Conjugation of nanoparticles (NP) to the surface of living cells is of interest in the context of exploiting the tissue homing properties of ex vivo engineered T cells for tumor‐targeted delivery of drugs loaded into NP. Cell surface conjugation requires either a covalent or non‐covalent reaction. Non‐covalent conjugation with ligand‐decorated NP (LNP) is challenging and involves a dynamic equilibrium between the bound and unbound state. Covalent NP conjugation results in a permanently bound state of NP, but the current routes for cell surface conjugation face slow reaction kinetics and random conjugation to proteins in the glycocalyx. To address the unmet need for alternative bioorthogonal strategies that allow for efficient covalent cell surface conjugation, we developed a 2‐step click conjugation sequence in which cells are first metabolically labeled with azides followed by reaction with sulfo‐6‐methyl‐tetrazine‐dibenzyl cyclooctyne (Tz‐DBCO) by SPAAC, and subsequent IEDDA with trans‐cyclooctene (TCO) functionalized NP. In contrast to using only metabolic azide labeling and subsequent conjugation of DBCO‐NP, our 2‐step method yields a highly specific cell surface conjugation of LNP, with very low non‐specific background binding.

Peptide-based subunit vaccines are attractive in view of personalized cancer vaccination with neo-antigens, as well as for the design of the newest generation of vaccines against infectious diseases. Key to mounting robust antigen-specific immunity is delivery of antigen to antigen-presenting (innate immune) cells in lymphoid tissue with concomitant innate immune activation to promote antigen presentation to T cells and to shape the amplitude and nature of the immune response. Nanoparticles that co-deliver both peptide antigen and molecular adjuvants are well suited for this task. However, in the context of peptide-based antigen, an unmet need exists for a generic strategy that allows for co-encapsulation of peptide and molecular adjuvants due to the stark variation in physicochemical properties based on the amino acid sequence of the peptide. These properties also strongly differ from those of many molecular adjuvants. Here, we devise a lipid nanoparticle (LNP) platform that addresses these issues. Key in our concept is poly­(l-glutamic acid) (PGA), which serves as a hydrophilic backbone for conjugation of, respectively, peptide antigen (Ag) and an imidazoquinoline (IMDQ) TLR7/8 agonist as a molecular adjuvant. Making use of the PGA’s polyanionic nature, we condensate PGA-Ag and PGA-IMDQ into LNP by electrostatic interaction with an ionizable lipid. We show in vitro and in vivo in mouse models that LNP encapsulation favors uptake by innate immune cells in lymphoid tissue and promotes the induction of Ag-specific T cells responses both after subcutaneous and intravenous administration.

Dendritic cells (DCs) mature in an immunogenic or tolerogenic manner depending on the context in which an antigen is perceived, preserving the balance between immunity and tolerance. Whereas the pathways driving immunogenic maturation in response to infectious insults are well-characterized, the signals that drive tolerogenic maturation during homeostasis are still poorly understood. We found that the engulfment of apoptotic cells triggered homeostatic maturation of type 1 conventional DCs (cDC1s) within the spleen. This maturation process could be mimicked by engulfment of empty, nonadjuvanted lipid nanoparticles (LNPs), was marked by intracellular accumulation of cholesterol, and was highly specific to cDC1s. Engulfment of either apoptotic cells or cholesterol-rich LNPs led to the activation of the liver X receptor (LXR) pathway, which promotes the efflux of cellular cholesterol, and repressed genes associated with immunogenic maturation. In contrast, simultaneous engagement of TLR3 to mimic viral infection via administration of poly(I:C)-adjuvanted LNPs repressed the LXR pathway, thus delaying cellular cholesterol efflux and inducing genes that promote T cell–mediated immunity. These data demonstrate that conserved cellular cholesterol efflux pathways are differentially regulated in tolerogenic versus immunogenic cDC1s and suggest that administration of nonadjuvanted cholesterol-rich LNPs may be an approach for inducing tolerogenic DC maturation.

Conjugation of nanoparticles (NP) to the surface of living cells is of interest in the context of exploiting the tissue homing properties of ex vivo engineered T cells for tumor‐targeted delivery of drugs loaded into NP. Cell surface conjugation requires either a covalent or non‐covalent reaction. Non‐covalent conjugation with ligand‐decorated NP (LNP) is challenging and involves a dynamic equilibrium between the bound and unbound state. Covalent NP conjugation results in a permanently bound state of NP, but the current routes for cell surface conjugation face slow reaction kinetics and random conjugation to proteins in the glycocalyx. To address the unmet need for alternative bioorthogonal strategies that allow for efficient covalent cell surface conjugation, we developed a 2‐step click conjugation sequence in which cells are first metabolically labeled with azides followed by reaction with sulfo‐6‐methyl‐tetrazine‐dibenzyl cyclooctyne (Tz‐DBCO) by SPAAC, and subsequent IEDDA with trans‐cyclooctene (TCO) functionalized NP. In contrast to using only metabolic azide labeling and subsequent conjugation of DBCO‐NP, our 2‐step method yields a highly specific cell surface conjugation of LNP, with very low non‐specific background binding.

The limited thermostability and need for (ultra)cold storage conditions is a major drawback of the current nucleoside-modified lipid nanoparticle (LNP) formulated messenger RNA (mRNA) vaccines and hampers distribution of these...