Revusiran is a 1st-generation short interfering RNA targeting transthyretin conjugated to an N-ac... more Revusiran is a 1st-generation short interfering RNA targeting transthyretin conjugated to an N-acetylgalactosamine ligand to facilitate delivery to hepatocytes via uptake by the asialoglycoprotein receptors. Revusiran, in development for the treatment of hereditary transthyretin-mediated amyloidosis, was discontinued after an imbalance in deaths in the ''ENDEAVOUR'' phase 3 clinical trial. Nonclinical safety assessments included safety pharmacology, acute and repeat-dose toxicity, genotoxicity, and carcinogenicity. There were no effects on cardiovascular or respiratory function in monkeys after single doses of up to 100 mg/kg. No neurological effects were noted in monkeys in repeat-dose studies up to 300 mg/kg. Revusiran was well tolerated in repeat-dose mouse (weekly doses) and rat and monkey (five daily doses followed by weekly doses) toxicity studies. The no observed adverse effect level (NOAEL) in rats was 30 mg/kg based on reversible microscopic changes in liver that were accompanied by correlating elevations in clinical chemistry at higher doses. Dose-limiting toxicity was absent in monkeys, and the NOAEL was 200 mg/kg. There was no evidence of genotoxicity in vitro or in vivo at limit doses or carcinogenicity in a 2-year study in rats at doses up to 100 mg/kg. Overall, these results demonstrate that revusiran had a favorable nonclinical safety profile.
G28-5 sFv-PE40 is a single-chain immunotoxin targeted to CD40, which is highly expressed on human... more G28-5 sFv-PE40 is a single-chain immunotoxin targeted to CD40, which is highly expressed on human hematologic malignancies, including non-Hodgkin's lymphoma, B-lineage leukemias, multiple myeloma, and Hodgkin's disease, as well as certain carcinomas. In vitro analysis showed that this monovalent immunotoxin had a binding affinity of 3 nmol/L, within 15-fold of the bivalent parental monoclonal antibody. G28-5 sFv-PE40 was stable when incubated in mouse serum at 37°C for 6 hours and cleared from the circulation of mice with a half-life of 16.7 minutes. This immunotoxin was effective in treating human Burkitt's lymphoma xenografted SCID mice with complete responses, defined by an asymptomatic phenotype for greater than 120 days, obtained at doses of 0.13 to 0.26 mg/kg. The efficacy of treatment was dependent on the schedule used, with every three days for five injections being the most effective tested. The toxicity of G28-5 sFv-PE40 was examined in SCID mice, rats, and mon...
Revusiran is a 1st-generation short interfering RNA targeting transthyretin conjugated to an N-ac... more Revusiran is a 1st-generation short interfering RNA targeting transthyretin conjugated to an N-acetylgalactosamine ligand to facilitate delivery to hepatocytes via uptake by the asialoglycoprotein receptors. Revusiran, in development for the treatment of hereditary transthyretin-mediated amyloidosis, was discontinued after an imbalance in deaths in the ''ENDEAVOUR'' phase 3 clinical trial. Nonclinical safety assessments included safety pharmacology, acute and repeat-dose toxicity, genotoxicity, and carcinogenicity. There were no effects on cardiovascular or respiratory function in monkeys after single doses of up to 100 mg/kg. No neurological effects were noted in monkeys in repeat-dose studies up to 300 mg/kg. Revusiran was well tolerated in repeat-dose mouse (weekly doses) and rat and monkey (five daily doses followed by weekly doses) toxicity studies. The no observed adverse effect level (NOAEL) in rats was 30 mg/kg based on reversible microscopic changes in liver that were accompanied by correlating elevations in clinical chemistry at higher doses. Dose-limiting toxicity was absent in monkeys, and the NOAEL was 200 mg/kg. There was no evidence of genotoxicity in vitro or in vivo at limit doses or carcinogenicity in a 2-year study in rats at doses up to 100 mg/kg. Overall, these results demonstrate that revusiran had a favorable nonclinical safety profile.
G28-5 sFv-PE40 is a single-chain immunotoxin targeted to CD40, which is highly expressed on human... more G28-5 sFv-PE40 is a single-chain immunotoxin targeted to CD40, which is highly expressed on human hematologic malignancies, including non-Hodgkin's lymphoma, B-lineage leukemias, multiple myeloma, and Hodgkin's disease, as well as certain carcinomas. In vitro analysis showed that this monovalent immunotoxin had a binding affinity of 3 nmol/L, within 15-fold of the bivalent parental monoclonal antibody. G28-5 sFv-PE40 was stable when incubated in mouse serum at 37°C for 6 hours and cleared from the circulation of mice with a half-life of 16.7 minutes. This immunotoxin was effective in treating human Burkitt's lymphoma xenografted SCID mice with complete responses, defined by an asymptomatic phenotype for greater than 120 days, obtained at doses of 0.13 to 0.26 mg/kg. The efficacy of treatment was dependent on the schedule used, with every three days for five injections being the most effective tested. The toxicity of G28-5 sFv-PE40 was examined in SCID mice, rats, and mon...
