mRNA-LNP vaccines tuned for systemic immunization induce strong antitumor immunity by engaging splenic immune cells.

mRNA vaccines have recently proved to be highly effective against SARS-CoV-2. Key to their success is the lipid-based nanoparticle (LNP), which enables efficient mRNA expression and endows the vaccine with adjuvant properties that drive potent antibody responses. Effective cancer vaccines require long-lived, qualitative CD8 T cell responses instead of antibody responses. Systemic vaccination appears to be the most effective route, but necessitates adaptation of LNP composition to deliver mRNA to antigen-presenting cells. Using a design-of-experiments methodology, we tailored mRNA-LNP compositions to achieve high-magnitude tumor-specific CD8 T cell responses within a single round of optimization. Optimized LNP compositions resulted in enhanced mRNA uptake by multiple splenic immune cell populations. Type I interferon and phagocytes were found to be essential for the T cell response. Surprisingly, we also discovered a yet unidentified role of B cells in stimulating the vaccine-elicited CD8 T cell response. Optimized LNPs displayed a similar, spleen-centered biodistribution profile in non-human primates and did not trigger histopathological changes in liver and spleen, warranting their further assessment in clinical studies. Taken together, our study clarifies the relationship between nanoparticle composition and their T cell stimulatory capacity and provides novel insights into the underlying mechanisms of effective mRNA-LNP-based antitumor immunotherapy.

VHH antibody targeting the chemokine receptor CX3CR1 inhibits progression of atherosclerosis.

CX3CR1 has been identified as a highly attractive target for several therapeutic interventions. Despite this potential, no potent antagonists, either small molecule or monoclonal antibody, have been identified. Here we describe the lead finding and engineering approach that lead to the identification of BI 655088, a potent biotherapeutic antagonist to CX3CR1. BI 655088 is a potent CX3CR1 antagonist that, upon therapeutic dosing, significantly inhibits plaque progression in the standard mouse model of atherosclerosis. BI 655088 represents a novel and highly selective biotherapeutic that could reduce inflammation in the atherosclerotic plaque when added to standard of care treatment including statins, which could result in a significant decrease in atherothrombotic events in patients with existing cardiovascular disease.

The constitutive activity of the virally encoded chemokine receptor US28 accelerates glioblastoma growth.

Glioblastoma (GBM) is the most aggressive and an incurable type of brain cancer. Human cytomegalovirus (HCMV) DNA and encoded proteins, including the chemokine receptor US28, have been detected in GBM tumors. US28 displays constitutive activity and is able to bind several human chemokines, leading to the activation of various proliferative and inflammatory signaling pathways. Here we show that HCMV, through the expression of US28, significantly enhanced the growth of 3D spheroids of U251- and neurospheres of primary glioblastoma cells. Moreover, US28 expression accelerated the growth of glioblastoma cells in an orthotopic intracranial GBM-model in mice. We developed highly potent and selective US28-targeting nanobodies, which bind to the extracellular domain of US28 and detect US28 in GBM tissue. The nanobodies inhibited chemokine binding and reduced the constitutive US28-mediated signaling with nanomolar potencies and significantly impaired HCMV/US28-mediated tumor growth in vitro and in vivo. This study emphasizes the oncomodulatory role of HCMV-encoded US28 and provides a potential therapeutic approach for HCMV-positive tumors using the nanobody technology.

Modulating ion channel function with antibodies and nanobodies.

Immune cells express various voltage-gated and ligand-gated ion channels that mediate the influx and efflux of charged ions across the plasma membrane, thereby controlling the membrane potential and mediating intracellular signal transduction pathways. These channels thus present potential targets for experimental modulation of immune responses and for therapeutic interventions in immune disease. Small molecule drugs and natural toxins acting on ion channels have illustrated the potential therapeutic benefit of targeting ion channels on immune cells. Unwanted side effects and immunogenicity have however hampered the application of these molecules. Owing to their high specificity, low immunogenicity and beneficial pharmacodynamics, antibodies targeting membrane and secretory proteins have emerged as potent therapeutics in oncology and inflammation. Nanobodies-single domain fragments derived from heavy chain antibodies naturally occurring in camelids-offer additional benefits versus antibodies, including protrusion into cryptic epitopes and easy formatting of multi-specific reagents. Here we review recent progress in the development and application of antibodies and Nanobodies targeting ion channels on immune cells.

Kv channel gating requires a compatible S4-S5 linker and bottom part of S6, constrained by non-interacting residues.

Voltage-dependent K(+) channels transfer the voltage sensor movement into gate opening or closure through an electromechanical coupling. To test functionally whether an interaction between the S4-S5 linker (L45) and the cytoplasmic end of S6 (S6(T)) constitutes this coupling, the L45 in hKv1.5 was replaced by corresponding hKv2.1 sequence. This exchange was not tolerated but could be rescued by also swapping S6(T). Exchanging both L45 and S6(T) transferred hKv2.1 kinetics to an hKv1.5 background while preserving the voltage dependence. A one-by-one residue substitution scan of L45 and S6(T) in hKv1.5 further shows that S6(T) needs to be alpha-helical and forms a "crevice" in which residues I422 and T426 of L45 reside. These residues transfer the mechanical energy onto the S6(T) crevice, whereas other residues in S6(T) and L45 that are not involved in the interaction maintain the correct structure of the coupling.

Differential modulation of Kv4 kinetics by KCHIP1 splice variants.

The beta-subunits of the KChIP family modulate properties and expression level of Kv4 channels. We report the cloning of the first splice variant of KChIP1 (KChIP1b) which contains an extra exon, rich in aromatic residues, in the amino terminus. Both splice variants interacted equally well with Kv4.2 subunits based on confocal imaging and upregulation of current density (more than five-fold). No effects on the voltage dependence of activation or inactivation were noted. However, the effects on the kinetics of recovery from inactivation were opposite: KChIP1b induced a slow component in the recovery (tau approximately 1.2 s), in contrast to the increased recovery rate (tau = 125 ms) with KChIP1a. Accordingly, frequency-dependent accumulation of inactivation was enhanced by KChIP1b but reduced by KChIP1a. Since Kv4.2 channels are involved in protection against back propagating action potentials in dendritic spines, a differential expression of either splice variant could shape the dendritic function.