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For successful chimeric antigen receptor T (CAR-T) cell therapy, CAR-T cells must be manufactured without failure caused by suboptimal expansion. In order to determine risk factors for CAR-T cell manufacturing failure, we performed a nationwide cohort study in Japan and analysed patients with diffuse large B-cell lymphoma (DLBCL) who underwent tisagenlecleucel production. We compared clinical factors between 30 cases that failed (7.4%) with those that succeeded (n = 378). Among the failures, the proportion of patients previously treated with bendamustine (43.3% vs. 14.8%; p, CAR-T細胞製造を成功させるためのレシピ --アフェレーシス前の下ごしらえでの工夫--. 京都大学プレスリリース. 2023-04-27.

pRetroX-TetOne vector sequence with CD19CAR and tEGFR

T cells genetically modified with a CD19 chimeric antigen receptor (CD19CAR) are remarkably effective against B-cell malignancies in clinical trials. However, major concerns remain regarding toxicities, such as hypogammaglobulinemia, due to B-cell aplasia or severe cytokine release syndrome after overactivation of CAR T cells. To resolve these adverse events, we aimed to develop an inducible CAR system by using a tetracycline regulation system that would be activated only in the presence of doxycycline (Dox). In this study, the second-generation CD19CAR was fused into the third-generation Tet-On vector (Tet-CD19CAR) and was retrovirally transduced into primary CD8+ T cells. Tet-CD19CAR T cells were successfully generated and had minimal background CD19CAR expression without Dox. Tet-CD19CAR T cells in the presence of Dox were equivalently cytotoxic against CD19+ cell lines and had equivalent cytokine production and proliferation upon CD19 stimulation, compared with conventional CD19CAR T cells. The Dox(+) Tet-CD19CAR T cells also had significant antitumor activity in a xenograft model. However, without Dox, Tet-CD19CAR T cells lost CAR expression and CAR T-cell functions in vitro and in vivo, clearly segregating the “On” and “Off” status of Tet-CD19CAR cells by Dox administration. In addition to suicide-gene technology, controlling the expression and the functions of CAR with an inducible vector is a potential solution for CAR T-cell therapy–related toxicities, and may improve the safety profile of CAR T-cell therapy. This strategy might also open the way to treat other malignancies in combination with other CAR or TCR gene–modified T cells. Cancer Immunol Res; 4(8); 658–68. ©2016 AACR.See related Spotlight by June, p. 643.

Adoptively transferred CD19 chimeric antigen receptor (CAR) T cells have led to impressive clinical outcomes in B cell malignancies. Beyond induction of remission, the persistence of CAR-T cells is required to prevent relapse and provide long-term disease control. To improve CAR-T cell function and persistence, we developed a composite co-stimulatory domain of a B cell signaling moiety, CD79A/CD40, to induce a nuclear translocating signal, NF-κB, to synergize with other T cell signals and improve CAR-T cell function. CD79A/CD40 incorporating CD19CAR-T cells (CD19.79a.40z) exhibited higher NF-κB and p38 activity upon CD19 antigen exposure compared with the CD28 or 4-1BB incorporating CD19CAR-T cells (CD19.28z and CD19.BBz). Notably, we found that CD19.79a.40z CAR-T cells continued to suppress CD19(+) target cells throughout the co-culture assay, whereas a tendency for tumor growth was observed with CD19.28z CAR-T cells. Moreover, CD19.79a.40z CAR-T cells exhibited robust T cell proliferation after culturing with CD19(+) target cells, regardless of exogenous interleukin-2. In terms of in vivo efficiency, CD19.79a.40z demonstrated superior anti-tumor activity and in vivo CAR-T cell proliferation compared with CD19.28z and CD19.BBz CD19CAR-T cells in Raji-inoculated mice. Our data demonstrate that the CD79A/CD40 co-stimulatory domain endows CAR-T cells with enhanced proliferative capacity and improved anti-tumor efficacy in a murine model.

Although anti-retroviral therapy (ART) is successful in suppressing HIV-1 replication, HIV latently infected reservoirs are not eliminated, representing a major hurdle in efforts to eradicate the virus. Current strategies to eradicate HIV involve two steps: (1) the reactivation of latently infected cells with latency reversing agents (LRAs) to expose persisting HIV, and (2) the elimination of these cells with immune effectors while continuing ART to prevent reinfection. HIV-specific T cells (HSTs) can kill reactivated HIV-infected cells and are currently being evaluated in early-stage immunotherapy trials. HIV can mutate sequences in T cell epitopes and evade T cell-mediated killing of HIV-infected cells. However, by directing T cells to target multiple conserved, non-escaped HIV epitopes, the opportunity for viral escape can be reduced. Using a good manufacturing practice (GMP)-compliant platform, we manufactured HSTs against non-escape epitope targets (HST-NEETs) from HIV+ and HIV-seronegative donors. HST-NEETs expanded to clinically relevant numbers, lysed autologous antigen-pulsed targets, and showed a polyfunctional pro-inflammatory cytokine response. Notably, HST-NEETs recognized multiple conserved, non-escaped HIV epitopes and their common variants. We propose that HST-NEETs could be used to eliminate reactivated virus from latently infected cells in HIV+ individuals following LRA treatment. Additionally, HST-NEETs derived from HIV-negative individuals could be used post-transplant for HIV+ individuals with hematologic malignancies to augment anti-viral immunity and destroy residual infected cells. Keywords: HIV-specific T cells, immunotherapy, latent reservoir, viral escape, conserved epitopes

Genomic deletion of donor-patient-mismatched HLA alleles in leukemic cells is a major cause of relapse after allogeneic hematopoietic stem cell transplantation (HSCT). Mismatched HLA is frequently lost as an individual allele or a whole region in HLA-class I, however, it is downregulated in HLA-class II. We hypothesized that there might be a difference in T cell recognition capacity against epitopes associated with HLA-class I and HLA-class II and consequently such allogeneic immune pressure induced HLA alterations in leukemic cells. To investigate this, we conducted in vitro experiments with T cell receptor-transduced T (TCR-T) cells. The cytotoxic activity of NY-ESO-1-specific TCR-T cells exhibited similarly against K562 cells with low HLA-A*02:01 expression. However, we demonstrated that the cytokine production against low HLA-DPB1*05:01 expression line decreased gradually from the HLA expression level approximately 2-log lower than normal expressors. Using sort-purified leukemia cells before and after HSCT, we applied the next-generation sequencing, and revealed that there were several marked downregulations of HLA-class II alleles which demonstrated consistently low expression from pre-transplantation. The marked downregulation of HLA-class II may lead to decreased antigen recognition ability of antigen-specific T cells and may be one of immune evasion mechanism associated with HLA-class II downregulation.

Owing to the difficulty in isolating T cells from human biopsy samples, the characteristics of T cells that are infiltratinghuman acute graft-versus-host disease (GVHD) tissues remain largely uninvestigated. In the present study, TCR-β deep sequencing of various GVHD tissue samples and concurrent peripheral blood obtained from transplant recipients was performed in combination with functional assays of tissue-infiltrating T cell clones. The T cell repertoire was more skewed in GVHD tissues than in the peripheral blood. The frequent clonotypes differed from tissue to tissue in the same patient, and the frequent clonotypes in the same tissue differed from patient to patient. Two T cell clones were successfully isolated from GVHD skin of a patient. In a cytotoxicity assay, both Tcell clones lysed patient peripheral blood mononuclear cells, but not donor-derived Epstein-Barr virus-transformed lymphoblastoid cells. Their clonotypes were identical to the most and second most frequent T cell clonotypes in the original GVHD skin and accounted for almost all of the skin-infiltrating T cells. These results suggest that human acute GVHD may result from only a few different alloreactive cytotoxic T cell clones, which differ from tissue to tissue and from patient to patient. The characterization of T cells infiltrating human GVHD tissues should be further investigated.

