Periodic Reporting for period 1 - LNP-DECODE (LNP-DECODE: Broadening the therapeutic window of LNP-based vaccination)
Periodo di rendicontazione: 2024-06-01 al 2025-11-30
Dysregulation of immune tolerance leads to the development of allergies and autoimmune diseases. The current standard of care comprises the use of broad-acting immunosuppressive drugs that are neither disease-specific nor antigen-specific, and that are not curative. Hence, there is an unmet need for novel antigen-specific therapeutic strategies.
Dendritic cells (DCs) are antigen-presenting cells that initiate and regulate the adaptive immune system functions, and that are crucial as gatekeepers between immunity and tolerance. Ever since the discovery of their potential as modulators of immune responses, the utilization and targeting of DCs for immunotherapy approaches to induce tolerance to certain autoimmune antigens, has been envisioned. Most therapies rely on the adoptive transfer of patient-derived blood monocytes that are differentiated in vitro to tolerogenic DCs in the presence of specific antigens and a cocktail of tolerogenic agents. While phase I trials proved that the therapy is safe and well tolerable, it seldomly leads to improved clinical symptoms. One of the major limitations is that in vitro generated DC cultures hardly reflect in vivo conventional DCs (cDCs) and lack migratory and antigen presentation capacity. Therefore, it would be more efficient to induce immune tolerance in vivo by targeting the relevant cDC subsets directly. Attempts towards targeted delivery by conjugating antigens to antibodies recognizing surface epitopes on cDC1s and cDC2s unfortunately had only limited success.
The success of ionizable lipid nanoparticles (iLNPs) as vehicles for mRNA as a safe and highly effective vaccine might be a gamechanger. The SARS-CoV-2 pandemic pushed the LNP technology to the forefront of medicine and launched worldwide interest in their potential as a therapeutic vaccine against numerous pathogens or tumor antigens. Still, at this point we do not fully understand the molecular principles behind their mechanism of action. One of the most remarkable properties of mRNA-LNPs is the fact that they do not require any additional adjuvants to induce potent immune responses. It has been postulated that the ionizable lipid component of the iLNPs themselves can serve as stand-alone driver of adjuvanticity without the need for additional RNA components.
Recent data from my lab challenged this hypothesis as empty (non-mRNA complexed) iLNPs (eLNPs) do not trigger innate immune activation, but rather induce immune tolerance. The aim of the ERC PoC study was to explore this finding and decode the adjuvanticity of LNPs (objective 1). In addition, we wanted to test the premise that by using LNPs loaded with non-immunogenic cargo, we would be able to target the relevant DC subsets directly in vivo and instruct naïve T cells towards tolerance against autoimmune antigens or allergens (objective 2).
Dendritic cells (DCs) are antigen-presenting cells that initiate and regulate the adaptive immune system functions, and that are crucial as gatekeepers between immunity and tolerance. Ever since the discovery of their potential as modulators of immune responses, the utilization and targeting of DCs for immunotherapy approaches to induce tolerance to certain autoimmune antigens, has been envisioned. Most therapies rely on the adoptive transfer of patient-derived blood monocytes that are differentiated in vitro to tolerogenic DCs in the presence of specific antigens and a cocktail of tolerogenic agents. While phase I trials proved that the therapy is safe and well tolerable, it seldomly leads to improved clinical symptoms. One of the major limitations is that in vitro generated DC cultures hardly reflect in vivo conventional DCs (cDCs) and lack migratory and antigen presentation capacity. Therefore, it would be more efficient to induce immune tolerance in vivo by targeting the relevant cDC subsets directly. Attempts towards targeted delivery by conjugating antigens to antibodies recognizing surface epitopes on cDC1s and cDC2s unfortunately had only limited success.
The success of ionizable lipid nanoparticles (iLNPs) as vehicles for mRNA as a safe and highly effective vaccine might be a gamechanger. The SARS-CoV-2 pandemic pushed the LNP technology to the forefront of medicine and launched worldwide interest in their potential as a therapeutic vaccine against numerous pathogens or tumor antigens. Still, at this point we do not fully understand the molecular principles behind their mechanism of action. One of the most remarkable properties of mRNA-LNPs is the fact that they do not require any additional adjuvants to induce potent immune responses. It has been postulated that the ionizable lipid component of the iLNPs themselves can serve as stand-alone driver of adjuvanticity without the need for additional RNA components.