The heavy metals Pb, Ni and Zn have previously been demonstrated to stimulate the proliferation o... more The heavy metals Pb, Ni and Zn have previously been demonstrated to stimulate the proliferation of an Lytl+2 , L3T4÷T-cell. This proliferation required the presence of Ia ÷ cells and was blocked by monoclonal antibodies directed against self I-A, self I-E and L3T4a. The work reported here examined the role of T-cell factors (IL-2 and y-IFN) and their receptors in the metal-induced lymphoproliferation. The metals Pb, Ni and Zn, at concentrations (100 /~M) which stimulate T-cell proliferation, had little effect on the ability of the IL-2-dependent cell line HT-2 to respond to exogenous IL-2. This suggests that Pb, Ni and Zn do not modulate the ability of IL-2 to interact with the IL-2 receptor. Ni and Zn significantly enhanced the synthesis/secretion of IL-2 by cultured splenocytes and the expression of the receptor for IL-2; however, Pb produced only slight enhancement. Likewise, anti-IL-2 receptor (7D4) antibodies were able to inhibit a significant portion of the Ni-and Zn-, but not Pb-, induced lymphoproliferation. The residual 3H-thymidine incorporation observed in the presence of anti-IL-2 and anti-IL-2R may represent cycling B-cells induced to proliferate by activated, but non-cycling, T-cells. Monoclonal anti-y-tFN (R4/6A2) equally inhibited all metal-induced lymphoproliferation, suggesting that metal-induced lymphoproliferation is dependent on the induction of y-IFN as well as IL-2 synthesis. The Pbinduced response being the least dependent on IL-2 lends support to the hypothesis that Pb, Ni and Zn may activate T-cells and/or B-cells by different mechanisms.
Journal of immunology (Baltimore, Md. : 1950), Jan 15, 1997
Immunotoxins have shown promise as antitumor agents in clinical trials. However, they have not be... more Immunotoxins have shown promise as antitumor agents in clinical trials. However, they have not become part of standard cancer therapy because of factors that include their inherent immunogenicity, which limits the duration of therapy. To address this issue, we evaluated in preclinical models the concomitant use of the immunosuppressive agent CTLA4Ig and BR96 sFv-PE40, a single-chain immunotoxin that binds to carcinoma cells expressing Le(y). Cotreatment with CTLA4Ig, an inhibitor of the CD28/CTLA4-CD80/CD86 costimulation pathway, blocked the production of Abs against BR96 sFv-PE40 in immunocompetent rodents and dogs. It also blocked hypersensitivity reactions in rats carrying colon carcinoma allografts during a second course of BR96 sFv-PE40 therapy, and the cotreatment with CTLA4Ig resulted in enhanced antitumor activity. Cotreatment with CTLA4Ig also prevented hypersensitivity reactions induced by repeat dosing of BR96 sFv-PE40 (q3dx5) in dogs. The production of anti-BR96-sFv-PE40...
Journal of immunology (Baltimore, Md. : 1950), Jan 15, 1990
PG of the E series are generally known to suppress immune responses, however, we have found that ... more PG of the E series are generally known to suppress immune responses, however, we have found that PGE synergizes with IL-4 to induce IgE and IgG1 production in LPS-stimulated murine B lymphocytes. PGE2 and PGE1 (10(-6) to 10(-8) M) significantly increase IgE and IgG1 production (up to 26-fold) at all concentrations of IL-4 tested. In addition to its effects on IgE and IgG1, PGE also causes a significant decrease in IgM and IgG3 synthesis, suggesting that PGE may promote IL-4-induced class switching. The specificity of the E series PG effect is demonstrated by the fact that PGF2 alpha (10(-6) M) does not alter production of any of these isotypes. Because PGE can mediate its effects through cAMP in some cases, we investigated the importance of cAMP levels in regulation of isotype expression. Other agents that increase intracellular cAMP levels (cholera toxin and dibutyryl cAMP) were assessed for their ability to regulate isotype differentiation. Cholera toxin (100 pg/ml) and dibutyryl ...
Journal of immunology (Baltimore, Md. : 1950), Jan 15, 1989
We have observed that a 2-h pretreatment of murine B cells with cholera toxin (CT) renders the B ... more We have observed that a 2-h pretreatment of murine B cells with cholera toxin (CT) renders the B cell incapable of receiving an activation signal via surface Ig as measured by cell volume increase and entry into the S phase of the cell cycle. In contrast, CT pretreatment does not inhibit the delivery of a signal by IL-4, as measured by increase in cell volume. In fact, CT pretreated B cells are able to respond to anti-Ig in the presence of IL-4, as measured by both an increase in cell size and entry into S suggesting that IL-4 overcomes the effects of CT on normal B cell activation. Despite blocking the anti-Ig-mediated entry into the cell cycle, CT was not able to interfere with the induction of nonresponsiveness by anti-Ig in normal B cells or with the delivery of growth-inhibitory signal to the B cell lymphoma WEHI-231. These results suggest that there are two signaling pathways mediated by cross-linking of surface Ig: one pathway sensitive and the other insensitive to modulation...
Journal of immunology (Baltimore, Md. : 1950), 1991
We have developed a simple and adaptable, polyclonal model for B cell nonresponsiveness that is b... more We have developed a simple and adaptable, polyclonal model for B cell nonresponsiveness that is based on the inhibitory activity of anti-Ig as a surrogate for Ag. In our system the induction phase (treatment with anti-Ig) is separated from the challenge phase (Ag or mitogen), so that the critical events in each phase can be evaluated. Our results show that T cell-depleted B cells precultured for 18 to 24 h with rabbit anti-Ig reagents are rendered unresponsive to challenge with either Ag, fluorescein coupled to Brucella abortus (FL-BA), or mitogen (LPS). This state of nonresponsiveness (anergy) is reflected by an inhibition of a prototype response to the fluorescein hapten, as well as total Ig and IgG synthesis, but no reduction in proliferation to LPS. Interestingly, mitogen-induced polyclonal antibody formation was consistently reduced by 90% by treatment with either F(ab')2 or intact IgG anti-Ig. In contrast, the Ag-driven (FL-BA) response of pretreated B cells was inhibited ...
