Impact of a third dose of anti-SARS-CoV-2 vaccine in hematopoietic cell transplant recipients: A Japanese multicenter observational study.

This prospective observational study aimed to assess the serological response and safety after the third booster shot of SARS-CoV-2 mRNA vaccines in 292 hematopoietic cell transplant (HCT) recipients. In our patients, mild systemic reactions were present in 10-40% and GVHD aggravation in 1.1%. Overall, clinically relevant response (>250 U/mL) was observed in 93.1% of allogeneic (allo)-HCT recipients and 70.6% of autologous (auto)-HCT recipients, respectively. Of note, detectable antibody response with any titer following the first two doses was a powerful predictor for adequate response after booster shot in both cohorts. For such patients, 98.8% of allo- and 92.3% of auto-HCT recipients obtained clinically relevant response after dose 3. In addition, continued systemic steroid and/or calcineurin inhibitors at the booster shot significantly correlated with serological response. These findings highlighted that booster vaccination efficiently improved serological response without safety concerns and thus recommended for the majority of HCT recipients.

Reinforced antimyeloma therapy via dual-lymphoid activation mediated by a panel of antibodies armed with bridging-BiTE.

Immunotherapy using bispecific antibodies including bispecific T-cell engager (BiTE) has the potential to enhance the efficacy of treatment for relapsed/refractory multiple myeloma. However, myeloma may still recur after treatment because of downregulation of a target antigen and/or myeloma cell heterogeneity. To strengthen immunotherapy for myeloma while overcoming its characteristics, we have newly developed a BiTE-based modality, referred to as bridging-BiTE (B-BiTE). B-BiTE was able to bind to both a human immunoglobulin G-Fc domain and the CD3 molecule. Clinically available monoclonal antibodies (mAbs) were bound with B-BiTE before administration, and the mAb/B-BiTE complex induced antitumor T-cell responses successfully while preserving and supporting natural killer cell reactivity, resulting in enhanced antimyeloma effects via dual-lymphoid activation. In contrast, any unwanted off-target immune-cell reactivity mediated by mAb/B-BiTE complexes or B-BiTE itself appeared not to be observed in vitro and in vivo. Importantly, sequential immunotherapy using 2 different mAb/B-BiTE complexes appeared to circumvent myeloma cell antigen escape, and further augmented immune responses to myeloma relative to those induced by mAb/B-BiTE monotherapy or sequential therapy with 2 mAbs in the absence of B-BiTE. Therefore, this modality facilitates easy and prompt generation of a broad panel of bispecific antibodies that can induce deep and durable antitumor responses in the presence of clinically available mAbs, supporting further advancement of reinforced immunotherapy for multiple myeloma and other refractory hematologic malignancies.

Predictors of impaired antibody response after SARS-CoV-2 mRNA vaccination in hematopoietic cell transplant recipients: A Japanese multicenter observational study.

HCT recipients reportedly have a high mortality rate after developing COVID-19. SARS-CoV-2 vaccination is generally useful to prevent COVID-19. However, its safety and efficacy among HCT recipients remain elusive. This large-scale prospective observational study including 543 HCT recipients with 37-months interval from transplant demonstrated high safety profiles of mRNA vaccine: only 0.9% of patients avoided the second dose due to adverse event or GVHD aggravation following the first dose. Regarding the efficacy, serological response with a clinically relevant titer (≥250 BAU/mL) was obtained in 397 (73.1%) patients. We classified the remaining 146 patients as impaired responders and compared the clinical and immunological parameters between two groups. In allogeneic HCT recipients, multivariable analysis revealed the risk factors for impaired serological response as follows: age (≥60, 1 points), HLA-mismatched donor (1 points), use of systemic steroids (1 points), absolute lymphocyte counts (<1000/µL, 1 points), absolute B-cell counts (<100/µL, 1 points), and serum IgG level (<500 mg/dL, 2 points). Notably, the incidence of impaired serological response increased along with the risk scores: patients with 0, 1-3, and 4-7 points were 3.9%, 21.8%, and 74.6%, respectively. In autologous HCT recipients, a shorter interval from transplant to vaccination was the only risk factor for impaired serological response. Our findings indicate that two doses of SARS-CoV-2 vaccine are safe but insufficient for a part of HCT recipients with higher risk scores. To improve this situation, we should consider additional treatment options, including booster vaccination and prophylactic neutralizing antibodies during the SARS-CoV-2 pandemic.