A 56-year-old woman was referred to our hospital with symptoms of swelling, purpura, and pain in her limbs. Prior to referral, bleeding in her limbs had spontaneously appeared and disappeared several times. Prothrombin time (PT) and activated partial thromboplastin time (aPTT) were prolonged, and the factor II level was 17%. The plasma-mixing test indicated lupus anticoagulant (LA), which was confirmed using aPTT-LA and dilute Russell's viper venom time (dRVVT). Therefore, she was diagnosed with lupus anticoagulant-hypoprothrombinemia syndrome (LAHPS). During screening for underlying disorders, chest computed tomography (CT) revealed a retrosternal mass. Biopsy was not performed because the administration of freshly frozen plasma failed to correct her coagulopathy. Prednisolone (PSL) treatment (1 mg/kg) was initiated, which normalized the coagulation tests. The retrosternal mass also disappeared. PSL was tapered without LAHPS recurrence; however, the follow-up CT revealed systemic lymphadenopathy. Follicular lymphoma was diagnosed using lymph-node biopsy. Considering the subsequent LAHPS recurrence, six cycles of bendamustine + rituximab were administered. Complete response with no LAHPS recurrence was observed at the time of drafting this report. LAHPS is rare and distinct from antiphospholipid syndrome because it can cause severe bleeding. Underlying disorders should be evaluated in cases of LAHPS.

Introduction: Chimeric antigen receptor-modified T cells targeting CD19 (CD19.CAR-T cells) have shown clinical success in patients with hematological malignancies. Despite the encouraging results obtained with this novel therapy, a major concern to its global spread, particularly in developing countries, is its high cost. We developed a method of non-viral gene transfer using piggyBac transposon to reduce the cost of CAR-T therapy. In preclinical study, the median number and transduction efficiency of CAR-T cells obtained from 2x10 7 PBMC in 9 donors were 1.0x10 8 (range, 0.58-1.8x10 8) and 51% (range, 29-73%), respectively. The major subset of CAR-T cells was phenotypically CD8+CD45RA+CCR7+, closely related T-memory stem cells. Ex vivo, CD19.CAR-T cells showed cytotoxic effect on CD19 positive tumor cell lines. In NSG mice model, CD19.CAR-T cells successfully inhibit tumor growth. CAR gene integration sites were determined by inverse polymerase chain reaction and subsequent next-generation sequencing using MiSeq and equally distributed throughout the genome without preference for specific sites. The pre-clinical testing in mouse demonstrated safe toxicity profile at the 50 times dose of CD19.CAR-T cells. We started a human clinical trial to define feasibility, toxicity, maximum tolerated dose and clinical response of CD19.CAR-T cells (jRCTa040190099). Methods: We report the results of cohort 1 of the study in which the safety and efficacy of autologous CD19.CAR-T in patients with relapsed or refractory B-precursor acute lymphoblastic leukemia were evaluated. We engineered autologous T cells via the piggyBac transposon system with CD19.CAR-expression transposon vector and piggyBac transposase-expression vector to express CD19.CAR incorporating CD28 costimulatory domain. We designed this phase I trial using a modified 3 + 3 design to enroll 3-12 patients with relapsed or refractory acute lymphoblastic leukemia in both children and adults. In this study, patients in cohorts 1 (16-60 years old) and 2 (1-15 years old) receive 1 × 10 5 CAR-transduced T cells per kg. Patients in cohorts 3 and 4 (both 1-60 years old) receive 3 × 10 5 and 1 × 10 6 CAR-transduced T cells per kg, respectively. All patients receive 25mg/m 2/d of fludarabine and 250mg/m 2/d of cyclophosphamide for 3 days followed by a single infusion of CAR-T cells. Results: Three patients were enrolled in cohort 1 and infused with 1 × 10 5 CAR-transduced T cells per kg. All patients had previously undergone allogeneic hematopoietic stem cell transplantation. All patients had achieved a hematological complete response with salvage treatment before CAR-T therapy. None of the patients had dose-limiting toxicities (DLT) defined as nonhematological toxicities above grade 4 or cytokine release syndrome (CRS) above grade 4 or graft versus host disease (GVHD) above 4, or grade 3 nonhematological toxicities and GVHD not improved to grade 2 within 4 weeks after CAR-T infusion. There was no occurrence of non-hematological adverse events above grade 3. CRS was observed in one patient (grade 1) who also developed headache due to infiltration of CAR-T cells into the spinal fluid. In two patients, B cell aplasia lasted 2 and 9 months, respectively. Elevation of serum cytokine levels was observed in all patients and the peak time point was 7-21 days after CAR-T cell infusion. Conclusions: CD19.CAR-T cell infusion produced by the piggyBac transposon gene engineering system was safe in cohort 1 of our study. As no patients had DLT in cohort 1, we are enrolling the patients in further cohorts. Disclosures Murata: MSD: Honoraria; Kyowa Kirin: Honoraria; Sumitomo Dainippon Pharma: Honoraria; FUJIFILM: Honoraria; Toyama Chemical: Honoraria; Novartis: Honoraria; JCR Pharmaceutical: Honoraria; Astellas: Honoraria; Miyarisan Pharmaceutical: Honoraria; Asahi Kasei: Honoraria; GlaxoSmithKline: Honoraria; Celgene: Honoraria; Otsuka Pharmaceutical: Honoraria.

Objective/background: Here, we described the clinical characteristics and outcomes of central nervous system (CNS) infections occurring after allogeneic hematopoietic stem cell transplantation (allo-HSCT) in a single institution over the previous 6 years. Methods: Charts of 353 consecutive allogeneic transplant recipients were retrospectively reviewed for CNS infection. Results: A total of 17 cases of CNS infection were identified at a median of 38 days (range, 10–1028 days) after allo-HSCT. Causative pathogens were human herpesvirus-6 (n = 6), enterococcus (n = 2), staphylococcus (n = 2), streptococcus (n = 2), varicella zoster virus (n = 1), cytomegalovirus (n = 1), John Cunningham virus (n = 1), adenovirus (n = 1), and Toxoplasma gondii (n = 1). The cumulative incidence of CNS infection was 4.1% at 1 year and 5.5% at 5 years. Conclusion: Multivariate analysis revealed that high-risk disease status was a risk factor for developing CNS infection (p = .02), and that overall survival at 3 years after allo-HSCT was 33% in patients with CNS infection and 53% in those without CNS infection (p = .04). Keywords: Allogeneic hematopoietic stem cell transplantation, Central nervous system infections, Human herpesvirus 6

PURPOSE Tumor-associated antigen cytotoxic T cells (TAA-Ts) represent a new, potentially effective and nontoxic therapeutic approach for patients with relapsed or refractory solid tumors. In this first-in-human trial, we investigated the safety of administering TAA-Ts that target Wilms tumor gene 1, preferentially expressed antigen of melanoma, and survivin to patients with relapsed/refractory solid tumors. MATERIALS AND METHODS TAA-T products were generated from autologous peripheral blood and infused over three dose levels: 1, 2, and 4 × 107 cells/m2. Patients were eligible for up to eight infusions administered 4 to 7 weeks apart. We assessed dose limiting toxicity during the first 45 days after infusion. Disease response was determined within the context of a phase I trial. RESULTS There were no dose-limiting toxicities. Of 15 evaluable patients, 11 (73%) with stable disease or better at day 45 postinfusion were defined as responders. Six responders remain without progression at a median of 13.9 months (range, 4.1 to 19.9 months) after initial TAA-Ts. Patients who were treated at the highest dose level showed the best clinical outcomes, with a 6-month progression-free survival of 73% after TAA-T infusion compared with a 38% 6-month progression-free survival with prior therapy. Antigen spreading and a reduction in circulating tumor-associated antigens using digital droplet polymerase chain reaction was observed in patients after TAA-T infusion. CONCLUSION TAA-Ts safely induced disease stabilization, prolonged time to progression, and were associated with antigen spreading and a reduction in circulating tumor-associated antigen DNA levels in patients with relapsed/refractory solid tumors without lymphodepleting chemotherapy before infusion. TAA-Ts are a promising new treatment approach for patients with solid tumors.

