Generation of a Nebulizable CDR-Modified MERS-CoV Neutralizing Human Antibody

Middle East respiratory syndrome coronavirus (MERS-CoV) induces severe aggravating respiratory failure in infected patients, frequently resulting in mechanical ventilation. As limited therapeutic antibody is accumulated in lung tissue following systemic administration, inhalation is newly recognized as an alternative, possibly better, route of therapeutic antibody for pulmonary diseases. The nebulization process, however, generates diverse physiological stresses, and thus, the therapeutic antibody must be resistant to these stresses, remain stable, and form minimal aggregates. We first isolated a MERS-CoV neutralizing antibody that is reactive to the receptor-binding domain (RBD) of spike (S) glycoprotein. To increase stability, we introduced mutations into the complementarity-determining regions (CDRs) of the antibody. In the HCDRs (excluding HCDR3) in this clone, two hydrophobic residues were replaced with Glu, two residues were replaced with Asp, and four residues were replaced with positively charged amino acids. In LCDRs, only two Leu residues were replaced with Val. These modifications successfully generated a clone with significantly greater stability and equivalent reactivity and neutralizing activity following nebulization compared to the original clone. In summary, we generated a MERS-CoV neutralizing human antibody that is reactive to recombinant MERS-CoV S RBD protein for delivery via a pulmonary route by introducing stabilizing mutations into five CDRs.

Therapy-related myeloid neoplasms as a concerning complication in acute promyelocytic leukemia

Acute promyelocytic leukemia (APL) has become a highly curable malignant disease after the introduction of all transretinoic acid (ATRA) to chemotherapy treatment. However, the risk to develop therapy-related myeloid neoplasms (t-MN) has become a matter of concern, as APL patients are otherwise expected to have a good prognosis. We report a patient with APL who achieved complete remission after chemotherapy induction with anthracycline and ATRA, followed by consolidation and maintenance chemotherapy. Two years later, the patient developed t-AML, with MLL rearrangements, without any evidence of relapse of the APL original clone. The increasing incidence of t-MN in oncohematological patients is partly due to the development of safer, more efficient or targeted therapies, which allow better outcomes and lengthened survival amongst treated patients. The identification of genetic factors, mechanisms or prognostic biomarkers in t-MN might open new windows for the development of personalized targeted therapy regimes in this underserved patient population.

A Rare Population of Stem Cell Memory T Cells is an Apex of the Hierarchy of Adult T-Cell Leukemia

Adult T-cell leukemia (ATL) is a mature T cell neoplasm causatively associated with HTLV-1. It takes a long period, around 50 years, to develop ATL after the transmission of HTLV-1 in infancy through breastfeeding, suggesting the pool for the generation of ATL clone may exist somewhere. Recent reports showed human CD34+ bone marrow cells infected by HTLV-1 ex vivo can generate CD4+ T-cell lymphomas in immunodeficiency mice or that CD117+ early hematopoietic progenitors can develop lymphoma in transgenic mice model of Tax which is an oncogenic protein of HTLV-1. These results suggest the model of leukemia initiating cells, that leukemia is organized hierarchically and sustained by a small subset of stem/progenitors at the apex, might be acceptable in ATL. A more recent report demonstrated that a novel compartment of normal