Multivalent pIX phage display selects for distinct and improved antibody properties

Høydahl, Lene Støkken; Nilssen, Nicolay Rustad; Gunnarsen, Kristin Støen; Pré, M. Fleur du; Iversen, Rasmus; Roos, Norbert; Chen, Xi; Michaelsen, T. E.; Sollid, Ludvig M.; Sandlie, Inger; Løset, Geir Åge

doi:10.1038/srep39066

Cited by 16 publications

(26 citation statements)

References 60 publications

(81 reference statements)

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“…H and L chain KI mice were crossed to obtain 14E06 double KI mice. Only B cells of the double KI mice and not the single H chain or single L chain KI mice stained with an antibody (anti-14E06; Høydahl et al, 2016) specific for the H and L chain combination of 14E06 ( Fig. 1 C).…”

Section: Resultsmentioning

confidence: 99%

B cell tolerance and antibody production to the celiac disease autoantigen transglutaminase 2

Pré

Blaževski

Dewan

et al. 2019

Journal of Experimental Medicine

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Autoantibodies to transglutaminase 2 (TG2) are hallmarks of celiac disease. To address B cell tolerance and autoantibody formation to TG2, we generated immunoglobulin knock-in (Ig KI) mice that express a prototypical celiac patient–derived anti-TG2 B cell receptor equally reactive to human and mouse TG2. We studied B cell development in the presence/absence of autoantigen by crossing the Ig KI mice to Tgm2−/− mice. Autoreactive B cells in Tgm2+/+ mice were indistinguishable from their naive counterparts in Tgm2−/− mice with no signs of clonal deletion, receptor editing, or B cell anergy. The autoreactive B cells appeared ignorant to their antigen, and they produced autoantibodies when provided T cell help. The findings lend credence to a model of celiac disease where gluten-reactive T cells provide help to autoreactive TG2-specific B cells by involvement of gluten–TG2 complexes, and they outline a general mechanism of autoimmunity with autoantibodies being produced by ignorant B cells on provision of T cell help.

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Section: Resultsmentioning

confidence: 99%

B cell tolerance and antibody production to the celiac disease autoantigen transglutaminase 2

Pré

Blaževski

Dewan

et al. 2019

Journal of Experimental Medicine

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“…Selection and rescue was performed essentially as described 18 with the following specifications: pre-blocked phage samples were incubated 1 h with 80 nM biotinylated HLA-DQ2.5:CLIP2 before capture onto Dynabeads MyOne Streptavidin T1 beads for 30 min; unbound phage was transferred to tubes containing 80 nM biotinylated HLA-DQ2.5:DQ2.5-glia-α1a for 1 h before transfer to beads; R4 stringency included 100x less antigen and 20 + 20 washes. Selection alternative 1 was performed as previously described for the OMV selection 18 ; alternative 2 included 16.6 nM non-biotinylated HLA-DQ2.5:DQ2.5-glia-α1a as competitor in solution. Phage rescue (E. coli XL1-Blue and M13K07), PEG/NaCl precipitation, spot titration, reformatting and soluble expression was performed as described 18 .…”

Section: Selection and Rescue Of Scfv Phage Libraries And Reformattinmentioning

confidence: 99%

Plasma Cells Are the Most Abundant Gluten Peptide MHC-expressing Cells in Inflamed Intestinal Tissues From Patients With Celiac Disease