Background Chronic norovirus infection in immunocompromised patients can be severe, and presently there is no effective treatment. Adoptive transfer of virus-specific T cells has proven to be safe and effective for the treatment of many viral infections, and this could represent a novel treatment approach for chronic norovirus infection. Hence, we sought to generate human norovirus-specific T cells (NSTs) that can recognize different viral sequences. Methods Norovirus-specific T cells were generated from peripheral blood of healthy donors by stimulation with overlapping peptide libraries spanning the entire coding sequence of the norovirus genome. Results We successfully generated T cells targeting multiple norovirus antigens with a mean 4.2 ± 0.5-fold expansion after 10 days. Norovirus-specific T cells comprised both CD4+ and CD8+ T cells that expressed markers for central memory and effector memory phenotype with minimal expression of coinhibitory molecules, and they were polyfunctional based on cytokine production. We identified novel CD4- and CD8-restricted immunodominant epitopes within NS6 and VP1 antigens. Furthermore, NSTs showed a high degree of cross-reactivity to multiple variant epitopes from clinical isolates. Conclusions Our findings identify immunodominant human norovirus T-cell epitopes and demonstrate that it is feasible to generate potent NSTs from third-party donors for use in antiviral immunotherapy.

Background Zika virus (ZIKV) infection can cause severe birth defects in newborns with no effective currently available treatment. Adoptive transfer of virus-specific T cells has proven to be safe and effective for the prevention or treatment of many viral infections, and could represent a novel treatment approach for patients with ZIKV infection. However, extending this strategy to the ZIKV setting has been hampered by limited data on immunogenic T-cell antigens within ZIKV. Hence, we have generated ZIKV-specific T cells and characterized the cellular immune responses against ZIKV antigens. Methods T-cell products were generated from peripheral blood of ZIKV-exposed donors, ZIKV-naive adult donors and umbilical cord blood by stimulation with pentadecamer (15mer) overlapping peptide libraries spanning four ZIKV polyproteins (C, M, E and NS1) using a Good Manufacturing Practice–compliant protocol. Results We successfully generated T cells targeting ZIKV antigens with clinically relevant numbers. The ex vivo –expanded T cells comprised both CD4 + and CD8 + T cells that were able to produce Th1-polarized effector cytokines and kill ZIKV-infected HLA-matched monocytes, confirming functionality of this unique T-cell product as a potential “off-the-shelf” therapeutic. Epitope mapping using peptide arrays identified several novel HLA class I and class II–restricted epitopes within NS1 antigen, which is essential for viral replication and immune evasion. Discussion Our findings demonstrate that it is feasible to generate potent ZIKV-specific T cells from a variety of cell sources including virus naive donors for future clinical use in an “off-the-shelf” setting.

Programmed death-ligand 1 (PD-L1) binds to programmed death-1 (PD-1) on activated T cells and contributes to T-cell exhaustion. PD-L1 expressed on antigen-presenting cells (APCs) could be thought to inhibit the induction of Ag-specific cytotoxic T lymphocytes (CTLs) by transducing negative signal into T cells; however, the roles of PD-L1 on APCs have not yet been well examined. Therefore, we evaluated the roles of PD-L1 on APCs in the induction of Ag-specific CTLs. CD3 T cells isolated from cytomegalovirus (CMV)-seropositive healthy donors were stimulated with mature dendritic cells pulsed with CMV pp65-derived HLA-restricted peptides in the presence of anti-PD-L1 blocking antibody. Unexpectedly, PD-L1 blockade resulted in a less efficient induction of CMV-specific CTLs, suggesting that PD-L1 play a positive role in the induction of Ag-specific CTLs. For further evaluations and application to adoptive immunotherapy, we generated K562-based artificial APCs, which were retrovirally transduced with HLA class I molecules and various combinations of CD80/86 and PD-L1. K562/HLA+CD80/86+PD-L1 cells produced significantly higher induction of CMV-specific CTLs than K562/HLA or K562/HLA+CD80/86 cells without causing excessive differentiation or functional exhaustion of the induced CTLs, whereas PD-L1 itself did not have a stimulatory effect. Furthermore, only K562/HLA+CD80/86+PD-L1 cells pulsed with HLA-A*24:02-restricted Wilms tumor 1 (WT1) peptide clearly expanded WT1-specific CTLs from healthy donors. Our findings presumed that PD-L1 expressed on APCs along with CD80/86 enhanced the induction of Ag-specific CTLs probably depending on fine-tuning excessive stimulation of CD80/86, and that K562/HLA+CD80/86+PD-L1 cells has therapeutic potential as a novel type of artificial APCs for adoptive immunotherapy.

T cells genetically modified with a CD19 chimeric antigen receptor (CD19CAR) are remarkably effective against B-cell malignancies in clinical trials. However, major concerns remain regarding toxicities, such as hypogammaglobulinemia, due to B-cell aplasia or severe cytokine release syndrome after overactivation of CAR T cells. To resolve these adverse events, we aimed to develop an inducible CAR system by using a tetracycline regulation system that would be activated only in the presence of doxycycline (Dox). In this study, the second-generation CD19CAR was fused into the third-generation Tet-On vector (Tet-CD19CAR) and was retrovirally transduced into primary CD8+ T cells. Tet-CD19CAR T cells were successfully generated and had minimal background CD19CAR expression without Dox. Tet-CD19CAR T cells in the presence of Dox were equivalently cytotoxic against CD19+ cell lines and had equivalent cytokine production and proliferation upon CD19 stimulation, compared with conventional CD19CAR T cells. The Dox(+) Tet-CD19CAR T cells also had significant antitumor activity in a xenograft model. However, without Dox, Tet-CD19CAR T cells lost CAR expression and CAR T-cell functions in vitro and in vivo, clearly segregating the “On” and “Off” status of Tet-CD19CAR cells by Dox administration. In addition to suicide-gene technology, controlling the expression and the functions of CAR with an inducible vector is a potential solution for CAR T-cell therapy–related toxicities, and may improve the safety profile of CAR T-cell therapy. This strategy might also open the way to treat other malignancies in combination with other CAR or TCR gene–modified T cells. Cancer Immunol Res; 4(8); 658–68. ©2016 AACR. See related Spotlight by June, p. 643.

Background Zika virus (ZIKV) infection during pregnancy can cause severe birth defects, with no effective treatment. Adoptive transfer of virus-specific T-cells has proven to be safe and effective for the prevention or treatment of many viral infections, and could represent a novel treatment approach for ZIKV infection. However, this approach for ZIKV infection has been hampered by limited data on immunogenic T-cell antigens within ZIKV. Hence, we have generated ZIKV-specific T-cells and characterized the cellular immune responses against ZIKV antigens. Methods T-cell products were generated from blood of ZIKV-exposed donors (n=3), ZIKV-naive adult donors (n=6), and umbilical cord blood (n=7) by stimulation with overlapping peptide libraries (15mers overlapping by 11 amino acids) spanning four ZIKV polyproteins (C, M, E, and NS1) using a GMP-compliant protocol. Results We successfully generated T-cells targeting ZIKV antigens with a mean 56.0-fold T cell expansion at 17 days for ZIKV-exposed donors and 21 days for ZIKV-naive donors and cord blood donors. The resultant products specifically recognized C, M, E, and NS1 proteins by IFN-γ ELISpot, with mean 142.5 spot forming cells (SFCs)/1 × 105 cells, 81.7 SFCs/1 × 105 cells, 132.1 SFCs/1 × 105 cells, and 180.7 SFCs/1 × 105 cells, respectively, compared to 5.4 SFCs/1 × 105 cells for irrelevant antigen (Fig 1). ZIKV-specific T-cells were polyclonal (16.2±2.8% CD3+CD4+ and 32.4±2.4% CD3+CD8+ T-cells) and expressed markers for both central and effector memory phenotype (3.5±0.6% CD45RO+CD62L+ and 48.1±1.9% CD45RO+CD62L–), with minimal expression of PD-1, Tim-3, Lag-3, and CTLA-4. Notably, both CD4+ and CD8+ subsets were polyfunctional comprising ZIKV-directed T-cell populations, which secreted multiple cytokines (16.7±0.6% CD4+IFN-γ+TNF-α+ and 7.4±0.4% CD8+IFN-γ+TNF-α+) as measured by intracellular cytokine staining. Moreover, multiplex cytokine assay demonstrated specific production of IFN-γ (928.7±239.8 pg/mL), TNF-α (1495.5±453.3 pg/mL), IL-2 (38.2±8.4 pg/mL), and GM-CSF (67.0±22.2 pg/mL) in response to ZIKV antigens. Importantly, ZIKV-specific T-cells selectively killed autologous monocytes infected with ZIKV confirming functionality of this unique T cell therapeutic. Epitope mapping using peptide arrays in IFN-γ ELISpot identified several novel HLA class I or class II-restricted epitopes within NS1, which is essential for viral replication and immune evasion. Conclusions Our findings demonstrate that it is feasible to generate potent ZIKV-specific T-cells from a variety of cell sources including virus naive donors for future clinical use in an “off the shelf’ setting.

