A tractable alent linker strategy for the production of immunogenic antigen-TLR7/8L bioconjugates (1)
Direct covalent attachment of Toll-like receptor (TLR) agonists to antigenic proteins has emerged as an effective strategy to enhance immunity while minimizing adjuvant toxicity. TLR7/8 ligands (TLR7/8Ls) in particular have been utilized in this approach due to the broad expression of their receptors in dendritic cells (DCs) and other antigen presenting cells (APCs).
The conjugation of TLR7/8Ls with antigenic proteins has since been extended to viral, bacterial, parasitic,and allergic disease models, as well as fundamental studies.
One synthetic imidazoquinoline (UM-3013) were selected because they possess amine nucleophilic handles and exhibit moderate (UM-3007), sparing (UM-3013), and negligible (UM-3006) water solubilities upon amide functionalization. These three molecules had biological activity to activate TLR7 and TLR8 receptors using HEK-293 reporter cells.
Conjugate C2 induced a significantly higher anti-BSA serum antibody response with an average total IgG titer more than 14 times greater than that of mice vaccinated with unconjugated antigen and adjuvant.
As was the case with conjugate C2, CRM-197 conjugate C3 induced superior antigen-specific antibody responses in mice compared to the admixed/unconjugated controls. Two doses of the CRM-197 antigen were used (1 or 10 μg), and at both doses the use of conjugated antigen resulted in significantly greater anti-CRM-197 antibody titers as compared to using the equivalent doses of protein admixed with adjuvant.
With our UM-3006 conjugates C4 and C1 in hand, we conducted head-to-head studies comparing their biological activities. The new conjugate, C4, proved to be similarly or somewhat more active in vitro than conjugate C1 both in HEK-293 cells expressing human TLR7, and in human peripheral blood mononuclear cells (PBMCs), producing cytokines interferon alpha (IFNα) and tumor necrosis factor alpha. These conjugates were also tested in vivo by vaccinating mice with 1 or 10 μg of CRM-197 either alone, conjugated with UM-3006 (at a copy # of 4.4), or admixed with free UM-3006 at the corresponding molar ratio. Both C4 and C1 elicited significantly higher antibody responses than the admixed control groups, and conjugate C4 performed similarly or slightly better than C1.
The production of antibody subtypes IgG1 and IgG2a was assessed to provide information regarding the nature of the CD4+ T cell responses. Both IgG1, generally associated with a T helper 2 (Th2) response, and IgG2a, associated with a T helper 1 (Th1) response, were significantly increased by the use of either conjugate. However, conjugation favored the Th1-associated response, as the fold-increase in IgG2a was greater in the conjugate groups compared to the admixed groups. T cells from mice immunized with either conjugate, C4 or C1, demonstrated an increased antigen-specific release of IFNγ upon in vitro restimulation along with an undetectable level of IL-5. In contrast, antigen alone induced primarily an IL-5 response following antigen re-stimulation in vitro. Together, these data suggest that the conjugation of UM-3006 with an antigen results in a shift towards a Th1-dominated immune response, which has been reported to be helpful for protection against intracellular pathogens such as viruses and certain bacteria.
By minimizing the number of linker attachments via direct NHS-mediated cross-linking, T and B cell epitope masking can be minimized. The resulting bioconjugates are remarkably immunogenic and drive robust Th1-polarized immune responses in vivo.