A single-chain antibody generation system yielding CAR-T cells with superior antitumor function.

Cancer immunotherapy using T cells redirected with chimeric antigen receptor (CAR) has shown a lot of promise. We have established a single-chain antibody (scFv) generation system in which scFv library-expressing CAR-T cells can be screened appropriately based on their antitumor functions. A variable region library containing the variable and J regions of the human immunoglobulin light or heavy chain was fused with the variable region of a heavy or light chain encoded by an existing tumor-specific antibody to generate a new scFv library. Then, scFv library-expressing CAR-T cells were generated and stimulated with target cells to concentrate the antigen-specific population. Using this system, target-specific recognition of CAR-T cells appeared to be finely tuned by selecting a new variable region. Importantly, we have demonstrated that the newly optimized scFv-expressing CAR-T cells had better proliferation capacity and durable phenotypes, enabling superior reactivity against advanced tumors in vivo in comparison with the original CAR-T cells. Therefore, the optimization of an scFv is needed to maximize the in vivo antitumor functions of CAR-T cells. This system may allow us to adjust an immunological synapse formed by an scFv expressed by CAR-T cells and a target antigen, representing an ideal form of CAR-T-cell immunotherapy.

Direct comparison of target-reactivity and cross-reactivity induced by CAR- and BiTE-redirected T cells for the development of antibody-based T-cell therapy.

The development of chimeric antigen receptor (CAR) and bispecific T-cell engager (BiTE) has led to the successful application of cancer immunotherapy. The potential reactivity mediated by CAR- and BiTE-redirected T cells needs to be assessed to facilitate the application of these treatment options to a broader range of patients. Here, we have generated CAR and BiTE possessing the same single chain fragment variable (scFv) specific for the HLA-A2/NY-ESO-1157-165 complex (A2/NY-ESO-1157). Using HLA-A2+NY-ESO-1+ myeloma cells and peptides presented by HLA-A2 molecules as a model, both sets of redirected T cells recognized and killed HLA-A2+NY-ESO-1+ myeloma cells in an A2/NY-ESO-1157-specific manner in vitro. Moreover, CAR- and BiTE-activated T cells showed similar functional avidity, as assessed by cytokine production and killing activity, both displaying antitumor reactivity against HLA-A2+NY-ESO-1+ myeloma cells in vivo. Interestingly, cross-reactivity for homologous peptides presented by HLA-A*02:01 and NY-ESO-1157 peptide presented by HLA-A2 alleles was not identical between CAR- and BiTE-redirected T cells, probably due to structural differences of modified antibodies. These results have demonstrated that both antitumor CAR- and BiTE-activated T cells have comparable potential to recognize tumors, while paying attention to unknown off-target reactivity that would differ for each antibody-based modality even if the same scFv was employed.

Safety and persistence of WT1-specific T-cell receptor gene-transduced lymphocytes in patients with AML and MDS.