Although recent studies of virus-specific T-cell (VST) therapy for viral infections after allogeneic hematopoietic stem cell transplantation have shown promising results, simple and less time-intensive and labor-intensive methods are required to generate VSTs for the wider application of VST therapy. We investigated the efficacy of anti-CD28 and anti-4-1BB antibodies, which can provide T cells with costimulatory signals similar in strength to those of antigen-presenting cells, in generating VSTs. When peripheral blood mononuclear cells were stimulated with viral peptides together with isotype control, anti-CD28, or anti-4-1BB antibodies, anti-4-1BB antibodies yielded the highest numbers of VSTs, which were on an average 7.9 times higher than those generated with isotype control antibody. The combination of anti-CD28 and anti-4-1BB antibodies did not result in increased numbers of VSTs compared with anti-4-1BB antibody alone. Importantly, the positive effect of anti-4-1BB antibody was observed regardless of the epitopes of the VSTs. In contrast, the capacity of dendritic cells (DCs) to generate VSTs differed considerably depending on the epitopes of the VSTs. Furthermore, the numbers of VSTs generated with DCs were at most similar to those generated with the anti-4-1BB antibody. Generation of VSTs with anti-4-1BB antibody did not result in excessive differentiation or deteriorated function of the generated VSTs compared with those generated with control antibody or DCs. In conclusion, VSTs can be generated rapidly and efficiently by simply stimulating peripheral blood mononuclear cells with viral peptide and anti-4-1BB antibody without using antigen-presenting cells. We propose using anti-4-1BB antibody as a novel strategy to generate VSTs for adoptive therapy.

Tobacco smoking is associated with infectious pulmonary complications after allogeneic hematopoietic stem cell transplantation

We retrospectively reviewed the medical records of 45 patients with relapsed acute leukemia after initial allogeneic hematopoietic stem cell transplantation (allo-HSCT). Among 45 patients, a total of 11 patients eventually underwent second allo-HSCT (HSCT-2). Median survival after relapse was 294 days (range, 135-942 days) for HSCT-2. Multivariate analysis showed significantly better survival for recipients of second allo-HSCT than for other patients (hazard ratio, 4.38; 95 % confidence interval, 1.45-13.2; P = 0.009). Although outcomes for patients with relapsed leukemia were generally poor, our results suggest that second HSCT could offer a survival advantage over other conventional salvage strategies.

Regenerating islet-derived protein 3 alpha (REG3A) is a biomarker of lower gastrointestinal graft-versus-host disease (GVHD); however, the biological role of REG3A in the pathophysiology of GVHD is not understood. Here, we examined the association between a single nucleotide polymorphism in the REG3A gene, rs7588571, which is located upstream and within 2 kb of the REG3A gene, and transplant outcomes including the incidence of GVHD. The study population consisted of 126 adult Japanese patients who had undergone bone marrow transplantation from a HLA-matched sibling. There was no association between rs7588571 polymorphism and the incidence of acute GVHD. However, a significantly higher incidence of extensive chronic GVHD was observed in patients with the rs7588571 non-GG genotype than in those with the GG genotype (Odds ratio 2.6; 95% confidence interval, 1.1–6.0; P = 0.029). Semi-quantitative reverse transcription PCR demonstrated that the rs7588571 non-GG genotype exhibited a significantly lower REG3A mRNA expression level than the GG genotype (P = 0.032), and Western blot analysis demonstrated that the rs7588571 non-GG genotype exhibited a trend toward lower REG3A protein expression level than the GG genotype (P = 0.053). Since REG proteins have several activities that function to control intestinal microbiota, and since intestinal dysbiosis is in part responsible for the development of GVHD, our findings lead to the novel concept that REG3A could have some protective effect in the pathogenesis of GVHD through the regulation of gut microbiota.

In allo-stem cell transplantation (SCT), it is unclear whether donor-specific anti-HLA Abs (DSAs) can actually mediate graft rejection or if they are simply surrogate markers for the cellular immunity that causes graft rejection. Here, we first analyzed a case of cord blood allograft rejection in which DSA and cytotoxic T lymphocyte (CTL) specific for donor HLA-B*54:01 were detected at the time of graft rejection. Both the DSA and CTL inhibited colony formation by unrelated bone marrow mononuclear cells sharing HLA-B*54:01, suggesting that the humoral and cellular immune responses were involved in the graft rejection. Interestingly, the DSA and CTL were also detected in cryopreserved pre-transplant patient blood, raising a hypothesis that the presence of anti-HLA Abs could be an indicator for corresponding HLA-specific T cells. We then evaluated the existence of HLA-specific CD8(+) T cells in other patient blood specimens having anti-HLA class I Abs. Interferon-γ enzyme-linked immunospot assays clearly confirmed the existence of corresponding HLA-specific T-cell precursors in three of seven patients with anti-HLA Abs. In conclusion, our data demonstrate that integrated humoral and cellular immunity recognizing the same alloantigen of the donor can mediate graft rejection in DSA-positive patients undergoing HLA-mismatched allo-SCT. Further studies generalizing our observation are warranted.

[Introduction] Regenerating islet-derived protein 3 alpha (REG3A), a protein produced by Paneth cells, has been reported as a biomarker of lower gastrointestinal graft-versus-host disease (GVHD). The single nucleotide polymorphism (SNP) rs7588571 is located at the upstream of REG3A gene within 2kb, suggesting that the expression of REG3A might be affected by the SNP. In this study, we analyzed the association between the rs7588571 genotype in recipients and transplant outcomes. [Patients and methods] The study population included 118 adult Japanese patients who had undergone T cell replete allogeneic bone marrow transplantation from an HLA-identical sibling donor with GVHD prophylaxis consisting of short-term methotrexate and cyclosporine between 1987 and 2006. Sixty-eight (58%) male and 50 (42%) female patients, with a median age of 35 years (range 15-56), were included. Eighty-eight (75%) patients were classified as having standard risk disease defined as acute myeloid leukemia in first or second complete remission, acute lymphoblastic leukemia without Philadelphia chromosome in first complete remission, chronic myeloid leukemia in chronic phase, myelodysplastic syndrome without excess of blasts, aplastic anemia and paroxysmal nocturnal hemoglobinuria, while other patients were classified as having high risk disease. Conditioning regimen was myeloablative in 113 (96%) patients and reduced-intensity in other patients. Genomic DNA was extracted from patient's peripheral blood or bone marrow by standard methods. Genotyping was performed using TaqMan SNP Genotyping Assays (Applied Biosystems, Foster City, California, USA) on ABI 7300 Real-Time PCR systems (Applied Biosystems). Multiple logistic regression model was used to identify risk factors associated with acute and chronic GVHD, and multivariate Cox model was created for overall survival (OS), non-relapse mortality (NRM), and relapse. Variables with a P-value < 0.1 in the univariate analysis were entered into the stepwise selection method. All tests were two-sided, and P [Results] The frequencies of the GG, AG, and AA genotypes were 41%, 43%, and 16%, respectively, which were compatible with those reported in the HapMap-JPT database. There was no significant association between the distribution pattern of the rs7588571 genotypes and each patient characteristic. Grade II-IV acute GVHD developed in 12% of all patients, 14% in those with GG genotype, 14% with AG genotype and 11% with AA genotype, respectively. In univariate analysis, the higher patient age (≥35) was the only significant factor for higher incidence of grade II-IV acute GVHD (OR: 5.4, 95% CI: 1.3-32, P=0.011). Extensive chronic GVHD developed in 38% of all patients, 27% in those with GG genotype, 45% with AG genotype and 50% with AA genotype, respectively. The incidence of extensive chronic GVHD in non-GG genotype (46%) was significantly higher than that in GG genotype (27%) (P=0.05). In multivariate analysis, non-GG genotype (OR: 2.5, 95% CI: 1.0-5.8, P=0.04), female to male transplant (2.8, 1.2-7.0, 0.023), and high risk disease (2.8, 1.1-7.2, 0.038) were significant factors for higher incidence of extensive chronic GVHD. The relapse rates at 4-year were 26% in non-GG and 17% in GG genotype (HR: 1.16, 95% CI: 0.49-2.7, P=0.73). High risk disease was the only significant factor for higher relapse rate. The OS and NRM at 4-year were 57% and 20% in non-GG, and 65% and 17% in GG genotype, respectively. The rs7588571 genotypes affected neither OS nor NRM. [Discussion] REG3A polymorphism was associated with the incidence of extensive chronic GVHD. Given its capacity to control the tissue regeneration and intestinal microbiota, REG3A may not only reflect the intestinal damage caused by GVHD but also have a certain role in the pathogenesis of GVHD. Our findings need to be confirmed in larger cohort, and further investigation is required in order to clarify the biological effect of rs7588571 genotypes and the exact role of REG3A in the pathophysiology of GVHD. Disclosures Kiyoi: MSD K.K.: Research Funding; Kyowa-Hakko Kirin Co.LTD.: Research Funding; Nippon Shinyaku Co., Ltd.: Research Funding; Fujifilm Corporation: Patents & Royalties, Research Funding; Yakult Honsha Co.,Ltd.: Research Funding; Zenyaku Kogyo Co.LTD.: Research Funding; Eisai Co., Ltd.: Research Funding; Novartis Pharma K.K.: Research Funding; Takeda Pharmaceutical Co., Ltd.: Research Funding; Phizer Japan Inc.: Research Funding; Alexion Pharmaceuticals: Research Funding; Toyama Chemikal Co.,Ltd.: Research Funding; Sumitomo Dainippon Pharma Co., Ltd.: Research Funding; AlexionpharmaLLC.: Research Funding; Astellas Pharma Inc.: Consultancy, Research Funding; Mochida Pharmaceutical Co., Ltd.: Research Funding; JCR Pharmaceutlcals Co.,Ltd.: Research Funding; Nippon Boehringer Ingelheim Co., Ltd.: Research Funding; Celgene Corporation: Consultancy; Chugai Pharmaceutical Co. LTD.: Research Funding.

