scholarly journals The Instability of Dimeric Fc-Fusions Expressed in Plants Can Be Solved by Monomeric Fc Technology

2021 ◽  
Vol 12 ◽  
Author(s):  
Pia Gattinger ◽  
Shiva Izadi ◽  
Clemens Grünwald-Gruber ◽  
Somanath Kallolimath ◽  
Alexandra Castilho
Keyword(s):  
Monoclonal Antibodies ◽  
Fusion Proteins ◽  
Degradation Products ◽  
Therapeutic Proteins ◽  
Cost Effective ◽  
Full Length ◽  
Peptide Sequence ◽  
Reporter Protein ◽  
The Stability

The potential therapeutic value of many proteins is ultimately limited by their rapid in vivo clearance. One strategy to limit clearance by metabolism and excretion, and improving the stability of therapeutic proteins, is their fusion to the immunoglobulin fragment crystallizable region (Fc). The Fc region plays multiple roles in (i) dimerization for the formation of “Y”-shaped structure of Ig, (ii) Fc-mediated effector functions, (iii) extension of serum half-life, and (iv) a cost-effective purification tag. Plants and in particular Nicotiana benthamiana have proven to be suitable expression platforms for several recombinant therapeutic proteins. Despite the enormous success of their use for the production of full-length monoclonal antibodies, the expression of Fc-fused therapeutic proteins in plants has shown limitations. Many Fc-fusion proteins expressed in plants show different degrees of instability resulting in high amounts of Fc-derived degradation products. To address this issue, we used erythropoietin (EPO) as a reporter protein and evaluated the efforts to enhance the expression of full-length EPO-Fc targeted to the apoplast of N. benthamiana. Our results show that the instability of the fusion protein is independent from the Fc origin or IgG subclass and from the peptide sequence used to link the two domains. We also show that a similar instability occurs upon the expression of individual heavy chains of monoclonal antibodies and ScFv-Fc that mimic the “Y”-shape of antibodies but lack the light chain. We propose that in this configuration, steric hindrance between the protein domains leads to physical instability. Indeed, mutations of critical residues located on the Fc dimerization interface allowed the expression of fully stable EPO monomeric Fc-fusion proteins. We discuss the limitations of Fc-fusion technology in N. benthamiana transient expression systems and suggest strategies to optimize the Fc-based scaffolds on their folding and aggregation resistance in order to improve the stability.

scholarly journals In Vivo Glycan Engineering via the Mannosidase I Inhibitor (Kifunensine) Improves Efficacy of Rituximab Manufactured in Nicotiana benthamiana Plants

2018 ◽  
Author(s):  
Vally Kommineni ◽  
Matthew Markert ◽  
Zhongjie Ren ◽  
Sreenath Palle ◽  
Berenice Carrillo ◽  
...  
Keyword(s):  
Monoclonal Antibodies ◽  
Transient Expression ◽  
Nicotiana Benthamiana ◽  
Cost Effective ◽  
Fold Increase ◽  
Expression Platform ◽  
Dependent Cell ◽  
Anti Cd20

N-glycosylation has been shown to affect the pharmacokinetic properties of several classes of biologics including monoclonal antibodies, blood factors, and lysosomal enzymes. In the last two decades, N-glycan engineering has been employed to achieve a N-glycosylation profile that is either more consistent or aligned with a specific improved activity (i.e. effector function or serum half-life). In particular, attention has focused on engineering processes in vivo or in vitro to alter the structure of the N-glycosylation of the Fc region of anti-cancer monoclonal antibodies in order to increase antibody-dependent cell-mediated cytotoxicity (ADCC). Here we applied the mannosidase I inhibitor kifunensine to the Nicotiana benthamiana transient expression platform to produce an afucosylated anti-CD20 antibody (rituximab). We determined the optimal concentration of kifunensine used in the infiltration solution, 0.375 µM, which was sufficient to produce exclusively oligomannose glycoforms, at a concentration 14 times lower than previously published levels. The resulting afucosylated rituximab revealed a 14-fold increase in ADCC activity targeting the lymphoma cell line Wil2-S when compared with rituximab produced in the absence of kifunensine. When applied to the cost-effective and scalable N. benthamiana transient expression platform, the use of kifunensine allows simple in-process glycan engineering without the need for transgenic hosts.