Wilms' tumor 1 (WT1) is constantly expressed in leukemic cells of acute leukemia and myelodysplastic syndrome (MDS). A T-cell receptor (TCR) that specifically reacts with WT1 peptide in the context of HLA-A*24:02 has been identified. We conducted a first-in-human trial of TCR-gene transduced T-cell (TCR-T-cell) transfer in patients with refractory acute myeloblastic leukemia (AML) and high-risk MDS to investigate the safety and cell kinetics of the T cells. The WT1-specific TCR-gene was transduced to T cells using a retroviral vector encoding small interfering RNAs for endogenous TCR genes. The T cells were transferred twice with a 4-week interval in a dose-escalating design. After the second transfer, sequential WT1 peptide vaccines were given. Eight patients, divided into 2 dose cohorts, received cell transfer. No adverse events of normal tissue were seen. The TCR-T cells were detected in peripheral blood for 8 weeks at levels proportional to the dose administered, and in 5 patients, they persisted throughout the study period. The persisting cells maintained ex vivo peptide-specific immune reactivity. Two patients showed transient decreases in blast counts in bone marrow, which was associated with recovery of hematopoiesis. Four of 5 patients who had persistent T cells at the end of the study survived more than 12 months. These results suggest WT1-specific TCR-T cells manipulated by ex vivo culture of polyclonal peripheral lymphocytes survived in vivo and retained the capacity to mount an immune reaction to WT1. This trial was registered at www.umin.ac.jp as #UMIN000011519.

Adoptive immunotherapy utilizing cancer antigen-specific T-cell receptors.

Synthetic immunology based on rapidly-advancing gene-engineering and immunobiology has made novel anticancer adoptive immunotherapies, using gene-modified T lymphocytes to express cancer antigen-specific receptors, a reality. Various technological innovations have overcome recent difficulties and achieved clear and long-lasting clinical efficacy against tumors, while seeking more powerful effector gene-modified T cells has yielded serious treatment-related adverse events. In this article, along with introducing our clinical trial for a novel anti-leukemia adoptive immunotherapy regimen using gene-modified autologous lymphocytes to express leukemia antigen Wilms Tumor 1(WT1)-specific T cell receptor (TCR) against refractory acute myeloid leukemia (AML) and high-risk myelodysplastic syndrome (MDS), we provide an overview of the current status of this emerging treatment option and discuss its future form in the context of neoantigens encoded by mutated genes in cancer cells and immune checkpoint inhibitors.

A Functionally Superior Second-Generation Vector Expressing an Aurora Kinase-A-Specific T-Cell Receptor for Anti-Leukaemia Adoptive Immunotherapy.

Aurora Kinase A is a cancer-associated protein normally involved in the regulation of mitosis. Being over-expressed in a range of cancers, it is a suitable target for cell-based immunotherapy. Gene transfer of T-cell receptor sequences cognisant of HLA-A*0201-restricted Aurora Kinase A antigen has previously been shown to transfer specific immunoreactivity against the target peptide in a Human Lymphocyte Antigen-restricted manner. While T cell receptor gene-transfer has great potential in overcoming the difficulties of isolating and expanding tumour-reactive lymphocytes from a patient's own cells, one hurdle is potential mispairing and competition between exogenous and endogenous T cell receptor chains. We have used a retroviral vector design bearing a short-interfering RNA that downregulates endogenous T cell receptor chains, without affecting expression of the transgenic T cell receptor sequences. The T cell receptor expression cassette also includes a 2A self-cleaving peptide, resulting in equimolar expression of the T cell receptor alpha and beta chains, further enhancing formation of the desired T cell receptor. Via a simple, modular cloning method, we have cloned the alpha and beta chains of the anti-Aurora Kinase A-reactive T cell receptor into this 'siTCR' vector. We then compared the activity of this vector against the original, 'conventional' vector across a panel of assays. T cell receptors expressed from the siTCR-vector retained the cytotoxic functionality of the original vector, with evidence of reduced off-target reactivity. The rate of expression of correctly-formed T cell receptors was superior using the siTCR design, and this was achieved at lower vector copy numbers. Maintaining T cell receptor efficacy with a reduced vector copy number reduces the risk of genotoxicity. The siTCR design also reduces the risk of mispairing and cross-reactivity, while increasing the functional titre. Such improvements in the safety of T cell receptor gene-transfer will be crucial for clinical applications of this technology.