Cell growth & differentiation : the molecular biology journal of the American Association for Cancer Research, 1992
Cross-linking of B-cell membrane immunoglobulin (Ig) receptors induces growth arrest at G1-S, lea... more Cross-linking of B-cell membrane immunoglobulin (Ig) receptors induces growth arrest at G1-S, leading to apoptosis and cell death in the immature lymphomas WEHI-231 and CH31, but not in the CH12 B-cell line. In this system, which has been used as a model for B-cell tolerance, we have established that these lymphomas produce active transforming growth factor beta (TGF-beta) when treated with anti-Ig and that their hierarchy of sensitivity to TGF-beta generally correlates with their growth inhibition by anti-Ig. TGF-beta, in turn, has been shown to interfere with the phosphorylation of the retinoblastoma gene product, pRB. Herein, we also demonstrate that in WEHI-231 B-lymphoma cells treated with anti-Ig for 24 h, the pRB protein is found to be predominantly in the underphosphorylated form, as previously reported for cells arrested by the exogenous addition of TGF-beta. However, neutralizing antibodies to TGF-beta failed to prevent growth inhibition by anti-Ig in WEHI-231 and CH31. Wh...
The pharmacokinetics of CTLA4Ig, a novel immunosuppressive therapeutic protein, were investigated... more The pharmacokinetics of CTLA4Ig, a novel immunosuppressive therapeutic protein, were investigated in cynomolgus monkeys following intravenous administration of single and multiple doses of, CTLA4Ig. In the single-dose study, two monkeys of each sex received a dose of either 10 or 33 mg kg-CTLA4Ig by an intravenous bolus injection into a sa4henous vein. In the multiple-dose study, three monkeys of each sex received either 10,22.4, or 50 mg kg-of CTLA4Ig as an intravenous bolus injection once every other day for 15 doses over 29 days. Serial blood samples were collected at appropriate time points following the single dose and the last multiple dose. The serum samples were analysed for CTLA4Ig using an enzyme immunoassay procedure. The serum concentration data were evaluated by a noncompartmental method. Following single and multiple doses, both peak plasma concentration (Cma) and area under the curve (AUC) values for CTLA4Ig increased in a proportion comparable with the dose increment ratio. The clearance values (C b) for CTLA4Ig were not altered following the administration of single doses. The terminal elimination half-life values (tf) showed a tendency to increase as the administered dose increased. The steady-state volume of distribution (Vdss) of CTLA4Ig was about 2-3 times the blood volume in monkeys. On the basis of the derived and simulated pharmacokinetic parameters (using superposition principles), it was concluded that the pharmacokinetics of CTLA4Ig was not altered by repeated dosing.
The purpose of this research was to determine whether prostaglandin E, (PGE,), a major product of... more The purpose of this research was to determine whether prostaglandin E, (PGE,), a major product of macrophages which can kill certain murine B cell lymphomas, induces death by a necrotic mechanism or by an alternate pathway called apoptosis. CH31 is a phenotypically "immature" B cell lymphoma which resembles immature neonatal B cells in its susceptibility to killing by reagents which cross-link surface immunoglobulin (slg). In the present study we first show that PGE,, but not the closely related prostanoid, PGF,,, kills CH31 lymphoma cells. In contrast, CH12, a phenotypically "mature" lymphoma which is not negatively affected by slg cross-linking, is not induced to die after exposure to PGE,. Agarose gel electrophoresis demonstrated that the DNA of PGE,-treated CH31, but not CH12 cells, is cleaved into characteristic 200 base pair oligonucleosomal fragments indicative of an apoptotic mechanism of death. However, a necrotic form of death, indicated by random DNA cleavage which produces a smear following electrophoresis, could be induced by treatment of CH12 or CH31 with anti-class II MHC antibodies and complement. The apoptotic mechanism of CH31 cell killing by PGE, was confirmed using scanning electron microscopy which demonstrated the unique membrane blebbing and bubbling pathognomonic of this form of death. Finally, using a recently devised flow cytometric method to study apoptosis in heterogeneous cell populations, we compared the ability of anti-IgM, PGE,, or PGF,, to induce apoptosis in B lymphocytes from neonatal or adult mice. Anti-IgM, and to a lesser extent PGE,, but not PGF,,, induces apoptosis in a fraction of neonatal B cells. None of these treatments induced cell death in B lymphocytes from mature mice. Overall, these observations sug
Engagement of the B7 family of molecules on antigen-presenting cells with their T cell-associated... more Engagement of the B7 family of molecules on antigen-presenting cells with their T cell-associated ligands, CD28 and CD152 (cytotoxic T lymphocyte-associated antigen-4 [CTLA-4]), provides a pivotal costimulatory signal in T-cell activation. We investigated the role of the CD28/CD152 pathway in psoriasis in a 26-week, phase I, open-label dose-escalation study. The importance of this pathway in the generation of humoral immune responses to T cell-dependent neoantigens, bacteriophage φX174 and keyhole limpet hemocyanin, was also evaluated. Forty-three patients with stable psoriasis vulgaris received 4 infusions of the soluble chimeric protein CTLA4Ig (BMS-188667). Forty-six percent of all study patients achieved a 50% or greater sustained improvement in clinical disease activity, with progressively greater effects observed in the highest-dosing cohorts. Improvement in these patients was associated with quantitative reduction in epidermal hyperplasia, which correlated with quantitative reduction in skin-infiltrating T cells. No markedly increased rate of intralesional T-cell apoptosis was identified, suggesting that the decreased number of lesional T cells was probably likely attributable to an inhibition of T-cell proliferation, T-cell recruitment, and/or apoptosis of antigen-specific T cells at extralesional sites. Altered antibody responses to T cell-dependent neoantigens were observed, but immunologic tolerance to these antigens was not demonstrated. This study illustrates the importance of the CD28/CD152 pathway in the pathogenesis of psoriasis and suggests a potential therapeutic use for this novel immunomodulatory approach in an array of T cell-mediated diseases.
