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Although coagulation factors play a role in host defense for \"living fossils\" such as horseshoe crabs, the role of the coagulation system in immunity in higher organisms remains unclear. We modeled the interface of human species C adenovirus (HAdv) interaction with coagulation factor X (FX) and introduced a mutation that abrogated formation of the HAdv-FX complex. In vivo genome-wide transcriptional profiling revealed that FX-binding-ablated virus failed to activate a distinct network of nuclear factor kB-dependent early-response genes that are activated by HAdv-FX complex downstream of TLR4/MyD88/TRIF/TRAF6 signaling. Our study implicates host factor \"decoration\" of the virus as a mechanism to trigger an innate immune sensor that responds to a misplacement of coagulation FX from the blood into intracellular macrophage compartments upon virus entry into the cell.

NRC publication: Yes

Abstract Background Production of chimeric antigen receptor T cell (CAR-T) therapies depends on antibody reagents to label, isolate, and expand T cell products. We sought to create antibody tools specific for the variable domain of heavy-chain only antibodies (VHHs), also known as nanobodies, used in some CARs. Methods We immunized a mouse with VHH and selected two murine monoclonal antibodies (mAbs) that bind to distinct epitopes in conserved framework regions of llama-derived VHHs, and not to human VH domains. Anti-VHH mAbs were characterized by enzyme-linked immunosorbent assay, surface plasmon resonance, and hydrogen-deuterium exchange mass spectrometry; were then tested for cell/tissue labeling and for modulating cellular activity in VHH-CAR-T cells. Results We produced a high-quality dual-clonal anti-VHH antibody product and confirmed reactivity to over 98% of VHH proteins regardless of their antigenic specificity, with no reactivity to human or mouse IgG and reduced reactivity to conventional llama or alpaca IgG. Anti-VHH binding did not disrupt VHH/antigen interaction, and thus was appropriate for secondary labeling to assess cellular or tissue reactivity of VHH molecules. Despite not interfering with antigen binding, anti-VHH antibodies (Abs) potently blocked VHH-CAR-T activation and cytolytic killing of target cells. When immobilized, anti-VHH Abs induced strong activation and expansion of VHH CAR-T cells; with 730-fold mean expansion, >94% CAR purity, and retained CD8/CD4 heterogeneity. Functionally, anti-VHH antibody-expanded CAR-T cells maintained strong antigen-specific activity without functional exhaustion. Conclusions Overall, these data identify useful anti-VHH mAbs that can be applied to better understand and manipulate VHH-based CAR-T cells or other VHH-based immunotherapies. Statement of Significance Antibody tools are foundational to discovery and development of engineered cell therapies; here we report broadly reactive anti-variable domain of heavy-chain only antibodies that can be used to quantify, isolate, inhibit, or activate T cells expressing llama nanobody chimeric antigen receptors.

Fluorescence spectroscopy of tryptophan residues is a valuable tool for dissecting the structure, function and dynamic behaviour of proteins. An important step in method development is the use of tryptophan analogues with unique photophysical properties that extend the utility of intrinsic protein fluorescence. Two difficulties limit the use of these analogues. Their structure, while similar to that of tryptophan, can perturb native protein structure resulting in misfolding or inactivity. There are also problems from an inability of tryptophanyl-tRNA synthetase (TrpRS) to use these analogues as a substrate in the tRNA charging reaction. The tryptophan residue in rat parvalbumin F102W is buried in the highly hydrophobic and conformationally restrained environment of the protein's core. Differences in hydrophobicity, size and hydrogen bonding capability of tryptophan analogues were expected to affect both the structure and stability of this protein once incorporated at position 102. All analogues were successfully incorporated. Incorporation of 5-hydroxytryptophan proved difficult and required an optimization of incubation temperature during induction. 7-azatryptophan and 5-hydroxytryptophan destabilized the protein while 4-fluorotryptophan, 5-fluorotryptophan and 6-fluorotryptophan stabilized the protein. Single tryptophan mutants of Bacillus stearothermophilis TrpRS showed large structural changes when forming the 4-fluorotryptophan-AMP intermediate. These changes were limited to the formation of the first intermediate. This is rationalized as a large global change occurring across both subunits that could affect substrate binding. H. sapien tryptophanyl-tRNA synthetase was expressed and found to be active when using tryptophan analogues as substrates. Differences in fluorescence properties of the intermediate suggest a unique environment at the active site.

Production of chimeric antigen receptor T cell (CAR-T) therapies is dependent on the use of antibody reagents to label, isolate, and/or expand T cell products. We sought to create antibody-based tools that directly target the variable domain of heavy-chain only antibodies (VHH or nanobody) used in some CAR molecules. Two murine antibodies were identified which bind to distinct epitopes in the conserved framework regions of llama-derived VHHs, and not to human VH domains. We produced a high-quality dual-clonal anti-VHH antibody product which reacts with over 98% of VHH proteins, regardless of their antigenic specificity. Anti-VHH binding did not disrupt VHH/antigen interaction, and thus could be used for secondary labeling to assess cellular or tissue reactivity of VHH molecules. Despite not interfering with antigen binding, anti-VHH antibodies potently inhibited VHH-CAR function, blocking CAR-T activation and cytolytic killing of target cells. When immobilized, anti-VHH antibodies could also be applied for activation and expansion of VHH CAR-T cells, inducing 730-fold mean expansion, >94% CAR purity, with retained CD8/CD4 heterogeneity. Functionally, anti-VHH antibody-expanded CAR-T cells maintained strong antigen specific activity without functional exhaustion. Overall, these data identify a useful new tool for understanding and manipulating VHH-based CAR-T cells. This work was funded by the National Research Council Canada Disruptive Technology Solutions Cell and Gene Therapy challenge program, and BioCanRx The anti-VHH antibodies reported here are the subject of a provisional patent application by the National Research Council of Canada