Jasmin Dülfer

Background: Immune exclusion (IE) where tumors deter the infiltration of immune cells into the tumor microenvironment has emerged as a key mechanism underlying immunotherapy resistance. We recently reported a novel role of discoidin domain-containing receptor 1 (DDR1) in promoting IE in breast cancer and validated its critical role in IE using neutralizing rabbit monoclonal antibodies (mAbs) in multiple mouse tumor models. Methods: To develop a DDR1-targeting mAb as a potential cancer therapeutic, we humanized mAb9 with a complementarity-determining region grafting strategy. The humanized antibody named PRTH-101 is currently being tested in a Phase 1 clinical trial. We determined the binding epitope of PRTH-101 from the crystal structure of the complex between DDR1 extracellular domain (ECD) and the PRTH-101 Fab fragment with 3.15 Å resolution. We revealed the underlying mechanisms of action of PRTH-101 using both cell culture assays and in vivo study in a mouse tumor model. Results: PRTH-101 has subnanomolar affinity to DDR1 and potent antitumor efficacy similar to the parental rabbit mAb after humanization. Structural information illustrated that PRTH-101 interacts with the discoidin (DS)-like domain, but not the collagen-binding DS domain of DDR1. Mechanistically, we showed that PRTH-101 inhibited DDR1 phosphorylation, decreased collagen-mediated cell attachment, and significantly blocked DDR1 shedding from the cell surface. Treatment of tumor-bearing mice with PRTH-101 in vivo disrupted collagen fiber alignment (a physical barrier) in the tumor extracellular matrix (ECM) and enhanced CD8⁺ T cell infiltration in tumors. Conclusions: This study not only paves a pathway for the development of PRTH-101 as a cancer therapeutic, but also sheds light on a new therapeutic strategy to modulate collagen alignment in the tumor ECM for enhancing antitumor immunity.

Norovirus capsids are icosahedral particles composed of 90 dimers of the major capsid protein VP1. The C-terminus of the VP1 proteins forms a protruding (P)-domain, mediating receptor attachment, and providing a target for neutralizing antibodies. NMR and native mass spectrometry directly detect P-domain monomers in solution for murine (MNV) but not for human norovirus (HuNoV). We report that the binding of glycochenodeoxycholic acid (GCDCA) stabilizes MNV-1 P-domain dimers (P-dimers) and induces long-range NMR chemical shift perturbations (CSPs) within loops involved in antibody and receptor binding, likely reflecting corresponding conformational changes. Global line shape analysis of monomer and dimer cross-peaks in concentration-dependent methyl TROSY NMR spectra yields a dissociation rate constantk_offof about 1 s⁻¹for MNV-1 P-dimers. For structurally closely related HuNoV GII.4 Saga P-dimers a value of about 10⁻⁶s⁻¹is obtained from ion-exchange chromatography, suggesting essential differences in the role of GCDCA as a cofactor for MNV and HuNoV infection.

Noroviruses are the major cause of viral gastroenteritis and re-emerge worldwide every year, with GII.4 currently being the most frequent human genotype. The norovirus capsid protein VP1 is essential for host immune response. The P domain mediates cell attachment via histo blood-group antigens (HBGAs) in a strain-dependent manner but how these glycan-interactions actually relate to cell entry remains unclear. Here, hydrogen/deuterium exchange mass spectrometry (HDX-MS) is used to investigate glycan-induced protein dynamics in P dimers of different strains, which exhibit high structural similarity but different prevalence in humans. While the almost identical strains GII.4 Saga and GII.4 MI001 share glycan-induced dynamics, the dynamics differ in the emerging GII.17 Kawasaki 308 and rare GII.10 Vietnam 026 strain. The structural aspects of glycan binding to fully deamidated GII.4 P dimers have been investigated before. However, considering the high specificity and half-life of N373D under physiological conditions, large fractions of partially deamidated virions with potentially altered dynamics in their P domains are likely to occur. Therefore, we also examined glycan binding to partially deamidated GII.4 Saga and GII.4 MI001 P dimers. Such mixed species exhibit increased exposure to solvent in the P dimer upon glycan binding as opposed to pure wildtype. Furthermore, deamidated P dimers display increased flexibility and a monomeric subpopulation. Our results indicate that glycan binding induces strain-dependent structural dynamics, which are further altered by N373 deamidation, and hence hint at a complex role of deamidation in modulating glycan-mediated cell attachment in GII.4 strains.

