Fab Fragment of VHH-Based Antibody Netakimab: Crystal Structure and Modeling Interaction with Cytokine IL-17A

Interleukin 17A (IL-17A) is a proinflammatory cytokine produced by Th17 cells. Antibody BCD-085 (netakimab) against human IL-17A is one of the new inhibitors of this cytokine. In netakimab, the VH domain is replaced by the VHH domain of Lama glama possessing a long complementarity determining region (CDR-H3) in its heavy chain. Here we demonstrate the high affinity of IL-17A to the Fab fragment of netakimab and to its integral part, the VHH domain. We have determined the crystal structure of the Fab fragment of netakimab at 1.9 Å resolution. High variability in the orientation of light and heavy chains of the Fab fragment of netakimab was shown, which is determined by the peculiarity of the structural organization of the CDR-H3. As the high conformational plasticity of the molecule hampers modeling the Fab fragment of netakimab complexed to IL-17A, we have carried out modeling the complex between the antigen and the integral part of the Fab fragment, the VHH domain. We explain the high netakimab Fab fragment affinity for IL-17A by a large number of protein–protein contacts due to additional interactions between CDR-H3 and the cytokine dimer.

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Crystal Structure of Fcγ Receptor I and Its Implication in High Affinity γ-Immunoglobulin Binding

Journal of Biological Chemistry ◽

10.1074/jbc.m111.257550 ◽

2011 ◽

Vol 286 (47) ◽

pp. 40608-40613 ◽

Cited By ~ 54

Author(s):

Jinghua Lu ◽

Jeff L. Ellsworth ◽

Nels Hamacher ◽

Si Won Oak ◽

Peter D. Sun

Keyword(s):

Crystal Structure ◽

Fcγ Receptor ◽

High Affinity ◽

Immunoglobulin Binding

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Expression of β‐NGF and high‐affinity NGF receptor (TrKA) in llama ( Lama glama ) male reproductive tract and spermatozoa

Molecular Reproduction and Development ◽

10.1002/mrd.23075 ◽

2018 ◽

Vol 85 (12) ◽

pp. 934-944 ◽

Cited By ~ 5

Author(s):

Luciana M. Sari ◽

Renato Zampini ◽

Martin E. Argañaraz ◽

María I. Carretero ◽

Fernanda G. Fumuso ◽

...

Keyword(s):

Reproductive Tract ◽

Male Reproductive Tract ◽

High Affinity ◽

Ngf Receptor ◽

Lama Glama

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Rapid identification of anti-idiotypic mAbs with high affinity and diverse epitopes by rabbit single B-cell sorting-culture and cloning technology

PLoS ONE ◽

10.1371/journal.pone.0244158 ◽

2020 ◽

Vol 15 (12) ◽

pp. e0244158

Author(s):

WeiYu Lin ◽

Wei-Ching Liang ◽

Trung Nguy ◽

Mauricio Maia ◽

Tulika Tyagi ◽

...

Keyword(s):

B Cells ◽

B Cell ◽

Cell Sorting ◽

Assay Development ◽

Enzyme Linked Immunosorbent Assay ◽

Fab Fragment ◽

Variable Regions ◽

High Affinity ◽

Clinical Assay ◽

Cloning Technology

The proactive generation of anti-idiotypic antibodies (anti-IDs) against therapeutic antibodies with desirable properties is an important step in pre-clinical and clinical assay development supporting their bioanalytical programs. Here, we describe a robust platform to generate anti-IDs using rabbit single B cell sorting-culture and cloning technology by immunizing rabbits with therapeutic drug Fab fragment and sorting complementarity determining regions (CDRs) specific B cells using designed framework control as a negative gate to exclude non-CDRs-specific B cells. The supernatants of cultured B cells were subsequently screened for binding to drug-molecule by enzyme-linked immunosorbent assay and the positive hits of B cell lysates were selected for cloning of their immunoglobulin G (IgG) variable regions. The recombinant monoclonal anti-IDs generated with this method have high affinity and specificity with broad epitope coverage and different types. The recombinant anti-IDs were available for assay development to support pharmacokinetic (PK) and immunogenicity studies within 12 weeks from the start of rabbit immunization. Using this novel rapid and efficient in-house approach we have generated a large panel of anti-IDs against a series of 11 therapeutic antibody drugs and successfully applied them to the clinical assay development.