scholarly journals Production of the Biocommodities Butanol and Acetone from Methanol with Fluorescent FAST-tagged Proteins using Metabolically Engineered Strains of Eubacterium Limosum

2021 ◽  
Author(s):  
Maximilian Flaiz ◽  
Gideon Ludwig ◽  
Frank R. Bengelsdorf ◽  
Peter Dürre
Keyword(s):  
Fusion Proteins ◽  
Reporter Protein ◽  
Gene Encoding ◽  
Fluorescent Reporter ◽  
Recombinant Production ◽  
Acetoacetate Decarboxylase ◽  
Genes Encoding ◽  
Eubacterium Limosum ◽  
Fluorescent Cells ◽  
The Stability

Abstract Background: The interest in using methanol as a substrate to cultivate acetogens increased in recent years since it can be sustainably produced from syngas and has the additional benefit of reducing greenhouse gas emissions. Eubacterium limosum is one of the few acetogens that can utilize methanol, is genetically accessible and, therefore, a promising candidate for the recombinant production of biocommodities from this C1 carbon source. Although several genetic tools are already available for certain acetogens including E. limosum, the use of brightly fluorescent reporter proteins is still limited.Results: In this study, we expanded the genetic toolbox of E. limosum by implementing the fluorescence-activating and absorption shifting tag (FAST) as a fluorescent reporter protein. Recombinant E. limosum strains that expressed the gene encoding FAST in an inducible and constitutive manner were constructed. Cultivation of these recombinant strains resulted in brightly fluorescent cells even under anaerobic conditions. Moreover, we produced the biocommodities butanol and acetone from methanol with recombinant E. limosum strains. Therefore, we used E. limosum cultures that produced FAST-tagged fusion proteins of the bifunctional acetaldehyde/alcohol dehydrogenase or the acetoacetate decarboxylase, respectively, and determined the fluorescence intensity and product yields during growth.Conclusions: The addition of FAST as an oxygen-independent fluorescent reporter protein expands the genetic toolbox of E. limosum. Moreover, our results show that FAST-tagged fusion proteins can be constructed without negatively impacting the stability, functionality, and productivity of the resulting enzyme. Finally, butanol and acetone can be produced from methanol using recombinant E. limosum strains expressing genes encoding fluorescent FAST-tagged fusion proteins.

Binding Properties of Monoclonal Antibodies against Human Fragment D-Dimer of Cross-Linked Fibrin to Human Plasma Clots in an In Vivo Model in Rabbits

1989 ◽  
Vol 61 (02) ◽  
pp. 307-313 ◽  
Author(s):  
P Holvoet ◽  
J M Stassen ◽  
Y Hashimoto ◽  
D Spriggs ◽  
P Devos ◽  
...  
Keyword(s):  
Monoclonal Antibodies ◽  
Human Plasma ◽  
Jugular Vein ◽  
Degradation Products ◽  
Control Group ◽  
Human Fibrinogen ◽  
Fab Fragments ◽  
Vein Segment ◽  
D Dimer

