<div>Abstract<p>Chimeric antigen receptor T (CAR-T) cells targeting multiple antigens (Ag), may reduce the risk of immune escape following the loss of the target Ag and further increase the efficacy of treatment. We developed dual-targeting CAR-T cells that target CD19 and CD37 Ags and evaluated their antitumor effects. CD19/CD37 dual CAR-T cells were generated using cotransduction and simultaneous gene transfer of two types of lentiviral vectors transferring CD19CAR or CD37CAR genes, including the intracellular domains of CD28 and CD3ζ signaling domains. These dual CAR-T cells contained three fractions: CD19/CD37 bispecific CAR-T cells, single CD19CAR-T cells, and single CD37CAR-T cells. In the functional evaluation of CAR-T cells <i>in vitro</i>, CD19/CD37 dual CAR-T cells showed adequate proliferation and cytokine production in response to CD19 and CD37 antigen stimulation alone or in combination. Evaluation of intracellular signaling revealed that dual CAR-T cell–mediated signals were comparable with single CAR-T cells in response to CD19- and CD37-positive B-cell tumors. Although the cytotoxicity of CD19/CD37 dual CAR-T cells in both CD19- and CD37-positive B-cell tumors was similar to that of single CD19 and CD37CAR-T cells, against CD19 and CD37 Ag-heterogeneous tumor, dual CAR-T cells demonstrated significantly superior tumor lysis compared with single CAR-T cells. Furthermore, CD19/CD37 dual CAR-T cells effectively suppressed Ag-heterogeneous Raji cells in a xenograft mouse model. Collectively, these results suggest that CD19/CD37 dual CAR-T cells may be effective target-Ag-loss B-cell tumor models <i>in vitro</i> and <i>in vivo</i>, which represents a promising treatment for patients with relapsed/refractory B-cell malignancies.</p></div>
A 55-year-old Asian man was diagnosed as mantle cell lymphoma in 2004. He received conventional chemotherapy followed by autologous peripheral blood stem cell transplantation and achieved complete remission in 2004. The disease relapsed in 2007, and he underwent bone marrow transplantation from an unrelated donor after conditioning with fludarabine 30 mg/m once daily i.v. for 5 days (total dose 150 mg/m) and cyclophosphamide 1 g/m once daily i.v. for 2 days (total dose 2 g/m). He received tacrolimus and shortterm methotrexate for graft-vs-host disease (GVHD) prophylaxis and achieved complete remission on day 89. On day 663, at the age of 61, he was referred to the emergency department of Nagoya University Hospital because of a 2day history of fever and appetite loss. On initial evaluation, he was febrile (temperature : 38.5C) with a pulse rate of 113 beats/min, blood pressure of 97/69 mmHg, respiratory rate of 20 breaths/min, and saturation of 88% at room air. He required 3 L of supplemental oxygen, which was supplied using a face mask, to maintain an oxygen saturation of 99%>A nasopharyngeal swab collected in the emergency department was negative for influenza A by rapid antigen testing. The patient had been immunized against seasonal influenza, but not against H1N1 influenza. On arrival at the hematology-oncology department, lymphadenopathy or skin rash was not observed in the patient. He did not take any immunosuppressive agents, having no sign of chronic GVHD at that time. Initial laboratory findings showed a hemoglobin level of 12.4 g/dL, hematocrit of 35. 9%, platelet count of 141,000/mm , and white blood cell (WBC) count of 13,200/mm with an absolute neutrophil count of 6,700/mm. The chemistry profile showed that sodium, potassium, chloride, blood urea nitrogen, creatinine, glucose, asparate aminotransferase (AST), alanine aminotransferase (ALT), and lactate dehydrogenase (LDH) were all within normal limits. The C-reactive protein (CRP) level was 7.6 mg/dL. Serum endotoxin and b-D glucan were negative. Serum immunoglobulin G, A, M level was 2,242, 27 and 156 mg/dL, respectively, and maintains the comparable level throughout the course. Soluble interleukin-2 receptor level was 1,360 U/mL. A chest radiograph was normal. A computed tomography scan of the chest was also normal. The patient showed no evidence of recurrence of lymphoma. A blood culture was obtained, and he received a dosage of intravenous imipenem/cilastatin (0.5 g×2/day). On hospital day 9, the patient’s clinical condition worsened with progressive dyspnea and hypoxia. The nasopharyngeal swab tested positive for influenza A by rapid antigen testing. A definite diagnosis was based on a positive result for pandemic H1N1 influenza virus by real-time reverse transcription-PCR (RT-PCR) for a nasopharyngeal swab. Oseltamivir (75 mg×2/day) was started on hospital day 9. Repeat laboratory data showed pancytopenia with a hemoglobin level of 9.3 g/dL, hematocrit of 28.1%, platelet count of
Umbilical cord blood (UCB) transplantation is limited by the low number of hematopoietic stem cells in UCB units, which results in a low engraftment rate in transplant recipients. Here, we measured the total nucleated cell count and CD34+, CD3+, CD4+, CD8+, CD14+, and CD16+/56+ cell doses in each UCB unit and evaluated their influence on engraftment and other outcomes in 146 recipients. Multivariate analysis showed a significant association between a higher incidence of successful engraftment and a dose of CD34+ and CD8+ cells above the median (1.4 × 105 and 15.7 × 105 cells/kg, respectively). Engraftment occurred 4 days earlier in patients who received UCB with more than the median dose of CD34+ cells than those receiving UCB at or below the median. Stratification of the group according to CD34+ cell dose revealed a significant influence of the CD8+ cell dose on the time to achieve neutrophil engraftment in patients receiving a lower CD34+ cell dose, whereas there was no significant influence in the patients receiving a higher CD34+ cell dose. These results suggest that consideration of CD34+ and CD8+ cell doses in UCB units may improve the engraftment in recipients of UCB transplantation.
