (signal 2), and cytokine (signal 3) signals, are essential. The conventional second and third generation CARs containing CD3ζ and a co-stimulatory domain such as a signal domain of CD28 and 4-1BB can conduct signal 1 and 2, but not signal 3. Recently, we have developed a new generation JAK-STAT CAR composed of a truncated cytoplasmic domain of the IL-2 receptor β chain and STAT3/5 binding motifs, CD28 co-stimulatory domain, and CD3ζ domain. The novel anti-CD19 JAK-STAT CAR-T cells showed antigen-specific activation of the JAK-STAT signaling pathway, enhanced proliferation, and limited terminal differentiation in vitro compared to second generation 28ζ CAR or 4-1BBζ CAR-transduced T cells. Furthermore, the anti-CD19 JAK-STAT CAR-T cells demonstrated superior in vivo persistence and antitumor effect in mouse models.1 In addition, we previously showed that a hinge region and the composition of a single chain variable fragment (scFv) such as the order of VH and VL regions critically influence not only antigen-dependent activation but also undesired antigen-independent activation known as tonic signaling.2 Methods In this study, we have optimized the scFv design in 28ζ CAR and JAK-STAT CAR constructs to show superior antigen-specific activation and reduced tonic signaling for several targets (CD19, CD20, Mesothelin, and GD2). And we have evaluated the feature of JAK-STAT CAR-T cells compared to 28ζ CAR-T cells. Results JAK-STAT CAR-T cells showed superior antigen-specific proliferation with less differentiated status, whereas 28ζ CAR-T cells showed antigen-independent proliferation and displayed higher exhaustion marker expression after repetitive stimulations. Conclusions These results suggest that our JAK-STAT-CARs with enhanced antigen-specific response with minimized tonic signaling targeting various antigens has the potential to demonstrate improved clinical efficacy. References Kagoya Y, et al. A novel chimeric antigen receptor containing a JAK–STAT signaling domain mediates superior antitumor effects. Nat Med 2018;24:p352–359. Okamoto S, et al. Detail analysis of non-specific activation of CD19 CAR-T cells caused by CAR design. ASGCT ( 2015)

Background Adoptive immunotherapy using chimeric antigen receptor (CAR) is recently reported as one of the effective cancer therapy. Especially CAR-T cell therapy targeting CD19 antigen in B-cell tumors have shown impressive clinical results and CAR-T cell products targeting CD19 have already approved. However as the high relapse rate is still the problem and the clinical efficacy of CAR-T cell therapy for solid tumors is currently inadequate, further improvement of CAR design is required.It is known that the design of CAR construct affects the function of CAR-T cells. For example co-stimulatory domain such as CD28 and 4-1BB is used in the second generation CARs, CD28z-CAR-T cells show higher anti-tumor activity, whereas 4-1BBz-CAR-T cells demonstrate superior in vivo persistence. To enhance survival of T cells, several attempts had been made to optimize the signaling domains. Recently, we have developed the novel CARs incorporated GITR (glucocorticoid-induced tumor necrosis factor receptor family-related protein) intracellular domain for T cell survival prolongation and inhibition of regulatory T cells’ suppressive activity. It is also reported that the antigen-nonspecific activation of CAR-T cells (tonic signaling) is influenced by the CAR design, and excessive T cell activation leads exhaustion of CAR-T cells. Previously, we have found that the design of CAR, not only single chain variable fragments (scFvs), affect the strength of tonic signaling. Thus, the optimization of CAR construct is essential to induce antigen-specific response with minimal non-specific activation, which results in maximal efficacy. Methods We have optimized the structure of anti-CEA-GITR-CAR targeting CEA antigen expressing solid tumor such as gastric and pancreatic cancer. We have constructed several CARs with the different composition such as hinge region, transmembrane domain, and the order of VL/VH in scFV region, and compared the tonic signaling and antigen-specific activity in CAR-T cells. Results The property of CAR-T cells was largely affected by the CAR constructs, especially the hinge region. The CAR-T cells with CD8α hinge showed strong tonic signaling, the CAR-T cells with short hinge-CAR lost antigen specificity, and elimination of hinge region lowered the CAR expression level and antigen reactivity. Furthermore, GITR-CAR-T cells showed higher proportion of CCR7+CD45RA+ cells and lower expression of exhaustion markers (PD1, Tim3, and LAG3) compared to CD28z-CAR-T cells. Conclusions Our CEA-GITR-CAR with the optimized scFV design and CD28-hinge demonstrated improved antigen-specific response with reduced tonic signaling, potentially indicating that our novel CAR-T cells may show improved clinical efficacy on solid tumor.

