The RD114/simian type D retroviruses, which include the feline endogenous retrovirus RD114, all strains of simian immunosuppressive type D retroviruses, the avian reticuloendotheliosis group ...including spleen necrosis virus, and baboon endogenous virus, use a common cell-surface receptor for cell entry. We have used a retroviral cDNA library approach, involving transfer and expression of cDNAs from highly infectable HeLa cells to nonpermissive NIH 3T3 mouse cells, to clone and identify this receptor. The cloned cDNA, denoted RDR, is an allele of the previously cloned neutral amino acid transporter ATB0(SLC1A5). Both RDR and ATB0serve as retrovirus receptors and both show specific transport of neutral amino acids. We have localized the receptor by radiation hybrid mapping to a region of about 500-kb pairs on the long arm of human chromosome 19 at q13.3. Infection of cells with RD114/type D retroviruses results in impaired amino acid transport, suggesting a mechanism for virus toxicity and immunosuppression. The identification and functional characterization of this retrovirus receptor provide insight into the retrovirus life cycle and pathogenesis and will be an important tool for optimization of gene therapy using vectors derived from RD114/type D retroviruses.
Pseudomonas aeruginosa, of medical, environmental, and industrial importance, depends on inorganic polyphosphate (poly P) for a wide range of functions, especially survival. Mutants of PAO1 lacking ...poly P kinase 1, PPK1, the enzyme responsible for most poly P synthesis in Escherichia coli and other bacteria, are defective in motility, quorum sensing, biofilm formation, and virulence. We describe here multiple defects in the ppk1 mutant PAOM5, including a striking compaction of the nucleoid, distortion of the cell envelope, lack of planktonic motility and exopolymer production, and susceptibility to the β-lactam antibiotic carbenicillin as well as desiccation. We propose that P. aeruginosa with reduced poly P levels undergoes ultrastructural changes that contribute to profound deficiencies in cellular functions.
The cell surface adhesion molecule LFA-1 coordinates leukocyte trafficking and is a costimulatory molecule for T cell activation. We developed a panel of mAbs that recognize activation epitopes on ...the CD18 subunit, and show that stimulation of T lymphocytes appears to be accompanied by a conformational change in a subpopulation of LFA-1 that does not require ligand binding. Activation epitope up-regulation requires divalent cations, is sensitive to cellular signal transduction events, and correlates with cell adhesion. In addition, the stimulated appearance of these activation epitopes is absent in cell lines from patients with leukocyte adhesion deficiency-1/variant that has previously been shown to be defective in LFA-1 activation. Thus, these activation epitope Abs can be used to dissect signal transmission to CD18. Evidence suggests that these CD18 activation epitopes are induced early in cellular activation and are independent of actin rearrangement necessary for avid adhesion. We have also determined that function-blocking CD18 Abs inhibit the induction of activation epitopes. One activation epitope Ab binds to a site on CD18 distinct from that of the blocking Abs, indicating that the blocking Abs suppress a conformational change in LFA-1. We also find that these neoepitopes are present on rLFA-1 with high affinity for ICAM-1 and their binding is modulated in parallel with the affinity of LFA-1 for ICAM-1. Collectively, these neoepitope Abs identify a subpopulation of LFA-1 most likely with high affinity for ICAM-1 and necessary for LFA-1 function.