et al. 2019

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Background & aims: The pathogenesis of celiac disease (CD) is thought to be driven by a transglutaminase 2 (TG2)-dependent inflammatory CD4 + T-cell response in the gut towards deamidated gluten peptides in the context of disease-associated HLA-DQ molecules. We aimed to gain insight into the antigen presentation process underlying this mucosal immune response. Methods: We generated monoclonal antibodies (mAbs) specific for the peptide-MHC (pMHC) complex HLA-DQ2.5 and the immunodominant gluten epitope DQ2.5-glia-α1a using phage display. Using these mAbs we assessed gluten peptide presentation in freshly prepared single-cell suspensions of patient intestinal biopsies. Results: The mAbs allowed specific detection of in vivo generated pMHC complexes on the cells of gut biopsies from CD patients consuming gluten. Surprisingly, we identified B cells and plasma cells (PCs) as the most abundant cells presenting DQ2.5-glia-α1a in the inflamed mucosa. Further, we demonstrate that a group of these PCs expresses B-cell receptors (BCRs) specific for either gluten peptides or the autoantigen TG2. MHC class II (MHCII) expression was not restricted to these specific PCs associated with CD, but was observed at an average of 30% of the gut PCs both in CD patients as well as in non-inflamed tissue. Conclusions: A population of PCs in the gut expresses MHCII and is the most abundant cell type presenting the immunodominant gluten peptide DQ2.5-glia-α1a. These results suggest an important and previously unappreciated role of PCs in the gut as antigen presenting cells (APCs). PCs may thus be responsible for promoting and sustaining intestinal inflammation such as in CD.

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“…We retrieved clones with higher affinity and thermostability in direct comparison to both LV and HV display on pIII (42), suggesting that HV display on pIX could be particularly valuable for affinity engineering.…”

Section: Discussionmentioning

confidence: 99%

“…Briefly, first-generation HLA-DQ2.5:DQ2.5-glia-α2-specific antibodies were isolated from a human naïve scFv library (19) selected on recombinant pMHC essentially as described before (18,42).…”

Section: Methodsmentioning

confidence: 99%

Affinity-engineered human antibodies detect celiac disease gluten pMHC complexes and inhibit T-cell activation

Frick

Høydahl

Hodnebrug

et al. 2019

Preprint

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Antibodies specific for antigenic peptides bound to major histocompatibility complex (MHC) molecules are valuable tools for studies of antigen presentation. Such T-cell receptor (TCR)-like antibodies may also have therapeutic potential in human disease due to their ability to target disease-associated antigens with high specificity. We previously generated celiac disease (CeD) relevant TCR-like antibodies that recognize the prevalent gluten epitope DQ2.5-glia-α1a in complex with HLA-DQ2.5. Here, we report on second-generation high-affinity antibodies towards this epitope as well as a panel of novel TCR-like antibodies to another immunodominant gliadin epitope, DQ2.5-glia-α2. The strategy for affinity engineering was based on Rosetta modeling combined with pIX phage display and is applicable to similar protein engineering efforts. We isolated picomolar affinity binders and validated them in Fab and IgG format. Flow cytometry experiments with CeD biopsy material confirm the unique disease specificity of these TCR-like antibodies and reinforce the notion that B cells and plasma cells have a dominant role in gluten antigen presentation in the inflamed CeD gut. Further, the lead candidate 3.C11 potently inhibited CD4 + T-cell activation and proliferation in vitro in an HLA and epitope specific manner, pointing to a potential for targeted disease interception without compromising systemic immunity. Significance StatementConsumption of gluten-containing food drives celiac disease in genetically predisposed individuals. The underlying disease mechanism is not fully understood, but it is strictly dependent on activation of pathogenic T cells. We have engineered high-affinity human antibodies recognizing the T-cell target HLA-DQ2.5 in complex with gluten epitopes and studied cell-specific antigen presentation in patients, which shows that plasma cells and not dendritic cells dominate the inflamed tissue. The only available treatment is lifelong adherence to a gluten-free diet, which is difficult and not effective in all cases. We show that at least one of our antibodies can specifically inhibit activation of pathogenic T-cells in vitro and therefore shows promise for therapy.

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Multivalent pIX phage display selects for distinct and improved antibody properties

Cited by 16 publications

References 60 publications

B cell tolerance and antibody production to the celiac disease autoantigen transglutaminase 2

B cell tolerance and antibody production to the celiac disease autoantigen transglutaminase 2

Plasma Cells Are the Most Abundant Gluten Peptide MHC-expressing Cells in Inflamed Intestinal Tissues From Patients With Celiac Disease

Affinity-engineered human antibodies detect celiac disease gluten pMHC complexes and inhibit T-cell activation

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