To fight against the worldwide COVID-19 pandemic, the development of an effective and safe vaccine against SARS-CoV-2 is required. As potential pandemic vaccines, DNA/RNA vaccines, viral vector vaccines and protein-based vaccines have been rapidly developed to prevent pandemic spread worldwide. In this study, we designed plasmid DNA vaccine targeting the SARS-CoV-2 Spike glycoprotein (S protein) as pandemic vaccine, and the humoral, cellular, and functional immune responses were characterized to support proceeding to initial human clinical trials. After intramuscular injection of DNA vaccine encoding S protein with alum adjuvant (three times at 2-week intervals), the humoral immunoreaction, as assessed by anti-S protein or anti-receptor-binding domain (RBD) antibody titers, and the cellular immunoreaction, as assessed by antigen-induced IFNγ expression, were up-regulated. In IgG subclass analysis, IgG2b was induced as the main subclass. Based on these analyses, DNA vaccine with alum adjuvant preferentially induced Th1-type T cell polarization. We confirmed the neutralizing action of DNA vaccine-induced antibodies via two different methods, a binding assay of RBD recombinant protein with angiotensin-converting enzyme 2 (ACE2), a receptor of SARS-CoV-2, and pseudovirus assay. Further B cell epitope mapping analysis using a peptide array showed that most vaccine-induced antibodies recognized the S2 and RBD subunits, but not the S1 subunit. In conclusion, DNA vaccine targeting the spike glycoprotein of SARS-CoV-2 might be an effective and safe approach to combat the COVID-19 pandemic.

The recent success of chimeric antigen receptor (CAR)-T cell therapy for treatment of hematologic malignancies supports further development of treatments for both liquid and solid tumors. However, expansion of CAR-T cell therapy is limited by the availability of surface antigens specific for the tumor while sparing normal cells. There is a rich diversity of tumor antigens from intracellularly expressed proteins that current and conventional CAR-T cells are unable to target. Furthermore, adoptively transferred T cells often suffer from exhaustion and insufficient expansion, in part, because of the immunosuppressive mechanisms operating in tumor-bearing hosts. Therefore, it is necessary to develop means to further activate and expand those CAR-T cells in vivo. The Wilms tumor 1 (WT1) is an intracellular oncogenic transcription factor that is an attractive target for cancer immunotherapy because of its overexpression in a wide range of leukemias and solid tumors, and a low level of expression in normal adult tissues. In the present study, we developed CAR-T cells consisting of a single chain variable fragment (scFv) specific to the WT1235-243/HLA-A*2402 complex. The therapeutic efficacy of our CAR-T cells was demonstrated in a xenograft model, which was further enhanced by vaccination with dendritic cells (DCs) loaded with the corresponding antigen. This enhanced efficacy was mediated, at least partly, by the expansion and activation of CAR-T cells. CAR-T cells shown in the present study not only demonstrate the potential to expand the range of targets available to CAR-T cells, but also provide a proof of concept that efficacy of CAR-T cells targeting peptide/major histocompatibility complex can be boosted by vaccination.

Carcinoembryonic antigen (CEA) is a cell surface antigen highly expressed in various cancer cell types and in healthy tissues. It has the potential to be a target for chimeric antigen receptor (CAR)-modified T-cell therapy; however, the safety of this approach in terms of on-target/off-tumor effects needs to be determined. To address this issue in a clinically relevant model, we used a mouse model in which the T cells expressing CEA-specific CAR were transferred into tumor-bearing CEA-transgenic (Tg) mice that physiologically expressed CEA as a self-antigen. The adoptive transfer in conjunction with lymphodepleting and myeloablative preconditioning mediated significant tumor regression but caused weight loss in CEA-Tg, but not in wild-type mice. The weight loss was not associated with overt inflammation in the CEA-expressing gastrointestinal tract but was associated with malnutrition, reflected in elevated systemic levels of cytokines linked to anorexia, which could be controlled by the administration of an anti-IL-6 receptor monoclonal antibody without compromising efficacy. The apparent relationship between lymphodepleting and myeloablative preconditioning, efficacy, and off-tumor toxicity of CAR-T cells would necessitate the development of CEA-specific CAR-T cells with improved signaling domains that require less stringent preconditioning for their efficacy. Taken together, these results suggest that CEA-specific CAR-based adoptive T-cell therapy may be effective for patients with CEA+ solid tumors. Distinguishing the fine line between therapeutic efficacy and off-tumor toxicity would involve further modifications of CAR-T cells and preconditioning regimens.

