Spacer Length Modification Facilitates Discrimination between Normal and Neoplastic Cells and Provides Clinically Relevant CD37 CAR T Cells.

Despite the remarkable initial efficacy of CD19 chimeric Ag receptor T (CAR-T) cell therapy, a high incidence of relapse has been observed. To further increase treatment efficacy and reduce the rate of escape of Ag-negative cells, we need to develop CAR-T cells that target other Ags. Given its restricted expression pattern, CD37 was considered a preferred novel target for immunotherapy in hematopoietic malignancies. Therefore, we designed a CD37-targeting CAR-T (CD37CAR-T) using the single-chain variable fragment of a humanized anti-CD37 Ab, transmembrane and intracellular domains of CD28, and CD3ζ signaling domains. High levels of CD37 expression were confirmed in B cells from human peripheral blood and bone marrow B cell precursors at late developmental stages; by contrast, more limited expression of CD37 was observed in early precursor B cells. Furthermore, we found that human CD37CAR-T cells with longer spacer lengths exhibited high gene transduction efficacy but reduced capacity to proliferate; this may be due to overactivation and fratricide. Spacer length optimization resulted in a modest transduction efficiency together with robust capacity to proliferate. CD37CAR-T cells with optimized spacer length efficiently targeted various CD37+ human tumor cell lines but had no impact on normal leukocytes both in vitro and in vivo. CD37CAR-T cells effectively eradicated Raji cells in xenograft model. Collectively, these results suggested that spacer-optimized CD37CAR-T cells could target CD37-high neoplastic B cells both in vitro and in vivo, with only limited interactions with their normal leukocyte lineages, thereby providing an additional promising therapeutic intervention for patients with B cell malignancies.

Composite CD79A/CD40 co-stimulatory endodomain enhances CD19CAR-T cell proliferation and survival.

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.

[Development of autoimmune hemolytic anemia after BNT162b2 mRNA COVID-19 vaccination].

A 75-year-old woman with a history of postoperative chemotherapy for lung adenocarcinoma and a history of Helicobacter pylori eradication for idiopathic thrombocytopenic purpura (ITP) was admitted to the department of hematology and oncology for the treatment of anemia 2 weeks after BNT162b2 mRNA COVID-19 vaccination. Her blood examination revealed direct and indirect Coombs test-positive hemolytic anemia and elevation of serum LDH and indirect bilirubin levels. No obvious trigger other than BNT162b2 mRNA COVID-19 vaccination was found. She was diagnosed with autoimmune hemolytic anemia (AIHA), and oral prednisolone therapy was administered. The anemia improved soon after the administration of prednisolone. Although vaccination is considered to be very important for suppressing the spread of COVID-19, there have been reports of increasing risk of ITP development and deterioration caused by BNT162b2 mRNA COVID-19 vaccination. Because the number of vaccinated people is increasing rapidly, hematologists must be vigilant to the development of AIHA after BNT162b2 mRNA COVID-19 vaccination although case reports of this phenomenon have been very rare thus far.

Quantitative Assessment of T Cell Clonotypes in Human Acute Graft-versus-Host Disease Tissues.

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-ß 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.

Cervical epidural hematoma in a healthy donor presenting stroke mimic symptoms: a rare adverse event following peripheral blood stem cell apheresis.

Peripheral blood stem cell apheresis from a healthy donor is indispensable for allogeneic peripheral blood stem cell transplantation. Here, we report a rare adverse event following peripheral blood stem cell apheresis. A female sibling donor, aged 61 years with an unremarkable medical history, complained of pain in the left neck and shoulder and numbness in the left upper limb 1 h after the end of peripheral blood stem cell apheresis. Paralysis of the left upper and lower limbs appeared consecutively. Computed tomography and magnetic resonance imaging of the head showed no abnormalities. Anticoagulant therapy was initiated according to the standard treatment of atherothrombotic brain infarction. Magnetic resonance imaging of the cervical cord on the following day revealed a cervical epidural hematoma. An emergency C4-C5 laminectomy was performed, and the paralysis was improved immediately after surgery. This report is the first case of cervical epidural hematoma in a healthy donor who underwent peripheral blood stem cell apheresis and presented symptoms confusingly similar to those of brain infarction.