T cells, termed stem cell memory T cells (TSCM), possesses stem cell-like capacity including relatively enhanced self-renewal capacity and multipotent ability to generate all memory and effector T cell subsets. This concept prompts us to hypothesize that TSCM subset might contribute to the development of ATL. First, to investigate whether hematopoietic stem/progenitor cells in primary samples of ATL patients are infected with HTLV-1, we sorted lineage(CD3, CD4, CD8, CD11b, CD14, CD19, CD20, CD56, CD235)-negative CD34+ cells from bone marrow samples of ATL patients by FACS AriaII, purified genomic DNA, and then performed PCR of pX region which is a virus specific gene. No amplification of pX was detected in all samples we studied. Next, to focus on TSCM population, we established the eight-color staining method with lineage of NK/Monocyte/DC (CD11b, CD14 and CD56), CD4, CD45RA, CD45RO, CCR7, CD95, CD122 and CD25. We successfully separated CD4+ T cells from ATL patients into four subsets; naïve T (TN: CD4+CD45RA+CD45RO-CCR7+CD95-CD122-), stem cell memory T (TSCM: CD4+CD45RA+CD45RO-CCR7+CD95+CD122dim), central memory T (TCM: CD4+CD45RA-CD45RO+CCR7+), and effector memory T (TEM: CD4+CD45RA-CD45RO+CCR7-). Although ATL cells are generally CD45RO positive, so that TCM and TEM were extremely dominant, CD45RA positive populations did exist in all ATL samples we studied. To assess the frequency of infected cells, we performed quantitative PCR with the sensitivity of 5 copies detectable at the minimum and calculated by dividing copy number of pX in HTLV-1 provirus with that of RNase P in human genome in genomic DNA from 1,000 cells of each subset. Almost all cells in TCM and TEM and more than 10% of cells in TSCM were infected in most of cases while none of infected cells were detected in TN in our experiment design. Since ATL is caused by monoclonal expansion from plenty of HTLV-1 infected clones, we focused on the existence of ATL original clone in each subset. We determined the ATL original clone by inverse PCR and the clone-specific provirus integration site by sequencing of the inverse PCR product in each case, and then constructed PCR primers being capable of detecting each ATL original clone by regular PCR. Interestingly we found each ATL clone was detected in TSCM, TCM, and TEM but not in TN subset in all studied cases, indicating TSCM could be an origin of ATL cells. To investigate whether there is a hierarchy in ATL cells, we next performed in vitro culture with IL-7 and examined the status of CD45RA and CCR7 after 2 weeks. Intriguingly TSCM generated TCM and TEM while TCM and TEM did not generate TSCM, suggesting TSCM is an apex in HTLV-1 infected T cells. To investigate tumorigenicity of TSCM, we adopted xenogeneic transplantation assay. When we inoculated 3x104 cells of each subset from one ATL patient into immune deficient mice (NOG mice) intraperitoneally, we observed high number of cells in peripheral blood and spleen of recipient mice transplanted with TSCM compared to TCM and TEM. Phenotypically TCM and TEM were generated in mice transplanted with TSCM as well as TCM and TEM while TSCM was only generated from TSCM but not TCM and TEM. Furthermore we analyzed the clonality of engrafted cells by ATL clone-specific PCR and inverse PCR, and found that the patient original clone becomes dominant in recipient mice inoculated with TSCM but not in those with TCM and TEM. Taken together, our findings provide new insights that TSCM could be an apex of the hierarchy of ATL and play an important role in development and maintenance of ATL clone. Disclosures: No relevant conflicts of interest to declare.

