Straight to the Point: Targeted mRNA Delivery to Immune Cells for Improved Vaccine Design

Feb 24, 2024 06:02 CST Updated 06:02

GSK

Pharmaceutical R&D Manufacturer

Introduction:With the understanding of adaptive immunity at the cellular and molecular levels, targeting antigens to immune cells has proven to be a successful strategy for developing innovative and potent vaccines, as it may enhance vaccine efficacy and/or modulate the quality of immune responses while reducing off-target effects. With the development of mRNA vaccines, several methods for targeting immune cells (dendritic cells, DCs), which are the most effective antigen-presenting cells, using nanoparticle delivery systems encapsulating mRNA have been explored in recent years.Antigen-presenting cells, as well as B cells and T cellsThe key medium of the epidemic is considered to be the ideal target for cell-specific antigen delivery.

So, how to design mRNA vaccine delivery systems to target immune cells? In November 2023, the GSK team published a review article in the journal Front Immunol:《Straight to the point: targeted mRNA-delivery to immune cells for improved vaccine design》The article summarizes the potential specific targets for mRNA targeted delivery to the immune system, categories of targeted receptor-ligands, and research progress in delivery systems.

1. Immunocyte Targeting

Due to the long development cycle (5-10 years) of conventional vaccines (antigens, inactivated pathogens) and the significant challenges in developing vaccines for emerging and re-emerging diseases, scientists have been dedicated to exploring new vaccine platforms. Recently, mRNA vaccines have achieved substantial breakthroughs in design and efficacy, which is why they have stood out.Related to the following characteristics:

1、Safety:The non-infectious and non-integrative nature of mRNA avoids the potential risks of infection or insertional mutagenesis.

2、Stability:mRNA vaccines can achieve in vivo delivery through nanocarriers (such as LNPs), protecting them from degradation, enabling rapid uptake and cytoplasmic delivery, and avoiding anti-carrier immunity.

3、High Yield: Due to the high yield of in vitro transcription reactions, mRNA vaccines have the potential for cost-effective and scalable production.

4、Modifiability: Can be rapidly modified to adjust half-life and immunogenicity characteristics for personalized treatment or urgent adaptation to emerging pathogens.

But at the same time, mRNA vaccines have the following shortcomings:

1、Potential off-target effects: After intramuscular injection, a small portion will reach the bloodstream, and ultimately, most of it will enter the liver and spleen, resulting in off-target effects.

2、Non-specificAdministration of mRNA-LNPs induces rapid and local infiltration of neutrophils, monocytes, and DCs into the administration site and draining lymph nodes (LNs), but mainly monocytes and DCs translate mRNA and upregulate key costimulatory receptors (CD80 and CD86) and cytokine production.

3. Antibody response to certain proteins (e.g., spike protein)Shorter persistence, requiring continuous administration of booster doses to maintain protection against infection.

mRNA Selective Targeting of Immune Cells Becomes Key Technology to Address Above Issues Due to Enhanced Immune Response, Reduced Vaccine Dosage, Prolonged Protection, and Minimized Off-Target Effects and Side Effects.And antigen-presenting cells (APCs), especially DCs, because they initiateThe unique ability of naive T cells to initiate adaptive immunity makes them a candidate cell for mRNA targeted delivery. Studies have shown that even at low antigen doses, DC targeting can enhance and accelerate specific antibody responses.

Targeting can be achieved by using ligands that specifically interact with receptors on the surface of specific cells. The involvement of different receptors can influence the type of immune response induced, thereby achieving specific outcomes. DCs can express a variety of receptors for targeted vaccine delivery, including C-type lectin receptors (CLRs), Toll-like receptors, scavenger receptors, chemokine receptors, complement receptors, and Fc receptors, among others. DC targeting not only enhances the efficacy of mRNA vaccines but also broadens the range of pathogens they can act against. In addition to DC subsets, other cell types involved in immune responses, such as macrophages, can also serve as targets.

Figure 1. Mechanism of APC-targeted LNP-mRNA action

2. Target Cells

The table below compares various antigen-presenting cells (APCs) in terms of origin, distribution, characteristics/functions, and other aspects:

Each type of APC has its advantages and limitations: compared with pDCs, cDCs are more efficient in antigen presentation and T-cell activation; compared with macrophages, DCs have a stronger ability to initiate antigen-specific immune responses due to their superior antigen presentation and capacity to migrate from peripheral tissues to lymph nodes, making them more advantageous for targeted delivery. The final choice of which APC to target also depends on the characteristics of the pathogen or antigen to be targeted. A combination of multiple APCs (such as DCs and macrophages) can trigger synergistic effects and elicit a stronger and more versatile immune response.

3. Target Receptor

The type and extent of the immune response depend on the environment in which the antigen is captured, including the receptors used for internalization. Therefore, the success of a targeted delivery platform depends on the correct selection of the ligand-receptor pair.

