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 Optimization of the crystallizability of a single-chain antibody fragment

2014 ◽  
Vol 70 (12) ◽  
pp. 1701-1706 ◽  
Author(s):  
Jana Škerlová ◽  
Vlastimil Král ◽  
Milan Fábry ◽  
Juraj Sedláček ◽  
Václav Veverka ◽  
...  
Keyword(s):  
Three Dimensional ◽  
Antibody Fragment ◽  
Binding Mode ◽  
Antibody Fragments ◽  
Dimensional Structure ◽  
Single Chain ◽  
Single Chain Antibody ◽  
Diagnostic Potential ◽  
Storage Buffer ◽  
Single Chain Antibody Fragment

Single-chain variable antibody fragments (scFvs) are molecules with immense therapeutic and diagnostic potential. Knowledge of their three-dimensional structure is important for understanding their antigen-binding mode as well as for protein-engineering approaches such as antibody humanization. A major obstacle to the crystallization of single-chain variable antibody fragments is their relatively poor homogeneity caused by spontaneous oligomerization. A new approach to optimization of the crystallizability of single-chain variable antibody fragments is demonstrated using a representative single-chain variable fragment derived from the anti-CD3 antibody MEM-57. A Thermofluor-based assay was utilized to screen for optimal conditions for antibody-fragment stability and homogeneity. Such an optimization of the protein storage buffer led to a significantly improved ability of the scFv MEM-57 to yield crystals.

T-cell activation and cytokine production via a bispecific single-chain antibody fragment targeted to blood-stage malaria parasites

Blood ◽  
2003 ◽  
Vol 101 (6) ◽  
pp. 2300-2306 ◽  
Author(s):  
Shigeto Yoshida ◽  
Tominari Kobayashi ◽  
Hiroyuki Matsuoka ◽  
Chisato Seki ◽  
William L. Gosnell ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell Activation ◽  
Cell Activation ◽  
Recombinant Baculovirus ◽  
Antibody Fragment ◽  
Antibody Fragments ◽  
Therapeutic Drug ◽  
Single Chain ◽  
Single Chain Antibody ◽  
Single Chain Antibody Fragment

A novel bispecific single-chain antibody fragment (biscFv) has been constructed to address the possibility of a new approach to malaria therapeutic drug development. The biscFv consists of 2 different single-chain antibody fragments linked by a flexible peptide linker (Gly4-Ser)3. Of the 2 scFv fragments, one is directed against a conserved epitope of the 19-kDa C-terminal fragment of the major surface protein of human malignant malaria parasite, Plasmodium falciparum, and the other is directed against the CD3 antigen of human T cells. The biscFv expressed by a recombinant baculovirus retained the antigen-binding properties of the corresponding univalent single-chain antibody fragments and formed a bridge between P falciparum and T cells. In cooperation with T cells, the biscFv specifically induced not only interferon γ and tumor necrosis factor α, but also a significant increase of merozoite phagocytosis and growth inhibition of P falciparum in vitro. Thus, the biscFv possesses highly selective malaria-targeting properties and stimulates T cells to induce cytokines, presumably resulting in activation of macrophages, neutrophils, and natural killer cells, and parasite killing in vivo.

scholarly journals Optimal Neutralization of Centruroides noxius Venom Is Understood through a Structural Complex between Two Antibody Fragments and the Cn2 Toxin

2015 ◽  
Vol 291 (4) ◽  
pp. 1619-1630 ◽  
Author(s):  
Lidia Riaño-Umbarila ◽  
Luis M. Ledezma-Candanoza ◽  
Hugo Serrano-Posada ◽  
Guillermo Fernández-Taboada ◽  
Timoteo Olamendi-Portugal ◽  
...  
Keyword(s):  
Neutralizing Antibodies ◽  
Recombinant Antibody ◽  
Current Trend ◽  
Cross Reactivity ◽  
Antibody Fragments ◽  
Crystallographic Structure ◽  
Single Chain ◽  
Single Chain Antibody ◽  
Neutralizing Capacity ◽  
Centruroides Noxius