Incidence, clinical course, and prognosis of secondary monoclonal gammopathy of undetermined significance in patients with multiple myeloma

During the course of multiple myeloma (MM), new monoclonal proteins of an isotype distinct from the original clone, referred to as secondary monoclonal gammopathy of undetermined significance (MGUS), have been described. We report on the frequency, characteristics, and outcome of secondary MGUS. Of the 1942 patients with MM, 128 (6.6%) developed a secondary MGUS, at a median of 12 months from the diagnosis of MM. The median duration of secondary MGUS was 5.9 months. Secondary MGUS was more common in patients after stem cell transplantation than in those who had not undergone such treatment (22.7% vs 1.6%, P < .001). Overall survival was significantly superior in MM patients who developed secondary MGUS compared with the rest of the cohort (73 vs 38 months, respectively; P < .001). The time of onset and the duration of secondary MGUS, as well as failure to resolve spontaneously, had an effect on overall survival and require further study.

Clonal Evolution of Malignant Populations In Potentially Biclonal Chronic Lymphocytic Leukemia Patients

Abstract 2412 Background: A majority of CLL cases represent monoclonal lymphoproliferation. On the other hand, simultaneous biclonal/biphenotypic disease has been sporadically recorded (Gonzales-Campos et al., 1997). Moreover, initially minor, aggressive subclone harboring p53 inactivation in monoclonal CLL can be selected after therapy (Malcikova el al., 2009). Here we present an analysis of patients expressing two or three functional IGH rearrangements. We also closely describe one case of a patient who developed another CLL population after therapy. In this case, the original malignant clone was forced out by the more aggressive CLL population that led to therapy resistance. Aims: We characterized clonal evolution in cases expressing two functional Ig rearrangements (potentially biclonal CLL) and described in detail the alteration of CLL populations in one case. Methods: B lymphocytes were separated from peripheral blood samples using Ficoll-Paque PLUS (GE Healthcare)/RosetteSep Kits (StemCell). IGHV status was assessed according to the ERIC recommendations (cDNA as an input material). A clonal rearrangement of IGH was confirmed by BIOMED-2 protocol, and Ig light chain utilization was assessed. Chromosomal alterations were analyzed by FISH (deletions 13q14, 17p13, 11q22 and trisomy 12) and metaphase cytogenetics with CpG/IL-2 stimulation. Results: From 1065 patients screened, 31 cases were identified as expressing two (28/31) or three (3/31) functional IGH in their disease history. 11 of them (35.5%) were analyzed repeatedly during the course of the disease with median follow-up between the first and the last IGHV examination 19.9 months (range 7.4 – 48.1). According to a number of light chains expressed and the presence of multiple IGH, all patients were divided as having a biclonal CLL (17/31) or a lack of IGH allelic exclusion (14/31). Patients lacking IGH allelic exclusion were omitted from further analysis. Interestingly, in five repeatedly analyzed cases, a mutated CLL clone vanished during a course of the disease and only unmutated clone VH1-69 (4/5) or VH4-34 (1/5) was still present; in one of these cases, therapy accelerated unmut VH1-69 clone selection. In another two patients, unmut CLL clone VH1-69 or VH3-30, respectively, was later selected beside the original one resulting to biclonal CLL. Importantly, we found a patient with an original monoclonal population who developed the second CLL clone after therapy. The original clone was, afterwards, totally overcome by the second one. In this case, Ig analysis of the sample prior to the first therapy showed only unmut VH3-21 rearrangement combined with VL1-51. Two distinct malignant populations were detected by flow-cytometry nine months after the therapy. Ig analysis assessed the original clone and a second unmut VH1-69/VK1-39 clone. IGH and IGL sequences were compared between both populations, and BCR editing was excluded as unlikely. The selection of distinct CLL was supported by cytogenetics which detected the second clone with complex karyotype including loss of chromosome 17 beside the original one with sole deletion in region 5q14.1-q21.3. Because of disease progression, second-line therapy was administered without therapeutic effect. Subsequent analysis of CLL cells revealed a total eradication of the original VH3-21 clone and persistence of the second aggressive clone with p53 inactivation. Conclusion: Frequency of biclonal cases in our cohort is 1.6% (17/1065). Our results show for the first time that aggressive CLL clones, especially VH1-69, can grow up beside and even displace more indolent mutated populations. Therapy can accelerate the clonal selection; this is documented by two cases. We also analyzed in detail one of these patients who developed the second aggressive CLL after therapy. We conclude that subsequent monitoring of CLL patients is necessary for understanding of malignant disease evolution and the role of therapy in the clone selection process. Supported by IGA MZ CR NS10439-3, NS9858-3, MSM0021622430, 204/09/H058 and MPO FR-TI2/254. Disclosures: No relevant conflicts of interest to declare.

Gliosarcoma With Features of Osteoblastic Osteosarcoma: A Review

Context.—Gliosarcoma is a rare tumor of the central nervous system characterized by a biphasic histologic pattern, consisting of a gliomatous and a sarcomatous component, respectively. In most instances the sarcomatous component is represented by a fibrosarcoma, but other stromal malignancies have also been described. Osteosarcomatous differentiation in gliosarcoma has been rarely reported. Objective.—To review characteristic radiologic and histopathologic features of this rare neoplasm, to debate about possible differential diagnoses that should be taken into consideration, and to provide an overview of the potential histopathogenesis of gliosarcomas. Data Sources.—Relevant articles indexed in PubMed (National Library of Medicine) and reference medical texts. Conclusions.—Recent molecular studies suggest that sarcomatous and gliomatous components of gliosarcoma might be derived from a single precursor cell clone, progressing in 2 subclones with distinct morphologic features during tumor evolution. Nonetheless, events determining splitting of the original clone into 2 histologic populations remain to be investigated.

Generation of Graft-Versus-Leukemia (GVL)-Effector T Cells through Targeting of Leukemia-Associated HY Antigens Via TCR Transfer.