3.1 C-type Lectins (CLRs) Receptors

3.1.1 General Properties of CLRs

CLRs are a superfamily composed of more than 1,000 proteins. They are highly expressed on the surface of various immune cells, including DCs, macrophages, and neutrophils, and play roles in recognizing self and non-self antigens, internalization, antigen processing, initiating immune responses, and regulating interactions between immune cells, making them targets for targeted antigens and mRNA delivery:

CLRs possess a conserved structural motif—the carbohydrate recognition domain (CRD)—which recognizes pathogen (virus, bacteria, and fungi)-associated carbohydrate structures through interactions with conserved calcium-chelating binding sites, promoting recognition and phagocytosis. CLR signaling can be divided into two groups: The first group transduces intracellular signals via an immunoreceptor tyrosine-based activation motif (ITAM)-like motif (Clec-2, Dectin-1) or by binding to FcRγ adaptor molecules carrying ITAMs (Dectin-2, Mincle, BDCA-2). After phosphorylation, the ITAM motif recruits and activates Syk, which induces the transcription of pro-inflammatory cytokines by activating subunits of the NF-kB transcription factor complex. The second group of CLRs has an immunoreceptor tyrosine-based inhibitory motif (ITIM) at their cytoplasmic tail (e.g., MICL).

3.1.2 Mannose Receptor

The mannose receptor (CD206, MR) is an endocytic receptor expressed by macrophages and DCs, mediating the cross-presentation of soluble ligands of mannans (from simple mannose to advanced mannan structures), fucose, and sulfated LacdiNAc. These characteristics make mannose-based targeting one of the most common CLR-targeting strategies. In 2006, White et al. used mannosylated liposomes in an in vitro model to enhance the uptake of OVA by monocyte-derived DCs (moDCs). Numerous reports indicate that mannose-based strategies increase the internalization and transfection of mRNA vaccines in immune cells through receptor-mediated mechanisms. The first clinical trial of an MR-targeted cancer vaccine was reported in 2011, but the clinical activity of MR-targeted mRNA vaccines remains unclear.

3.1.3 Other C-type Lectins

1、DC-SIGN: Calcium-dependent, present on human DCs and subsets of macrophages, can bind to highly mannosylated glycoconjugates and fucose-containing antigens, mediating DC-T cell interactions by promoting T cell adhesion to the DC surface to detect the presence of peptide antigens. DC-SIGN induces intracellular signaling, thereby modulating the signal transduction of other pathogen recognition receptors (PRRs) such as TLRs.

2、DEC-205（CD205）: Expressed on DCs, monocytes, and LCs, as well as low-level expression on B cells, T cells, and NK cells. It has the function of recognizing apoptotic and necrotic signals, participating in endocytosis, and enhancing MHC II presentation. Most DC205 targeting relies on the use of antibodies or antibody fragments, resulting in effective DC targeting and enhanced antigen cross-presentation. In 2011, a clinical trial for an anti-HIV DEC-205 targeted vaccine was conducted.

3、Dectin-1: Expressed by DCs, macrophages, monocytes, neutrophils, and T cell subsets, it specifically recognizes soluble and particulate β(1-3) and/or β(1-6) linked glucans with different affinities, promotes particle uptake through actin-dependent phagocytosis, and participates in cell activation via an ITAM-like motif. It is considered a co-stimulatory molecule as it can bind to CD4+ and CD8+ T cells and promote their proliferation.

4、Langerin（CD207）: Expressed by LCs, dermal DCs, and other DC subtypes, antibody or small-molecule glycans have been successfully used as targeting ligands for LC targeting in vitro. Wamhoff et al. discovered a Langerin glycomimetic ligand and demonstrated its selectivity for Langerin+ cells using functionalized liposomes. Schulze et al. used this Langerin ligand to selectively target nanoparticles (liposomes) to human LCs in vitro. This ligand can be used to functionalize nanoparticles encapsulating drugs, antigens, or toxins for delivery to LCs.

5、DNGR-1（Clec9a) and MGL (Clec10a)DNGR-1/Clec9a is selectively expressed on human cDC1. Specific interaction with this receptor can enhance humoral immunity in non-human primates. Recent studies have found that DNGR-1-specific peptides can target nanoparticles to Clec9a+ DCs. MGL is specifically expressed on human cDC2 (CD1c+ DCs) and selectively binds to GalNAc termini and mucin-1 peptides with different glycosylation profiles. Heger et al. discovered that using Clec10a-binding glycopeptides can selectively target CD1c+ cells (cDC2) in PBMCs, but no activation or cytokine secretion of CD1c+ DCs was observed with the ligand of Clec10 alone.

6、hDCIR（Clec4a）: Present on CD14+ monocytes, CD15+ granulocytes, all DC subsets (including pDCs), and B cells in peripheral blood; not found in T cells. Using antibodies to target antigens to DCIR can enhance cross-presentation by LCs, blood mDCs, and pDCs, and augment CD8+ T cell activation in human cells in vitro.

