Antigen improves binding of IgGs to FcγRs in SPR analysis

Wang, Wei; Chen, Qing

doi:10.1016/j.ab.2021.114411

Cited by 11 publications

(12 citation statements)

References 24 publications

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“…The MD simulations from which we extract structures for analysis in this paper are reported in detail in [ 32 ]. Conclusions obtained in [ 32 ] on antibody structure distributions were verified by independent experiments; as reported in [ 50 ], the MD simulation results in [ 32 ] reproduce experimentally-observed antibody structure distributions.…”

Section: Introductionsupporting

confidence: 64%

“…MD trajectories were saved by every 2 ps for analysis. The structures obtained from simulation reproduced experimentally-observed structure distribution [ 50 ]. The potential energy of the system was calculated using the generalized Born method with molecular volume (GBMV) after the steps of energy minimization to relax the local geometries caused by the thermal fluctuations that occurred in the MD simulations.…”

Section: Introductionmentioning

confidence: 74%

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Fewer Dimensions, More Structures for Improved Discrete Models of Dynamics of Free versus Antigen-Bound Antibody

Kabir

Nussinov

et al. 2022

Biomolecules

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Over the past decade, Markov State Models (MSM) have emerged as powerful methodologies to build discrete models of dynamics over structures obtained from Molecular Dynamics trajectories. The identification of macrostates for the MSM is a central decision that impacts the quality of the MSM but depends on both the selected representation of a structure and the clustering algorithm utilized over the featurized structures. Motivated by a large molecular system in its free and bound state, this paper investigates two directions of research, further reducing the representation dimensionality in a non-parametric, data-driven manner and including more structures in the computation. Rigorous evaluation of the quality of obtained MSMs via various statistical tests in a comparative setting firmly shows that fewer dimensions and more structures result in a better MSM. Many interesting findings emerge from the best MSM, advancing our understanding of the relationship between antibody dynamics and antibody–antigen recognition.

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Section: Introductionsupporting

confidence: 64%

Section: Introductionmentioning

confidence: 74%

Fewer Dimensions, More Structures for Improved Discrete Models of Dynamics of Free versus Antigen-Bound Antibody

Kabir

Nussinov

et al. 2022

Biomolecules

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“…The FcγRIa−IgG characterization studies reported K D values ranging from 0.1 to 100 nM with diverse immobilization strategies in which FcγRIa was usually immobilized to the surface as a ligand. 10,13,14 Our SPR studies indicate that the K D values vary significantly depending on the FcγRIa protein orientation and are susceptible to conjugation chemistry. The steric hindrance could partially explain this result where the orientation of the molecules on the surface may have changed the binding interactions, especially for the soluble FcγRIa ectodomain, which could easily find the Fc regions aligned on the chip.…”

Section: Igg1 Binding Capacity Analysis Withmentioning

confidence: 87%

“…It has been reported that the binding between FcγRs and antibodies depends on the IgG isotypes and their glycosylation profile. − The impact of the glycosylation profile of the monoclonal antibodies on FcγRs binding has been the core subject of many immune therapy-related reports where surface plasmon resonance (SPR) analyses were conducted to evaluate the corresponding binding characteristics. − The interaction between IgG and FcγRs occurs through the lower hinge in the Fc region, usually with a Langmuir 1:1 binding model where one ligand molecule interacts with a single analyte molecule. ,,, FcγRIa is the only IgG receptor with a notably high affinity on the order of 10 –8 and 10 –9 M, thus vital in immunotherapy. The crystal structure of the FcγRIa extracellular domain and Fc domain of human IgG suggests a binding scheme similar to those of low-affinity FcγRII and FcγRIII receptors, with additional hydrogen bonds and salt bridges in the lower hinge region. , The receptor D2 domain FG loop conformation also enables a unique charged KHR amino acid pattern that interacts with proximal carbohydrate units of the Fc glycans, whereas the third domain has been reported to increase specificity and affinity.…”

Section: Introductionmentioning

confidence: 99%

“… 4 − 11 The impact of the glycosylation profile of the monoclonal antibodies on FcγRs binding has been the core subject of many immune therapy-related reports where surface plasmon resonance (SPR) analyses were conducted to evaluate the corresponding binding characteristics. 12 − 14 The interaction between IgG and FcγRs occurs through the lower hinge in the Fc region, usually with a Langmuir 1:1 binding model where one ligand molecule interacts with a single analyte molecule. 1 , 10 , 15 , 16 FcγRIa is the only IgG receptor with a notably high affinity on the order of 10 –8 and 10 –9 M, 17 thus vital in immunotherapy.…”

