Albumin-binding nanobody-antigen fusions enhance antigen presentation and improve vaccine responses through pharmacokinetic modulation

Introduction: In the contexts of both activation and tolerance, antigen-specific immunotherapies have the potential to direct the adaptive immune system to achieve a desired therapeutic outcome. Many of these therapies utilize peptide antigens (~2 kDa), to enhance antigen presentation on either Class I or Class II Major Histocompatibility Complex (MHC) for display to CD8+ or CD4+ T cells, respectively. Unfortunately, the efficacy of peptide-based vaccines has been limited by multiple shortcomings, including inefficient trafficking to the local draining lymph node (dLN), rapid renal clearance, susceptibility to proteolysis, and presentation in an inappropriate context without the spatiotemporal co-delivery of an adjuvant. Although strategies have been developed to address these challenges, they are often complex either in their manufacturing or route of administration, motivating the development of novel solutions. Nanobodies (Nbs) are alpaca-derived single-domain antibodies of small size (~12-15 kDa) and high target specificity. About ten times smaller than conventional antibodies (Abs), Nbs exhibit efficient tissue penetration and clear rapidly when unbound to their target, making them effective delivery vehicles for conjugated therapeutic cargos. To this end, we hypothesized that the fusion of peptide epitopes with a Nb targeting mouse serum albumin (MSA, 69 kDa), termed nAlb, would enhance antigen trafficking to the dLN by limiting renal clearance and increasing the absolute bioavailability of the antigen. In this work, we synthesized multiple nAlb-antigen fusions and demonstrated that co-administration with appropriate nAlb-adjuvant conjugates produces either robust, antigen-specific immune activation with antitumoral effects or potent, antigen-specific immunosuppression capable of ameliorating autoimmune diseases.

Materials and

Methods: nAlb-antigen fusions were produced by designing gene cassettes in which antigenic peptides were placed at the C-terminus of nAlb along with a hexahistadine tag for purification and a sortase tag for site-specific conjugation of molecular adjuvants. We then ligated the nAlb-peptide gene into a pET28-b(+) plasmid. The plasmid was transformed into T7 Shuffle Express E. Coli for protein expression. Cultures were maintained in 500 mL of 2xYT media containing 50 μg/mL Kanamycin and shaken until the OD600 reached ~0.8. Bacteria were then cooled to ~16°C, induced with 2.5 mM IPTG, and cultured overnight. The following day, bacteria were collected, lysed, and incubated with Talon IMAC resin. nAlb-antigen fusions were collected by elution with a 10 mM imidazole buffer. Eluate was purified using an Akta FPLC and concentrated using centrifugal dialysis for subsequent use. For biodistribution studies, nAlb was labeled with Cy5 using a sortase reaction, and 2 nmol Cy5 was administered subcutaneously (SC). 24 h post injection, clearance organs and LNs were collected and imaged via IVIS. For activation studies, mice were administered nAlb-OVA251-270 or a mixture of nAlb-OVA251-270 and nAlb-diABZI. For tumor studies, mice were inoculated with 5 x 105 B16.F10-OVA cells, and tumor volumes were measured every other day and calculated as 0.5*L*W2. Mice were sacrificed when tumor volume reached 1500 mm3. For experimental autoimmune encephalomyelitis (EAE) studies, mice were administered nAlb-MOG35-55 on days -7, -5, and -3 prior to disease induction. Mice were monitored daily and disease severity was evaluated according to established scoring guidelines.

Results, Conclusions, and Discussions: Results and

Discussion:
Fusion to nAlb extended Cy5 half life (Fig. 1B), leading to increased Cy5 accumulation in all major clearance organs as well as the draining inguinal LN (iLN) (Fig. 1C), compared to an nGFP-Cy5 control. Flow cytometry of the iLNs and spleens (Fig. 1D) indicated that Cy5 was preferentially taken up by antigen presenting cells (APCs), including macrophages and dendritic cells. Collectively, these data suggest that nAlb enhances the lymphatic trafficking of fused moieties through binding to MSA. To test the ability of nAlb-antigen fusions to induce antigen specific immune tolerance through systemic antigen presentation, mice were prophylactically administered nAlb-MOG35-55 and challenged with experimental autoimmune encephalomyelitis (EAE) – a mouse model of multiple sclerosis (MS). Treatment with nAlb-MOG35-55 inhibited disease progression to a significant degree (Fig. 1E). To evaluate the ability of nAlb-OVA251-270 to induce an activating adaptive immune response, we treated C57Bl/6j mice with either PBS, nAlb-OVA251-270, or a mixture of nAlb-OVA251-270 and nAlb-diABZI. We then collected and processed blood for flow cytometric profiling of circulating T cells. We found that the mixture of nAlb-OVA251-270 and nAlb-diABZI significantly increased the population of CD8+ T cells specific to the Class I MHC restricted epitope of OVA, OVA257-264 (SIINFEKL), using a SIINFEKL/MHC tetramer that binds T cell receptors recognizing SIINFEKL in the context of H2-Kb (Fig. 1F). To test the antitumor efficacy of generated antigen-specific immune cells, mice were inoculated with B16.F10-OVA tumors and treated with nAlb-OVA251-270 and nAlb-diABZI. Treatment suppressed tumor growth and extended mouse survival (Fig. 1G).

Conclusions:
nAlb-antigen fusions drain to the lymphatics and are taken up by APCs. When combined with a STING-activating adjuvant, antigen epitopes are presented on MHC I alongside co-stimulatory markers, leading to clonal expansion of SIINFEKL-specific, antitumoral CD8+ T cells. Without an adjuvant, this platform is capable of inducing antigen specific immune tolerance through robust antigen presentation absent co-stimulation. This work introduces a platform for neoantigen targeted cancer therapy and autoimmune disease (AD) treatment. Future work aims to replace model antigens with disease specific neoantigens, utilize synthetic peptide antigens in lieu of cloning, and incorporate tolerizing adjuvants for AD applications.

Acknowledgements (Optional): ITED NIH T32 DK101003
NSF GRFP 2022305770
NIH 1R01CA266767-01A1