3.2 For mRNATargeted DeliveryOther receptors

1、Toll-like receptors (TLRs): Expressed by innate immune system cells (such as macrophages and DCs) and adaptive immune cell subsets, playing a key role in the innate immune system by recognizing pathogen-associated molecular patterns (PAMPs). The study uses Pam3CSK4 (a TLR-2 agonist) as a targeting ligand to direct nanoparticles to DCs.

2、X-C Motif Chemokine Receptor 1 (XCR1): A chemokine receptor that recognizes XCL1, selectively expressed on cDC1. Fossum et al. studied Clec9a, DEC-205, and XCR-1 as targets by injecting DNA vaccines encoding single-chain variable fragments (scVf) fused with antigens into mice. They found that targeting XCR-1 enhanced IFN-γ+CD8+ T cell responses in the spleen and lungs, with stronger cytotoxicity. Although both Clec9a and XCR1 are cDC1-specific, they led to different outcomes, indicating that not only the cell subtype but also the receptor itself determines the outcome and efficacy of targeting.

Table 1 Cases of Immune Cell Targeting, Targeting Strategies, and Outcomes

4. Nanoparticles as Targeted mRNA Delivery Carriers

mRNA is combined with carriers to protect it from degradation and achieve intracellular delivery. Current delivery systems are simply divided into viral and non-viral vectors. Viral vectors utilize their naturally evolved ability to efficiently transfer genetic material into cells, achieving high translation efficiency, but their inherent tropism does not always meet therapeutic needs, and the induced immune response can translate into reactogenicity that compromises treatment efficacy. Currently, nanoparticles (especially lipid nanoparticles, LNPs) are the preferred non-viral vectors, offering the possibility of customizing their properties to optimize their performance as transfection agents. Lipid nanoparticles (LNPs) have evolved from liposomes and lipid complexes into more effective mRNA carriers, giving rise to ionizable lipids, which have the following advantages:

1. Ionizable lipids are positively charged at acidic pH and neutral at physiological pH, which significantly improves the inherent drawbacks of permanently cationic lipids, such as poor tolerability and cytotoxicity.

2. After ionizable lipids are acidified in endosomes, the amine groups are protonated and promote chloride ion transport to balance membrane charge and osmotic pressure until the membrane is disrupted and genetic material is delivered into the cytosol.

3. By increasing the unsaturation of the hydrophobic tails of ionizable lipids and adjusting their pKa, endosomal escape can be enhanced, thereby strengthening vaccine efficacy;

4. Ionizable lipids can achieve high-rate mRNA encapsulation. Due to the low pH of the aqueous phase during production, ionizable lipids are positively charged, thereby promoting interaction with the negatively charged mRNA backbone.

Another component of LNPs is pegylated lipids, which are formed by the conjugation of hydrophilic PEG polymers with hydrophobic lipids. Pegylated lipids can prevent opsonin binding, increase LNP circulation time, and prevent particle fusion. By altering the anchor length of PEG lipids, their shedding rate from the LNP surface can be adjusted, which is crucial for promoting cellular uptake and endosomal escape. Additionally, phospholipids and cholesterol in LNPs help encapsulate mRNA and enhance stability.

Modifying the lipid components and/or ratios of LNPs to achieve their preferential accumulation in specific organs, Goswami et al. demonstrated that mannosylated cholesterol can enhance the internalization and potency of an anti-RSV self-amplifying mRNA vaccine (SAM). Examples of MR-targeted siRNA (ligand directly conjugated to siRNA or siRNA encapsulated particles) have also been reported.

Figure 2 LNP Structure, Composition, and Targeting Ligand Incorporation Strategies

5. Conclusion

Targeting specific receptors on innate immune-related cells can elicit a stronger and more durable immune response tailored to vaccines. A combined targeting strategy for different subsets of innate immune cells is even more promising, as they play unique and central roles in the immune response. For example, macrophages drive inflammatory responses, while dendritic cells play a crucial role in antigen presentation to T cells; simultaneously targeting both cell types may help initiate an improved antigen-specific immune response.

Further research is needed to identify potential common key receptors in different cell types or to formulate LNPs with combinations of multiple targeting ligands.

For the development of RNA delivery vectors, it is necessary to consider the compatibility of components during the formulation process, as well as the need to maintain effective endosomal escape and adequate multivalent ligand presentation.

eGFP mRNA and mCherry mRNA Achieve High Expression in 293T Cells

References

[1] Clemente B, Denis M, Silveira CP, Schiavetti F, Brazzoli M, Stranges D. Straight to the point: targeted mRNA-delivery to immune cells for improved vaccine design. Front Immunol. 2023 Nov 27;14:1294929. doi: 10.3389/fimmu.2023.1294929.

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