Recent data from my lab challenged this hypothesis as empty (non-mRNA complexed) iLNPs (eLNPs) do not trigger innate immune activation, but rather induce immune tolerance. The aim of the ERC PoC study was to explore this finding and decode the adjuvanticity of LNPs (objective 1). In addition, we wanted to test the premise that by using LNPs loaded with non-immunogenic cargo, we would be able to target the relevant DC subsets directly in vivo and instruct naïve T cells towards tolerance against autoimmune antigens or allergens (objective 2).
WP1: Decoding the adjuvants activity of LNPs
In the first WP we assessed the adjuvants activity of different types of LNPs by performing single cell sequencing on LNP+ mature DCs in the spleen and determining their maturation program. This was complemented with functional assays to analyze the capacity of LNP+ DCs to steer the adaptive immune response toward immunity or tolerance against non-immunogenic cargo incorporated in the LNPs.
This revealed that:
1) eLNPs are not perceived as “danger” by DCs and their uptake leads to induction of a homeostatic not immunogenic maturation program
2) The cargo and not the lipids determine the adjuvanticity of LNPs
3) LNPs can be exploited as vehicles to induce either immunity or tolerance against incorporated antigens
4) The immunogenicity of the cargo needs to be carefully assessed, especially when incorporated in the LNP: synthetic peptides are non-immunogenic while most mRNA purifications are immunogenic unless care is taken to remove all dsRNA contaminants
In addition, we designed a DC maturation tool box, a set of tools that can be used to assess the immunogenicity of newly developed LNP formulations. This comprises:
1) a flow cytometry panel for mice and humans to deconvolute homeostatic from immunogenic mature DCs. This is the first time such a panel has been designed and is meanwhile widely adapted by other labs
2) a transcriptional profiling strategy to monitor the DC maturation program. This can be defined by qPCR or by sequencing methods. We developed an open access web-based tool that allows researchers to enter their sequencing data of mature DCs in an algorithm that will assign the DC maturation state along a homeostatic-immunogenic spectrum and perform a meta-analysis
WP2: Tolerogenic potential of LNP vaccines
In WP2 we aimed to address whether LNPs can be used to induce tolerance against allergens and/or autoimmune disease. As a proof-of-concept, we used the house dust mite (HDM) protocol, a well-established model for allergic airway inflammation and asthma.
The major bottleneck appeared to be the incorporation of the antigen in the LNPs.
Originally we proposed to incorporate the main HDM antigens, Derp1 and Derp2, as synthetic peptides in the LNPs, as we knew this yielded non-immunogenic antigens. However, to improve the outcome of the response we switched to whole proteins, produced recombinantly either in E.coli or P.pastoris. We took special precautions to keep the LPS during the purification procedure as low as possible. Still, even the sligthest presence of LPS (which was inevitable) yielded in immunogenic DC maturation, showing that incorporation in LNPs amplifies any TLR contaminants.
Therefore, we turned our attention towards mRNA antigen incorporation, as was used for the original mRNA-LNPs of BioNTech. We applied different strategies to reduce the dsDNA content, both by using a mutated version of the T7 RNA polymerase and by applying additional columns to remove the dsDNA. The effects on DC maturation in vivo were assessed by using the strategies from WP1.
The optimized mRNA purification strategy had success and yielded the desired non-immunogenic profile. We are currently applying similar strategies for Derp1 and Derp constructs to assess their potential as a tool to induce tolerance and improving astma outcomes.
In the first WP we assessed the adjuvants activity of different types of LNPs by performing single cell sequencing on LNP+ mature DCs in the spleen and determining their maturation program. This was complemented with functional assays to analyze the capacity of LNP+ DCs to steer the adaptive immune response toward immunity or tolerance against non-immunogenic cargo incorporated in the LNPs.
