Expression of functional eGFP-fused antigen-binding fragment of ranibizumab in Pichia pastoris

Bioimpacts ◽  
2021 ◽  
Author(s):  
Shirin Movaghar Asareh ◽  
Tahereh Savei ◽  
Sareh Arjmand ◽  
Seyed Omid Ranaei Siadat ◽  
Fataneh Fatemi ◽  
...  
Keyword(s):  
Pichia Pastoris ◽  
Fluorescent Protein ◽  
Antibody Fragment ◽  
Binding Activity ◽  
Age Related Macular Degeneration ◽  
Light Chains ◽  
Antigen Binding ◽  
Active Structure ◽  
Age Related ◽  
Fragment Antigen Binding

Introduction: Ranibizumab is a mouse monoclonal antibody fragment antigen-binding (Fab) against human vascular endothelial growth factor-A (VEGF-A), inhibiting angiogenesis. This antibody is commercially produced in Escherichia coli host and used to treat wet age-related macular degeneration (AMD).Methods: In this study, the heavy and light chains of ranibizumab were expressed in Pichia pastoris. The expressed chains were incubated overnight at 4°C for interaction. The formation of an active structure was evaluated based on the interaction with substrate VEGF-A using an indirect ELISA, and an electrochemical setup. Furthermore, reconstruction of split enhanced green fluorescent protein (eGFP) reporter, chimerized at the C-terminus of the heavy and light chains, was used to characterize chains’ interaction. Results: P. pastoris efficiently expressed designed constructs and secreted them into the culture medium. The anti-Fab antibody detected the constructed Fab structure in western blot analysis. Reconstruction of the split reporter confirmed the interaction between heavy and light chains. The designed ELISA and electrochemical setup results verified the binding activity of the recombinant Fab structure against VEGF-A. Conclusion: In this work, we indicated that the heavy and light chains of ranibizumab Fab fragments (with or without linkage to split parts of eGFP protein) were produced in P. pastoris. The fluorescence of reconstructed eGFP was detected after incubating the equal ratio of chimeric-heavy and light chains. Immunoassay and electrochemical tests verified the bioactivity of constructed Fab. The data suggested that P. pastoris could be considered a potential efficient eukaryotic host for ranibizumab production.

scholarly journals Human rheumatoid factor crossidiotypes. I. WA and BLA are heat-labile conformational antigens requiring both heavy and light chains.

1986 ◽  
Vol 164 (5) ◽  
pp. 1809-1814 ◽  
Author(s):  
V Agnello ◽  
J L Barnes
Keyword(s):  
Rheumatoid Factor ◽  
Primary Structure ◽  
Binding Activity ◽  
Light Chains ◽  
Heat Denaturation ◽  
Antigen Binding ◽  
Rheumatoid Factors ◽  
Simple Method ◽  
Heat Labile

Evidence was obtained that both the WA and BLA crossidiotype (XId) groups are conformational antigens requiring both L and H chains and that with heat denaturation the antigens that define the XIds and antigen-binding activity are lost in parallel. In contrast, the primary structure-dependent crossreactive idiotype (CRI), PSL2, which is only weakly detected on native Wa and Bla monoclonal rheumatoid factors (mRFs), became prominently detected on the heated Wa and Bla mRFs. Heat denaturation may provide a simple method for distinguishing Ids determined by conformational antigen from primary structure-dependent Ids. In addition to heat denaturation, some acid conditions commonly used for preparation of RFs were also found to cause marked loss of Id antigen. The finding of PSL2-CRI on Bla mRF indicates that this Id is not unique to the WA XId.

scholarly journals Biofunctionalized Two-dimensional MoS2 Receptors for Rapid Response Modular Electronic SARS-CoV-2 and Influenza A Antigen Sensors

2020 ◽  
Author(s):  
C. Muratore ◽  
M.K. Muratore ◽  
D.R. Austin ◽  
P. Look ◽  
A.K. Benton ◽  
...  
Keyword(s):  
Influenza A ◽  
Antibody Fragment ◽  
Antibody Fragments ◽  
Thin Layers ◽  
Receptor Protein ◽  
Antigen Binding ◽  
Two Dimensional ◽  
Fab Fragment ◽  
Fragment Antigen Binding ◽  
Transducer Surface