A sensitive method has been developed for analyzing mitochondrial (mt) DNA content in multiple sp... more A sensitive method has been developed for analyzing mitochondrial (mt) DNA content in multiple species. Sequences specific for the mitochondrial cytochrome b gene (mtCytb) were selected as generic primers for use in the polymerase chain reaction (PCR), and were shown to amplify the expected 370-bp fragment in human, mouse, and rat. The specificity of the product was confirmed by hybridization analysis. Because of sequence diversity within the region defined by the primers, species-specific probes were synthesized to increase the sensitivity of detection. Because these mt sequences are not appreciably homologous to any nuclear chromosomal targets, total cellular DNA is a sufficient starting material for specific amplification. The relative amount of mtDNA from different tissues in mouse and rat assessed by PCR was in agreement with the histologic and biochemical characteristics of these tissues. The ability of the assay to measure previously described changes in mtDNA content in nucleoside analog-treated c...
The toxicity of BMS-182248, an immunoglobulin (cBR96)-cytotoxic drug (doxorubicin) conjugate, was... more The toxicity of BMS-182248, an immunoglobulin (cBR96)-cytotoxic drug (doxorubicin) conjugate, was investigated in Sprague-Dawley rats at single intravenous doses of 508, 1,200, and 2,550 mg/m2 (conjugated doxorubicin doses of 14.7, 34.8, and 74 mg/m 2, respectively) and compared to that obtained from administration of free doxorubicin at single doses of 33.6 and 72 mg/m2 (approximately equivalent to that contained in the 1,200- and 2,550-mg/m2 doses of BMS-182248, respectively). Necropsies were conducted on day 8, upon death/moribund sacrifice, or after an approximate 3-mo observation period following completion of treatment. Death/moribundity of all rats that received 72 mg/m2 and of 9 of 20 rats given 33.6 mg/m2 free doxorubicin were attributed primarily to delayed cardiotoxicity and glomerulonephropathy. With BMS-182248, death from glomerulonephropathy and cardiotoxicity occurred in only 4 of 20 rats given 2,550 mg/m2 (74 mg/m2 doxorubicin equivalent). No deaths or cardiotoxicity...
Proceedings of the National Academy of Sciences, 1988
The CH family of murine B-cell lymphomas includes several members that are sensitive to growth in... more The CH family of murine B-cell lymphomas includes several members that are sensitive to growth inhibition when their membrane IgM (mIgM) receptors are cross-linked by anti-mu chain, anti-kappa chain, or anti-idiotypic antibodies. These lymphomas are IgM+, Ia+, and IgD +/- and resemble neonatal B cells in terms of their exquisite sensitivity to anti-IgM-mediated negative signaling as a model for tolerance induction. In this report, we describe the properties of one of these lymphomas, CH33, which had been transfected with a construct containing an allotypically different delta chain constant region and the heavy chain variable region fragment from S107 (T15 idiotype positive). This transfected cell line allowed us to investigate the possibility that membrane IgD (mIgD) and mIgM can mediate different signals. Our results show that the transfected cells retained their exquisite sensitivity to anti-IgM-mediated growth inhibition; however, crosslinking of IgD with anti-delta chain antibo...
On page 2 of the original Correspondence article, under the subheading "Failure to reconcile disc... more On page 2 of the original Correspondence article, under the subheading "Failure to reconcile discordant preclinical data", the dose of 5.11A1 administered to HIS mice in the study by Legrand et al. was incorrectly stated as 0.3 mg per kg, when it should have been 0.3 mg per mouse or ~15 mg per kg. The authors apologize for this error and would like to clarify that it does not affect the conclusions that they drew from their discussion of this study, as the actual doses and dose-multiples were not their major point of concern. Indeed, the authors think that the surrogate end point of peripheral T cell depletion provided a clear signal that there should be concern regarding the risk to human safety. In addition, they think that the four key failures that they identified in the use of preclinical laboratory and animal data to 'predict' human responses and to address human safety risk are the main issue, and that those points are independent of dose.
Ly-1+ B cells have been reported to produce a number of autoantibodies, and to be involved in the... more Ly-1+ B cells have been reported to produce a number of autoantibodies, and to be involved in the selection and regulation of the conventional B cell repertoire. It is not known if these B cells, which are found in high numbers in the peritoneum of normal adult mice, themselves can be regulated. In this study, we evaluated the sensitivity of peritoneal B cells (PBCs) versus conventional splenic B cells to regulation in a model system for tolerance. Normal splenic (conventional) or PBCs (containing both CD5+ and CD5- 'sister' cells) were cultured overnight with either F(ab')2 or intact IgG anti-mouse Ig, washed, and then challenged with fluorescein(FL)-coupled to Brucella abortus (BA), trimethylammonium (TMA)-BA or lipopolysaccharide (LPS), and the IgM responses to the FL and TMA haptens measured. In contrast to spleen cells, which exhibited up to a 90% reduction in anti-FL responsiveness, pretreated PBCs were mostly resistant to this form of tolerance regardless of challenge. The anti-TMA response of PBCs, which reflects the skewed VH11 usage by peritoneal CD5 B cells, was also resistant to tolerance. However, splenic TMA-specific B cells appeared to be sensitive to unresponsiveness induced by anti-Ig. Signaling studies show that PBCs have a blunted initial Ca2+ response, suggesting that the consequence of anti-Ig crosslinking may be defective in these cells. Furthermore, phorbal myristate acetate and/or ionomycin treatment of both PB and splenic B cells led to hyporesponsiveness to LPS challenge. This suggests that PBCs may be defective in a signalling pathway, perhaps involving protein kinase C activation.(ABSTRACT TRUNCATED AT 250 WORDS)
... providing purified lymphokines, to Dr. Charles Sidman for several monoclonal anti-Igs, Dr. Da... more ... providing purified lymphokines, to Dr. Charles Sidman for several monoclonal anti-Igs, Dr. David Penney for electron micro-Page 17. LYMPHOKTNES IN B-CELL TOLERANCE 169 graphs, and Dr. Barbara Iglewski for cholera toxin. We also thank Daniella Livnat, Mary Holman ...