SummaryTwo (MA-15C5 and MA-8D3) out of approximately 500 monoclonal antibodies, obtained by fusion of P3X63-Ag8-6.5.3 myeloma cells with spleen cells of mice immunized with purified fragment D-dimer from human fibrin, demonstrated a more than 1,000-fold higher affinity for fragment D-dimer than for native fibrinogen. MA-15C5 was directed against a neoantigenic determinant only expressed in fragment D-dimer. MA-8D3 reacted equally well with fragment D-dimer of crosslinked fibrin and with fragment D of non-crosslinked fibrin but not with fragment D of fibrinogen. Both monoclonal antibodies did not crossreact with rabbit fibrin and its degradation products.The binding of 125I-labeled Fab fragments to human plasma clots, introduced and aged for 1 hr in the jugular vein of heparinized rabbits was studied. Following injection of an equimolar mixture of Fab fragments derived from MA-15C5 and MA-8D3, the clot to blood ratios of radioactivity increased from 3.2 ± 1.2(mean ± SD) at 4 hr to 7.2 ± 1.4 at 17 hr. The binding of Fab fragments of MA-15C5 and MA-8D3 was independent of the age (1 to 72hrs) of the clot and of heparin anticoagulation and was only slightly decreased (by 20%) in the presence of circulating human fibrinogen (90 mg/kg body weight) and of human crosslinked fibrin degradation products at a plasma concentration of 10 pg/ml. The binding of Fab fragments of MA-15C5 and MA-8D3 to occlusive human plasma clots in the femoral artery of rabbits was comparable to that of the non-occlusive human plasma clots in the jugular vein. The Fab fragments of MA-15C5 and MA-8D3, labeled with 123I to a specific activity of 10 μCi/pg were injected intravenously (3 μg/kg) in 72 rabbits with a nonocclusive 0.2 ml human plasma clot in the jugular vein and in 7 control rabbits that underwent the surgical procedure without clot formation. Total body scans performed at hourly intervals revealed a higher relative increase in gamma counts over the thrombus region in the group with thrombus as compared to controls: at 6 hr 54 ± 18 vs 16 ± 13% (mean ± SEM, p <0.1) and at12 hrs 35 ± 11 vs –7 ± 12 (p <0.05). The vein segment to blood ratios of 123I at 24 hrs were 6.6 ± 2.4 in the group with clot and 1.5 to 0.7 in the control group (p <0.01). We conclude that these Fab fragments may have a sufficiently high fibrin-affinity to allow in vivo thrombus localization by external scanning.

Unreliability of Current Serum Fibrin Degradation Product (FDP) Assays

1985 ◽  
Vol 53 (03) ◽  
pp. 301-302 ◽  
Author(s):  
P J Gaffney ◽  
M J Perry
Keyword(s):  
Monoclonal Antibodies ◽  
Serum Level ◽  
Degradation Product ◽  
Degradation Products ◽  
Polyclonal Antibodies ◽  
Fibrin Degradation Product ◽  
Serum Samples ◽  
Fibrinogen Degradation Products ◽  
A Value

SummaryPreviously, assays of fibrin-fibrinogen degradation products (FDP) had to be performed on serum samples. However, monoclonal antibodies (Mabs) are now available which permit the measurement of FDP directly in plasma. We have employed two Mabs, one monospecific for FDP originating from crosslinked fibrin and another panspecific for the FDP fraction, to determine normal FDP levels in plasma and serum. The monospecific Mab gave a value of 40 ng FDP/ml in plasma and 10 ng/ml in serum, while the serum level of FDP recorded using the panspecific Mab was >1000 ng/ml, at all the concentrations of thrombin employed. Similarly, when a solution of purified fibrinogen was treated with thrombin, the concentration of FDP present in the clot supernatant was >1000 ng/ml when assayed using the panspecific Mab. Thus during serum preparation as much as 75% of the native FDP is incorporated into the clot while in excess of 1000 ng/ml of laboratory generated FDP, probably incompletely polymerized fibrin, is measured using panspecific antisera. These data indicate that current FDP assays using polyclonal antibodies are not a reliable reflection of the FDP level generated in vivo. The use of FDP-specific Mabs which do not react with fibrinogen is recommended for future FDP assays performed directly on plasma.