Section: Introductionmentioning

confidence: 99%

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Characterization of FcγRIa (CD64) as a Ligand Molecule for Site-Specific IgG1 Capture: A Side-By-Side Comparison with Protein A

et al. 2022

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Fc γ receptors (FcγRs) are one of the structures that can initiate effector function for monoclonal antibodies. FcγRIa has the highest affinity toward IgG1-type monoclonal antibodies among all FcγRs. In this study, a comprehensive characterization was performed for FcγRIa as a potential affinity ligand for IgG1-type monoclonal antibody binding. The binding interactions were assessed with the SPR technique using different immobilization techniques such as EDC-NHS coupling, streptavidin–biotin interaction, and His-tagged FcγRIa capture. The His-tagged FcγRIa capture was the most convenient method based on assay repeatability. Next, a crude IgG1 sample and its fractions with different monomer contents obtained from protein A affinity chromatography were used to evaluate FcγRIa protein in terms of monoclonal antibody binding capacity. The samples were also compared with a protein A-immobilized chip (a frequently used affinity ligand) for IgG1 binding responses. The antibody binding capacity of the protein A-immobilized chip surface was significantly better than that of the FcγRIa-immobilized chip surface due to its 5 Ig binding domains. The antibody binding responses changed similarly with protein A depending on the monomer content of the sample. Finally, a different configuration was used to assess the binding affinity of free FcγRs (FcγRIa, FcγRIIa, and FcγRIIIa) to three different immobilized IgGs by immobilizing protein L to the chip surface. Unlike previous immobilization techniques tested where the FcγRIa was utilized as a ligand, nonimmobilized or free FcγRIa resulted in a significantly higher antibody binding response than free protein A. In this configuration, kinetics data of FcγRI revealed that the association rate (k a 50–80 × 105 M–1 s–1) increased in comparison to His capture method (1.9–2.4 × 105 M–1 s–1). In addition, the dissociation rate (k d 10–5 s–1) seemed slower over the His capture method (10–4 s–1) and provided stability on the chip surface during the dissociation phase. The K D values for FcγRIa were found in the picomolar range (2.1–10.33 pM from steady-state affinity analysis and 37.5–46.2 pM from kinetic analysis) for IgG1-type antibodies. FcγRIa possesses comparable ligand potential as well as protein A. Even though the protein A-immobilized surface bound more antibodies than the FcγRIa-captured surface, FcγRIa presented a significant antibody binding capacity in protein L configuration. The results suggest FcγRIa protein as a potential ligand for site-oriented immobilization of IgG1-type monoclonal antibodies, and it needs further performance investigation on different surfaces and interfaces for applications such as sensing and antibody purification.

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High-throughput optofluidic screening of single B cells identifies novel cross-reactive antibodies as inhibitors of uPAR with antibody-dependent effector functions

Lourenço

Chuo

Bohn

et al. 2023

mAbs

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The urokinase-type plasminogen activator receptor (uPAR) is an essential regulator for cell signaling in tumor cell proliferation, adhesion, and metastasis. The ubiquitous nature of uPAR in many aggressive cancer types makes uPAR an attractive target for immunotherapy. Here, we present a rapid and successful workflow for developing cross-reactive anti-uPAR recombinant antibodies (rAbs) using high-throughput optofluidic screening of single B-cells from human uPAR-immunized mice. A total of 80 human and cynomolgus uPAR cross-reactive plasma cells were identified, and selected mouse VH/VL domains were linked to the trastuzumab (Herceptin®) constant domains for the expression of mouse-human chimeric antibodies. The resulting rAbs were characterized by their tumor-cell recognition, binding activity, and cell adhesion inhibition on triple-negative breast cancer cells. In addition, the rAbs were shown to enact antibody-dependent cellular cytotoxicity (ADCC) in the presence of either human natural killer cells or peripheral blood mononuclear cells, and were evaluated for the potential use of uPAR-targeting antibody-drug conjugates (ADCs). Three lead antibodies (11857, 8163, and 3159) were evaluated for their therapeutic efficacy in vivo and were shown to suppress tumor growth. Finally, the binding epitopes of the lead antibodies were characterized, providing information on their unique binding modes to uPAR. Altogether, the strategy identified unique cross-reactive antibodies with ADCC, ADC, and functional inhibitory effects by targeting cell-surface uPAR, that can be tested in safety studies and serve as potential immunotherapeutics.

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Antigen improves binding of IgGs to FcγRs in SPR analysis

Cited by 11 publications

References 24 publications

Fewer Dimensions, More Structures for Improved Discrete Models of Dynamics of Free versus Antigen-Bound Antibody

Fewer Dimensions, More Structures for Improved Discrete Models of Dynamics of Free versus Antigen-Bound Antibody

Characterization of FcγRIa (CD64) as a Ligand Molecule for Site-Specific IgG1 Capture: A Side-By-Side Comparison with Protein A

High-throughput optofluidic screening of single B cells identifies novel cross-reactive antibodies as inhibitors of uPAR with antibody-dependent effector functions

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