Introduction: Endogenous or gene-modified T cells with T cell receptors (TCRs) are major designs of cytotoxic T lymphocytes (CTL) therapies. However, gene-modified TCR-CTL therapies targeting on malignant tumors have been generally unsuccessful in previous clinical trials. The major reasons were poor expansion and short persistence of CTL. To overcome these problems, TCR-affinity have been genetically enhanced, but often resulted in life-threatening toxicities. We aimed to modify TCR-CTL in order to improve the function by enhancing intracellular signaling after TCR ligation without affinity modulation. Method: We generated three artificial T cell activating adapter molecules (ATAM), which are CD3z, CD3z/CD28, and CD3z/4-1BB. CD28 or 4-1BB intracellular domain was inserted in the middle of CD3z intracellular domain, so that they can assemble with internal TCR complex when stimulated with corresponding antigen. ATAM-T2A-tEGFR was packaged into retrovirus vector to transduce the T cells. In order to evaluate the functional alteration, we established ATAM-CMV CTL model. We stimulated T cells with CMV pp65 peptide to induce endogenous CMV CTL. On day 4 after stimulation, ATAMs were retrovirally transduced into CMV CTL. After a selection with anti-EGFR mAb, we achieved more than 90% purity of ATAM positive CMV CTL and used in 51Cr release assay, cytokine release assay, and the growth assay. In the growth assay, we stimulated the ATAM CMV-CTL weekly and assayed the viable cell number, tetramer positivity, phenotype, and apoptosis. Regarding these assay, we used HLA transduced K562 (HLA-K562) as APC. Results: First, we confirmed the expression of each ATAM on CD8+ T cells, which showed similar expression among each ATAM. To determine the cytotoxicity of CMV CTL according to each ATAM or control tEGFR transduction, we performed 51Cr release assay against HLA-K562 pulsed with or without CMV peptide. In the presence of CMV peptide, each ATAM-CMV CTL showed similar cytolytic activities to control CMV CTL. [specific lysis (%): tEGFR 32.0+-6.0, CD3z 32.4+-2.6, CD3z/CD28 31.1+-8.8, and CD3z/4-1BB 37.4+-1.5: at an E:T ratio = 3:1, respectively}. In the intracellular cytokine assay against HLA-K562 pulsed with CMV peptide, similar proportions of responder were positive for IFN-g and IL-2 in control and ATAM-CMV CTL. [IFN-g (%): tEGFR, 67.3+-9.4, CD3z, 69.0+-7.4, CD3z/CD28, 66.5+-8.0, and CD3z/4-1BB, 69.6+-7.9; IL-2 (%): tEGFR, 17.5+-5.0, CD3z, 17.7+-4.3, CD3z/CD28, 15.9+-4.3, and CD3z/4-1BB, 18.9+-4.8, respectively]. The cytotoxicity and cytokine secretion upon irrelevant stimulation were completely negative among each ATAM-CMV CTL. Although the surface phenotype was not changed in control CMV-CTL, that of ATAMs-CMV CTL was significantly expanded after antigen stimulation [central memory (CD45RA-CD62L+) (%): tEGFR, 6.0+-2.9, CD3z, 26.8+-11.1, CD3z/CD28, 19.4+-9.5, and CD3z/4-1BB, 35.8+-26.8: at 7 days after stimulation]. Regarding the cell growth, CD3z/CD28 and CD3z/4-1BB showed greater CTL expansion upon weekly antigen stimulation. The fold expansion of tEGFR, CD3z, CD3z/CD28, and CD3z/4-1BB-CMV CTL were 4.9+-0.7, 8.2+-2.4, 12.3+-1.1, and 13.0+-1.6 at day7, 1.6+-0.4, 8.0+-2.5, 18.6+-3.9, and 21.3+-7.7 at day14, and 0.4+-0.2, 1.4+-0.7, 5.1+-3.0, and 30.4+-22.9 at day21, respectively (Fig). Comparing with control, CD3z/4-1BB-CMV CTL showed continuous greater expansion after several stimulations (p˂0.01: during day0 to day7, p˂0.05: during day7 to day14). To examine the reason for the growth advantage, we investigated the apoptosis after the stimulation with DAPI/Annexin V staining. It revealed that the live cells after specific stimulation elevated in ATAMs-CMV CTL compared with control CMV CTL. Conclusion: We generated three ATAMs and successfully transduced into CMV CTL. Particularly, the whole cell growth and continuance of growth after weekly stimulation were significantly improved in CD3z/4-1BB CTL. This greater cell expansion may improve in vivo persistence and further effectiveness of TCR-CTL therapy. ATAM-CMV CTL did not recognize any APC without corresponding peptide, indicating that ATAM transduction could improve the CTL effectiveness without altering CTL specificity. Thus we believe that we could potentially apply ATAM transduction to other TCR-CTLs targeting malignant tumors safely. Figure Figure. Disclosures Kiyoi: Alexion Pharmaceuticals: Research Funding; Novartis Pharma K.K.: Research Funding; Mochida Pharmaceutical Co., Ltd.: Research Funding; Toyama Chemikal Co.,Ltd.: Research Funding; Sumitomo Dainippon Pharma Co., Ltd.: Research Funding; AlexionpharmaLLC.: Research Funding; JCR Pharmaceutlcals Co.,Ltd.: Research Funding; Nippon Boehringer Ingelheim Co., Ltd.: Research Funding; MSD K.K.: Research Funding; Takeda Pharmaceutical Co., Ltd.: Research Funding; Phizer Japan Inc.: Research Funding; Yakult Honsha Co.,Ltd.: Research Funding; Eisai Co., Ltd.: Research Funding; Astellas Pharma Inc.: Consultancy, Research Funding; Nippon Shinyaku Co., Ltd.: Research Funding; Fujifilm Corporation: Patents & Royalties, Research Funding; Zenyaku Kogyo Co.LTD.: Research Funding; Kyowa-Hakko Kirin Co.LTD.: Research Funding; Chugai Pharmaceutical Co. LTD.: Research Funding; Celgene Corporation: Consultancy.