Antipeptide Monoclonal Antibodies to Defined Fibrinogen Aα Chain Regions: Anti-Aα 487-498, a Structural Probe for Fibrinogenolysis

Blood ◽  
1998 ◽  
Vol 91 (5) ◽  
pp. 1590-1598 ◽  
Author(s):  
Joan H. Sobel ◽  
Ilya Trakht ◽  
Nicolas Pileggi ◽  
Hong Qi Wu
Keyword(s):  
Monoclonal Antibodies ◽  
Degradation Products ◽  
Clinical Intervention ◽  
Molecular Forms ◽  
Enzyme Linked Immunosorbent Assay ◽  
Peptide Cleavage ◽  
Multiple Antigenic Peptide ◽  
Structural Probe

The fibrinogen αC domain (Aα 220-610) is one of the earliest targets attacked by plasmin following fibrinolytic system activation. Monoclonal antibodies (MoAbs) to defined sequences within the αC domain provide the opportunity to explore the structure-function relationships involved in plasmin's interaction with its Aα chain substrate at greater resolution and can serve as reagents with potential clinical use for detecting fibrinogenolysis in vivo. The MoAb F-104 was raised against a multiple antigenic peptide derivative modelled after the hydrophilic 12-residue sequence corresponding to Aα 487-498 within the αC domain. A sensitive solution phase competitive enzyme-linked immunosorbent assay (ELISA) was developed for MoAb F-104 that can be applied for the direct measurement of intact fibrinogen (purified or plasma; ED50%≈5 pmol Aα chain equivalents/mL), with negligible cross-reactive interference from peptide cleavage products released by plasmin from the COOH-terminal end of the Aα chain (<3%). Immunoblotting and ELISA studies to characterize the fate of the F-104 epitope during fibrinogenolysis in vitro indicated a rapid loss of fibrinogen-associated immunoreactivity that reflected the heterogeneity of plasmin cleavage sites within the αC domain; cleavage at the 493-494 arg-his bond destroyed the F-104 epitope, while cleavage at other sites released it in an altered, inaccessible, conformation within the structure of 35- to 40-kD and 17.5- to 18-kD Aα chain degradation products. Application of the F-104 ELISA to monitor the course of Aα chain proteolysis in a small study population of patients undergoing thrombolytic therapy for myocardial infarction (n = 14) showed that the loss of fibrinogen-associated F-104 immunoreactivity was a very early marker (within 15 to 30 minutes) of in vivo fibrinogenolysis. Additional data obtained suggest that MoAb F-104 may have promise as a reagent for evaluating the creation of an effective lytic state early during therapy, information that could help determine the need for further clinical intervention. Thus, these studies illustrate a rational, targeted, approach towards the development of a novel antifibrinogen MoAb whose application as a structural probe for the region Aα 487-498 in vitro and in vivo can provide new insights into the various molecular forms of fibrinogen that circulate under physiologic conditions and in disease.

scholarly journals The stability of rat liver ribonucleic acid in vivo after methylation with methyl methanesulphonate or dimethylnitrosamine

1971 ◽  
Vol 125 (3) ◽  
pp. 821-827 ◽  
Author(s):  
Marilyn J. McElhone ◽  
P. J. O'Connor ◽  
A. W. Craig
Keyword(s):  
Rat Liver ◽  
Half Life ◽  
Orotic Acid ◽  
Degradation Products ◽  
Rna Degradation ◽  
Biological Half Life ◽  
Extraction And Purification ◽  
The Stability ◽  
Methylating Agents

1. RNA was isolated from rat liver at selected times after the intraperitoneal injection of either [14C]methyl methanesulphonate (50mg/kg) or [14C]dimethylnitrosamine (2mg/kg). These doses were chosen to minimize effects due to toxicity. 2. Two methods of extraction and purification of RNA were used and an analysis of the radioactivity present was made by column chromatography of acid hydrolysates of the purified RNA. 3. The extent of methylation of guanine, the principal site of alkylation in rat liver RNA, was determined at times up to 14 days after injection. Although dimethylnitrosamine is a potent liver carcinogen and methyl methanesulphonate is not carcinogenic to rat liver, the rate of disappearance of 7-methylguanine from RNA was similar for both compounds, with a half-life of about 3.5 days. 4. An estimate of the biological half-life of rRNA was made by using [3H]orotic acid. A half-life of 5 days was obtained and this was not affected by injecting animals with unlabelled methyl methanesulphonate at the same dosage of 50mg/kg used in the studies of RNA methylation. 5. After administration of labelled orotic acid, reutilization of labelled RNA degradation products probably results in an overestimation of the biological half-life for rRNA. It is suggested that non-toxic doses of methylating agents such as methyl methanesulphonate and dimethylnitrosamine may prove to be a more effective way of accurately estimating the biological turnover of RNA species.

