Analysis of Protein Interactions by Surface Plasmon Resonance

Complement activation in human diseases is characterized by the local covalent deposition of the long-lived C3 fragments iC3b/C3dg/C3d. Previously, TT30, a complement alternative pathway (AP)-selective inhibitor, was designed as a fusion protein linking the first four short consensus repeats (SCRs) of human complement receptor type 2 (CR2) with the first five SCRs of human factor H (fH). TT30 acts by utilizing CR2 SCR1–4 to bind the initially formed iC3b/C3dg/C3d fragments and delivering surface-targeted inhibition of AP C3 and C5 convertases through fH SCR 1–5. In order to combine classical (CP) and lectin (LP) pathway inhibitory abilities employing CR2-mediated targeting, TT32 was developed. TT32 is a CR2-CR1 fusion protein using the first ten SCRs of CR1, chosen because they contain both C3 and C5 convertase inhibitory activity through utilization of decay-acceleration and cofactor activity for both AP and CP. In Wieslab assays, TT32 showed potent inhibition of the CP and AP with IC50 of 11 and 46 nM, respectively. The TT32 inhibitory activity is partially blocked with a molar excess of a competing anti-CR2 mAb, thus demonstrating the importance of the CR2 targeting. TT32 was studied in the type II (CII) collagen-induced arthritis (CIA), an active immunization model, and the CII antibody-induced arthritis (CAIA) passive transfer model. In CIA, injection of 2.0 mg TT32 at day 21 and 28 post disease induction, but not untargeted CR1 alone, resulted in a 51.5% decrease in clinical disease activity (CDA). In CAIA, treatment with TT32 resulted in a 47.4% decrease in CDA. Therefore, a complement inhibitor that targets both the AP and CP/LP C3/C5 convertases was shown to limit complement-mediated tissue damage and inflammation in disease models in which all three complement activation pathways are implicated.

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“…KD was calculated using steady state equilibrium analysis. Principles of SPR are reviewed in (Drescher et al, 2018).…”

Section: Methodsmentioning

confidence: 99%

The human complement receptor type 2 (CR2)/CR1 fusion protein TT32, a novel targeted inhibitor of the classical and alternative pathway C3 convertases, prevents arthritis in active immunization and passive transfer mouse models

Fridkis-Hareli

Storek

et al. 2019

Molecular Immunology

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“…The proportions of FVIII-specific immunoglobulin isotypes in patient samples have been analyzed semi-quantitatively by ELISAs employing isotype-specific MAbs, together with engineered MAbs consisting of the variable heavy chain domain of a FVIII-specific scFv fused to the constant CH1-CH2-CH3 fragments of IgG1, IgG2, IgG3 and IgG4 as internal controls [113]. As an alternative method, Lewis et al developed a novel SPR-based assay to quantify both the total anti-FVIII antibody titers, and the amount of each subclass comprising this total [114], exploiting the fact that the change in Response Units (RUs) upon saturation binding of an antibody to a ligand immobilized on a biosensor chip is easily converted to the total mass of the captured antibodies (Figure 2) [115]. The molecular weight of monoclonal or polyclonal IgG is ~150 kDa.…”

Section: Identifying and Modifying B-cell Epitopes In Biotherapeuticsmentioning

confidence: 99%

“…“Saturation SPR” method to characterize polyclonal anti-FVIII anti-drug antibodies (ADAs) in patient plasma samples [114]. First a high-affinity MAb specific for the FVIII A1 domain is covalently immobilized on the biosensor surface, and FVIII protein is then injected and captured by this MAb (0–700 s, not shown in figure).…”

Section: Figurementioning

confidence: 99%

Anti-Drug Antibodies: Emerging Approaches to Predict, Reduce or Reverse Biotherapeutic Immunogenicity