In this study we present two novel leukemia-associated antigens and their specific targeting through TCR-transfer. First, two CD8+ CTL clones were characterized that specifically recognized normal and malignant male B and T cell lymphoblasts, but not fibroblasts, resting lymphocytes or monocytes. One of clones recognized the HLA-B*5201-restricted TIRYPDPVI epitope of the RPS4Y protein, while the other recognized the HLA-B*5201-restricted MQQMRHKEV epitope of the UTY protein. Western blot analysis demonstrated the overexpression of RPS4 proteins in normal and malignant lymphoblasts. However, for UTY, real time RT-PCR failed to reveal differential expression of UTY mRNA in lymphoblasts, indicating that posttranscriptional regulation of UTY protein expression and/or improved processing and presentation of this epitope in lymphoblasts are likely causes of its differential recognition. These findings open up the possibility to specifically eliminate leukemic cells by targeting either RPS4Y or UTY. We evaluated the feasibility of transfer of the RPS4Y- and UTY-specific T cell receptors (TCRRPS4Y and TCRUTY, respectively) to naïve T cells to specifically eliminate RPS4Y- and UTY-expressing lymphoblasts. For each clone, the TCR α- and β-chains were cloned into separate pMX retroviral vectors. T cell transduction with either TCRRPS4Y or TCRUTY and subsequent purification resulted in 57–75% of cells expressing both α- and β-chain. TCRRPS4Y/TCRUTY-transduced T cells efficiently lysed B*5201-positive EBV-LCL (100%/92%), PHA blasts (72%/72%), and B*5201-transduced Raji (71%/56%), YT (21%/53%) and RPMI 8226 (28%/47%) cell lines, in the absence of significant recognition of B*5201-transduced fibroblasts. Killing of B*5201-positive primary leukemias could not be checked due to the paucity of B*5201-positive samples. Interestingly, despite the 1.4–1.7 fold lower expression of the exogenous TCRs on transduced T cells, both TCR-transduced populations had the same requirements for the minimal antigenic peptide concentration as the respective clones. This suggests that the limited peptide availability and not the TCR-affinity determines the selective pattern of recognition of these HY antigens. In addition, the amount of IFN-γ produced by TCRUTY-transduced T cells was significantly higher (5455±1286 pg/ml) than that of the original clone (721±134 pg/ml) (p=0.003). Similarly, IFN-γ production by the original RPS4Y-specific clone and TCRRPS4Y-transduced T cells was 391±35 vs. 1498±431 pg/ml (p=0.01). TCR-transduced T cells also displayed a significant increase in stimulation-induced production of IL-2 and TNF-α in comparison to T cell clones, produced less IL-10 and demonstrated great proliferative potential. This suggests that while preserving the fine specificity and differential pattern of recognition of the original clone, TCR-transduced T cells may have much more pronounced anti-tumor effect in vivo. This is likely due to their ‘freshness’ compared to the original clones, which had been in culture for more than 6 months. In conclusion, this is the first demonstration of the possibility to preserve the specific pattern of a T cell response to a differentially recognized antigen after TCR-transfer and to augment the amplitude of this response concomitantly. Our experiments support the feasibility of the selective targeting of male B and T cell lymphoblasts by T cells transduced with UTY- and RPS4Y-specific TCRs. This approach is currently being investigated in vivo in RAG2−/− γc−/− mice bearing male EBV-LCL.

The Concept of “Foundation Clones” in Source Selection for Vegetative Propagation of Almond

The term “clone” is a key biological term that designates a number of horticultural situations. In breeding, many cultivars are designated as clones, originating from consecutive vegetative propagation from individuals within a seedling population, from individual plants of a clone exhibiting “bud mutations,” and, more recently, from genetic engineering and biotechnology. Extensive vegetative propagation of a limited numbers of clones in modern horticultural systems has been accompanied by systemic incorporation by serious pathogens (viruses, viroids, phytoplasmas, etc.), and in some cases by horticultural deterioration (e.g., noninfectious bud-failure in almonds). Control of these problems in clonal propagation is achieved by 1) propagation source selection 2) maintenance of the source in a registered foundation block under protected conditions and 3)multipli-cation in controlled “mother blocks” or “increase blocks” from which commercial material is distributed after a minimum of consecutive generations of vegetative propagation. This system is the basis for Registration and Certification programs and “clean stock” in general. In many crops the selected propagation source is a single plant, its progeny constitutes a “clone,” and the new entity is given a unique name or number. To distinguish this “new” clone from the “original” clone, the designation of FOUNDATION CLONE is suggested. Biological and horticultural significance is illustrated in almond (Prunus dulcis).

Responses of Two Somaclonal Variants of Quince (Cydonia oblonga) to Iron Deficiency in the Greenhouse and Field

Somaclonal quince (Cydonia oblonga Mill.) variants IE-1 and IE-2 (Dolcet-Sanjuan et al., 1992) were more tolerant to Fe-deficient conditions in vitro than the original clone Quince A. The tolerance was evidenced by higher chlorophyll concentrations in leaves, higher Fe(III) reduction in roots, and increased acidification of the medium. In Summers 1993 and 1994, the two variants and Quince A were compared in the greenhouse, grown in normal potting soil (pH 5) and in soil obtained from an Fe-deficient orchard, with and without the addition of lime and KHCO3 solution to increase the pH (up to pH 8.3) and intensify Fe stress. In both years, the variants had significantly higher leaf chlorophyll concentrations than Quince A when grown in high-pH soils. The Fe+2 concentrations in leaves were also higher in the variants. Under field conditions at IRTA Reus, Spain, IE-1 and IE-2 had higher chlorophyll concentrations than the controls at some, but not all, analysis dates. These results suggest that IE-1 and IE-2, when grown as young plants in the greenhouse, have higher tolerance to Fe deficiency than Quince A, as observed in vitro, but that the tolerance is more variable under uncontrolled conditions in the field.