The aim of this study was to evaluate short-term safety and tolerability of fingolimod in a real-world population with relapsing multiple sclerosis, focusing on cardiac safety during treatment ...initiation. Patients received fingolimod 0.5 mg once daily for four months. Patients excluded from the pivotal studies with certain pre-existing cardiac conditions or baseline cardiac findings (PCCs), and those receiving beta blockers (BBs) and/or calcium channel blockers (CCBs), were eligible. Heart rate (HR) and electrical conduction events were monitored using ambulatory electrocardiography for at least 6 h after the first dose. Of 2,417 enrolled patients, 2,282 (94.4 %) completed the study. Fingolimod initiation was associated with a transient, mostly asymptomatic decrease in HR. Bradycardia adverse events occurred in 0.6 % of patients and were more frequent in individuals receiving BBs/CCBs (3.3 %) than in other patient subgroups (0.5–1.4 %); most events were asymptomatic, and all patients recovered without pharmacological intervention. In the 6 h post-dose, the incidences of Mobitz type I second-degree atrioventricular block (AVB) and 2:1 AVB were higher in patients with PCCs (4.1 and 2.0 %, respectively) than in those without (0.9 and 0.3 %, respectively); at pre-dose screening, patients with PCCs had the same incidence of Mobitz type I second-degree AVB (4.1 %) and a slightly lower incidence of 2:1 AVB (0.7 %) than 6 h post-dose. All recorded conduction abnormalities were asymptomatic. This study adds to the evidence showing that cardiac effects during fingolimod initiation remain consistent with those known from previous, controlled studies, even if patients with PCCs are included.
To investigate the effect of different natalizumab washout (WO) periods on recurrence of MRI and clinical disease activity in patients switching from natalizumab to fingolimod.
In this multicenter, ...double-blind, placebo-controlled trial (TOFINGO), patients with relapsing-remitting multiple sclerosis (RRMS) were randomized 1:1:1 to 8-, 12-, or 16-week WO followed by fingolimod treatment over 32 weeks from last natalizumab infusion (LNI). Brain MRI was performed at baseline and weeks 8, 12, 16, 20, and 24.
Of 142 enrolled and randomized patients, 112 (78.9%) completed the study (8 weeks, n = 41/50; 12 weeks, n = 31/42; 16 weeks, n = 40/50). Number (95% confidence interval CI) of active (new/newly enlarged T2) lesions from LNI through 8 weeks of fingolimod treatment (primary outcome) was similar in the 8-week (2.1 1.7-2.6) and 12-week WO groups (1.7 1.3-2.2) and higher in the 16-week WO group (8.2 7.3-9.1). During the WO period only, the number (95% CI) of active lesions increased with increasing WO duration (8 weeks, 0.4 0.2-0.6; 12 weeks, 2.1 1.6-2.6; 16 weeks, 3.6 3.0-4.2). Over the 24 weeks from LNI, gadolinium-enhancing T1 lesion counts were lower in the 8-week WO group (14.1 5.67-22.53) than in the 12-week (21.3 1.41-41.19) or 16-week (18.5 8.40-28.60) WO groups. More patients were relapse-free in the 8-week (88%) and 12-week (91%) WO groups than the 16-week WO group (84%). Sixty-eight percent of patients experienced adverse events (mostly mild/moderate), with similar incidence across groups. No unusually severe relapses or opportunistic infections occurred.
Initiating fingolimod therapy 8-12 weeks after natalizumab discontinuation is associated with a lower risk of MRI and clinical disease reactivation than initiation after 16-week WO.
This study provides Class II evidence that for patients with RRMS switching from natalizumab to fingolimod, shorter natalizumab WO periods are associated with less MRI disease activity than are longer WO periods.
Background: The IL-3 receptor CD123 is an attractive target for antibody-based therapies in acute myeloid leukemia (AML) because of its frequent expression on AML blasts and its reported ...overexpression on leukemic stem cells (LSCs) as compared to normal hematopoietic tissues. Previously, two CD123 x CD3 bispecific targeting ADAPTIR constructs (APVO436 and APVO437) have been shown to bind CD123 with high affinity, engage T-cells and, in the presence of effector T-cells, cause cytotoxicity in CD123-expressing cell lines as well as in an in vivo xenograft model. Here, we further investigated the determinants of anti-tumor efficacy of these constructs in human AML.