Pratt

2018

Antibodies

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Abstract:The development of anti-drug antibodies (ADAs) following administration of biotherapeutics to patients is a vexing problem that is attracting increasing attention from pharmaceutical and biotechnology companies. This serious clinical problem is also spawning creative research into novel approaches to predict, avoid, and in some cases even reverse such deleterious immune responses. CD4 + T cells are essential players in the development of most ADAs, while memory B-cell and long-lived plasma cells amplify and maintain these responses. This review summarizes methods to predict and experimentally identify T-cell and B-cell epitopes in therapeutic proteins, with a particular focus on blood coagulation factor VIII (FVIII), whose immunogenicity is clinically significant and is the subject of intensive current research. Methods to phenotype ADA responses in humans are described, including T-cell stimulation assays, and both established and novel approaches to determine the titers, epitopes and isotypes of the ADAs themselves. Although rational protein engineering can reduce the immunogenicity of many biotherapeutics, complementary, novel approaches to induce specific tolerance, especially during initial exposures, are expected to play significant roles in future efforts to reduce or reverse these unwanted immune responses.

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“…The affinities were around ten times higher than pdFH binding (data not shown). This increased affinity could be caused by the HIS-tag on the FH, which is known to cause artefactual enhanced binding on CM5 sensors, as used in this SPR setup (27). In addition, the tested FH mutants showed slight decrease in affinity for C3b compared to WT FH.…”

Section: Discussionmentioning

confidence: 99%

“…This studies indicates a ∼4-fold reduction in affinity towards C3b while we observe only moderate reduction. As described before, our recombinant proteins contain a HIS-tag which might have influenced our SPR based assay (27). However, as our WT FH also contains this HIS tag it might also be the sample preparation or other causes that affect the functionality of these mutants.…”

Section: Discussionmentioning

confidence: 99%

Unravelling the effect of a potentiating anti-Factor H antibody on atypical hemolytic uremic syndrome associated factor H variants

Dekkers

Brouwer

Jeremiasse

et al. 2020

Preprint

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The complement system plays an important role in our innate immune system. Complement activation results in clearance of pathogens, immune complex and apoptotic cells. The host is protected from complement-mediated damage by several complement regulators. Factor H (FH) is the most important fluid-phase regulator of the alternative pathway of the complement system. Heterozygous mutations in FH are associated with complement-related diseases such as atypical hemolytic uremic syndrome (aHUS) and age-related macular degeneration.We recently described an agonistic anti-FH monoclonal antibody that can potentiate the regulatory function of FH. This antibody could serve as a potential new drug for aHUS patients and alternative to C5 blockade by Eculizumab. However, it is unclear whether this antibody can potentiate FH mutant variants in addition to wild type FH. Here, the functionality and potential of the agonistic antibody in the context of pathogenic aHUS-related FH mutant proteins was investigated. The binding affinity of recombinant WT FH, and the FH variants, W1183L, V1197A, R1210C, and G1194D to C3b was increased upon addition of the potentiating antibody and similarly, the decay accelerating activity of all mutants is increased. The potentiating anti-FH antibody is able to restore the surface regulatory function of most of the tested FH mutants to WT FH levels. In conclusion, our potentiating anti-FH is broadly active and able to enhance both WT FH function as well as most aHUS-associated FH variants tested in this study.

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Analysis of Protein Interactions by Surface Plasmon Resonance

Cited by 57 publications

References 67 publications

The human complement receptor type 2 (CR2)/CR1 fusion protein TT32, a novel targeted inhibitor of the classical and alternative pathway C3 convertases, prevents arthritis in active immunization and passive transfer mouse models

The human complement receptor type 2 (CR2)/CR1 fusion protein TT32, a novel targeted inhibitor of the classical and alternative pathway C3 convertases, prevents arthritis in active immunization and passive transfer mouse models

Anti-Drug Antibodies: Emerging Approaches to Predict, Reduce or Reverse Biotherapeutic Immunogenicity

Unravelling the effect of a potentiating anti-Factor H antibody on atypical hemolytic uremic syndrome associated factor H variants

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