scholarly journals Converting monoclonal antibody-based immunotherapies from passive to active: bringing immune complexes into play

2016 ◽  
Vol 5 (1) ◽  
pp. 1-9 ◽  
Author(s):  
Jennifer Lambour ◽  
Mar Naranjo-Gomez ◽  
Marc Piechaczyk ◽  
Mireia Pelegrin
Keyword(s):  
Monoclonal Antibody ◽  
Immune Complexes

Making Weak Antigens Strong: Preparing Immune Complexes for Injection

2021 ◽  
Vol 2021 (9) ◽  
pp. pdb.prot099978
Author(s):  
Edward A. Greenfield ◽  
James DeCaprio ◽  
Mohan Brahmandam
Keyword(s):  
Monoclonal Antibody ◽  
Immune Complexes ◽  
Native Protein ◽  
Immunodominant Epitope ◽  
Strong Response ◽  
Polyclonal Antisera

If antibodies against a particular antigen are available, that antigen can be purified and used for further immunizations, and antigens thus purified can show enhanced immunogenicity. Purified immune complexes can be injected directly, or while coupled to beads; the presence of antibodies and/or beads stimulates phagocytosis and usually will not influence the response. This method provides a useful means of antigen enrichment for a variety of applications, such as using antibodies raised against a denatured antigen to harvest a native protein for further immunizations, or when using a monoclonal antibody as an intermediate to the preparation of polyclonal antisera. Injecting antibody-coated antigens has also been used to mask a particularly immunodominant epitope on an antigen, and thereby develop a response against other epitopes. The amount of antigen needed to elicit a strong response using immune complexes will vary from one compound to another. Doses as low as 50 ng of antigen have been used successfully when delivered this way.

Administration of a human monoclonal antibody (TUVIRUMAB) to chronic hepatitis B patients pre-treated with lamivudine: monitoring of serum TUVIRUMAB in immune complexes

2001 ◽  
Vol 64 (4) ◽  
pp. 427-434 ◽  
Author(s):  
R.A. Heijtink ◽  
A.B. van Nunen ◽  
P. van Bergen ◽  
L. Östberg ◽  
A.D.M.E. Osterhaus ◽  
...  
Keyword(s):  
Monoclonal Antibody ◽  
Chronic Hepatitis ◽  
Hepatitis B ◽  
Chronic Hepatitis B ◽  
Immune Complexes ◽  
Human Monoclonal Antibody

C1q-bearing immune complexes detected by a monoclonal antibody to human C1q in rheumatoid arthritis sera and synovial fluids

1991 ◽  
Vol 10 (6) ◽  
pp. 245-250 ◽  
Author(s):  
U. Antes ◽  
H. -P. Heinz ◽  
D. Schultz ◽  
D. Brackertz ◽  
M. Loos
Keyword(s):  
Rheumatoid Arthritis ◽  
Monoclonal Antibody ◽  
Immune Complexes ◽  
Synovial Fluids

Complement Regulates the Procoagulant Effects of HIT Immune Complexes

Blood ◽  
2020 ◽  
Vol 136 (Supplement 1) ◽  
pp. 11-12
Author(s):  
Sanjay Khandelwal ◽  
Lubica Rauova ◽  
Ayiesha Barnes ◽  
Ann Rux ◽  
Serge Yarovoi ◽  
...  
Keyword(s):  
Monoclonal Antibody ◽  
Cell Surface ◽  
Complement Activation ◽  
Immune Complexes ◽  
Cell Activation ◽  
Alternative Pathway ◽  
Classical Pathway ◽  
Platelet Factor ◽  
Neutrophil Degranulation ◽  
Pathway Inhibition