Development of Engineered T Cells Expressing a Chimeric CD16-CD3ζ Receptor to Improve the Clinical Efficacy of Mogamulizumab Therapy Against Adult T-Cell Leukemia.

PURPOSE: Mogamulizumab (Mog), a humanized anti-CC chemokine receptor 4 (CCR4) mAb that mediates antibody-dependent cellular cytotoxicity (ADCC) using FcγR IIIa (CD16)-expressing effector cells, has recently been approved for treatment of CCR4-positive adult T-cell leukemia (ATL) in Japan. However, Mog failure has sometimes been observed in patients who have accompanying chemotherapy-associated lymphocytopenia. In this study, we examined whether adoptive transfer of artificial ADCC effector cells combined with Mog would overcome this drawback. EXPERIMENTAL DESIGN: We lentivirally gene-modified peripheral blood T cells from healthy volunteers and ATL patients expressing the affinity-increased chimeric CD16-CD3ζ receptor (cCD16ζ-T cells). Subsequently, we examined the ADCC effect mediated by those cCD16ζ-T cells in the presence of Mog against ATL tumor cells both in vitro and in vivo RESULTS: cCD16ζ-T cells derived from healthy donors killed in vitro Mog-opsonized ATL cell line cells (n = 7) and primary ATL cells (n = 4) depending on both the number of effector cells and the dose of the antibody. cCD16ζ-T cells generated from ATL patients (n = 3) also exerted cytocidal activity in vitro against Mog-opsonized autologous ATL cells. Using both intravenously disseminated model (n = 5) and subcutaneously inoculated model (n = 4), coadministration of Mog and human cCD16ζ-T cells successfully suppressed tumor growth in xenografted immunodeficient mice, and significantly prolonged their survival (P < 0.01 and P = 0.02, respectively). CONCLUSIONS: These data strongly suggest clinical feasibility of the novel combined adoptive immunotherapy using cCD16ζ-T cells and Mog for treatment of aggressive ATL, particularly in patients who are ineligible for allogeneic hematopoietic stem cell transplantation. Clin Cancer Res; 22(17); 4405-16. ©2016 AACR.

Gene-modified human α/ß-T cells expressing a chimeric CD16-CD3ζ receptor as adoptively transferable effector cells for anticancer monoclonal antibody therapy.

The central tumoricidal activity of anticancer monoclonal antibodies (mAb) is exerted by FcγR IIIa (CD16)-expressing effector cells in vivo via antibody-dependent cell-mediated cytotoxicity (ADCC), as observed for natural killer (NK) cells. In practice, chemotherapy-induced leukopenia and exhaustion of NK cells resulting from ADCC often hamper the clinical efficacy of cancer treatment. To circumvent this drawback, we examined in vivo the feasibility of T cells, gene-modified to express a newly generated affinity-matured (158V/V) chimeric CD16-CD3ζ receptor (cCD16ζ-T cells), as a transferable alternative effector for cancer mAb therapy. cCD16ζ-T cells were readily expandable in ex vivo culture using anti-CD2/CD3/CD28 beads and recombinant human interleukin-2 (rhIL-2), and they successfully displayed ADCC-mediated tumoricidal activity in vitro. During ADCC, ligation of opsonized cancer cells to the introduced cCD16ζ-T cells stimulated the effector cells to produce proinflammatory cytokines and release toxic granules through the activation of the Nuclear factor of activated T cells (NFAT) pathway after phosphorylation of the CD3ζ chain. In parallel, these stimulated cCD16ζ-T cells transiently proliferated and differentiated into effector memory T cells. In contrast, NK cells activated by rhIL-2 displayed similar ADCC activity, but failed to proliferate. Human cCD16ζ-T cells infused concomitantly with anti-CD20 mAb synergistically inhibited the growth of disseminated Raji cells, a CD20(+) lymphoma cell line, in immunodeficient mice, whereas similarly infused rhIL-2-treated NK cells survived for a shorter time and displayed less effective tumor suppression. Our findings strongly suggest the clinical feasibility of cCD16ζ-T cells as adoptively transferable ADCC effector cells that could potentially enhance the clinical responses mediated by currently available anticancer mAbs.