Materials and Methods: Frozen aliquots of Ficoll-isolated mononuclear cells from peripheral blood or bone marrow specimens were obtained from adults with AML. CD123 expression on myeloblasts and cells lines and the percentage of endogenous CD3+ T-cells in AML specimens were quantified by flow cytometry. To determine specific cytotoxicity, cell lines or primary AML cells were incubated in culture medium containing various concentrations of ADAPTIR constructs APVO436 and APVO437. Exogenous T-cells isolated from healthy volunteers were labeled with CellVue membrane dye and added at different effector:target (E:T) cell ratios. After 48 hours, cell numbers and drug-induced cytotoxicity, using DAPI to detect non-viable cells, were determined by flow cytometry; target AML cells were identified by forward/side scatter properties and negativity for CellVue dye. Specific cytotoxicity was calculated as: 100 x (%DAPItreated - %DAPIcontrol)/(100-%DAPIcontrol) and are presented as mean±SEM of all samples tested. T-cell activation, proliferation and depletion of CD123+ cell populations in normal and AML specimens were assessed using multi-color flow cytometry.
Results: We examined the ability of APVO436 and APVO437 to induce cytotoxicity in primary human AML specimens. Of 20 samples tested, 10 had both >60% viability on thaw and >40% viability after 48 hours in cultures and were included for further analysis. The median percentage of myeloid blasts and CD3+ T-cells in the analyzed specimens was 78.5% (range 52-91.7%) and 2.95% (range 0.3-8.8%). With no exogenous T-cells, APVO436 and APVO437 induced modest cytotoxicity; at 1000 pM, APVO436 and APVO437 induced specific cytotoxicity of 6.8±3.8% and 3.9±3.6%, respectively. There was no statistically significant correlation between the percentage of endogenous T-cells present in the sample and the amount of cytotoxicity in the absence of exogenous T-cells. The addition of healthy donor T-cells increased cytotoxicity in a E:T-dependent manner (p <0.0001 for both APVO436 and APVO437) and increasing drug concentrations also increased cytotoxicity in a statistically-significant fashion (p <0.0001 for both APVO436 and APVO437). There was no consistent linear relationship between MFICD123 of blasts (MFICD123, arbitrary units) and cytotoxicity. Significant cytotoxicity was achieved even at the lowest levels of blast CD123 expression (MFICD123 range among patient samples: 349-3423). Inclusion of all 20 samples in cytotoxicity analysis did not qualitatively change results. APVO436 induced T-cell activation in both normal and AML samples at 24 hours as assessed by quantification of CD25 and CD69 expressing T-cells. T-cell activation was accompanied by proliferation of T-cells in normal and AML specimens at 96 hours in the presence of APVO436. APVO436 induced dose dependent depletion of CD123+ cells which was evident at 24 hours in both normal and AML samples.
Conclusion: APVO436 and APVO437 cause target cell cytolysis in a dose-dependent, target-antigen-dependent, and E:T-dependent manner. Broad activity was seen in primary human AML samples and both constructs were effective in inducing cytotoxicity across a range of blast CD123 expression levels, including specimens with very limited CD123 expression. Endogenous T-cell activation and proliferation accompanied by CD123+ cell depletion demonstrate similar activity of anti-CD123 x anti-CD3 ADAPTIRs in both normal and AML subject samples. These data are supportive of further investigation of anti-CD123 x anti-CD3 ADAPTIR molecules as a potential treatment option for AML.
Gottschalk:Aptevo Therapeutics: Employment, Equity Ownership. Comeau:Aptevo Therapeutics: Employment, Equity Ownership. Hoyos:Aptevo Therapeutics: Employment, Equity Ownership. Walter:ADC Therapeutics: Research Funding; Aptevo Therapeutics: Research Funding.
Abstract 3722
Despite advances in treatments for B-cell leukemias and lymphomas, many patients ultimately relapse and succumb to disease following multiple courses of therapy. Bispecific antibody ...fragments that can simultaneously engage T cells and tumor cells have been shown, in the literature, to destroy tumor cells by effectively redirecting the cytotoxic function of T cells. T-cell engaging bispecific molecules linking anti-CD19 and anti-CD3 binding domains in the context of novel SCORPION™ (multi-specific protein therapeutic) proteins were evaluated both in vitro and in vivo for function and stability.