Recent success in the treatment of patients with B cell malignancy by CD19-CAR encourages the development of successive CAR therapy targeting broad range of tumor-associated antigens. However, the search for the appropriate target molecule for CAR, other than B cell markers, has not met the needs. The molecules recognized by CAR is generally limited to the cellular surface molecules, making difficult the search for the tumor-specific targets. Inspired by the physiological recognition of epitope peptide and MHC molecule (pMHC) by T cells, we have generated a series of antibodies that recognize the pMHC complexes with peptides derived from tumor antigens expressed intracellularly. Screening a phage display library of human antibody scFv, we isolated an scFv antibody clone “WT#213” that can specifically recognize WT1 p235-243 peptide (CMTWNQMNL) complexed with HLA-A24:02 molecule. We have constructed a retroviral vector that encodes the CAR consists of WT#213 and intracellular signal transduction domains of CD3ζ and GITR (WT#213 CAR). We confirmed the specific recognition of endogenous WT1-expressing cells by the CAR-T cells with the intracellular cytokine staining and the 51Cr release cytotoxic assay. Moreover, we demonstrated the effectiveness of adoptive cell therapy with WT#213 CAR against the WT1 expressing HLA-A24:02 positive human leukemia cells, utilizing NOG immunodeficient mice. To evaluate the safety of the WT#213 CAR, we predicted the potential property of WT#213 CAR to cross-react to normal tissues in humans. We conducted alanine scan analysis of WT1p235-243 peptide that was recognized by WT#213 CAR as well as the TCR derived from CTL clone TAK-1 which recognizes same epitope peptide in association with HLA-A24:02 to define the amino acids that were critically important in the recognition by the WT#213 CAR or TAK-1-derived TCR. After BLAST search, we synthesized the normal protein-derived peptides with potential risk of cross-reactivity, and tested the recognition of these peptides by WT#213 CAR or TAK-1-derived TCR. Although the critical peptides, and therefore the peptides with potential risk, were quite different between the WT#213 CAR and TAK-1-derived TCR, none of these peptides showed the stimulation of WT#213 CAR or TAK-1-derived TCR. The results here suggest that the immunotherapy with WT#213 CAR will be effective for the treatment of the leukemia patients without the predicted risk in the evaluation we performed.View Large Image | Download PowerPoint Slide

Glucosylceramide and galactosylceramide were detected in three Aspergillus species: Aspergillus oryzae, Aspergillus sojae and Aspergillus. awamori, using borate-coated TLC. The cerebrosides from A. oryzae were further purified by ion exchange and iatrobeads column chromatographies with or without borate, and determined the composition of sugar, fatty acid and sphingoid base by GC/MS, MALDI-TOF/MS and (1)H-NMR. We identified them as β-glucosylceramide and β-galactosylceramide. The ceramide moiety of both cerebrosides consisted mainly of 2-hydroxystearic acid and either 9-methyl-octadeca-4, 8-sphingadienine or octadeca-4, 8-sphingadienine. To our knowledge, this is the first study to provide evidence for the presence of β-galactosylceramide in A. oryzae.

Adoptive immunotherapy using the chimeric antigen receptor (CAR) gene-modified T cells is a promising strategy to treat patients with malignancy and autoimmune diseases. The CAR-T cells can target any cell surface antigens in a HLA independent manner, therefore CAR-T therapy is applicable to a broad range of patients irrespective of HLA phenotype. Typically CARs have higher affinities compared to TCRs, and T cell activation and cytotoxic effect on target cells mediated by CARs correlated with the affinity until it reach the thresholds. The latest results of CD19 CAR-T immunotherapy clinical trials have demonstrated impressive potential in a range of B-lymphoid malignancies. In spite of the recent great success, serious adverse events occurred after infusion of CAR-T cells including “on-target off-organ” activation, and cytokine release syndrome has been observed in a number of CAR-T cell therapies as a result of excessive T cell activation. To improve the efficacy and safety of CAR-T cells, selection of the target tumor-associated antigen, that are expressed only on tumor cells, and the suitable CAR constructs showing the optimal T cell activities are essential, thereby minimizing the risk of side effects.Previously, we have found CAR-T cells acquired to express T cell activation marker CD25 without specific antigen stimulation during in vitro culture. To validate of consequence of CD25-expression, we have analyzed the immune phenotype and antigen specific activities of CAR-T cells, and found the excess activation of CAR-T cells caused T cells differentiation with much less Naive T cell populations, resulting reduction of T cell activities against specific tumor cells. All CAR-T cells we have tested were activated without specific antigen stimulation, although the intensity of non-specific activation were correlated with the expression levels of CARs and depended on the scFvs and the extracellular-spacer domains.In this study, aiming to select the optimal design of CD19 CAR for effective and safe immunotherapy, using anti-CD19 antibody, clone FMC63, we performed detail analysis of non-specific activation of CD19 CAR-T cells caused by the design of scFv (e.g.the leader sequences, the order of VH and VL, the spacer sequences between VH and VL, and the extracellular-spacer domains). In vitro culture of CD19-CAR T cells without antigen specific stimulation induced elevation of T cell activation marker expression, such as CD25 and CD69 on CAR T cells, and reduction of Naive phenotype T cell populations (CCR7+/CD45RA+), as the level of non-specific activation of CAR-T cells was depended on the structure of scFvs (the order of VH and VL, and the spacer sequences) and the extracellular-spacer domains. Especially, the CAR-T cells with CD8 hinge domain were highly activated without specific activation, and the excessive non-specific activation lowered the cytokine secretion and the cytotoxic activities against CD19-expressing tumor cells. Although the low level activation of CAR-T cells may contribute the long term survival of T cells in vivo, it is desired to select the optimal antibodies and CAR constructs which shows high antigen-specific activities with low non-specific activities for the safe and effective CAR-T cell therapy.