Ultra-low dose interleukin-2 promotes immune-modulating function of regulatory T cells and natural killer cells in healthy volunteers.

Low-dose interleukin-2 (IL-2) expands regulatory T cells (Tregs) and natural killer (NK) cells after stem cell transplantation (SCT) and may reduce graft-versus-host disease (GVHD). We hypothesized that ultra-low dose (ULD) IL-2 could serve as an immune-modulating agent for stem cell donors to prevent GVHD following SCT. However, the safety, dose level, and immune signatures of ULD IL-2 in immune-competent healthy subjects remain unknown. Here, we have characterized the phenotype and function of Tregs and NK cells as well as the gene expression and cytokine profiles of 21 healthy volunteers receiving 50,000 to 200,000 units/m(2)/day IL-2 for 5 days. ULD IL-2 was well tolerated and induced a significant increase in the frequency of Tregs with increased suppressive function. There was a marked expansion of CD56(bright) NK cells with enhanced interferon-γ (IFN-γ) production. Serum cytokine profiling demonstrated increase of IFN-γ induced protein 10 (IP-10). Gene expression analysis revealed significant changes in a highly restricted set of genes, including FOXP3, IL-2RA, and CISH. This is the first study to evaluate global immune-modulating function of ULD IL-2 in healthy subjects and to support the future studies administrating ULD IL-2 to stem cell donors.

Genetically engineered fixed K562 cells: potent "off-the-shelf" antigen-presenting cells for generating virus-specific T cells.

BACKGROUND AIMS: The human leukemia cell line K562 represents an attractive platform for creating artificial antigen-presenting cells (aAPC). It is readily expandable, does not express human leukocyte antigen (HLA) class I and II and can be stably transduced with various genes. METHODS: In order to generate cytomegalovirus (CMV) antigen-specific T cells for adoptive immunotherapy, we transduced K562 with HLA-A∗0201 in combination with co-stimulatory molecules. RESULTS: In preliminary experiments, irradiated K562 expressing HLA-A∗0201 and 4-1BBL pulsed with CMV pp65 and IE-1 peptide libraries failed to elicit antigen-specific CD8⁺ T cells in HLA-A∗0201⁺ peripheral blood mononuclear cells (PBMC) or isolated T cells. Both wild-type K562 and aAPC strongly inhibited T cell proliferation to the bacterial superantigen staphylococcal enterotoxin B (SEB) and OKT3 and in mixed lymphocyte reaction (MLR). Transwell experiments suggested that suppression was mediated by a soluble factor; however, MLR inhibition was not reversed using transforming growth factor-ß blocking antibody or prostaglandin E2 inhibitors. Full abrogation of the suppressive activity of K562 on MLR, SEB and OKT3 stimulation was only achieved by brief fixation with 0.1% formaldehyde. Fixed, pp65 and IE-1 peptide-loaded aAPC induced robust expansion of CMV-specific T cells. CONCLUSIONS: Fixed gene-modified K562 can serve as effective aAPC to expand CMV-specific cytotoxic T lymphocytes for therapeutic use in patients after stem cell transplantation. Our findings have implications for broader understanding of the immune evasion mechanisms used by leukemia and other tumors.

Activation of T-cell receptor signaling in peripheral T-cell lymphoma cells plays an important role in the development of lymphoma-associated hemophagocytosis.