Revusiran is a 1st-generation short interfering RNA targeting transthyretin conjugated to an N-ac... more Revusiran is a 1st-generation short interfering RNA targeting transthyretin conjugated to an N-acetylgalactosamine ligand to facilitate delivery to hepatocytes via uptake by the asialoglycoprotein receptors. Revusiran, in development for the treatment of hereditary transthyretin-mediated amyloidosis, was discontinued after an imbalance in deaths in the ''ENDEAVOUR'' phase 3 clinical trial. Nonclinical safety assessments included safety pharmacology, acute and repeat-dose toxicity, genotoxicity, and carcinogenicity. There were no effects on cardiovascular or respiratory function in monkeys after single doses of up to 100 mg/kg. No neurological effects were noted in monkeys in repeat-dose studies up to 300 mg/kg. Revusiran was well tolerated in repeat-dose mouse (weekly doses) and rat and monkey (five daily doses followed by weekly doses) toxicity studies. The no observed adverse effect level (NOAEL) in rats was 30 mg/kg based on reversible microscopic changes in liver that were accompanied by correlating elevations in clinical chemistry at higher doses. Dose-limiting toxicity was absent in monkeys, and the NOAEL was 200 mg/kg. There was no evidence of genotoxicity in vitro or in vivo at limit doses or carcinogenicity in a 2-year study in rats at doses up to 100 mg/kg. Overall, these results demonstrate that revusiran had a favorable nonclinical safety profile.
G28-5 sFv-PE40 is a single-chain immunotoxin targeted to CD40, which is highly expressed on human... more G28-5 sFv-PE40 is a single-chain immunotoxin targeted to CD40, which is highly expressed on human hematologic malignancies, including non-Hodgkin's lymphoma, B-lineage leukemias, multiple myeloma, and Hodgkin's disease, as well as certain carcinomas. In vitro analysis showed that this monovalent immunotoxin had a binding affinity of 3 nmol/L, within 15-fold of the bivalent parental monoclonal antibody. G28-5 sFv-PE40 was stable when incubated in mouse serum at 37°C for 6 hours and cleared from the circulation of mice with a half-life of 16.7 minutes. This immunotoxin was effective in treating human Burkitt's lymphoma xenografted SCID mice with complete responses, defined by an asymptomatic phenotype for greater than 120 days, obtained at doses of 0.13 to 0.26 mg/kg. The efficacy of treatment was dependent on the schedule used, with every three days for five injections being the most effective tested. The toxicity of G28-5 sFv-PE40 was examined in SCID mice, rats, and mon...
Revusiran is a 1st-generation short interfering RNA targeting transthyretin conjugated to an N-ac... more Revusiran is a 1st-generation short interfering RNA targeting transthyretin conjugated to an N-acetylgalactosamine ligand to facilitate delivery to hepatocytes via uptake by the asialoglycoprotein receptors. Revusiran, in development for the treatment of hereditary transthyretin-mediated amyloidosis, was discontinued after an imbalance in deaths in the ''ENDEAVOUR'' phase 3 clinical trial. Nonclinical safety assessments included safety pharmacology, acute and repeat-dose toxicity, genotoxicity, and carcinogenicity. There were no effects on cardiovascular or respiratory function in monkeys after single doses of up to 100 mg/kg. No neurological effects were noted in monkeys in repeat-dose studies up to 300 mg/kg. Revusiran was well tolerated in repeat-dose mouse (weekly doses) and rat and monkey (five daily doses followed by weekly doses) toxicity studies. The no observed adverse effect level (NOAEL) in rats was 30 mg/kg based on reversible microscopic changes in liver that were accompanied by correlating elevations in clinical chemistry at higher doses. Dose-limiting toxicity was absent in monkeys, and the NOAEL was 200 mg/kg. There was no evidence of genotoxicity in vitro or in vivo at limit doses or carcinogenicity in a 2-year study in rats at doses up to 100 mg/kg. Overall, these results demonstrate that revusiran had a favorable nonclinical safety profile.
G28-5 sFv-PE40 is a single-chain immunotoxin targeted to CD40, which is highly expressed on human... more G28-5 sFv-PE40 is a single-chain immunotoxin targeted to CD40, which is highly expressed on human hematologic malignancies, including non-Hodgkin's lymphoma, B-lineage leukemias, multiple myeloma, and Hodgkin's disease, as well as certain carcinomas. In vitro analysis showed that this monovalent immunotoxin had a binding affinity of 3 nmol/L, within 15-fold of the bivalent parental monoclonal antibody. G28-5 sFv-PE40 was stable when incubated in mouse serum at 37°C for 6 hours and cleared from the circulation of mice with a half-life of 16.7 minutes. This immunotoxin was effective in treating human Burkitt's lymphoma xenografted SCID mice with complete responses, defined by an asymptomatic phenotype for greater than 120 days, obtained at doses of 0.13 to 0.26 mg/kg. The efficacy of treatment was dependent on the schedule used, with every three days for five injections being the most effective tested. The toxicity of G28-5 sFv-PE40 was examined in SCID mice, rats, and mon...