Dual CAR-T Cells Targeting CD19 and CD37 Are Effective in Target Antigen Loss B-cell Tumor Models.

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 in vitro, 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 in vitro and in vivo, which represents a promising treatment for patients with relapsed/refractory B-cell malignancies.

Enhanced Coarse-Grained Molecular Dynamics Simulation with a Smoothed Hybrid Potential Using a Neural Network Model.

In all-atom (AA) molecular dynamics (MD) simulations, the rugged energy profile of the force field makes it challenging to reproduce spontaneous structural changes in biomolecules within a reasonable calculation time. Existing coarse-grained (CG) models, in which the energy profile is set to a global minimum around the initial structure, are unsuitable to explore the structural dynamics between metastable states far away from the initial structure without any bias. In this study, we developed a new hybrid potential composed of an artificial intelligence (AI) potential and minimal CG potential related to the statistical bond length and excluded volume interactions to accelerate the transition dynamics while maintaining the protein character. The AI potential is trained by energy matching using a diverse structural ensemble sampled via multicanonical (Mc) MD simulation and the corresponding AA force field energy, profile of which is smoothed by energy minimization. By applying the new methodology to chignolin and TrpCage, we showed that the AI potential can predict the AA energy with significantly high accuracy, as indicated by a correlation coefficient (R-value) between the true and predicted energies exceeding 0.89. In addition, we successfully demonstrated that CGMD simulation based on the smoothed hybrid potential can significantly enhance the transition dynamics between various metastable states while preserving protein properties compared to those obtained with conventional CGMD and AAMD.

Artificial T Cell Adaptor Molecule-Transduced TCR-T Cells Demonstrated Improved Proliferation Only When Transduced in a Higher Intensity.

An artificial T cell adaptor molecule (ATAM) was generated to improve persistence of T cell receptor (TCR) gene-transduced T (TCR-T) cells compared to such persistence in a preceding study. ATAMs are gene-modified CD3ζ with the intracellular domain of 4-1BB inserted in the middle of CD3ζ. NY-ESO-1 TCR-T cells transduced with an ATAM with two separated virus vectors demonstrated superior proliferation upon antigen stimulation. To further develop clinically applicable ATAM-transduced TCR-T cells, we attempted to make a single virus vector to transduce the TCR and ATAM simultaneously. Because we failed to observe improved proliferation capacity upon stimulation after one virus vector (1vv) transduction, we compared TCR-T cells transduced with 1vv and two virus vector (2vv) methods to elucidate the reason. In Jurkat reporter cells, an ATAM transduced by the 2vv method demonstrated a higher intensity than by the 1vv method, and the ATAM intensity was associated with increased nuclear factor κB (NF-κB) signals upon stimulation. In ATAM-transduced primary T cells, a transduced ATAM by the 2vv method showed higher intensity and better proliferation. ATAM-transduced TCR-T cells demonstrated improved proliferation only when the ATAM was transduced at a higher intensity. To create a simpler transduction method, we need to develop a strategy to make a higher ATAM expression to prove the efficacy of ATAM transduction in TCR-T therapy.

Donor single nucleotide polymorphism in ACAT1 affects the incidence of graft-versus-host disease after bone marrow transplantation.

Acyl-coenzyme A: cholesterol acyltransferase 1 (ACAT1) is an enzyme that converts cholesterol to cholesteryl esters. A recent in vivo study reported that inhibiting ACAT1 enzyme activity upregulates the membrane cholesterol levels of T cells, enhancing their cytotoxic function. In the present study, we investigated whether the presence of the ACAT1 single nucleotide polymorphism rs11545566 in transplant donors affected the risk of graft-versus-host disease (GVHD) in 116 adult patients who underwent bone marrow transplantation from human leukocyte antigen-identical sibling donors, and who received GVHD prophylaxis with short-term methotrexate and cyclosporine. The frequencies of the AA, AG, and GG genotypes in the donors were 31%, 45%, and 24%, respectively. The cumulative incidences of grade II-IV acute GVHD on day 100 in patients whose donors had AA vs. non-AA genotypes were 6% and 18%, respectively, and those of extensive chronic GVHD at 2 years were 7% and 32%, respectively. Multivariate analyses demonstrated that donor rs11545566 non-AA genotypes showed a trend toward a higher incidence of grade II-IV acute GVHD (P = 0.079), and were significantly associated with a higher incidence of extensive chronic GVHD (P = 0.021). These results suggest that donor ACAT1 rs11545566 genotype may be predictive of GVHD.