The murine norovirus (MNV) capsid protein is the target for various neutralizing antibodies binding to distal tips of its protruding (P)-domain. The bile acid glycochenodeoxycholic acid (GCDCA), an important co-factor for murine norovirus (MNV) infection, has recently been shown to induce conformational changes in surface-loops and a contraction of the virion. Here, we employ protein NMR experiments using stable isotope labeled MNV P-domains to shed light on underlying molecular mechanisms. We observe two separate sets of NMR resonance signals for P-domain monomers and dimers, permitting analysis of the corresponding exchange kinetics. Unlike human norovirus GII.4 P-dimers, which exhibit a half-life in the range of several days, MNV P-dimers are very short lived with a half-life of about 17 s. Addition of GCDCA shifts the equilibrium towards the dimeric form by tightly binding to the P-dimers. In MNV virions GCDCA-mediated stabilization of the dimeric arrangement of P-domains generates a more ordered state, which in turn may entropically assist capsid contraction. Numerous long-range chemical shift perturbations (CSPs) upon addition of GCDCA reflect allosteric conformational changes as a feature accompanying dimer stabilization. In particular, CSPs indicate rearrangement of the E’F’ loop, a target for various neutralizing antibodies. Indeed, treating MNV virions with GCDCA prior to neutralizing antibody exposure abolishes neutralization. These findings advance our understanding of GCDCA-induced structural changes of MNV capsids and experimentally support an intriguing viral immune escape mechanism relying on GCDCA-triggered conformational changes of the P-dimer. Significance Statement This study sheds light on the role of glycochenodeoxycholic acid (GCDCA) in promoting murine norovirus (MNV) infection and immune escape. Binding of GCDCA to the dimeric P-domain has been well characterized by crystallography and cryo EM studies, showing that upon GCDCA binding, a 90° rotation of the P-domain occurs, which results in its collapse onto the underlying shell of the virus. Our NMR experiments now reveal P-dimer stability as a new dimension of plasticity of MNV capsids and suggest that capsid contraction is entropically assisted. Conformational changes as a feature of P-dimer stabilization eliminate recognition by neutralizing antibodies, no longer being able to prevent infection. These findings highlight key differences between human and MNV capsid structures, promote our understanding of MNV infection on a molecular level, and reveal a novel immune escape mechanism.

Noroviruses cause immense sporadic gastroenteritis outbreaks worldwide. Emerging genotypes, which are divided based on the sequence of the major capsid protein VP1, further enhance this public threat. Self-assembling properties of the human norovirus major capsid protein VP1 are crucial for using virus-like particles (VLPs) for vaccine development. However, there is no vaccine available yet. Here, VLPs from different variants produced in insect cells were characterized in detail using a set of biophysical and structural tools. We used native mass spectrometry, gas-phase electrophoretic mobility molecular analysis, and proteomics to get clear insights into particle size, structure, and composition, as well as stability. Generally, noroviruses have been known to form mainly T = 3 particles. Importantly, we identified a major truncation in the capsid proteins as a likely cause for the formation of T = 1 particles. For vaccine development, particle production needs to be a reproducible, reliable process. Understanding the underlying processes in capsid size variation will help to produce particles of a defined capsid size presenting antigens consistent with intact virions. Next to vaccine production itself, this would be immensely beneficial for bio-/nano-technological approaches using viral particles as carriers or triggers for immunological reactions.

Noroviruses are the major cause of gastroenteritis and re-emerge worldwide every year, with GII.4 currently being the most frequent human genotype. The norovirus capsid protein VP1 is essential for host immune response. The P domain mediates cell attachment via histo blood-group antigens (HBGAs) in a strain-dependent manner but how these glycan-interactions actually relate to cell entry remains unclear. Here, hydrogen/deuterium exchange mass spectrometry (HDX-MS) is used to investigate glycan-induced protein dynamics in P dimers of different strains, which exhibit high structural similarity but different prevalence in humans. While the almost identical strains GII.4 Saga and GII.4 MI001 share glycan-induced dynamics, the dynamics differ in the emerging GII.17 Kawasaki 308 and rare GII.10 Vietnam 026 strain. We also further examine structural effects of N373 deamidation upon glycan binding in partially deamidated GII.4 P dimers, which are likely present during infection. Such mixed species exhibit increased exposure to solvent in the P dimer upon glycan binding as opposed to pure wildtype. Furthermore, deamidated P dimers display increased flexibility and a monomeric population. Our results indicate that glycan binding induces strain-dependent structural dynamics, which are further altered by N373 deamidation, and hence hint at a role of deamidation in modulating cell attachment and entry in GII.4 strains.

Attachment of human noroviruses to histo blood group antigens (HBGAs) is essential for infection, but how this binding event promotes the infection of host cells is unknown. Here, we employ protein NMR experiments supported by mass spectrometry and crystallography to study HBGA binding to the P-domain of a prevalent virus strain (GII.4). We report a highly selective transformation of asparagine 373, located in an antigenic loop adjoining the HBGA binding site, into an iso-aspartate residue. This spontaneous post-translational modification (PTM) proceeds with an estimated half-life of a few days at physiological temperatures, independent of the presence of HBGAs but dramatically affecting HBGA recognition. Sequence conservation and the surface-exposed position of this PTM suggest an important role in infection and immune recognition for many norovirus strains.