The heavy metals Pb, Ni and Zn have previously been demonstrated to stimulate the proliferation o... more The heavy metals Pb, Ni and Zn have previously been demonstrated to stimulate the proliferation of an Lytl+2 , L3T4÷T-cell. This proliferation required the presence of Ia ÷ cells and was blocked by monoclonal antibodies directed against self I-A, self I-E and L3T4a. The work reported here examined the role of T-cell factors (IL-2 and y-IFN) and their receptors in the metal-induced lymphoproliferation. The metals Pb, Ni and Zn, at concentrations (100 /~M) which stimulate T-cell proliferation, had little effect on the ability of the IL-2-dependent cell line HT-2 to respond to exogenous IL-2. This suggests that Pb, Ni and Zn do not modulate the ability of IL-2 to interact with the IL-2 receptor. Ni and Zn significantly enhanced the synthesis/secretion of IL-2 by cultured splenocytes and the expression of the receptor for IL-2; however, Pb produced only slight enhancement. Likewise, anti-IL-2 receptor (7D4) antibodies were able to inhibit a significant portion of the Ni-and Zn-, but not Pb-, induced lymphoproliferation. The residual 3H-thymidine incorporation observed in the presence of anti-IL-2 and anti-IL-2R may represent cycling B-cells induced to proliferate by activated, but non-cycling, T-cells. Monoclonal anti-y-tFN (R4/6A2) equally inhibited all metal-induced lymphoproliferation, suggesting that metal-induced lymphoproliferation is dependent on the induction of y-IFN as well as IL-2 synthesis. The Pbinduced response being the least dependent on IL-2 lends support to the hypothesis that Pb, Ni and Zn may activate T-cells and/or B-cells by different mechanisms.
Journal of immunology (Baltimore, Md. : 1950), Jan 15, 1997
Immunotoxins have shown promise as antitumor agents in clinical trials. However, they have not be... more Immunotoxins have shown promise as antitumor agents in clinical trials. However, they have not become part of standard cancer therapy because of factors that include their inherent immunogenicity, which limits the duration of therapy. To address this issue, we evaluated in preclinical models the concomitant use of the immunosuppressive agent CTLA4Ig and BR96 sFv-PE40, a single-chain immunotoxin that binds to carcinoma cells expressing Le(y). Cotreatment with CTLA4Ig, an inhibitor of the CD28/CTLA4-CD80/CD86 costimulation pathway, blocked the production of Abs against BR96 sFv-PE40 in immunocompetent rodents and dogs. It also blocked hypersensitivity reactions in rats carrying colon carcinoma allografts during a second course of BR96 sFv-PE40 therapy, and the cotreatment with CTLA4Ig resulted in enhanced antitumor activity. Cotreatment with CTLA4Ig also prevented hypersensitivity reactions induced by repeat dosing of BR96 sFv-PE40 (q3dx5) in dogs. The production of anti-BR96-sFv-PE40...
Journal of immunology (Baltimore, Md. : 1950), Jan 15, 1990
PG of the E series are generally known to suppress immune responses, however, we have found that ... more PG of the E series are generally known to suppress immune responses, however, we have found that PGE synergizes with IL-4 to induce IgE and IgG1 production in LPS-stimulated murine B lymphocytes. PGE2 and PGE1 (10(-6) to 10(-8) M) significantly increase IgE and IgG1 production (up to 26-fold) at all concentrations of IL-4 tested. In addition to its effects on IgE and IgG1, PGE also causes a significant decrease in IgM and IgG3 synthesis, suggesting that PGE may promote IL-4-induced class switching. The specificity of the E series PG effect is demonstrated by the fact that PGF2 alpha (10(-6) M) does not alter production of any of these isotypes. Because PGE can mediate its effects through cAMP in some cases, we investigated the importance of cAMP levels in regulation of isotype expression. Other agents that increase intracellular cAMP levels (cholera toxin and dibutyryl cAMP) were assessed for their ability to regulate isotype differentiation. Cholera toxin (100 pg/ml) and dibutyryl ...
Journal of immunology (Baltimore, Md. : 1950), Jan 15, 1989
We have observed that a 2-h pretreatment of murine B cells with cholera toxin (CT) renders the B ... more We have observed that a 2-h pretreatment of murine B cells with cholera toxin (CT) renders the B cell incapable of receiving an activation signal via surface Ig as measured by cell volume increase and entry into the S phase of the cell cycle. In contrast, CT pretreatment does not inhibit the delivery of a signal by IL-4, as measured by increase in cell volume. In fact, CT pretreated B cells are able to respond to anti-Ig in the presence of IL-4, as measured by both an increase in cell size and entry into S suggesting that IL-4 overcomes the effects of CT on normal B cell activation. Despite blocking the anti-Ig-mediated entry into the cell cycle, CT was not able to interfere with the induction of nonresponsiveness by anti-Ig in normal B cells or with the delivery of growth-inhibitory signal to the B cell lymphoma WEHI-231. These results suggest that there are two signaling pathways mediated by cross-linking of surface Ig: one pathway sensitive and the other insensitive to modulation...
Journal of immunology (Baltimore, Md. : 1950), 1991
We have developed a simple and adaptable, polyclonal model for B cell nonresponsiveness that is b... more We have developed a simple and adaptable, polyclonal model for B cell nonresponsiveness that is based on the inhibitory activity of anti-Ig as a surrogate for Ag. In our system the induction phase (treatment with anti-Ig) is separated from the challenge phase (Ag or mitogen), so that the critical events in each phase can be evaluated. Our results show that T cell-depleted B cells precultured for 18 to 24 h with rabbit anti-Ig reagents are rendered unresponsive to challenge with either Ag, fluorescein coupled to Brucella abortus (FL-BA), or mitogen (LPS). This state of nonresponsiveness (anergy) is reflected by an inhibition of a prototype response to the fluorescein hapten, as well as total Ig and IgG synthesis, but no reduction in proliferation to LPS. Interestingly, mitogen-induced polyclonal antibody formation was consistently reduced by 90% by treatment with either F(ab')2 or intact IgG anti-Ig. In contrast, the Ag-driven (FL-BA) response of pretreated B cells was inhibited ...