Membrane-active antimicrobial poly(amino-modified alkyl) ß-cyclodextrins synthesized via click reactions.

The emergence of drug-resistant bacteria has led to the high demand for new antibiotics. In this report, we investigated membrane-active antimicrobial ß-cyclodextrins. These contain seven amino-modified alkyl groups on a molecule, which act as functional moieties to permeabilize bacterial cell membranes. The polyfunctionalization of cyclodextrins was achieved through a click reaction assisted by microwave irradiation. A survey using derivatives with systematically varied functionalities clarified the unique correlation of the antimicrobial activity of these compounds with their molecular structure and hydrophobicity/hydrophilicity balances. The optimum hydrophobicity for the compounds being membrane-active was specific to bacterial strains and animal cells; this led to specific compounds having selective toxicity against bacteria including multidrug-resistant pathogens. The results demonstrate that cyclodextrin is a versatile molecular scaffold for rationally designed structures and can be used for the development of new antibiotics.

Introduction of Genetically Modified CD3ζ Improves Proliferation and Persistence of Antigen-Specific CTLs.

The clinical efficacy of T-cell therapies based on T cells transduced with genes encoding tumor-specific T-cell receptors (TCR-T) is related to the in vivo persistence of the T cells. To improve persistence without modifying TCR affinity, we instead modified intracellular signaling, using artificial T cell-activating adapter molecules (ATAM), generated by inserting the intracellular domain (ICD) of activating T-cell signaling moieties into CD3ζ. ATAMs with the ICD of either CD28 or 4-1BB were generated, assembled into the TCR complex as a part of CD3ζ, and enhanced downstream signaling from the supramolecular activation cluster. ATAMs were retrovirally introduced into human CMV-specific or NY-ESO-1-specific TCR-transduced CD8+ T lymphocytes, and downstream functionality was then examined. ATAM-transduced NY-ESO-1 TCR-T cells were also investigated using the U266-xenograft mouse model. ATAMs were successfully transduced and localized to the cell membrane. ATAM-transduced CMV-specific T cells retained their cytotoxic activity and cytokine production against peptide-pulsed target cells without altering antigen-specificity and showed resistance to activation-induced cell death. Upon both single and repeated stimulation, CD3ζ/4-1BB-transduced T cells had superior proliferation to the CD3ζ-transduced T cells in both the CMV-specific and the NY-ESO-1 TCR-T models and significantly improved antitumor activity compared with untransduced T cells both in vitro and in a mouse xenograft model. ATAM-transduced TCR-T cells demonstrated improved proliferation and persistence in vitro and in vivo This strategy to control the intracellular signaling of TCR-T cells by ATAM transduction in combination with various tumor-specific TCRs may improve the efficacy of TCR-T therapy. Cancer Immunol Res; 6(6); 733-44. ©2018 AACR.

Impact of Synchronous Multiple Primary Malignant Tumors on Newly Diagnosed Hematological Malignancies.

BACKGROUND: Hematological malignancies are occasionally observed with synchronous multiple primary malignant tumors (sMPMTs) at diagnosis. We aimed to clarify the impact of sMPMTs on newly diagnosed hematological malignancies and determine the optimal treatment strategies. PATIENTS AND METHODS: We analyzed the outcomes of 649 patients with hematological malignancies, including 19 patients with sMPMTs (2.9%), and compared the outcomes between patients with and without sMPMTs. RESULTS: The overall survival (OS) and disease-free survival (DFS) rates for patients with sMPMTs were 77% and 70%, respectively, at 2 years; these rates were not statistically different from those for patients without sMPMTs (P = .17 and P = .64, respectively). Multivariate analysis showed that the presence of sMPMTs was not a significant prognostic factor for OS, DFS, or relapse (hazard ratio [HR] 1.48, 95% confidence interval [CI] 0.65-3.38, P = .35; HR 0.97, 95% CI 0.46-2.10, P = .97; and HR 0.79, 95% CI 0.29-2.14, P = .65). In patients with sMPMTs, the order of treatment was not a significant prognostic factor. However, discontinuation of treatment was a marginally favorable factor and might reflect a selection bias. CONCLUSION: The presence of sMPMTs was not a significant risk factor for patients with newly diagnosed hematological malignancies. It is important to provide adequate treatment for both hematological malignancies and solid tumors at the physician's discretion.

