Abstract

CD40 is a potent activating receptor expressed on antigen-presenting cells (APCs) of the immune system. CD40 regulates many aspects of B and T cell immunity via interaction with CD40L expressed on activated T cells. Targeting antigens to CD40 via agonistic anti-CD40 antibody fusions promotes both humoral and cellular immunity, but current anti-CD40 antibody-antigen vaccine prototypes require co-adjuvant administration for significant in vivo efficacy. This may be a consequence of dulling of anti-CD40 agonist activity via antigen fusion. We previously demonstrated that direct fusion of CD40L to anti-CD40 antibodies confers superagonist properties. Here we show that anti-CD40-CD40L-antigen fusion constructs retain strong agonist activity, particularly for activation of dendritic cells (DCs). Therefore, we tested anti-CD40-CD40L antibody fused to antigens for eliciting immune responses in vitro and in vivo. In PBMC cultures from HIV-1-infected donors, anti-CD40-CD40L fused to HIV-1 antigens preferentially expanded HIV-1-specific CD8+ T cells versus CD4+ T cells compared to analogous anti-CD40-antigen constructs. In normal donors, anti-CD40-CD40L-mediated delivery of Influenza M1 protein elicited M1-specific T cell expansion at lower doses compared to anti-CD40-mediated delivery. Also, on human myeloid-derived dendritic cells, anti-CD40-CD40L-melanoma gp100 peptide induced more sustained Class I antigen presentation compared to anti-CD40-gp100 peptide. In human CD40 transgenic mice, anti-CD40-CD40L-HIV-1 gp140 administered without adjuvant elicited superior antibody responses compared to anti-CD40-gp140 antigen without fused CD40L. In human CD40 mice, compared to the anti-CD40 vehicle, anti-CD40-CD40L delivery of Eα 52-68 peptide elicited proliferating of TCR I-Eα 52-68 CD4+ T cells producing cytokine IFNγ. Also, compared to controls, only anti-CD40-CD40L-Cyclin D1 vaccination of human CD40 mice reduced implanted EO771.LMB breast tumor cell growth. These data demonstrate that human CD40-CD40L antibody fused to antigens maintains highly agonistic activity and generates immune responses distinct from existing low agonist anti-CD40 targeting formats. These advantages were in vitro skewing responses towards CD8+ T cells, increased efficacy at low doses, and longevity of MHC Class I peptide display; and in mouse models, a more robust humoral response, more activated CD4+ T cells, and control of tumor growth. Thus, the anti-CD40-CD40L format offers an alternate DC-targeting platform with unique properties, including intrinsic adjuvant activity.

Highlights

  • CD40 is a potent activating TNFR superfamily member expressed on antigen-presenting cells (APCs) [1]

  • Our studies show that anti-CD40-CD40Lantigen constructs alter the nature of expanded antigen-specific memory T cells in vitro, and in vivo can promote significant cellular and humoral responses without co-administered adjuvant, and have potential as adjuvant-intrinsic Dendritic Cell (DC)-targeting vaccine vehicles

  • We have previously described a convenient method for noncovalent assembly of anti-DC antibodies and antigens using a bacterial Dockerin (Doc) domain fused to the antibody heavy chain C-terminus, and antigen such as Flu M1 alone (Flu M1) fused to a Cohesin (Coh) counter-domain [6]

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Summary

Introduction

CD40 is a potent activating TNFR superfamily member expressed on antigen-presenting cells (APCs) [1]. Potent activation of CD40 is not required for efficient Class I and Class II presentation of antigens via CD40-targeting in vitro [7, 12]; in vivo efficacy requires co-administration of Toll-like receptor (TLR) activating agents such as poly IC [8,9,10,11] These in vitro and in vivo studies utilized anti-CD40 antibody-antigen complexes or fusions with low CD40 agonist activity [7, 10], and the clear benefit of agonistic anti-CD40 antibody combined with poly IC for peptide-based vaccination in non-human primates [3] suggests CD40-targeting of antigens may be further improved by utilizing fully agonistic anti-CD40 targeting vehicles

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