scholarly journals Unusual duplication mutation in a surface loop of human transthyretin leads to an aggressive drug-resistant amyloid disease

2018 ◽  
Vol 115 (28) ◽  
pp. E6428-E6436 ◽  
Author(s):  
Elena S. Klimtchuk ◽  
Tatiana Prokaeva ◽  
Nicholas M. Frame ◽  
Hassan A. Abdullahi ◽  
Brian Spencer ◽  
...  
Keyword(s):  
Protein Interactions ◽  
Amyloid Fibrils ◽  
Tertiary Structure ◽  
Full Length ◽  
Single Amino Acid ◽  
Surface Loop ◽  
Amyloid Disease ◽  
Poor Treatment ◽  
The Stability

Transthyretin (TTR) is a globular tetrameric transport protein in plasma. Nearly 140 single amino acid substitutions in TTR cause life-threatening amyloid disease. We report a one-of-a-kind pathological variant featuring a Glu51, Ser52 duplication mutation (Glu51_Ser52dup). The proband, heterozygous for the mutation, exhibited an unusually aggressive amyloidosis that was refractory to treatment with the small-molecule drug diflunisal. To understand the poor treatment response and expand therapeutic options, we explored the structure and stability of recombinant Glu51_Ser52dup. The duplication did not alter the protein secondary or tertiary structure but decreased the stability of the TTR monomer and tetramer. Diflunisal, which bound with near-micromolar affinity, partially restored tetramer stability. The duplication had no significant effect on the free energy and enthalpy of diflunisal binding, and hence on the drug–protein interactions. However, the duplication induced tryptic digestion of TTR at near-physiological conditions, releasing a C-terminal fragment 49–129 that formed amyloid fibrils under conditions in which the full-length protein did not. Such C-terminal fragments, along with the full-length TTR, comprise amyloid deposits in vivo. Bioinformatics and structural analyses suggested that increased disorder in the surface loop, which contains the Glu51_Ser52dup duplication, not only helped generate amyloid-forming fragments but also decreased structural protection in the amyloidogenic residue segment 25–34, promoting misfolding of the full-length protein. Our studies of a unique duplication mutation explain its diflunisal-resistant nature, identify misfolding pathways for amyloidogenic TTR variants, and provide therapeutic targets to inhibit amyloid fibril formation by variant TTR.

scholarly journals In Vivo Glycan Engineering via the Mannosidase I Inhibitor (Kifunensine) Improves Efficacy of Rituximab Manufactured in Nicotiana benthamiana Plants

2019 ◽  
Vol 20 (1) ◽  
pp. 194 ◽  
Author(s):  
Vally Kommineni ◽  
Matthew Markert ◽  
Zhongjie Ren ◽  
Sreenath Palle ◽  
Berenice Carrillo ◽  
...  
Keyword(s):  
Monoclonal Antibodies ◽  
Transient Expression ◽  
Nicotiana Benthamiana ◽  
Cost Effective ◽  
Fold Increase ◽  
Expression Platform ◽  
Dependent Cell ◽  
Anti Cd20