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Analysis of GPIIb/IIIa receptor number by quantification of 7E3 binding to human platelets

Blood ◽

10.1182/blood.v88.3.907.907 ◽

1996 ◽

Vol 88 (3) ◽

pp. 907-914 ◽

Cited By ~ 281

Author(s):

CL Wagner ◽

MA Mascelli ◽

DS Neblock ◽

HF Weisman ◽

BS Coller ◽

...

Keyword(s):

Quantitative Estimation ◽

Critical Factor ◽

Human Platelets ◽

Single Individual ◽

Binding Studies ◽

Fab Fragment ◽

High Affinity ◽

Fibrinogen Binding ◽

Receptor Number ◽

Derivatives Of

Abstract A large number of glycoprotein (GP) IIb/IIIa receptors are present on the surface of platelets. Studies to define precisely the number of GPIIb/IIIa receptors using specific monoclonal antibodies (MoAbs) or fibrinogen binding have, however, yielded varying estimates of receptor number. To refine the quantitative estimation of GPIIb/IIIa receptors on resting platelets, we have used the MoAb 7E3, which has high affinity for GPIIb/IIIa. Quantitative binding studies were performed using radiolabeled conjugates of 7E3 IgG, as well as fragments and derivatives of 7E3. For platelets obtained from any single individual, the numbers of 7E3 F(ab′)2 and IgG molecules bound per platelet were equivalent (approximately 40,000), whereas the number of Fab molecules bound per platelet was consistently approximately twofold higher (approximately 80,000). To investigate the basis of the quantitative disparity in binding of intact 7E3 and 7E3 F(ab′)2 versus 7E3 Fab, we studied the binding of a newly constructed, bispecific (Fab′)2 molecule containing only a single 7E3 combining site. Because this construct bound to the same extent as the Fab species, the larger size of the intact 7E3 and 7E3 F(ab′)2 molecules could not explain the reduced number of molecules that bound per platelet compared to the Fab fragment. Rather, it appears that the valency of the antibody is the critical factor determining the number of antibody molecules bound per platelet. Thus, we conclude that the binding of 7E3 Fab corresponds most closely with surface GPIIb/IIIa number and that the number of GPIIb/IIIa receptors is approximately 80,000 per platelet.

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Recognition of an α-helical hairpin in P22 large terminase by a synthetic antibody fragment

Acta Crystallographica Section D Structural Biology ◽

10.1107/s2059798320009912 ◽

2020 ◽

Vol 76 (9) ◽

pp. 876-888

Author(s):

Ravi K. Lokareddy ◽

Ying-Hui Ko ◽

Nathaniel Hong ◽

Steven G. Doll ◽

Marcin Paduch ◽

...

Keyword(s):

Crystal Structure ◽

Conformational Changes ◽

Recombinant Antibody ◽

Antibody Fragment ◽

Genome Packaging ◽

Synthetic Antibody ◽

High Affinity ◽

N Terminus ◽

Helical Hairpin ◽

Accessible Surface

The genome-packaging motor of tailed bacteriophages and herpesviruses is a multisubunit protein complex formed by several copies of a large (TerL) and a small (TerS) terminase subunit. The motor assembles transiently at the portal protein vertex of an empty precursor capsid to power the energy-dependent packaging of viral DNA. Both the ATPase and nuclease activities associated with genome packaging reside in TerL. Structural studies of TerL from bacteriophage P22 have been hindered by the conformational flexibility of this enzyme and its susceptibility to proteolysis. Here, an unbiased, synthetic phage-display Fab library was screened and a panel of high-affinity Fabs against P22 TerL were identified. This led to the discovery of a recombinant antibody fragment, Fab4, that binds a 33-amino-acid α-helical hairpin at the N-terminus of TerL with an equilibrium dissociation constant K d of 71.5 nM. A 1.51 Å resolution crystal structure of Fab4 bound to the TerL epitope (TLE) together with a 1.15 Å resolution crystal structure of the unliganded Fab4, which is the highest resolution ever achieved for a Fab, elucidate the principles governing the recognition of this novel helical epitope. TLE adopts two different conformations in the asymmetric unit and buries as much as 1250 Å2 of solvent-accessible surface in Fab4. TLE recognition is primarily mediated by conformational changes in the third complementarity-determining region of the Fab4 heavy chain (CDR H3) that take place upon epitope binding. It is demonstrated that TLE can be introduced genetically at the N-terminus of a target protein, where it retains high-affinity binding to Fab4.