Redirected T-cell cytotoxicity (RTCC) was measured by combining CD19 positive or negative cell lines with SCORPION proteins in the presence of human T cells. In a similar assay context, CFSE-labeled T cells were monitored for activation and proliferation. Functional RTCC assays were also used to analyze serum stability of SCORPION molecules in vitro and to complete an in vivo pharmacokinetic analysis. In vivo efficacy was assessed by monitoring the rate of tumor outgrowth of Ramos xenografts co-implanted with human peripheral blood mononuclear cells (PBMC) in NOD/SCID mice after treatment with SCORPION molecules.
SCORPION molecules potently mediate target-specific T-cell cytotoxicity toward tumor cell lines presenting cell surface CD19, with EC50 values for cytotoxicity at low pM concentrations. These molecules also demonstrate induction of T-cell activation and proliferation in the presence of target-bearing tumor cells but not in the absence of target expression. SCORPION molecules retain stable function following incubation at 37°C in mouse serum for up to a week in vitro, and pharmacokinetic analysis of SCORPION protein function in BALB/c mouse serum following intravenous administration resulted in half-life estimates of 69–84 hours. In efficacy studies conducted in NOD/SCID mice, SCORPION proteins significantly inhibited the outgrowth of Ramos tumor xenografts in the presence of human effector cells.
SCORPION molecules targeting CD19 and CD3 effectively harness the cytotoxic activity of T cells to kill CD19 positive tumor cells both in vitro and in vivo and show potential for further investigation as possible therapeutic agents for B-cell malignancies.
Chenault:Emergent BioSolutions: Employment. Gottschalk:Emergent BioSolutions: Employment. Hernandez-Hoyos:Emergent BioSolutions: Employment. Wiens:Emergent BioSolutions: Employment. Gordon:Emergent BioSolutions: Employment. Klee:Emergent BioSolutions: Employment, Equity Ownership. Bienvenue:Emergent BioSolutions: Employment. Dasovich:Emergent BioSolutions: Employment. Kumer:Emergent BioSolutions: Employment. Aguilar:Emergent BioSolutions: Employment. Bannink:Emergent BioSolutions: Employment, Equity Ownership. McMahan:Emergent BioSolutions: Employment, Equity Ownership. Natarajan:Emergent BioSolutions: Employment, Equity Ownership. Algate:Emergent BioSolutions: Employment, Equity Ownership. Blankenship:Emergent BioSolutions: Employment, Equity Ownership, Patents & Royalties.
•Lentiviral vectors displaying T-cell activation and costimulatory molecules (VivoVec) generate CAR T cells in vivo without lymphodepletion.•VivoVec administration in nonhuman primates generates ...anti-CD20 CAR T cells in vivo, leading to prolonged complete B-cell depletion.
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Chimeric antigen receptor (CAR) T-cell therapies have demonstrated transformative efficacy in treating B-cell malignancies. However, high costs and manufacturing complexities hinder their widespread use. To overcome these hurdles, we have developed the VivoVec platform, a lentiviral vector capable of generating CAR T cells in vivo. Here, we describe the incorporation of T-cell activation and costimulatory signals onto the surface of VivoVec particles (VVPs) in the form of a multidomain fusion protein and show enhanced in vivo transduction and improved CAR T-cell antitumor functionality. Furthermore, in the absence of lymphodepleting chemotherapy, administration of VVPs into nonhuman primates resulted in the robust generation of anti-CD20 CAR T cells and the complete depletion of B cells for >10 weeks. These data validate the VivoVec platform in a translationally relevant model and support its transition into human clinical testing, offering a paradigm shift in the field of CAR T-cell therapies.
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