Peripheral T-cell lymphoma (PTCL) is a biologically diverse lymphoid malignancy. The clinical aggressiveness associated with hemophagocytic syndrome (HS) is a characteristic of PTCL, being more distinctive in CD8(+) PTCL. However, the underlying mechanism of PTCL-associated HS has not yet been fully investigated. We newly established a novel IL-2-dependent CD8(+) PTCL lymphoma cell line (T8ML-1) from a patient with CD8(+) PTCL who suffered recurrent HS accompanying disease flare-up. Focusing on the lymphoma cell T-cell receptor (TCR), we examined the lymphoma cell functions responsible for such clinical manifestations. First, T8ML-1.1 in which endogenous TCR-α/ß chains were silenced by siRNAs, and T8ML-1.2 in which endogenous TCR-α/ß chains were replaced with HLA-A*24:02-restricted and WT1(235-243)-specific TCR-α/ß, were established. T8ML-1 exerted phytohemagglutinin (PHA)-dependent cytotoxicity via granular exocytosis. Additionally, soluble factors produced by PHA-stimulated T8ML-1, which included INF-γ and TNF-α, but not by simple-cultured T8ML-1, caused human monocytes to exhibit erythrophagocytosis and thrombophagocytosis in vitro. PHA binding induced phosphorylation of CD3ζ chain. Furthermore, both cytotoxicity and hemophagocytosis were completely inhibited by T8ML-1.1, but eventually restored by T8ML-1.2. These data suggest that exogenous activation of TCR signaling in PTCL cells might play an important role in the formation of PTCL-associated HS.

Human herpesvirus 6 infection impairs Toll-like receptor signaling.

Human herpesvirus 6 (HHV-6) has a tropism for immunocompetent cells, including T lymphocytes, monocytes/macrophages, and dendritic cells (DCs) suggesting that HHV-6 infection affects the immunosurveillance system. Toll-like receptor (TLR) system plays an important role in innate immunity against various pathogens. In the present study, we investigated the effect of HHV-6 infection on the expression and intracellular signaling of TLRs in DCs. Although expression levels of TLRs were not decreased or slightly elevated following HHV-6 infection, the amounts of cytokines produced following stimulation with ligands for TLRs appeared to be dramatically decreased in HHV-6-infected DCs as compared to mock-infected DCs. Similarly, phosphorylation levels of TAK-1, IkappaB kinase, and IkappaB-alpha following stimulation of HHV-6-infected DCs with lipopolysaccharide, which is the ligand for TLR4, appeared to be decreased. These data show that HHV-6 impairs intracellular signaling through TLRs indicating the novel mechanism of HHV-6-mediated immunomodulation.

Clinical significance of co-expression of CD21 and LFA-1 in B-cell lymphoma.

We previously reported that the prognosis of CD21-positive diffuse large B-cell lymphoma (DLBCL) is significantly favorable to that of CD21-negative DLBCL (Otsuka et al. in Br J Haematol 127:416-424, 2004). In this study, we attempted to clarify the biological significance of CD21 expression in B-cell lymphoma (BCL) by performing in vitro experiments using CD21 transfection into a CD21-negative lymphoma cell line and analyzing clinical data from lymphoma samples. Established clones of CD21 transfectants showed homotypic aggregation in suspension culture. Analysis of integrin expression revealed that LFA-1 appeared to be expressed on CD21 transfectants, and the cell aggregation was abrogated by anti-LFA-1 antibody. The CD21 transfectants could adhere to plastic plates coated with ICAM-1. Moreover, flow cytometry and/or immunohistochemical analyses of clinical BCL samples (n = 29) revealed positive for CD21 in all cases; LFA-1 was also expressed without exception. All BCL cells isolated from cavity fluids (n = 10) failed to express both CD21 and LFA-1. These data suggest that CD21 is tightly related to LFA-1 expression in BCL and the absence of CD21/LFA-1 expression is associated with pleural/peritoneal fluid involvement by BCL, a potential indicator of disease progression of BCL.

Absence of chlamydial infection in Japanese patients with ocular adnexal lymphoma of mucosa-associated lymphoid tissue.