Heparin induced thrombocytopenia (HIT) is a prothrombotic disorder mediated by ultra-large immune complexes (ULICs) containing IgG antibodies bound to multivalent complexes of platelet factor 4 (PF4) and heparin (H). HIT ULICs activate cellular FcγIIA receptors that initiate diverse cellular effector functions including neutrophil degranulation and monocyte expression of tissue factor (TF). Previous studies have shown that HIT ULICs also potently activate complement through the classical pathway (Cines et al., 1980). Whether complement activation contributes to FcγRIIA-dependent prothrombotic pathways has not been addressed in detail. In studies that follow, we describe: 1) robust complement activation by HIT ULICs in plasma and whole blood (WB), 2) cell-surface deposition of complement and IgG triggered by HIT ULICs, 3) complement-dependent neutrophil degranulation and monocyte TF expression, 4) efficacy of proximal, but not terminal, pathway inhibition in regulating monocyte TF expression, and 5) deposition of complement in thrombi formed in "HIT mice" that generate ULICs containing KKO, a HIT-like monoclonal antibody (Arepally et al., 2000). Consistent with prior studies showing involvement of the classical pathway in HIT (Cines et al., 1980), we observed that binding of C1q induced marked enlargement of HIT ULICs in buffer assessed by dynamic light scattering as well as in plasma using confocal microscopy (data not shown). To assess complement activation by HIT ULICs, we incubated WB and plasma with PF4 (25 µg/mL) ± heparin (1 U/mL) in the presence of KKO (or isotype, "ISO"; 50 µg/mL) or HIT IgG (or control IgG, "CON"; 500 µg/mL) and measured C3c with a capture immunoassay as previously described (Khandelwal et al., 2018). KKO (Figure 1A) or HIT ULICs (n=3; HIT1-3, Figure 1B), showed robust generation of C3c in the presence of PF4/heparin, but not antigens alone or with control IgG (ISO/CON). Complement activation by HIT ULICs leads to downstream generation of C5a and formation of sC5b-9 (data not shown). Pre-incubation of plasma or WB with a variety of classical pathway inhibitors, including a C1r inhibitor derived from Borrelia burgdorferi (BBK 32), C1 esterase inhibitor (Berinert, CSL Behring) and anti-C1q antibody (α-C1q Ab; Annexon Biosciences) inhibited C3c generation by KKO ULICs (p <0.001), whereas inhibitors of the alternative pathway (anti-properdin antibody) or C5 inhibitor (α-C5 Ab; Eculizumab, Alexion Pharmaceuticals) did not (data not shown). Incubation of WB with KKO or HIT ULICs, but not ISO or CON IgG, markedly increased deposition of C3 and IgG on neutrophils, monocytes and B cells (data not shown) and lead to cell activation assessed by neutrophil degranulation (MMP9 release) and monocyte TF expression (data not shown). To examine the contribution of complement activation in monocyte TF expression, WB was pre-incubated with α-C1q, α-C5 or IV.3 (a monoclonal antibody to FcγRIIA) or isotype controls prior to addition of HIT ULICs. As shown in Figure 2, the classical pathway inhibitor, α-C1q Ab markedly diminished TF expression (about 70% reduction; p<0.001 vPF4/H/ KKO), as did IV.3 (about 85% reduction; p<0.001 vPF4/H/ KKO) but not α-C5 Ab or ISO antibodies, demonstrating: 1) FcγRIIA independent mechanism of monocyte TF expression and 2) a requirement for proximal rather than terminal complement pathway components in the induction of monocyte TF. We next asked if complement activation facilitates binding of ULICs and promotes subsequent ULIC engagement of FcγRIIA. To examine complement dependent binding of HIT ULICs, we incubated WB with α-C1q Ab prior to addition of KKO ULICs and measured ULIC binding to monocytes and TF expression. As shown in Figure 3, classical pathway inhibition markedly reduced cell-surface IgG (Figure 3A) and monocyte TF expression (Figure 3B). The effects of complement inhibition could not be overcome with increasing amounts of KKO IgG (2-4 fold excess). We observed significant co-localization of complement with KKO ULICs in a cremaster-laser injury model in "HIT mice" and in in situ thrombi formed in uninjured vessels (data not shown). Together, these studies demonstrate an independent role for complement activation in regulating the binding and procoagulant effects of HIT ULICs and identify new non-anticoagulant therapeutic targets that could improve clinical outcomes in this otherwise potentially devastating thrombotic disorder. Disclosures Arepally: Novartis: Consultancy; Alexion: Other; Annexon Biosciences: Consultancy, Other; Veralox Therapeutics: Consultancy, Membership on an entity's Board of Directors or advisory committees; Biokit: Consultancy, Patents & Royalties; Apotex: Consultancy, Research Funding.

scholarly journals Efficacy and safety of NI-0101, an anti-toll-like receptor 4 monoclonal antibody, in patients with rheumatoid arthritis after inadequate response to methotrexate: a phase II study

2019 ◽  
Vol 79 (3) ◽  
pp. 316-323 ◽  
Author(s):  
Emmanuel Monnet ◽  
Ernest H Choy ◽  
Iain McInnes ◽  
Tamta Kobakhidze ◽  
Kathy de Graaf ◽  
...  
Keyword(s):  
Rheumatoid Arthritis ◽  
Monoclonal Antibody ◽  
Immune Complexes ◽  
Inadequate Response ◽  
Toll Like Receptor ◽  
Double Blind ◽  
Reactive Protein ◽  
American College Of Rheumatology ◽  
Subgroup Analyses