N-glycosylation has been shown to affect the pharmacokinetic properties of several classes of biologics, including monoclonal antibodies, blood factors, and lysosomal enzymes. In the last two decades, N-glycan engineering has been employed to achieve a N-glycosylation profile that is either more consistent or aligned with a specific improved activity (i.e., effector function or serum half-life). In particular, attention has focused on engineering processes in vivo or in vitro to alter the structure of the N-glycosylation of the Fc region of anti-cancer monoclonal antibodies in order to increase antibody-dependent cell-mediated cytotoxicity (ADCC). Here, we applied the mannosidase I inhibitor kifunensine to the Nicotiana benthamiana transient expression platform to produce an afucosylated anti-CD20 antibody (rituximab). We determined the optimal concentration of kifunensine used in the infiltration solution, 0.375 µM, which was sufficient to produce exclusively oligomannose glycoforms, at a concentration 14 times lower than previously published levels. The resulting afucosylated rituximab revealed a 14-fold increase in ADCC activity targeting the lymphoma cell line Wil2-S when compared with rituximab produced in the absence of kifunensine. When applied to the cost-effective and scalable N. benthamiana transient expression platform, the use of kifunensine allows simple in-process glycan engineering without the need for transgenic hosts.

scholarly journals Antipeptide Monoclonal Antibodies to Defined Fibrinogen Aα Chain Regions: Anti-Aα 487-498, a Structural Probe for Fibrinogenolysis

Blood ◽  
1998 ◽  
Vol 91 (5) ◽  
pp. 1590-1598 ◽  
Author(s):  
Joan H. Sobel ◽  
Ilya Trakht ◽  
Nicolas Pileggi ◽  
Hong Qi Wu
Keyword(s):  
Monoclonal Antibodies ◽  
Degradation Products ◽  
Clinical Intervention ◽  
Molecular Forms ◽  
Enzyme Linked Immunosorbent Assay ◽  
Peptide Cleavage ◽  
Multiple Antigenic Peptide ◽  
Structural Probe

AbstractThe fibrinogen αC domain (Aα 220-610) is one of the earliest targets attacked by plasmin following fibrinolytic system activation. Monoclonal antibodies (MoAbs) to defined sequences within the αC domain provide the opportunity to explore the structure-function relationships involved in plasmin's interaction with its Aα chain substrate at greater resolution and can serve as reagents with potential clinical use for detecting fibrinogenolysis in vivo. The MoAb F-104 was raised against a multiple antigenic peptide derivative modelled after the hydrophilic 12-residue sequence corresponding to Aα 487-498 within the αC domain. A sensitive solution phase competitive enzyme-linked immunosorbent assay (ELISA) was developed for MoAb F-104 that can be applied for the direct measurement of intact fibrinogen (purified or plasma; ED50%≈5 pmol Aα chain equivalents/mL), with negligible cross-reactive interference from peptide cleavage products released by plasmin from the COOH-terminal end of the Aα chain (<3%). Immunoblotting and ELISA studies to characterize the fate of the F-104 epitope during fibrinogenolysis in vitro indicated a rapid loss of fibrinogen-associated immunoreactivity that reflected the heterogeneity of plasmin cleavage sites within the αC domain; cleavage at the 493-494 arg-his bond destroyed the F-104 epitope, while cleavage at other sites released it in an altered, inaccessible, conformation within the structure of 35- to 40-kD and 17.5- to 18-kD Aα chain degradation products. Application of the F-104 ELISA to monitor the course of Aα chain proteolysis in a small study population of patients undergoing thrombolytic therapy for myocardial infarction (n = 14) showed that the loss of fibrinogen-associated F-104 immunoreactivity was a very early marker (within 15 to 30 minutes) of in vivo fibrinogenolysis. Additional data obtained suggest that MoAb F-104 may have promise as a reagent for evaluating the creation of an effective lytic state early during therapy, information that could help determine the need for further clinical intervention. Thus, these studies illustrate a rational, targeted, approach towards the development of a novel antifibrinogen MoAb whose application as a structural probe for the region Aα 487-498 in vitro and in vivo can provide new insights into the various molecular forms of fibrinogen that circulate under physiologic conditions and in disease.

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