This revealed that:
1) eLNPs are not perceived as “danger” by DCs and their uptake leads to induction of a homeostatic not immunogenic maturation program
2) The cargo and not the lipids determine the adjuvanticity of LNPs
3) LNPs can be exploited as vehicles to induce either immunity or tolerance against incorporated antigens
4) The immunogenicity of the cargo needs to be carefully assessed, especially when incorporated in the LNP: synthetic peptides are non-immunogenic while most mRNA purifications are immunogenic unless care is taken to remove all dsRNA contaminants
In addition, we designed a DC maturation tool box, a set of tools that can be used to assess the immunogenicity of newly developed LNP formulations. This comprises:
1) a flow cytometry panel for mice and humans to deconvolute homeostatic from immunogenic mature DCs. This is the first time such a panel has been designed and is meanwhile widely adapted by other labs
2) a transcriptional profiling strategy to monitor the DC maturation program. This can be defined by qPCR or by sequencing methods. We developed an open access web-based tool that allows researchers to enter their sequencing data of mature DCs in an algorithm that will assign the DC maturation state along a homeostatic-immunogenic spectrum and perform a meta-analysis
WP2: Tolerogenic potential of LNP vaccines
In WP2 we aimed to address whether LNPs can be used to induce tolerance against allergens and/or autoimmune disease. As a proof-of-concept, we used the house dust mite (HDM) protocol, a well-established model for allergic airway inflammation and asthma.
The major bottleneck appeared to be the incorporation of the antigen in the LNPs.
Originally we proposed to incorporate the main HDM antigens, Derp1 and Derp2, as synthetic peptides in the LNPs, as we knew this yielded non-immunogenic antigens. However, to improve the outcome of the response we switched to whole proteins, produced recombinantly either in E.coli or P.pastoris. We took special precautions to keep the LPS during the purification procedure as low as possible. Still, even the sligthest presence of LPS (which was inevitable) yielded in immunogenic DC maturation, showing that incorporation in LNPs amplifies any TLR contaminants.
Therefore, we turned our attention towards mRNA antigen incorporation, as was used for the original mRNA-LNPs of BioNTech. We applied different strategies to reduce the dsDNA content, both by using a mutated version of the T7 RNA polymerase and by applying additional columns to remove the dsDNA. The effects on DC maturation in vivo were assessed by using the strategies from WP1.
The optimized mRNA purification strategy had success and yielded the desired non-immunogenic profile. We are currently applying similar strategies for Derp1 and Derp constructs to assess their potential as a tool to induce tolerance and improving astma outcomes.
Our data have been published in Rennen et al. (Cell Rep, 2024) and Van Lil et al. (STAR Protocols, 2026, in press). They were (or will be) orally presented at the ICI (Chemical Immunology) Meeting in Leiden, the ESGCT meeting in Rome, and the VIB Next generation mRNA vaccines meeting in March 2026 to probe interest from key actors in the LNP field.
As a result, we secured a contract research grant with the biotech company Etherna (Gent) to assess the immunogenicity of several newly developed LNP formulations, which were used in collaboration with UHasselt to induce tolerance in the autoimmune model EAE (Baeten et al., manuscript in preparation).
Further research is needed to fully work out objective 2, and this may demand substantially more time than the 18 months that were foreseen for this ERC PoC. Postdoc Greta Webb has been hired on the project and meanwhile obtained an MSCA postdoctoral fellowship to continue this research line in our lab, starting as from April 1, 2026. She will work in close collaboration with the Lambrecht/Hammad Lab at IRC (VIB UGent), world reknown experts in mucosal immunology and astma.
As a result, we secured a contract research grant with the biotech company Etherna (Gent) to assess the immunogenicity of several newly developed LNP formulations, which were used in collaboration with UHasselt to induce tolerance in the autoimmune model EAE (Baeten et al., manuscript in preparation).
Further research is needed to fully work out objective 2, and this may demand substantially more time than the 18 months that were foreseen for this ERC PoC. Postdoc Greta Webb has been hired on the project and meanwhile obtained an MSCA postdoctoral fellowship to continue this research line in our lab, starting as from April 1, 2026. She will work in close collaboration with the Lambrecht/Hammad Lab at IRC (VIB UGent), world reknown experts in mucosal immunology and astma.