ObjectivesAnti-citrullinated protein antibodies (ACPAs) form immune complexes with citrullinated proteins binding toll-like receptor (TLR) 4, which has been proposed as a mediator of rheumatoid arthritis (RA). NI-0101 is a first-in-class humanised monoclonal antibody blocking TLR4, as confirmed by inhibition of in vivo lipopolysaccharide-induced cytokine release in healthy volunteers. This study was design to confirm preclinical investigations supporting a biomarker-driven approach for treatment of patients with RA who present positive for these immune complexes.MethodsPlacebo-controlled, double-blind, randomised (2:1) trial of the tolerability and efficacy of NI-0101 (5 mg/kg, every 2 weeks for 12 weeks) versus placebo in ACPA-positive RA patients with inadequate response to methotrexate. Efficacy measures included Disease Activity Score (28-joint count) with C reactive protein (DAS28-CRP), European League Against Rheumatism (EULAR) good and moderate responses, and American College of Rheumatology (ACR) 20, ACR50 and ACR70 responses. Subgroup analyses defined on biomarkers were conducted. Pharmacokinetics, pharmacodynamics and safety were reported.Results90 patients were randomised (NI-0101 (61) and placebo (29)); 86 completed the study. No significant between-group difference was observed for any of the efficacy endpoints. Subgroup analyses using baseline parameters as covariants did not reveal any population responding to NI-0101. Treatment-emergent adverse events occurred in 51.7% of patients who received placebo versus 52.5% for NI-0101.ConclusionsWe demonstrate for the first time that in RA, a human immune-mediated inflammatory disease, blocking the TLR4 pathway alone does not improve disease parameters. Successful targeting of innate immune pathways in RA may require broader and/or earlier inhibitory approaches.

Erythrocyte CR1 Determination Using Monoclonal Antibody in a Microtiter Plate ELISA; Receptors Are Not Masked by Immune Complexes

Allergy ◽  
1986 ◽  
Vol 41 (7) ◽  
pp. 479-486 ◽  
Author(s):  
B. S. Thomsen ◽  
H. Nielsen ◽  
G. Bendixen
Keyword(s):  
Monoclonal Antibody ◽  
Immune Complexes ◽  
Microtiter Plate

An Algorithm for “Indeterminate” Test Results in the Platelet Serotonin Release Assay for Heparin-Induced Thrombocytopenia (HIT).

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 1048-1048
Author(s):  
Carol A. Smith ◽  
Andrew E. Warkentin ◽  
Theodore E. Warkentin ◽  
Donald M. Arnold ◽  
Jane C. Moore ◽  
...  
Keyword(s):  
Monoclonal Antibody ◽  
Platelet Activation ◽  
Immune Complexes ◽  
Fc Receptors ◽  
Fc Receptor ◽  
Serotonin Release ◽  
Indeterminate Result ◽  
Receptor Blocking ◽  
Release Assay ◽  
Test Result

Abstract HIT is a prothrombotic complication of heparin caused by antibodies that recognize complexes of platelet factor 4 (PF4) bound to heparin or certain other polyanions. These antibodies produce thrombocytopenia by activating platelets via their Fc receptors. A “functional” (platelet activation) assay that utilizes washed platelets, known as the 14C-serotonin release assay (SRA), has the highest reported sensitivity-specificity tradeoff for detecting clinically-significant antibodies that recognize PF4/heparin complexes (“HIT antibodies”). Platelet activation (% serotonin release) induced by patient (or control) serum is assessed under several different reaction conditions, including absence of heparin, therapeutic concentrations of unfractionated heparin (UFH, 0.1 to 0.3 U/mL) and low-molecular-weight heparin (LMWH, 0.2 U/mL), supratherapeutic concentrations of UFH (100 U/mL), and with 0.1 U/mL UFH in the presence of a platelet Fc receptor-blocking monoclonal antibody. Various technical aspects of the assay optimize test sensitivity and specificity (e.g., using heat-inactivated patient serum; washing the platelets with apyrase; resuspending the platelets in albumin-free Tyrode’s buffer; using platelets from healthy volunteers known to react well to IgG platelet agonists, etc.). The classic HIT platelet activation profile is strong platelet activation (>50% serotonin release) in the presence of therapeutic UFH or LMWH that is inhibited by supratherapeutic heparin and the platelet Fc receptor-blocking monoclonal antibody. One drawback to the assay is that some patient sera activate platelets via the Fc receptors in a heparin-independent fashion, i.e., the platelets are activated at all heparin concentrations. This is known as an “indeterminate” reaction profile, since the presence of in vitro immune complexes (generated by the heat-inactivation process) or in vivo immune complexes or other platelet-activating factors could “mask” the presence of a true HIT antibody. We developed an algorithm for dealing with such indeterminate reaction profiles. First, we repeat the SRA using another aliquot of patient serum that is newly heat-inactivated, and also use different platelet donors to perform the assay. Often, this results in an interpretable test result. However, if the repeat SRA also gives an indeterminate result, we then use an in-house anti-PF4/heparin ELISA (that detects only IgG class antibodies) to determine whether HIT antibodies could be present. From 2091 patient serum samples tested for HIT antibodies using the SRA, we identified 199 (9.5%) samples that gave an initial indeterminate result. Using our algorithm, 81 samples subsequently gave clearly negative results, and 35 samples gave clearly positive results. However, 83 samples (representing 41.7% of the retested samples, or 4.0% of the total samples) gave a repeat indeterminate test result. When this last group of samples was tested using the anti-PF4/heparin-IgG ELISA, 53 of the samples tested negative (OD<0.45) and 30 tested positive (OD >0.45). With this algorithmic approach, about 96% of patients can be classified as negative or positive using the SRA. However, 4% of patients require the use of a complementary assay- the anti-PF4/heparin-IgG ELISA, to evaluate for the presence of HIT antibodies. Further studies are required to determine the causes of persistent indeterminate results in the SRA, which may lead to new approaches to further optimize this assay.