Ocular adnexal extranodal marginal zone B-cell lymphomas of mucosa-associated lymphoid tissue (ocular adnexal MALT lymphoma) are predominately low-grade, small B-cell types and may be caused by several putative inflammatory agents. Twenty-three ocular adnexal MALT lymphoma cases, the monoclonality of which was confirmed by examination of immunoglobulin heavy chain gene rearrangement and/or cell surface antigens, were analyzed for evidence of several causative factors. A serologic evaluation of our series of patients showed no evidence of infection by Epstein-Barr virus, hepatitis C virus, or Chlamydophila psittaci. Two patients tested positive for serum antibodies for autoimmunity, and another 2 patients tested positive for antibodies against Chlamydia trachomatis. Polymerase chain reaction analysis did not reveal the presence of the chlamydial 16S ribosomal RNA (rRNA) gene or the 16S-23S spacer rRNA gene. These results indicate that the inflammatory agents in our series of ocular adnexal MALT lymphomas are still unknown and that some types of chlamydial infections are not associated with orbital adnexal MALT lymphoma in southern regions of Japan.

Single-dose daily infusion of cyclosporine for prevention of Graft-versus-host disease after allogeneic bone marrow transplantation from HLA allele-matched, unrelated donors.

Peak blood concentration of cyclosporine (CsA) in renal transplantation patients was recently reported to be associated with clinical efficacy. We therefore evaluated the toxicity and efficacy of a regimen of once-daily infusion of CsA plus a short course of methotrexate as prophylaxis of graft-versus-host disease (GVHD) after allogeneic bone marrow transplantation from an HLA allele-matched, unrelated donor. Nineteen patients with hematologic malignancies received CsA, 3 mg/kg per day, as a 4-hour intravenous (IV) infusion from day -1. After engraftment, patients received CsA orally at twice the IV dose. The CsA dose was adjusted to maintain the blood trough level between 150 and 200 ng/mL. Methotrexate was administered IV at doses of 10 mg/m(2) on day 1 and 7 mg/m(2) on days 3, 6, and 11. Bone marrow engraftment occurred in all patients. Grade 1 and grade 2 GVHD occurred in 6 (31.6%) and 7 (36.8%) of the 19 patients, respectively. No patient had grade 3 or 4 GVHD. Acute nephrotoxicity developed in 1 (5.3%) of the 19 patients, and hypertension developed in 3 (15.8%) of the 19 patients. We evaluated the pharmacokinetics of 4-hour CsA infusion in 10 patients. The mean trough concentration, mean peak concentration, mean time to peak concentration, and area under the curve (24 hours) were 161 +/- 43 ng/mL, 1498 +/- 387 ng/mL, 3.2 +/- 1.0 hours, and 10,848 +/- 1,991 ng +/- h/mL, respectively. This regimen was well tolerated and did not enhance the risk of severe GVHD in patients undergoing allogeneic bone marrow transplantation from an HLA allele-matched, unrelated donor.

Expression of SOCS3 mRNA in bone marrow cells from CML patients associated with cytogenetic response to IFN-alpha.

Previously, we have demonstrated that constitutive expression of suppressor of cytokine signaling-3 (SOCS3) affects the sensitivity of chronic myelogenous leukemia (CML) cell lines to interferon-alpha (IFN-alpha). In the present study, we analyzed the expression of SOCS3 mRNA in bone marrow cells from patients with CML at diagnosis, with the aid of real-time polymerase chain reaction. SOCS3 mRNA expression in bone marrow cells from CML patients who responded well to IFN-alpha therapy was significantly lower than that in cells from healthy volunteers and patients who were resistant to IFN-alpha therapy. Methylation of SOCS3 promoter was absent in bone marrow cells from all CML patients examined. These results indicate that the expression of SOCS3 mRNA is inversely associated with the sensitivity to IFN-alpha both in vitro and in vivo and that differences in SOCS3 mRNA expression are not due to the methylation status of SOCS3 promoters.