AbstractMultiplex electronic antigen sensors for detection of SARS-Cov-2 spike glycoproteins or hemagglutinin from Influenza A in liquid samples with characteristics resembling extracted saliva were fabricated using scalable processes with potential for economical mass-production. The sensors utilize the sensitivity and surface chemistry of a two-dimensional MoS2 transducer for attachment of antibody fragments in a conformation favorable for antigen binding. Ultra-thin layers (3 nm) of amorphous MoS2 were directly sputtered over the entire sensor chip at room temperature and laser annealed to create an array of semiconducting 2H-MoS2 active sensor regions between metal contacts. The semiconducting region was functionalized with monoclonal antibody Fab (fragment antigen binding) fragments derived from whole antibodies complementary to either SARS-CoV-2 S1 spike protein or Influenza A hemagglutinin using a papain digestion to cleave the antibodies at the disulfide hinges. The high affinity for the MoS2 transducer surface with some density of sulfur vacancies for the antibody fragment base promoted chemisorption with antigen binding regions oriented for interaction with the sample. The angiostatin converting enzyme 2 (ACE2) receptor protein for the SARS-CoV-2 spike glycoprotein, was tethered to a hexa-histidine (his6) tag at its c-terminus both for purification purposes, as well as a motif for binding to MoS2. This modified protein was also investigated as a bio-recognition element. Electrical resistance measurements of sensors functionalized with antibody fragments and exposed to antigen concentrations ranging from 2-20,000 picograms per milliliter revealed selective responses in the presence of complementary antigens with sensitivity to SARS-CoV-2 or influenza A on the order of pg/mL and comparable to gold-standard diagnostics such as Polymerase Chain Reaction (PCR) analysis. Lack of antigen sensitivity for the larger ACE2 BRE further demonstrates the utility of the engineered antibody fragment/transducer interface in bringing the target antigen closer to the transducer surface for sensitivity required for early detection viral diagnostics.

scholarly journals Ranibizumab in Treatment for Age-related Macular Degeneration

2013 ◽  
Vol 07 (02) ◽  
pp. 109
Author(s):  
Javier Araiz ◽  
Luis Arias ◽  
Alfredo García-Layana ◽  
José María Ruiz-Moreno ◽  
◽  
...  
Keyword(s):  
Macular Degeneration ◽  
Significant Proportion ◽  
Antibody Fragment ◽  
Age Related Macular Degeneration ◽  
Long Term Results ◽  
Office Visits ◽  
Intravitreal Ranibizumab ◽  
Anti Vegf ◽  
Age Related ◽  
Number Of Injections

Ranibizumab is an antibody fragment (Fab) that binds and inhibits all isoforms of vascular endothelial growth factor (VEGF), which is considered the main target in neovascular age-related macular degeneration (nAMD). Based on scientific evidence of the Anti-VEGF Antibody for the Treatment of Predominantly Classic Choroidal Neovascularization in AMD (ANCHOR)/Minimally Classic/Occult Trial of the Anti-VEGF Antibody Ranibizumab in the Treatment of Neovascular AMD (MARINA) studies, intravitreal ranibizumab is indicated in all typical types of nAMD. Nevertheless, specific forms such as retinal angiomatous proliferation (RAP), polypoidal choroidal vasculopathy (PCV) and bad or non-responders may need combination therapies to increase the efficacy and reduce the number of injections. Due to the frequent office visits and injections required with a monthly ranibizumab therapy, several dosing strategies have been evaluated to maintain optimal efficacy while reducing the number of injections and visits in the clinical practice.Pro re nata(PRN) and ‘Treat and Extend’ are the most popular individualised therapeutic regimens. Few studies address long-term results of ranibizumab. It remains controversial whether initial good results can be maintained over time. AMD is a chronic disease and a significant proportion of patients require continued treatment.

scholarly journals Loxoscelism: Advances and Challenges in the Design of Antibody Fragments with Therapeutic Potential