Cell growth & differentiation : the molecular biology journal of the American Association for Cancer Research, 1992
Cross-linking of B-cell membrane immunoglobulin (Ig) receptors induces growth arrest at G1-S, lea... more Cross-linking of B-cell membrane immunoglobulin (Ig) receptors induces growth arrest at G1-S, leading to apoptosis and cell death in the immature lymphomas WEHI-231 and CH31, but not in the CH12 B-cell line. In this system, which has been used as a model for B-cell tolerance, we have established that these lymphomas produce active transforming growth factor beta (TGF-beta) when treated with anti-Ig and that their hierarchy of sensitivity to TGF-beta generally correlates with their growth inhibition by anti-Ig. TGF-beta, in turn, has been shown to interfere with the phosphorylation of the retinoblastoma gene product, pRB. Herein, we also demonstrate that in WEHI-231 B-lymphoma cells treated with anti-Ig for 24 h, the pRB protein is found to be predominantly in the underphosphorylated form, as previously reported for cells arrested by the exogenous addition of TGF-beta. However, neutralizing antibodies to TGF-beta failed to prevent growth inhibition by anti-Ig in WEHI-231 and CH31. Wh...
The pharmacokinetics of CTLA4Ig, a novel immunosuppressive therapeutic protein, were investigated... more The pharmacokinetics of CTLA4Ig, a novel immunosuppressive therapeutic protein, were investigated in cynomolgus monkeys following intravenous administration of single and multiple doses of, CTLA4Ig. In the single-dose study, two monkeys of each sex received a dose of either 10 or 33 mg kg-CTLA4Ig by an intravenous bolus injection into a sa4henous vein. In the multiple-dose study, three monkeys of each sex received either 10,22.4, or 50 mg kg-of CTLA4Ig as an intravenous bolus injection once every other day for 15 doses over 29 days. Serial blood samples were collected at appropriate time points following the single dose and the last multiple dose. The serum samples were analysed for CTLA4Ig using an enzyme immunoassay procedure. The serum concentration data were evaluated by a noncompartmental method. Following single and multiple doses, both peak plasma concentration (Cma) and area under the curve (AUC) values for CTLA4Ig increased in a proportion comparable with the dose increment ratio. The clearance values (C b) for CTLA4Ig were not altered following the administration of single doses. The terminal elimination half-life values (tf) showed a tendency to increase as the administered dose increased. The steady-state volume of distribution (Vdss) of CTLA4Ig was about 2-3 times the blood volume in monkeys. On the basis of the derived and simulated pharmacokinetic parameters (using superposition principles), it was concluded that the pharmacokinetics of CTLA4Ig was not altered by repeated dosing.
The purpose of this research was to determine whether prostaglandin E, (PGE,), a major product of... more The purpose of this research was to determine whether prostaglandin E, (PGE,), a major product of macrophages which can kill certain murine B cell lymphomas, induces death by a necrotic mechanism or by an alternate pathway called apoptosis. CH31 is a phenotypically "immature" B cell lymphoma which resembles immature neonatal B cells in its susceptibility to killing by reagents which cross-link surface immunoglobulin (slg). In the present study we first show that PGE,, but not the closely related prostanoid, PGF,,, kills CH31 lymphoma cells. In contrast, CH12, a phenotypically "mature" lymphoma which is not negatively affected by slg cross-linking, is not induced to die after exposure to PGE,. Agarose gel electrophoresis demonstrated that the DNA of PGE,-treated CH31, but not CH12 cells, is cleaved into characteristic 200 base pair oligonucleosomal fragments indicative of an apoptotic mechanism of death. However, a necrotic form of death, indicated by random DNA cleavage which produces a smear following electrophoresis, could be induced by treatment of CH12 or CH31 with anti-class II MHC antibodies and complement. The apoptotic mechanism of CH31 cell killing by PGE, was confirmed using scanning electron microscopy which demonstrated the unique membrane blebbing and bubbling pathognomonic of this form of death. Finally, using a recently devised flow cytometric method to study apoptosis in heterogeneous cell populations, we compared the ability of anti-IgM, PGE,, or PGF,, to induce apoptosis in B lymphocytes from neonatal or adult mice. Anti-IgM, and to a lesser extent PGE,, but not PGF,,, induces apoptosis in a fraction of neonatal B cells. None of these treatments induced cell death in B lymphocytes from mature mice. Overall, these observations sug
Engagement of the B7 family of molecules on antigen-presenting cells with their T cell-associated... more Engagement of the B7 family of molecules on antigen-presenting cells with their T cell-associated ligands, CD28 and CD152 (cytotoxic T lymphocyte-associated antigen-4 [CTLA-4]), provides a pivotal costimulatory signal in T-cell activation. We investigated the role of the CD28/CD152 pathway in psoriasis in a 26-week, phase I, open-label dose-escalation study. The importance of this pathway in the generation of humoral immune responses to T cell-dependent neoantigens, bacteriophage φX174 and keyhole limpet hemocyanin, was also evaluated. Forty-three patients with stable psoriasis vulgaris received 4 infusions of the soluble chimeric protein CTLA4Ig (BMS-188667). Forty-six percent of all study patients achieved a 50% or greater sustained improvement in clinical disease activity, with progressively greater effects observed in the highest-dosing cohorts. Improvement in these patients was associated with quantitative reduction in epidermal hyperplasia, which correlated with quantitative reduction in skin-infiltrating T cells. No markedly increased rate of intralesional T-cell apoptosis was identified, suggesting that the decreased number of lesional T cells was probably likely attributable to an inhibition of T-cell proliferation, T-cell recruitment, and/or apoptosis of antigen-specific T cells at extralesional sites. Altered antibody responses to T cell-dependent neoantigens were observed, but immunologic tolerance to these antigens was not demonstrated. This study illustrates the importance of the CD28/CD152 pathway in the pathogenesis of psoriasis and suggests a potential therapeutic use for this novel immunomodulatory approach in an array of T cell-mediated diseases.