Adoptive immunotherapy using TCR gene-modified T-lymphocytes is an attractive strategy for targeting malignancies. However, TCR mispairings between endogenous and introduced TCR chains are a major concern, as they may induce mixed TCRs with unknown specificities and may reduce the expression of therapeutic TCRs. To overcome these problems, we have recently established a novel retroviral siTCR vector encoding small-interfering RNAs (siRNAs) to knockdown endogenous TCR genes for the efficient expression of therapeutic TCRs. In this study, to improve the efficacy of siTCR vectors, we developed 2A peptide-based siTCR vectors that could increase the expression levels of transduced TCRs compared with internal promoter-based siTCR vectors. We also evaluated the efficacy of an siTCR strategy and the addition of a new interchain disulfide bond created by cysteine modification. We found that the effect of the cysteine modification depended on TCR variations, while the siTCR strategy improved the expression of all TCRs tested. Furthermore, the combined effect of the siTCR and cysteine modification strategies was highly significant for certain TCRs. Therefore, our novel siTCR technology, in isolation or in combination with another strategy, may open the door to effective immunotherapy for cancer patients.Molecular Therapy - Nucleic Acids (2012) 1, e63. doi:10.1038/mtna.2012.52; published online 18 December 2012.

Adoptive cell therapy with lymphocytes transduced with chimeric antigen receptor (CAR) is a promising strategy to treat cancer patients. Recent success in the treatment of patients with B cell malignancy by CD19-CAR encourages the development of successive CAR therapy targeting other tumor-associated antigens. However, the search for the appropriate target molecule for CAR, other than B cell markers, is a serious question. The target of CAR is generally limited to the cellular surface molecules, making difficult to expand CAR therapy for broad range of cancer patients. Inspired by the physiological recognition of epitope peptide and MHC molecule (pMHC) by T cells, we have generated a series of antibodies that recognize the pMHC complexes with peptides derived from tumor antigens expressed intracellularly. We isolated an scFv antibody clone WT#213 that can specifically recognize WT1 p235-243 peptide (CMTWNQMNL) complexed with HLA-A*24:02 molecule by the screening of human antibody scFv phage display library. We have constructed retrovirus that encodes the CAR consists of WT#213 and intracellular signal transduction domains of CD3z and GITR (WT#213 CAR). We confirmed the specific recognition of endogenous WT1-expressing cells by the CAR-T cells with the intracellular cytokine staining and the 51Cr release cytotoxic assay. Utilizing NOG immunodeficient mice, we demonstrated the effectiveness of adoptive cell therapy with WT#213 CAR against the WT1 expressing HLA-A*24:02 positive human leukemia cells. To evaluate the safety of the WT#213 CAR, we predicted the potential property of WT#213 CAR to cross-react to normal tissues in humans. We conducted alanine scan analysis of WT1p235-243 peptide that was recognized by WT#213 CAR as well as the TCR derived from CTL clone TAK-1 which recognizes same epitope peptide in association with HLA-A*24:02 to define the amino acids that were critically important in the recognition by the WT#213 CAR or TAK-1-derived TCR. After BLAST search, we synthesized the normal protein-derived peptides with potential risk of cross-reactivity, and tested the recognition of these peptides by WT#213 CAR or TAK-1-derived TCR. Although the critical peptides, and therefore the peptides with potential risk, were quite different between the WT#213 CAR and TAK-1-derived TCR, none of these peptides showed the stimulation of WT#213 CAR or TAK-1-derived TCR. The results here suggest that the immunotherapy with WT#213 CAR will be effective for the treatment of the leukemia patients without the predicted risk at least in the evaluation we performed. Figure 1. Figure 1. Disclosures Ikeda: Takara Bio Inc.: Research Funding. Akahori:Takara Bio Inc.: Research Funding. Miyahara:Takara Bio Inc.: Research Funding. Amaishi:Takaa Bio Inc.: Employment. Okamoto:Takara Bio Inc.: Employment. Mineno:Takara Bio Inc.: Employment. Takesako:TAKARA BIO INC.: Employment. Fujiwara:Celgene: Honoraria, Other: Travel, Acomodations, Expenses.