Pertussis toxin (PTx) is a major protective antigen produced by Bordetella pertussis that is included in all current acellular vaccines. Of several well-characterized monoclonal antibodies binding this toxin, the humanised hu1B7 and hu11E6 antibodies are highly protective in multiple in vitro and in vivo assays. In this study, we determine the molecular mechanisms of protection mediated by these antibodies. Neither antibody directly binds the B. pertussis bacterium nor supports antibody-dependent complement cytotoxicity. Both antibodies, either individually or as a cocktail, form multivalent complexes with soluble PTx that bind the FcγRIIb receptor more tightly than antibody alone, suggesting that the antibodies may accelerate PTx clearance via immune complex formation. However, a receptor binding assay and cellular imaging indicate that the main mechanism used by hu11E6 is competitive inhibition of PTx binding to its cellular receptor. In contrast, the main hu1B7 neutralising mechanism appears to be inhibition of PTx internalisation and retrograde trafficking. We assessed the effects of hu1B7 on PTx retrograde trafficking in CHO-K1 cells using quantitative immunofluorescence microscopy. In the absence of hu1B7 or after incubation with an isotype control antibody, PTx colocalizes to organelles in a manner consistent with retrograde transport. However, after preincubation with hu1B7, PTx appears restricted to the membrane surface with colocalization to organelles associated with retrograde transport significantly reduced. Together, these data support a model whereby hu11E6 and hu1B7 interfere with PTx receptor binding and PTx retrograde trafficking, respectively.
Despite high vaccination rates, the incidence of whooping cough has steadily been increasing in developing countries for several decades. The current acellular pertussis (aP) vaccines all include the major protective antigen pertussis toxin (PTx) and are safer, but they appear to be less protective than infection or older, whole-cell vaccines. To better understand the attributes of individual antibodies stimulated by aP, we isolated plasmablast clones recognizing PTx after booster immunization of two donors. Five unique antibody sequences recognizing native PTx were recovered and expressed as recombinant human IgG1 antibodies. The antibodies all bind different epitopes on the PTx S1 subunit, B oligomer, or S1-B subunit interface, and just one clone neutralized PTx in an in vitro assay. To better understand the epitopes bound by the nonneutralizing S1-subunit antibodies, comprehensive mutagenesis with yeast display provided a detailed map of the epitope recognized by antibodies A8 and E12. Residue R76 is required for antibody A8 binding and is present on the S1 surface but is only partially exposed in the holotoxin, providing a structural explanation for A8's inability to neutralize holotoxin. The B-subunit-specific antibody D8 inhibited PTx binding to a model receptor and neutralized PTx in vitro as well as in an in vivo leukocytosis assay. This is the first study, to our knowledge, to identify individual human antibodies stimulated by the acellular pertussis vaccine and demonstrates the feasibility of using these approaches to address outstanding issues in pertussis vaccinology, including mechanisms of accelerated waning of protective immunity despite repeated aP immunization.
Tumor resident regulatory T cells (Tregs) are important mediators of an immunosuppressive tumor microenvironment (TME) promoting tumor immune evasion. The presence of Tregs, and a higher ratio of Tregs to effector T cells in the TME, are associated with poor prognosis. The depletion of Tregs in the TME is expected to re-expose the tumor to the immune system to allow for tumor control. CLTA-4 is expressed on the cell surface of Tregs and is a clinically validated target. Better responses to CTLA-4 monoclonal antibody (mAb) treatment are correlated with stronger ADCC-mediated Treg depletion in preclinical models, and patient FcγR polymorphism has been reported to correlate with response to CTLA-4 mAb therapy. Towards improving on current therapies, many efforts to increase effective depletion of Tregs (such as by Fc effector modification) are being pursued. Engineered toxin bodies (ETBs) are comprised of a proprietarily engineered form of Shiga-like Toxin A subunit (SLT-A) genetically fused to antibody-like binding domains. ETBs work through novel mechanisms of action and are capable of forcing internalization, self-routing through intracellular compartments to the cytosol, and inducing potent cell-kill via the enzymatic and permanent inactivation of ribosomes. Targeted ETBs are being developed to specifically target and destroy CTLA-4 expressing cells. In vitro, cells expressing CTLA-4 are effectively and specifically killed by targeted ETBs. This direct cell kill activity of the ETB has the potential to deplete Tregs in the TME without a requirement for ADCC mechanisms. ETBs have been designed to bind various CTLA-4 epitopes, and to comprise of different targeting domains formats, including monomeric and diabody single chain variable fragments (scFvs) as well as single domain and biparatopic antibody fragments. The development of a CTLA-4 targeted ETB is ongoing. The entry of a protein with direct cell kill properties into the therapeutic space represents a differentiated mechanism of action to deplete Tregs for ultimate re-invigoration of the anti-tumor immune response, and has the potential to provide benefit to patients, including in the relapsed or refractory setting. Citation Format: Aimee Iberg, Edith Acquaye-Seedah, Lilia A. Rabia, Garrett L. Robinson, Hilario J. Ramos, Joseph D. Dekker, Jay Zhao, Erin K. Willert. CTLA-4 targeted engineered toxin bodies designed to deplete regulatory T cells (Tregs) [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 2278.
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