Programmed death-ligand 1 (PD-L1) binds to programmed death-1 (PD-1) on T cells and contributes to T cell exhaustion during chronic infections. Moreover, it has been shown that engagement of PD-1 on T cells by PD-L1 on tumor cells is associated with the immune escape of tumors, and recent clinical trials have highlighted the anti-tumor efficacy of blockade of the PD-1/PD-L1 pathway. Along with costimulatory molecules such as CD80 and CD86, PD-L1 is also highly expressed on dendritic cells (DCs), which are professional antigen-presenting cells (APCs) that are widely used to generate antigen-specific cytotoxic T lymphocytes (CTLs) for adoptive immunotherapy. However, the roles of PD-L1 on APCs have not been well examined. Thus, we evaluated the roles of PD-L1 on APCs in the induction of cytomegalovirus (CMV)-specific CTLs (CMV-CTLs). First, peripheral blood mononuclear cells (PBMCs) were obtained from five CMV-seropositive healthy donors (HLA-A*02:01, n = 3; A*24:02, n = 1; B*07:02, n = 1). Mature DCs were generated from adherent PBMCs by stimulation with GM-CSF/IL-4 and maturational cytokines (TNF-a, IL-1b, IL-6, and PGE2), and freshly isolated CD3+ T cells were stimulated with mature DCs pulsed with CMV pp65-derived HLA-restricted peptides (NLVPMVATV for HLA-A*02:01, QYDPVAALF for A*24:02, and TPRVTGGGAM for B*07:02) in the presence or absence of anti-PD-L1 blocking antibody. After 14 days of culture, the presence of anti-PD-L1 antibody resulted in a less efficient induction of CMV-CTLs (Figure 1), suggesting that PD-L1 might play a positive role in the induction of antigen-specific CTLs. For further evaluation, we generated K562-based artificial APCs (aAPCs), which were retrovirally transduced with HLA class I molecules and various combinations of CD80/86 and PD-L1 (named K562, K562+CD80/86, K562+PD-L1, and K562+CD80/86+PD-L1). CD3+ T cells isolated from nine CMV-seropositive healthy donors (HLA-A*02:01, n = 4; A*24:02, n = 4; B*07:02, n = 1) were stimulated weekly for 28 days using HLA-restricted CMV peptide-pulsed K562-based aAPCs. K562+CD80/86+PD-L1 led to significantly higher induction of CMV-CTLs than K562 or K562+CD80/86 (Figure 2). Since the original K562 cells slightly express CD80, we completely knocked out the CD80 expression in the cells using the CRISPR/Cas9 system, and then we transduced the cells with or without PD-L1 (named CD80KO K562+PD-L1 and CD80KO K562, respectively) to determine whether PD-L1 itself has a stimulatory effect on the induction of CMV-CTLs. CD80KO K562 and CD80KO K562+PD-L1 induced the same amount of CMV-CTLs, suggesting that PD-L1 itself does not have a stimulatory effect for the induction of CMV-CTLs. Phenotypic analysis showed that CMV-CTLs induced by K562+CD80/86+PD-L1 contained more CD45RA- CD62L+ central memory T cells, which persist longer and are more effective in vivo after adoptive transfer in CTL therapy, than CMV-CTLs induced by other K562-based aAPCs. In addition, when compared with CMV-CTLs induced by other K562-based aAPCs, CMV-CTLs induced by K562+CD80/86+PD-L1 did not express higher levels of exhaustion markers such as PD-1 and TIM-3, and exhibited similar levels of IFN-gamma and IL-2 production and CD107a expression in response to the specific peptide stimulation. Furthermore, to evaluate the effect of K562-based aAPCs on the induction of tumor antigen-specific CTLs, CD3+ T cells were isolated from three HLA-A*24:02-positive healthy donors and stimulated with HLA-A*24:02-restricted WT1 peptide-pulsed K562-based aAPCs weekly. After four stimulations, only K562+CD80/86+PD-L1 could clearly expand the WT1-specific CTLs from all of the three donors (Figure 3). In conclusion, our findings demonstrate that PD-L1 expressed on APCs along with CD80/86 enhanced the induction of antigen-specific CTLs without causing excessive differentiation or functional exhaustion of the induced CTLs. These results suggest that stimulation with K562+CD80/86 might result in the activation-induced cell death of antigen-specific CTLs and that PD-L1 might be involved in fine-tuning excessive stimulation of CD80/86. Further analyses to determine the mechanisms of our findings are warranted. Our results also highlight the possibility that K562+CD80/86+PD-L1 has therapeutic potential as novel aAPCs for the generation of CTLs for adoptive immunotherapy. Disclosures Kiyoi: Takeda Pharmaceutical Co., Ltd.: Research Funding; Pfizer Inc.: Research Funding; Teijin Ltd.: Research Funding; Taisho Toyama Pharmaceutical Co., Ltd.: Research Funding; Eisai Co., Ltd.: Research Funding; Yakult Honsha Co.,Ltd.: Research Funding; MSD K.K.: Research Funding; Alexion Pharmaceuticals: Research Funding; Novartis Pharma K.K.: Research Funding; Astellas Pharma Inc.: Consultancy, Research Funding; Japan Blood Products Organization: Research Funding; Nippon Shinyaku Co., Ltd.: Research Funding; FUJIFILM RI Pharma Co.,Ltd.: Research Funding; Nippon Boehringer Ingelheim Co., Ltd.: Research Funding; FUJIFILM Corporation: Patents & Royalties, Research Funding; Zenyaku Kogyo Co., Ltd.: Research Funding; Sumitomo Dainippon Pharma Co., Ltd.: Research Funding; Kyowa Hakko Kirin Co., Ltd.: Consultancy, Research Funding; Bristol-Myers Squibb: Research Funding; Chugai Pharmaceutical Co., Ltd.: Research Funding; Mochida Pharmaceutical Co., Ltd.: Research Funding.