Extending Mass Spectrometry Contribution to Therapeutic Monoclonal Antibody Lead Optimization: Characterization of Immune Complexes Using Noncovalent ESI-MS

2009 ◽  
Vol 81 (15) ◽  
pp. 6364-6373 ◽  
Author(s):  
Cédric Atmanene ◽  
Elsa Wagner-Rousset ◽  
Martine Malissard ◽  
Bertrand Chol ◽  
Alain Robert ◽  
...  
Keyword(s):  
Mass Spectrometry ◽  
Monoclonal Antibody ◽  
Immune Complexes ◽  
Lead Optimization ◽  
Esi Ms ◽  
Therapeutic Monoclonal Antibody

scholarly journals An Investigation of the Antigenic Characteristics of the Renal Glomerulus in the Rat

2021 ◽  
Author(s):  
◽  
Maurice James Nicol
Keyword(s):  
Monoclonal Antibody ◽  
Monoclonal Antibodies ◽  
Animal Models ◽  
Basement Membrane ◽  
Glomerular Basement Membrane ◽  
Immune Complexes ◽  
Capillary Wall ◽  
Glomerular Capillary Wall ◽  
Glomerular Capillary

<p>The finding of a granular deposition of immunoglobulin in the kidney in experimental animal models of glomerulonephritis has been been interpreted as resulting from the random deposition of immune complexes in the glomeruli. Recent data suggests that although immune complex deposition may be an important factor in some forms of glomerulonephritis, the in situ formation of immune complexes between circulating anti-kidney antibodies and fixed glomerular capillary wall antigens may also be a significant factor in the pathogenesis of some animal models of glomerulonephritis. To examine the characteristics of discontinuously represented glomerular capillary wall antigens in the rat, monoclonal antibodies were generated against a glomerular plasma membrane fraction, depleted of glomerular basement membrane, prepared from isolated Lewis rat glomeruli. A total of 17 hybridomas, generated from the fusion of splenocytes obtained from mice immunised with the glomerular membrane fraction produced monoclonal antibodies which reacted with discontinuously represented antigens in the glomerulus and renal tubules. One further hybridoma secreted a monoclonal antibody which reacted with an antigen present on glomerular and tubular nuclear membranes. No hybridomas were produced which secreted a monoclonal antibody which reacted with linearly arrayed glomerular basement membrane antigens. Two of these monoclonal antibodies, both of the IgM subclass and code-named PH7 and SC5, produced a heavy granular glomerular staining pattern when examined by indirect immunofluorescence microscopy. Neither monoclonal antibody was kidney specific, with reactivity being demonstrated with a number of non-renal tissues. When administered intravenously to normal Lewis rats both SC5 and PH7 induced a mild proteinuric lesion. The proteinuria was not associated with histopathological changes at the light or electron microscope level. Immunoblotting experiments revealed that SC5 reacted predominantly with a protein band of 96 kDa present in detergent extracts of isolated glomeruli and glomerular plasma membranes. PH7 was shown to react with three low molecular weight proteins of 14, 13 and 11 kDa The findings of this study demonstrate the potential for a nephritogenic response to occur following the in situ formation of immune complexes between circulating anti-kidney antibodies and discontinuously arrayed non-glomerular, basement membrane glomerular capillary wall antigens characterised by granular immunofluorescence patterns,in animal models of glomerulonephritis.</p>

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