Structure-Guided Combinatorial Engineering Facilitates Affinity and Specificity Optimization of Anti-CD81 Antibodies

Nelson, Bryce; Adams, Jarrett; Kuglstatter, A.; Z, Li; Harris, S.F.; Liu, Yang; Bohini, Sandya; Ma, Han; Klumpp, Klaus; Gao, Jun; Sidhu, Sachdev S.

doi:10.1016/j.jmb.2018.05.018

Cited by 16 publications

(19 citation statements)

References 53 publications

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“…In both nanobody-bound structures, CD9EC2 adopts an arrangement that is globally similar to previously reported EC2-structures of CD81 (23,24,(40)(41)(42)(43). 4C8 and 4E8 bind nearly identical epitopes that span the C and D loops of CD9EC2 ( Fig.…”

Section: Crystal Structures Of Cd9ec2 In Complex With Nanobodiessupporting

confidence: 83%

Implications for tetraspanin-enriched microdomain assembly based on structures of CD9 with EWI-F

Oosterheert

Xenaki

Neviani

et al. 2020

Preprint

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Tetraspanins are ubiquitous eukaryotic membrane proteins that contribute to a variety of signaling processes by spatially organizing partner-receptor molecules in the plasma membrane. How tetraspanins bind and cluster partner receptors into so-called tetraspaninenriched microdomains is unknown. Here we present crystal structures of the large extracellular loop of CD9 in complex with nanobodies 4C8 and 4E8; and, the cryo-EM structure of 4C8-bound CD9 in complex with its prototypical partner EWI-F. The CD9 -EWI-F complex displays a tetrameric arrangement with two centrally positioned EWI-F molecules, dimerized through their ectodomains, and two CD9 molecules, one bound to each EWI-F single-pass transmembrane helix through CD9-helices h3 and h4. In the crystal structures, nanobodies 4C8 and 4E8 bind CD9 at the C and D loop, in agreement with 4C8 binding at the ends of the CD9 -EWI-F cryo-EM complex. Overall, the 4C8 -CD9 -EWI-F -EWI-F -CD9 -4C8 complexes varied from nearly two-fold symmetric (i.e. with the two CD9 -4C8 copies in nearly anti-parallel orientation) to ca. 50º bent arrangements. Since membrane helices h1 and h2 and the EC2 D-loop have been previously identified as sites for tetraspanin homodimerization, the observed linear but flexible arrangement of CD9 -EWI-F with potential CD9 -CD9 homo-dimerization at either end provides a new 'concatenation model' for forming short linear or circular assemblies, which may explain the occurrence of tetraspanin-enriched microdomains.

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Section: Crystal Structures Of Cd9ec2 In Complex With Nanobodiessupporting

confidence: 83%

Implications for tetraspanin-enriched microdomain assembly based on structures of CD9 with EWI-F

Oosterheert

Xenaki

Neviani

et al. 2020

Preprint

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“…The targeted libraries were designed to primarily introduce new interactions in loops that were predicted to interact poorly with the pMHC. By comparison, other studies have successfully chosen to randomize suboptimal side chains directly neighboring hot spot residues in the main contributing loops (35,38,39). We found that our strategy is likely capable of introducing new interactions and increasing the interface SASA considerably, which is not the case for strategies aiming exclusively at improving existing interactions.…”

Section: Discussionmentioning

confidence: 73%

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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“…To further characterize the CD19-CD81 complex, we constructed a fusion protein in which the C-terminus of CD19 is directly connected to the N-terminus of CD81 with a short intervening linker (Figure 2A). To assess the integrity of this fusion protein, we evaluated its abundance on the cell surface by flow cytometry, and examined the reactivity of the fusion protein with a panel of anti-CD19 and anti-CD81 antibodies (Nelson et al, 2018). The abundance of the CD19-CD81 fusion protein on the cell surface is comparable to that observed when full-length CD19 is coexpressed with wild type CD81, indicating that CD81 in the fusion protein is functional in trafficking CD19 to the cell surface ( Figure 2B).…”

Section: The Epitope Of the 5a6 Cd81 Antibody Is Masked When Cd81 Is mentioning

confidence: 99%

“…Surprisingly, however, one anti-CD81 antibody, called 5A6 (Levy et al, 2017;Oren et al, 1990), showed significantly decreased binding of the fusion protein compared to the other anti-CD81 antibodies ( Figure 2C). Although all four antibodies bind the large extracellular loop of CD81, only 5A6 is unable to detect the CD19-CD81 fusion protein, suggesting that its epitope overlaps with the region(s) of CD81 that contact CD19 in the native complex (Nelson et al, 2018). A prior co-immunoprecipitation experiment also showed that the 5A6 antibody cannot be used to pull down the components of CD21/CD19 complex in a B cell line, providing further evidence the 5A6 epitope is masked by CD19 (Matsumoto et al, 1993).…”

Section: The Epitope Of the 5a6 Cd81 Antibody Is Masked When Cd81 Is mentioning

confidence: 99%

“…CD19 showed no difference in surface staining between resting and activated cells ( Figure 4B). When surface CD81 is detected using Ab21 (Nelson et al, 2018), which recognizes both free and complexed CD81 ( Supplementary Figure 3), there is no difference between resting and activated B cells ( Figure 4B, 4C), but when CD81 is detected using the 5A6 antibody, which selectively recognizes free CD81, there is a two-fold increase in surface staining on activated B cells ( Figure 4B, 4C). Western blotting of whole cell lysates from resting and activated cells with Ab21 and 5A6 revealed no significant difference in protein levels, indicating that the increase in CD81 surface staining is not due to increased production of CD81 in activated B cells ( Figure 4D) but instead reflects a change in the accessibility of the 5A6 epitope.…”

Section: The Cd19-cd81 Complex Dissociates In Activated B Cellsmentioning

confidence: 99%

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A dynamic interaction between CD19 and the tetraspanin CD81 controls B cell co-receptor trafficking

Susa

Seegar

Blacklow

et al. 2019

Preprint

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SUMMARYCD81 and its binding partner CD19 are core subunits of the B cell co-receptor complex. While CD19 is a single-pass transmembrane protein belonging to the extensively studied Ig superfamily, CD81 belongs to a conserved but poorly understood family of four-pass transmembrane proteins called tetraspanins. These functionally diverse proteins play important roles in a wide variety of different organ systems by controlling protein trafficking and other cellular processes. Here, we show that CD81 relies on its ectodomain to control trafficking of CD19 to the cell surface. Moreover, the anti-CD81 antibody 5A6, which binds selectively to activated B cells, recognizes a conformational epitope on CD81 that is masked when CD81 is in complex with CD19. Mutations of CD81 in this contact interface suppress its CD19 surface-export activity. Taken together, these data indicate that the CD81 - CD19 interaction is dynamically regulated upon B cell activation, suggesting that this dynamism can be exploited to regulate B cell function. These results are not only important for understanding B cell biology, but also have important implications for understanding tetraspanin function more generally.

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Structure-Guided Combinatorial Engineering Facilitates Affinity and Specificity Optimization of Anti-CD81 Antibodies

Cited by 16 publications

References 53 publications

Implications for tetraspanin-enriched microdomain assembly based on structures of CD9 with EWI-F

Implications for tetraspanin-enriched microdomain assembly based on structures of CD9 with EWI-F

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

A dynamic interaction between CD19 and the tetraspanin CD81 controls B cell co-receptor trafficking

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