Introduction: Genetic modification of T cells with chimeric antigen receptor (CAR) has emerged with astonishing treatment outcomes for B cell malignancies. Clinical trials of CAR-T therapy demonstrated toxicities such as hypogammaglobulinemia due to B cell aplasia or hemophagocytic syndrome after overactivation of CAR-T cells. These toxicities are considered as major drawbacks for broader application of CAR-T therapy. To overcome these serious adverse events, further modification of CAR-T technology to control CAR expression arbitrary is needed. Therefore we aimed to develop inducible CAR expressing T cells based on tetracycline-regulation system. Methods: We developed a novel inducible CD19CAR system by infusing anti-CD19-CD3z-CD28-tEGFR into pRetroX-TetOne vector (Tet-19CAR). By using Tet-19CAR transduced SUPT1 (T cell line), expression and disappearance kinetics of CAR were determined. We also retrovirally transduced Tet-19CAR into human CD8+ T cells, and achieved more than 90% purity of CAR positive T cells after a selection with anti-EGFR mAb. These CAR-T cells were again expanded with anti-CD3/28 beads and used in 51 Cr release assay, coculture assay, cytokine release assay and T cell proliferation assay. Regarding coculture assay, CD19 transduced K562-CD19 (K562-CD19) was labeled with 0.1 nM CFSE and plated with CAR-T cells at a ratio of 1:1 without IL-2 supplementation and incubated for 96 hours. Finally we examined this system in NOG mice. We injected 0.5 x 106 Raji-ffluc (fire-fly luciferase) followed by 5.0 x 106 CAR-T cells from the tail vein, then we evaluated the tumor flux by in vivo imaging system on days 7, 14, 21, and 30. Results: With more than 100 ng/mL of Doxycycline (Dox), CD19CAR was successfully expressed on both of SUPT1 and CD8+ T cells. For maximum and minimum expression, 24 and 72 hours were needed after addition and discontinuation of Dox, respectively. To determine the cytotoxicity of Tet-19CAR-T cells according to presence or absence of Dox, we performed 51 Cr release assay and coculture assay against K562-CD19. In the presence of Dox, Tet-19CAR showed an equivalent lytic activity to conventional CD19CAR-T cells (c19CAR). In contrast, Tet-19CAR without Dox exhibited significantly lower cytotoxicity against CD19+ target cells. (Dox (-) Tet-19CAR, Dox (+) Tet-19CAR and c19CAR: 14.0±4.0%, 38.0±4.0% and 37.0±2.0% at an E:T ratio = 10:1, respectively). In the coculture assay, Tet-19CAR with Dox eradiated K562-CD19, while they failed to suppress the target cells without Dox. In the intracellular IFN-g assay against K562-CD19, a similar proportion of responder was IFN-g + in Tet-19CAR with Dox and c19CAR. On the other hand, a significantly low proportion of IFN-g + cells were observed in Tet-19CAR without Dox. (Dox (-) Tet-19CAR, 1.0%±0%, Dox (+) Tet-19CAR, 19.1%±6.0% and c19CAR 21.5%±4.0%, respectively) Similar to intracellular IFN-g assay, ELISA revealed that Tet-19CAR with Dox and c19CAR produced IL-2 and IFN-g equally well. However, Tet-19CAR without Dox hardly did. [IL-2 (ng/ml): Dox (-) Tet-19CAR, 1.00±0.060, Dox (+) Tet-19CAR, 9.25±0.30 and c19CAR 8.75±0.68; IFN-g (ng/ml): 2.32±1.24, 57.96±6.95 and 62.42±5.95] (Fig). We next analyzed CAR-T cell proliferation upon stimulation with K562-CD19 over 96 hours. Tet-19CAR with Dox showed 6-7 fold expansion, whereas Tet-19CAR without Dox failed to proliferate. Regarding in vivo model, the mice treated with c19CAR or Tet-19CAR with Dox showed significantly low tumor flux but the mice treated with Tet-19CAR without Dox showed higher tumor burden at day 21 of CAR-T cell infusion [Photons/sec: Dox (-) Tet-19CAR, 2.5 x 1010, Dox (+) Tet-19CAR, 6.4 x 108 and c19CAR, 8.4 x 108 ]. Conclusions: We generated tetracycline-inducible CAR-T cells and successfully controlled the CAR expression with Dox administration. Tet-19CAR without Dox still demonstrated some CD19CAR expression and subsequent cytotoxicity against CD19 positive cells. Nonetheless the CAR expression level of Tet-19CAR without Dox was lower than the threshold for exhibiting positive responses in the function assays such as cytokine production and proliferation. This phenomenon was also confirmed in the xenograft model. To regulate CAR expression more precisely and pursue clinical translations in combinations with other CARs, further efforts are needed to reduce any leaky CAR expression by modification of the system. Figure 1. Figure 1. Disclosures Kiyoi: Pfizer Inc.: Research Funding; Eisai Co., Ltd.: Research Funding; Yakult Honsha Co.,Ltd.: Research Funding; Alexion Pharmaceuticals: Research Funding; MSD K.K.: Research Funding; Takeda Pharmaceutical Co., Ltd.: Research Funding; Taisho Toyama Pharmaceutical Co., Ltd.: Research Funding; Teijin Ltd.: Research Funding; Astellas Pharma Inc.: Consultancy, Research Funding; Japan Blood Products Organization: Research Funding; Nippon Shinyaku Co., Ltd.: Research Funding; FUJIFILM RI Pharma Co.,Ltd.: Research Funding; Nippon Boehringer Ingelheim Co., Ltd.: Research Funding; FUJIFILM Corporation: Patents & Royalties, Research Funding; Zenyaku Kogyo Co., Ltd.: Research Funding; Sumitomo Dainippon Pharma Co., Ltd.: Research Funding; Kyowa Hakko Kirin Co., Ltd.: Consultancy, Research Funding; Bristol-Myers Squibb: Research Funding; Chugai Pharmaceutical Co., Ltd.: Research Funding; Novartis Pharma K.K.: Research Funding; Mochida Pharmaceutical Co., Ltd.: Research Funding.

We report a case of Epstein-Barr virus (EBV)-associated lymphoproliferative disease (LPD) following unrelated bone marrow transplantation (UBMT) for severe aplastic anemia treated with a conditioning regimen that included anti-thymocyte globulin (ATG). The patient showed signs of EBV reactivation as early as 34 days after UBMT. Our weekly schedule for EBV monitoring failed to trace rapid changes in EBV viral load and the patient eventually developed EBV-LPD. However, early intervention with monoclonal antibody against CD20, rituximab, stopped the further progression of EBV-LPD. As several recent reports have suggested, the safety and efficacy of rituximab treatment for EBV-LPD is supported by our limited experience with post transplant EBV-LPD.

Recent statistical analyses of the clinical outcome have revealed that the presence of donor-specific anti-HLA antibodies (DSAs) in pre-transplant recipient is correlated with increased risk for graft rejection after allogeneic hematopoietic stem cell (HSC) transplantation. However, while cytotoxic T lymphocytes (CTLs) recognizing mismatched HLA molecule of the donor have been shown to be involved in graft rejection, there has been no biological evidence in humans that graft rejection is mediated by DSAs. In the present study, we demonstrate a case of cord blood allograft rejection in which DSAs acted to mediate graft rejection. Interestingly, CTL clones specific for donor HLA molecule were also isolated, suggesting that humoral and cellular immune responses were together responsible for allograft rejection. A patient with graft rejection after HLA-mismatched cord blood transplantation was studied. The white blood cell count increased transiently, subsequently decreased to undetectable level, and finally graft rejection was diagnosed on day 34. Screening for pre-transplant anti-HLA antibodies was not routinely performed at that time. We initially presumed that DSA would be responsible for graft rejection. The patient serum on day 36 were screened for HLA antibodies and further evaluated to determine HLA specificities using a LABScreen Single Antigen Kit. An antibody against HLA-B* 54:01, which was expressed in donor cells but not in patient cells, was detected. To test the effect of the DSA against HLA-B* 54:01 on HSC engraftment, antibody-dependent cellular cytotoxicity (ADCC) activity was assessed using unrelated HLA-B* 54:01-positive bone marrow mononuclear cells (BMMNCs) pre-cultured with patient serum and pre-transplant NK cells. The patient serum clearly showed an inhibitory effect on colony formation. Complement-dependent cytotoxicity activity of the DSA could not be detected. These data suggest that the DSA impaired the cord blood engraftment through ADCC activities. We next determined if cellular immunity was also responsible for allograft rejection. Two independent CTL clones, CD4-CD8+ and CD4+CD8-, were isolated from the patient blood at the time of graft rejection by limiting dilution. Both CTL clones were of patient origin by using short tandem repeat analysis and showed cytotoxicity against donor cells but not patient cells in Cr release assay. A CTL stimulation assay using COS cells transfected with various mismatched donor HLA cDNA constructs demonstrated that CD8+ and CD4+CTL clones recognized HLA-B* 54:01 and -DRB1* 15:02 molecules, respectively, both of which were expressed in donor cells but not in patient cells. To test the effect of the CTL clones on engraftment, a colony forming assay using either HLA-B* 54:01 or -DRB1* 15:02-positive unrelated BMMNCs pre-cultured with the corresponding CTL clones was performed. Each of these CTL clones inhibited colony formation from unrelated BMMNCs sharing target HLA in a dose-dependent manner. These data suggest that the CTLs also impaired the cord blood engraftment. Furthermore, anti-HLA-B* 54:01 antibody was detected in cryopreserved pre-transplant patient serum, and in nested PCR assays specific for the HLA-B* 54:01-specific CTL clone’s uniquely rearranged T cell receptor V beta chain, PCR product was detected by amplification of cDNA from pre-transplant patient peripheral blood mononuclear cells. Thus, both HLA-B* 54:01-specific antibody and CTL clone developed in the patient prior to transplantation. The present data provide the first direct evidence showing that humoral and cellular immune responses were involved in the graft rejection. Of note is the recognition of the same mismatched HLA-B* 54:01 molecule by the DSA and CTL, suggesting that humoral and cellular immune processes acted together to mediate allograft rejection. Given the difficulty in detecting HLA-specific CTLs in pre-transplant patient blood in contrast to the easiness in screening for DSAs, the presence of DSA not only means a direct deleterious effect on donor cells but it may also reflect the presence of CTLs that causes allograft rejection. Further studies are warranted to clarify whether the present observation can be duplicated in other patients with DSA. We are conducting the study to clarify whether patients with anti-HLA antibodies have CTLs that recognize the same HLA molecules before transplantation. Disclosures: No relevant conflicts of interest to declare.