The current trend of using recombinant antibody fragments in research to develop novel antidotes against scorpion stings has achieved excellent results. The polyclonal character of commercial antivenoms, obtained through the immunization of animals and which contain several neutralizing antibodies that recognize different epitopes on the toxins, guarantees the neutralization of the venoms. To avoid the use of animals, we aimed to develop an equivalent recombinant antivenom composed of a few neutralizing single chain antibody fragments (scFvs) that bind to two different epitopes on the scorpion toxins. In this study, we obtained scFv RU1 derived from scFv C1. RU1 showed a good capacity to neutralize the Cn2 toxin and whole venom of the scorpion Centruroides noxius. Previously, we had produced scFv LR, obtained from a different parental fragment (scFv 3F). LR also showed a similar neutralizing capacity. The simultaneous administration of both scFvs resulted in improved protection, which was translated as a rapid recovery of previously poisoned animals. The crystallographic structure of the ternary complex scFv LR-Cn2-scFv RU1 allowed us to identify the areas of interaction of both scFvs with the toxin, which correspond to non-overlapping sites. The epitope recognized by scFv RU1 seems to be related to a greater efficiency in the neutralization of the whole venom. In addition, the structural analysis of the complex helped us to explain the cross-reactivity of these scFvs and how they neutralize the venom.

scholarly journals Characterization of a Monoclonal Antibody and Its Single-Chain Antibody Fragment Recognizing the Nucleoside Triphosphatase/Helicase Domain of the Hepatitis C Virus Nonstructural 3 Protein

2000 ◽  
Vol 7 (1) ◽  
pp. 58-63 ◽  
Author(s):  
Zhu-Xu Zhang ◽  
Una Lazdina ◽  
Margaret Chen ◽  
Darrell L. Peterson ◽  
Matti Sällberg
Keyword(s):  
Monoclonal Antibody ◽  
Hepatitis C Virus ◽  
Hepatitis C ◽  
Antibody Fragment ◽  
Expression Vectors ◽  
Helicase Domain ◽  
Single Chain ◽  
Single Chain Antibody ◽  
Variable Regions

ABSTRACT We have produced a murine monoclonal antibody (MAb), ZX10, recognizing the NTPase/helicase domain of the hepatitis C virus (HCV) nonstructural 3 protein (NS3), from which we designed a single-chain variable fragment (ScFv). The ZX10 MAb recognized a discontinuous epitope of the NTPase/helicase domain, of which the linear sequence GEIPFYGKAIPL at residues 1371 to 1382 constitutes one part. cDNAs from variable regions coding for the heavy and light chains were cloned, sequenced, and assembled into the NS3-ScFv, which was inserted into procaryotic and eucaryotic expression vectors.Escherichia coli-expressed NS3-ScFv inhibited the binding of the ZX10 MAb to NS3, confirming a retained specificity. However, the ability to bind the peptide 1371–1382 had been lost. In vitro-translated NS3-ScFv and HCV NS3/NS4A were coprecipitated by antibodies to HCV NS4A, confirming the in vitro activity of the NS3 ScFv. Thus, we have designed a functional NS3 NTPase/helicase domain-specific ScFv which should be evaluated further with respect to disturbing enzymatic functions of the NS3 protein.

Improved antigen binding by a CD20-specific single-chain antibody fragment with a mutation in CDRH1

2006 ◽  
Vol 43 (6) ◽  
pp. 550-558 ◽  
Author(s):  
P.J. Adamson ◽  
D.J. Millard ◽  
A.W. Hohmann ◽  
C. Mavrangelos ◽  
P.J. Macardle ◽  
...  
Keyword(s):  
Antibody Fragment ◽  
Antigen Binding ◽  
Single Chain ◽  
Single Chain Antibody ◽  
Single Chain Antibody Fragment

scholarly journals Research on the Mechanism of Action of a Citrinin and Anti-Citrinin Antibody Based on Mimotope X27

Toxins ◽  
2020 ◽  
Vol 12 (10) ◽  
pp. 655
Author(s):  
Yanping Li ◽  
Yucheng Hu ◽  
Zhui Tu ◽  
Zhenqiang Ning ◽  
Qinghua He ◽  
...  
Keyword(s):  
Amino Acids ◽  
Molecular Docking ◽  
Mechanism Of Action ◽  
3D Structure ◽  
Antibody Fragment ◽  
Binding Activity ◽  
Single Chain ◽  
Single Chain Antibody ◽  
Binding Model ◽  
Modeling Strategy