Toxins ◽  
2020 ◽  
Vol 12 (4) ◽  
pp. 256
Author(s):  
Sabrina Karim-Silva ◽  
Alessandra Becker-Finco ◽  
Isabella Gizzi Jiacomini ◽  
Fanny Boursin ◽  
Arnaud Leroy ◽  
...  
Keyword(s):  
Monoclonal Antibody ◽  
Therapeutic Potential ◽  
Antibody Fragment ◽  
Binding Activity ◽  
Antibody Fragments ◽  
Antigen Binding ◽  
Single Chain ◽  
Single Chain Antibody ◽  
Humanized Antibody ◽  
Neutralizing Capacity

Envenoming due to Loxosceles spider bites still remains a neglected disease of particular medical concern in the Americas. To date, there is no consensus for the treatment of envenomed patients, yet horse polyclonal antivenoms are usually infused to patients with identified severe medical conditions. It is widely known that venom proteins in the 30–35 kDa range with sphingomyelinase D (SMasesD) activity, reproduce most of the toxic effects observed in loxoscelism. Hence, we believe that monoclonal antibody fragments targeting such toxins might pose an alternative safe and effective treatment. In the present study, starting from the monoclonal antibody LimAb7, previously shown to target SMasesD from the venom of L. intermedia and neutralize its dermonecrotic activity, we designed humanized antibody V-domains, then produced and purified as recombinant single-chain antibody fragments (scFvs). These molecules were characterized in terms of humanness, structural stability, antigen-binding activity, and venom-neutralizing potential. Throughout this process, we identified some blocking points that can impact the Abs antigen-binding activity and neutralizing capacity. In silico analysis of the antigen/antibody amino acid interactions also contributed to a better understanding of the antibody’s neutralization mechanism and led to reformatting the humanized antibody fragment which, ultimately, recovered the functional characteristics for efficient in vitro venom neutralization.

scholarly journals Single-Chain Antibody Fragment VEGF Inhibitor RTH258 for Neovascular Age-Related Macular Degeneration

Ophthalmology ◽  
2016 ◽  
Vol 123 (5) ◽  
pp. 1080-1089 ◽  
Author(s):  
Frank G. Holz ◽  
Pravin U. Dugel ◽  
Georges Weissgerber ◽  
Robin Hamilton ◽  
Rufino Silva ◽  
...  
Keyword(s):  
Macular Degeneration ◽  
Antibody Fragment ◽  
Age Related Macular Degeneration ◽  
Single Chain ◽  
Single Chain Antibody ◽  
Vegf Inhibitor ◽  
Age Related ◽  
Single Chain Antibody Fragment

scholarly journals Brolucizumab: A Newly Developed Anti-VEGF Molecule for the Treatment of Neovascular Age-Related Macular Degeneration

2020 ◽  
Author(s):  
Ramin Tadayoni ◽  
Laura Sararols ◽  
Georges Weissgerber ◽  
Rohini Verma ◽  
Andreas Clemens ◽  
...  
Keyword(s):  
Disease Control ◽  
Macular Degeneration ◽  
Antibody Fragment ◽  
Age Related Macular Degeneration ◽  
Phase Iii ◽  
Single Chain ◽  
Single Chain Antibody ◽  
Pivotal Phase ◽  
Anti Vegf ◽  
Age Related

Background Despite the success of anti-vascular endothelial growth factors (anti-VEGF), there is currently a need for highly effective compounds that can alleviate the burden of managing neovascular age-related macular degeneration (nAMD). Purpose To review the milestones in the molecular and clinical development of brolucizumab, the first single-chain antibody fragment designed specifically for intraocular use in humans. Methods In this article, we summarize the pre-clinical and current clinical evidence for brolucizumab, with an outlook to other treatment regimens and additional indications under investigation. Results The unique molecular design of brolucizumab led to a low molecular weight of only 26 kDa, allowing for a concentrated molar dosing in one intra-vitreal injection compared with other anti-VEGF agents. The Phase I and II clinical trial outcomes validated the efficacy of brolucizumab in the treatment of nAMD with signals of a more durable treatment effect. The pivotal Phase III trials HAWK and HARRIER, which included a total of 1,817 patients, established that brolucizumab can be administered every 3 months while maintaining disease control. Conclusions The pre-clinical and clinical data for brolucizumab provide evidence of sustained disease control with longer injection intervals, thus potentially reducing the treatment burden in patients with nAMD.

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