Background In chimeric antigen receptor transduced T-cell (CAR-T cell) therapy, setting of the target antigen is critical in terms of both the efficacy and the possible adverse effects. However, how low expressing antigen CAR-T cells can recognize and present cytotoxicity, and how low expressing antigen would be a candidate for the target or should be avoided for the concern of off-organ effect is still unclear. Thus we developed novel anti-CD20 CAR-T cells, and investigated the threshold antigen expression level required to activate CAR-T cells. Methods In this study, we generated a retrovirus vector that encodes a novel CAR consisting of anti-CD20 single chain variable fragments linked to a CD3-zeta chain, a CD28 costimulatory domain, and a truncated epidermal growth factor receptor (tEGFR) as a transduction and selection marker. CD3 positive T cells from a healthy donor were activated with anti-CD3/28 beads, transduced with the CAR on day 3, enriched by selection with anti-EGFR mAb, and expanded by stimulation with gamma-irradiated B-lymphoblastoid cells. CD20 expression level of the target cells was evaluated with CD20 mean fluorescence intensity (CD20-MFI) and was quantified as CD20 specific antibody biding capacity (CD20-ABC). Results To determine the threshold expression level of CD20 antigen to induce cytotoxicity, we performed 51Cr release assay by anti-CD20 CAR-T cells against 30 clones of CD20 transduced CCRF-CEM (CD20-CEM) expressing variable levels of CD20 (CD20-MFI: 126-6,924/ CD20-ABC: 240-230,546 molecules) (kindly provided by Dr. A.C.M. Martens, University Medical Center Utrecht). CAR-T cells lysed CD20-CEMs equally well from low to high level of CD20 (CD20MFI: 157/ CD20ABC: 5,172 molecules or more, 40-60% of lysis at E:T ratio of 10:1), and lysed the clone expressing the lowest level of CD20 (126/ 240 molecules, 22.8±2% of lysis). However, in rituximab-induced CDC assay against the same 30 clones, a minimum CD20 MFI of 600 (ABC of 65,000 molecule) was required to induce CDC. Next, cytokine production and proliferation upon stimulation were investigated by using four representative CD20-CEM clones out of the 30 clones that cover a wide range of CD20 expression levels; Very low (VL-CEM) (MFI: 126/ ABC:240 molecules), Low (L-CEM)(252/ 26,990 molecules), Mid (M-CEM)(826/ 91,567 molecules), High (H-CEM)(6,924/ 142,722 molecules). In intracellular IFN-gamma assay, CAR-T cells produced IFN-gamma equally well (L-CEM, 45.9%, M-CEM, 35.4% and H-CEM, 38.0%, respectively), except against VL-CEM (0.2%). In CFSE division assay, CAR-T cells proliferated upon stimulation with L-CEM, M-CEM, H-CEM (31.4%, %36.1, 37.3% at 72h and, 88.9%, 90.2%, 91.1% at 96h), except with LV-CEM (0% at 72h/ 96h). In intracellular signaling assay, phosphorylation of downstream signaling molecule, CD3-zeta and ERK were evaluated. Whereas only minimal phosphorylations of CD3-zeta and ERK were observed upon VL-CEM stimulation, other 3 CEMs induced similar strength of signals in the majority of CAR-T cells, and induced phosphorylation of proximal signaling molecule, CD3-zeta according to the CD20 expression level. Finally, we evaluated cytotoxicity against low-CD20 expressing primary tumor cells and cell lines. CAR-T cells lysed RRBL1 which is a cell line established from a patient who exhibited relapse of B-cell lymphoma with very weak CD20 expression and became resistant to rituximab (47.8±3% of lysis at E:T ratio 10:1) and primary DLBCL cells isolated from pleural effusion with low CD20 expression (32.6±2.9% of lysis). Conclusions We observed that CAR-T cells recognize CD20 antigen with considerably low expression. A minimum number of antigen molecules required to activate CAR-T cells is very low; the threshold is a few hundreds of target antigen. For the future search of a novel target of CAR-T therapy, antigens with expression above the threshold, even if that is below the range of mAb therapy, could be also considered. Conversely, antigens with off-organ expression above the threshold may induce off-organ effect, thus should be avoided. CD20-CAR therapy may be also effective for the patients with CD20 down-regulated lymphoma and refractory to CD20 mAb therapy. Disclosures: No relevant conflicts of interest to declare.

Abstract 1150 Poster Board I-172 Introduction ABO-incompatibility is not generally considered to be a risk factor of hematopoietic stem cell transplantation (HSCT). However, in our single-institute study of 568 patients, the incidence of acute GVHD was higher in minor-ABO incompatible HSCT than others (45.3% vs 35.2%, p=0.019). We made a hypothesis that there is a potential association between donor-derived isohemagglutinin (IH) and the outcomes of minor-ABO incompatible HSCT. Patients and method There were 1052 patients undergone HSCT between 1988 and 2009 at Komagome hospital. We analyzed 130 of 1052 patients (12.4%), who had received minor- ABO incompatible HSCT. Blood type and anti-A/B antibody titers were evaluated at least weekly in first one month after HSCT, and were repeated monthly until blood type had completely changed to the donor type. Our analysis was especially focused on the impact of IH on the outcome of minor-ABO incompatible HSCT. Result Minor ABO-incompatible HSCT were 130 cases that included AML (n=40), ALL (n=28), CML (n=27), MDS (n=15), SAA (n=9), NHL (n=7), MM (n=2) and ATL (n=2). Median age was 37 (range: 16-67) years old. Seventy-seven patients were male, 53 were female. Stem cell sources were bone marrow (n=96), peripheral blood (n=16) and umbilical cord blood (n=18). Eleven cases were HLA-matched and 9 were HLA-mismatched transplants. Anti-host IgG and IgM IH had been detected in 20 of 130 transplants undergoing minor ABO-incompatible HSCT (15.4%). Median time to IH detection was 13 days after HSCT (12-39days). There was a higher incidence of IH production in the HLA-mismatched group than in the HLA-matched group (p=0.007).There was also a higher incidence of IH production in the unrelated transplants group than in the related transplants group. (p=0.021). None of 18 patients receiving umbilical cord blood transplantation showed evidence of IH production. The incidence of grade II-IV acute GVHD was significantly higher (90% vs 60%, p Discussion and Conclusion Our study showed that IH production had association with HLA-mismatched and unrelated transplants in minor-ABO incompatible HSCT. We also showed that early detection of IH was related with the onset of grade II-IV acute GVHD. These findings indicate that strong allo-immunity induces not only severe acute GVHD caused by T lymphocytes but also antibody-production by B lymphocytes. In conclusion, we suggest that early detection of IH has impact on severity of acute GVHD. Disclosures No relevant conflicts of interest to declare.

Hematopoietic stem cell transplantation (HSCT) recipients have an increased risk of acute kidney injury (AKI) or chronic kidney disease (CKD). However, serum creatinine level may underestimate the prevalence of these renal complications because of decreased lean body mass or concurrent liver disease, which was frequently observed in a HSCT setting. Cystatin C measurement may be more sensitive for detecting impaired kidney function. We retrospectively reviewed the medical records of 70 HSCT (54 allogeneic and 16 autologous) recipients who had at least one chance to monitor serum cystatin C level during last 2 years in our institution, and evaluated cystatin C as a possible new marker which can predict subsequent renal dysfunction. The occurrence of AKI was defined by the RIFLE classification and CKD staging was based on KDOQI criteria. Of 70 transplant recipients, 20 patients developed AKI after median 50.5 days (range 0–283 days) after HSCT, while worsening CKD stage was observed in 21 patients during observational periods. Cystatin C level was not influenced by pretransplant disease status (P=0.153) or autologous transplant (P=0.311), but significantly elevated after allogeneic transplantation (P0.90mg/l) and the episode of AKI after transplantation were a great risk for substantially worsening CKD stage (Hazard ratio 4.21 and 16.0, P=0.042 and P