Immunoassays are developed based on antigen–antibody interactions. A mimotope is an effective recognition receptor used to study the mechanism of action of antigens and antibodies, and is used for improving the sensitivity of the antibody. In this study, we built a 3D structure of the citrinin (CIT) mimotope X27 and anti-CIT single-chain antibody fragment (ScFv) through a “homologous modeling” strategy. Then, CIT and X27 were respectively docked to anti-CIT ScFv by using the “molecular docking” program. Finally, T28, F29, N30, R31, and Y32 were confirmed as the key binding sites in X27. Furthermore, the result of the phage-ELISA showed that the mutational phage lost the binding activity to the anti-CIT ScFv when the five amino acids were mutated to “alanine”, thereby proving the correctness of the molecular docking model. Lastly, a site-directed saturation strategy was adopted for the sites (T28, F29, N30, R31, and Y32). Eighteen different amino acids were introduced to each site on average. The activities of all mutants were identified by indirect competitive ELISA. The sensitivities of mutants T28F, T28I, F29I, F29V, N30T, and N30V were 1.83-, 1.37-, 1.70-, 2.96-, 1.31-, and 2.01-fold higher than that of the wild-type, respectively. In conclusion, the binding model between the CIT and antibody was elaborated for the first time based on the mimotope method, thereby presenting another strategy for improving the sensitivity of citrinin detection in immunoassays.

scholarly journals Functional Production and Characterization of a Fibrin-Specific Single-Chain Antibody Fragment from Bacillus subtilis: Effects of Molecular Chaperones and a Wall-Bound Protease on Antibody Fragment Production

2002 ◽  
Vol 68 (7) ◽  
pp. 3261-3269 ◽  
Author(s):  
Sau-Ching Wu ◽  
Jonathan C. Yeung ◽  
Yanjun Duan ◽  
Ruiqiong Ye ◽  
Steven J. Szarka ◽  
...  
Keyword(s):  
Monoclonal Antibody ◽  
Bacillus Subtilis ◽  
Molecular Chaperones ◽  
Blood Clot ◽  
Expression System ◽  
Antibody Fragment ◽  
High Sensitivity ◽  
Single Chain ◽  
Single Chain Antibody ◽  
Major Factors

ABSTRACT To develop an ideal blood clot imaging and targeting agent, a single-chain antibody (SCA) fragment based on a fibrin-specific monoclonal antibody, MH-1, was constructed and produced via secretion from Bacillus subtilis. Through a systematic study involving a series of B. subtilis strains, insufficient intracellular and extracytoplasmic molecular chaperones and high sensitivity to wall-bound protease (WprA) were believed to be the major factors that lead to poor production of MH-1 SCA. Intracellular and extracytoplasmic molecular chaperones apparently act in a sequential manner. The combination of enhanced coproduction of both molecular chaperones and wprA inactivation leads to the development of an engineered B. subtilis strain, WB800HM[pEPP]. This strain allows secretory production of MH-1 SCA at a level of 10 to 15 mg/liter. In contrast, with WB700N (a seven-extracellular-protease-deficient strain) as the host, no MH-1 SCA could be detected in both secreted and cellular fractions. Secreted MH-1 SCA from WB800HM[pMH1, pEPP] could be affinity purified using a protein L matrix. It retains comparable affinity and specificity as the parental MH-1 monoclonal antibody. This expression system can potentially be applied to produce other single-chain antibody fragments, especially those with folding and protease sensitivity problems.

scholarly journals Single Chain Variable Fragment Fused to Maltose Binding Protein: A Modular Nanocarrier Platform for the Targeted Delivery of Antitumorals

2021 ◽  
Author(s):  
Francisco J. Reche-Perez ◽  
Simona Plesselova ◽  
Eduardo De los Reyes-Berbel ◽  
Mariano Ortega-Muñoz ◽  
F. Javier Lopez-Jaramillo ◽  
...  
Keyword(s):  
Monoclonal Antibody ◽  
Targeted Delivery ◽  
Specific Binding ◽  
Protein A ◽  
Antibody Fragments ◽  
Single Chain Variable Fragment ◽  
Single Chain ◽  
Binding Properties ◽  
Single Chain Variable Fragments ◽  
Selective Delivery

The use of the specific binding properties of monoclonal antibody fragments such as single-chain variable fragments (ScFv) for the selective delivery of antitumor therapeutics for cancer cells is attractive due...

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