High incidence of extensive chronic graft-versus-host disease in patients with the REG3A rs7588571 non-GG genotype.

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.

Successful treatment with allogeneic stem cell transplantation followed by DLI and TKIs for e6a2 BCR-ABL-positive acute myeloid leukaemia: A case report and literature review.

RATIONALE: Patients with the e6a2 BCR-ABL transcript, 1 of the atypical transcripts, have been reported to have a poor prognosis, and allogeneic stem cell transplantation (ASCT) can be considered as additional therapy. However, long-term survival after ASCT for this disease is rare. PATIENT CONCERNS: This report concerns a 55-year-old female patient with e6a2 BCR-ABL-positive acute myeloid leukemia including the outcome of ASCT followed by donor lymphocyte infusion (DLI). DIAGNOSES: The breakpoint was confirmed by direct sequencing. Her minimal residual disease could be detected by nested reverse-transcription polymerase chain reaction using primers for the minor BCR-ABL (e1a2) transcript. INTERVENTIONS: Treatment with tyrosine kinase inhibitors (TKIs) and ASCT followed by DLI. OUTCOMES: Despite multiple cytogenetic and molecular relapses after ASCT, she remains in molecular remission at 46 months after ASCT. LESSONS: This case indicates the efficacy of the combination of the graft-versus-leukemia effect and TKIs for e6a2 BCR-ABL-positive acute leukemia. When the Philadelphia chromosome with an unusual chromosomal breakpoint is suggested, we should clarify the breakpoint because that information can aid molecular assessments and decisions to provide an additional or alternative therapy.

A new prognostic index to make short-term prognoses in MDS patients treated with azacitidine: A combination of p53 expression and cytogenetics.

TP53 mutation is associated with various hematological malignancies and immunohistochemistry of p53 has been used as a simple method to establish the presence of a TP53 mutation. Since the significance of p53 expression is controversial in myelodysplastic syndrome (MDS) patients treated with azacitidine (Aza), we analyzed the prevalence of p53 expression as a prognostic factor in 60 MDS patients treated with Aza. To assess p53 expression, immunohistochemical analyses of bone marrow clot sections were performed. Overall survival (OS) was significantly lower in p53-positive patients compared with p53-negetive patients (59% vs. 85% at 12 months; P=0.006). Multivariate analysis demonstrated that p53-positive was a significant prognostic factor for OS along with poor cytogenetics. Here, we propose a new prognostic index to make short-term prognoses of MDS patients in the era of Aza treatment; high: p53-positive and poor cytogenetics, low: p53-negative and absence of poor cytogenetics, and intermediate: the others. OS was significantly different among the three groups according to this index (Low 92%, Intermediate 65% and High 27% at 12 months; P<0.0001). In conclusion, p53 expression was a significant prognostic factor in MDS patients treated with Aza. In combination with cytogenetic abnormalities, it is possible to make short-term prognoses.

Molecular response to nilotinib in a patient with imatinib-intolerant e19a2-positive chronic myeloid leukemia.

The BCR-ABL1 fusion gene is the molecular marker of chronic myeloid leukemia (CML). The e19a2 transcript is a rare variant associated with various clinical presentations and courses of CML. We herein present a case of e19a2-positive CML who was intolerant to initial treatment with imatinib and successfully responded to subsequent nilotinib therapy. She achieved a major molecular response and has since be able to sustain it. According to the literature, achieved molecular response by imatinib monotherapy has not yet been reported in e19a2-positive CML patients. Second generation tyrosine kinase inhibitors may therefore be a more effective treatment for e19a2-positive CML patients.