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Inter-residue coupling contributes to high-affinity subtype-selective binding of α-bungarotoxin to nicotinic receptors

Biochemical Journal ◽

10.1042/bj20130638 ◽

2013 ◽

Vol 454 (2) ◽

pp. 311-321 ◽

Cited By ~ 14

Author(s):

Steven M. Sine ◽

Sun Huang ◽

Shu-Xing Li ◽

Corrie J. B. daCosta ◽

Lin Chen

Keyword(s):

Crystal Structure ◽

Ligand Binding ◽

Nicotinic Receptors ◽

Radioligand Binding ◽

Binding Domain ◽

Tyrosine Residue ◽

Selective Binding ◽

Conserved Residues ◽

High Affinity ◽

Subtype Selective

On the basis of the crystal structure of a pentameric α7 ligand-binding domain chimaera with bound α-bungarotoxin, mutagenesis and radioligand-binding measurements show that high-affinity target-selective binding depends on interactions between a single conserved tyrosine residue in α7 and nearby conserved and non-conserved residues.

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Crystal structure of oleate hydratase from Stenotrophomonas sp. KCTC 12332 reveals conformational plasticity surrounding the FAD binding site

Biochemical and Biophysical Research Communications ◽

10.1016/j.bbrc.2018.03.220 ◽

2018 ◽

Vol 499 (4) ◽

pp. 772-776 ◽

Cited By ~ 10

Author(s):

Ae Kyung Park ◽

Gyeong Hweon Lee ◽

Do Wan Kim ◽

Eun Hyuk Jang ◽

Ha Taek Kwon ◽

...

Keyword(s):

Crystal Structure ◽

Binding Site ◽

Conformational Plasticity ◽

Fad Binding ◽

Oleate Hydratase

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Structures of the antibody 64M-5 Fab and its complex with dT(6–4)T indicate induced-fit and high-affinity mechanisms

Acta Crystallographica Section F Structural Biology Communications ◽

10.1107/s2053230x18017661 ◽

2019 ◽

Vol 75 (2) ◽

pp. 80-88 ◽

Cited By ~ 1

Author(s):

Hideshi Yokoyama ◽

Ryuta Mizutani ◽

Shuji Noguchi ◽

Naoki Hayashida

Keyword(s):

Crystal Structures ◽

Ultraviolet Light ◽

Electrostatic Interaction ◽

Side Chain ◽

Induced Fit ◽

Phosphate Moiety ◽

High Affinity ◽

Dna Photoproducts ◽

Complementarity Determining Region

DNA photoproducts with (6–4) pyrimidine–pyrimidone adducts produced by ultraviolet light are mutagenic and carcinogenic. The crystal structures of the anti-(6–4) photoproduct antibody 64M-5 Fab and of its complex with dT(6–4)T were determined at 2.5 and 2.0 Å resolution, respectively. A comparison between the dT(6–4)T-liganded and unliganded structures indicates that the side chain of His93L is greatly rotated and shifted on binding to dT(6–4)T, leading to the formation of an electrostatic interaction with the phosphate moiety of dT(6–4)T, which shows a remarkable induced fit. Based on a comparison of the dT(6–4)T-liganded structures of the 64M-5 and 64M-2 Fabs, the electrostatic interaction between the side chain of His93L in 64M-5 and the phosphate moiety of dT(6–4)T is lost for Leu93L in 64M-2, while Arg90L in 64M-5 instead of Gln90L in 64M-2 stabilizes the conformation of complementarity-determining region (CDR) L3. These differences contribute to the higher affinity of 64M-5 for dT(6–4)T compared with that of 64M-2.

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