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 and extend pharmacokinetic half-life as molecules > 40 kDa exhibit lymphatic drainage (Fig. 1A). In this work, we synthesized an nAlb-OVA_251-270 fusion and demonstrated that co-administration with nAlb conjugated to the adjuvant diamidobenzimidazole (diABZI) produces a robust antigen-specific immune response with antitumoral effects.


Materials and Methods:: The nAlb-OVA_251-270 fusion was produced by designing a gene cassette in which OVA_251-270 was 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 PCR amplified, gel purified, and ligated the nAlb-OVA_251-270 gene into a pET28-b(+) plasmid. Following ligation and plasmid expansion, the plasmid was transformed into T7 Shuffle Express E. Coli for protein expression. T7 Shuffle Express cultures were maintained in 500 mL of 2xYT media containing 50 μg/mL Kanamycin and shaken until the OD₆₀₀ reached ~0.8. The bacteria were then cooled to ~20°C, induced with 2.5 mM IPTG, and cultured overnight. The following day, bacteria were collected, lysed, and incubated with Talon IMAC resin. nAlb-OVA_251-270 was 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 μg/kg Cy5 was administered subcutaneously (SC). 24 h post injection, spleen and LN were collected and imaged via IVIS. For tetramer studies, mice were administered 1 nmol of nAlb-OVA_251-270 or a mixture of nAlb-OVA_251-270 and 2.5 μg of nAlb-diABZI three times 7 days apart. 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.


Results, Conclusions, and Discussions:: Results and Discussion:

As hypothesized (Fig. 1A), Cy5 fused nAlb (nAlb-Cy5) localized to the draining inguinal lymph node (iLN) and irrelevant axillary LN (aLN), compared to both PBS and an EGFR-targeting control Nb (nEGFR-Cy5) (Fig. 1B, 1C). Flow cytometry of the spleens (Fig. 1D) and iLNs (Fig. 1E) 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. Lymphatic drainage is an important attribute of vaccine platforms as LNs are major cites of T cell priming. To evaluate the ability of nAlb-OVA_251-270 to induce an adaptive immune response, we treated C57Bl/6j mice with either PBS, nAlb-OVA_251-270, or a mixture of nAlb-OVA_251-270 and nAlb-diABZI. We then collected and processed blood for flow cytometric profiling of circulating T cells. We found that the mixture of nAlb-OVA_251-270 and nAlb-diABZI significantly increased the population of CD8⁺ T cells specific to the Class I MHC restricted epitope of OVA, OVA_257-264 (SIINFEKL), using a SIINFEKL/MHC tetramer that binds T cell receptors recognizing SIINFEKL in the context of H2-Kb (Fig. 1F). These data suggest that nAlb-OVA_251-270 facilitates antigen delivery to APCs and that diABZI is necessary to induce cross presentation. We then challenged the vaccinated mice with OVA expressing B16.F10 (B16.F10-OVA) tumors and found that on Day 17, mice treated with the mixture of nAlb-OVA_251-270 and nAlb-diABZI exhibited delayed tumor growth compared to relevant controls (Fig. 1G). Although this study is still ongoing, it suggests that the clonal expansion of OVA-specific CD8⁺ T cells induced by our vaccine exhibits antitumoral effects.

Conclusions:

nAlb-OVA_251-270 fusions drain to the lymphatics and are taken up by APCs. When combined with an adjuvant, the SIINFEKL epitope is presented on MHC I along with co-stimulatory markers, leading to clonal expansion of SIINFEKL-specific CD8⁺ T cells, and these T cells exhibit antitumoral effects. This work introduces an exciting platform for neoantigen targeted cancer therapy. Future work aims to better characterize the vaccine response, conjugate diABZI directly to nAlb-OVA_251-270, and replace OVA_251-270 for peptide neoantigens.


Acknowledgements (Optional): : NSF FMSG 2036809
NIH ITED T32 DK101003
NSF GRFP 2022305770


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