A sensitive method has been developed for analyzing mitochondrial (mt) DNA content in multiple sp... more A sensitive method has been developed for analyzing mitochondrial (mt) DNA content in multiple species. Sequences specific for the mitochondrial cytochrome b gene (mtCytb) were selected as generic primers for use in the polymerase chain reaction (PCR), and were shown to amplify the expected 370-bp fragment in human, mouse, and rat. The specificity of the product was confirmed by hybridization analysis. Because of sequence diversity within the region defined by the primers, species-specific probes were synthesized to increase the sensitivity of detection. Because these mt sequences are not appreciably homologous to any nuclear chromosomal targets, total cellular DNA is a sufficient starting material for specific amplification. The relative amount of mtDNA from different tissues in mouse and rat assessed by PCR was in agreement with the histologic and biochemical characteristics of these tissues. The ability of the assay to measure previously described changes in mtDNA content in nucleoside analog-treated c...
The toxicity of BMS-182248, an immunoglobulin (cBR96)-cytotoxic drug (doxorubicin) conjugate, was... more The toxicity of BMS-182248, an immunoglobulin (cBR96)-cytotoxic drug (doxorubicin) conjugate, was investigated in Sprague-Dawley rats at single intravenous doses of 508, 1,200, and 2,550 mg/m2 (conjugated doxorubicin doses of 14.7, 34.8, and 74 mg/m 2, respectively) and compared to that obtained from administration of free doxorubicin at single doses of 33.6 and 72 mg/m2 (approximately equivalent to that contained in the 1,200- and 2,550-mg/m2 doses of BMS-182248, respectively). Necropsies were conducted on day 8, upon death/moribund sacrifice, or after an approximate 3-mo observation period following completion of treatment. Death/moribundity of all rats that received 72 mg/m2 and of 9 of 20 rats given 33.6 mg/m2 free doxorubicin were attributed primarily to delayed cardiotoxicity and glomerulonephropathy. With BMS-182248, death from glomerulonephropathy and cardiotoxicity occurred in only 4 of 20 rats given 2,550 mg/m2 (74 mg/m2 doxorubicin equivalent). No deaths or cardiotoxicity...
Proceedings of the National Academy of Sciences, 1988
The CH family of murine B-cell lymphomas includes several members that are sensitive to growth in... more The CH family of murine B-cell lymphomas includes several members that are sensitive to growth inhibition when their membrane IgM (mIgM) receptors are cross-linked by anti-mu chain, anti-kappa chain, or anti-idiotypic antibodies. These lymphomas are IgM+, Ia+, and IgD +/- and resemble neonatal B cells in terms of their exquisite sensitivity to anti-IgM-mediated negative signaling as a model for tolerance induction. In this report, we describe the properties of one of these lymphomas, CH33, which had been transfected with a construct containing an allotypically different delta chain constant region and the heavy chain variable region fragment from S107 (T15 idiotype positive). This transfected cell line allowed us to investigate the possibility that membrane IgD (mIgD) and mIgM can mediate different signals. Our results show that the transfected cells retained their exquisite sensitivity to anti-IgM-mediated growth inhibition; however, crosslinking of IgD with anti-delta chain antibo...
On page 2 of the original Correspondence article, under the subheading "Failure to reconcile disc... more On page 2 of the original Correspondence article, under the subheading "Failure to reconcile discordant preclinical data", the dose of 5.11A1 administered to HIS mice in the study by Legrand et al. was incorrectly stated as 0.3 mg per kg, when it should have been 0.3 mg per mouse or ~15 mg per kg. The authors apologize for this error and would like to clarify that it does not affect the conclusions that they drew from their discussion of this study, as the actual doses and dose-multiples were not their major point of concern. Indeed, the authors think that the surrogate end point of peripheral T cell depletion provided a clear signal that there should be concern regarding the risk to human safety. In addition, they think that the four key failures that they identified in the use of preclinical laboratory and animal data to 'predict' human responses and to address human safety risk are the main issue, and that those points are independent of dose.
Ly-1+ B cells have been reported to produce a number of autoantibodies, and to be involved in the... more Ly-1+ B cells have been reported to produce a number of autoantibodies, and to be involved in the selection and regulation of the conventional B cell repertoire. It is not known if these B cells, which are found in high numbers in the peritoneum of normal adult mice, themselves can be regulated. In this study, we evaluated the sensitivity of peritoneal B cells (PBCs) versus conventional splenic B cells to regulation in a model system for tolerance. Normal splenic (conventional) or PBCs (containing both CD5+ and CD5- 'sister' cells) were cultured overnight with either F(ab')2 or intact IgG anti-mouse Ig, washed, and then challenged with fluorescein(FL)-coupled to Brucella abortus (BA), trimethylammonium (TMA)-BA or lipopolysaccharide (LPS), and the IgM responses to the FL and TMA haptens measured. In contrast to spleen cells, which exhibited up to a 90% reduction in anti-FL responsiveness, pretreated PBCs were mostly resistant to this form of tolerance regardless of challenge. The anti-TMA response of PBCs, which reflects the skewed VH11 usage by peritoneal CD5 B cells, was also resistant to tolerance. However, splenic TMA-specific B cells appeared to be sensitive to unresponsiveness induced by anti-Ig. Signaling studies show that PBCs have a blunted initial Ca2+ response, suggesting that the consequence of anti-Ig crosslinking may be defective in these cells. Furthermore, phorbal myristate acetate and/or ionomycin treatment of both PB and splenic B cells led to hyporesponsiveness to LPS challenge. This suggests that PBCs may be defective in a signalling pathway, perhaps involving protein kinase C activation.(ABSTRACT TRUNCATED AT 250 WORDS)
... providing purified lymphokines, to Dr. Charles Sidman for several monoclonal anti-Igs, Dr. Da... more ... providing purified lymphokines, to Dr. Charles Sidman for several monoclonal anti-Igs, Dr. David Penney for electron micro-Page 17. LYMPHOKTNES IN B-CELL TOLERANCE 169 graphs, and Dr. Barbara Iglewski for cholera toxin. We also thank Daniella Livnat, Mary Holman ...
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