Structural and functional characterization of IgG- and non-IgG-based T-cell-engaging bispecific antibodies.

Bispecific T-cell-engaging antibodies are a growing class of therapeutics with numerous molecules being tested in clinical trials and, currently, seven of them have received market approval. They are structurally complex and function as adaptors to redirect the cytotoxicity of T cells to kill tumor cells. T-cell-engaging bispecific antibodies can be generally divided into two categories: IgG/IgG-like and non-IgG-like formats. Different formats may have different intrinsic potencies and physiochemical properties, and comprehensive studies are needed to gain a better understanding of how the differences in formats impact on structural and functional characteristics. In this study, we designed and generated bispecific T-cell-engaging antibodies with IgG-like (DVD-Ig) and non-IgG (BiTE) formats. Both target the same pair of antigens (EGFR and CD3) to minimize the possible influence of targets on functional characterization. We performed a side-by-side comparison to assess differences in the physiochemical and biological properties of these two bispecific T-cell-engaging antibodies using a variety of breast and ovarian cancer cell-based functional assays to delineate the structural-functional relationships and anti-tumor activities/potency. We found that the Fc portion of T-cell-engaging bispecific antibodies can significantly impact antigen binding activity, potency, and stability in addition to eliciting different mechanisms of action that contribute the killing of cancer cells.

Croquemort elicits activation of the immune deficiency pathway in ticks.

The immune deficiency (IMD) pathway directs host defense in arthropods upon bacterial infection. In Pancrustacea, peptidoglycan recognition proteins sense microbial moieties and initiate nuclear factor-κB-driven immune responses. Proteins that elicit the IMD pathway in non-insect arthropods remain elusive. Here, we show that an Ixodes scapularis homolog of croquemort (Crq), a CD36-like protein, promotes activation of the tick IMD pathway. Crq exhibits plasma membrane localization and binds the lipid agonist 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoglycerol. Crq regulates the IMD and jun N-terminal kinase signaling cascades and limits the acquisition of the Lyme disease spirochete B. burgdorferi. Additionally, nymphs silenced for crq display impaired feeding and delayed molting to adulthood due to a deficiency in ecdysteroid synthesis. Collectively, we establish a distinct mechanism for arthropod immunity outside of insects and crustaceans.

Dome1-JAK-STAT signaling between parasite and host integrates vector immunity and development.

Ancestral signaling pathways serve critical roles in metazoan development, physiology, and immunity. We report an evolutionary interspecies communication pathway involving a central Ixodes scapularis tick receptor termed Dome1, which acquired a mammalian cytokine receptor motif exhibiting high affinity for interferon-gamma (IFN-γ). Host-derived IFN-γ facilitates Dome1-mediated activation of the Ixodes JAK-STAT pathway. This accelerates tick blood meal acquisition and development while upregulating antimicrobial components. The Dome1-JAK-STAT pathway, which exists in most Ixodid tick genomes, regulates the regeneration and proliferation of gut cells-including stem cells-and dictates metamorphosis through the Hedgehog and Notch-Delta networks, ultimately affecting Ixodes vectorial competence. We highlight the evolutionary dependence of I. scapularis on mammalian hosts through cross-species signaling mechanisms that dually influence arthropod immunity and development.

Controlled Proteolysis of an Essential Virulence Determinant Dictates Infectivity of Lyme Disease Pathogens.

The Borrelia burgdorferi BB0323 protein undergoes a complex yet poorly defined proteolytic maturation event that generates N-terminal and C-terminal proteins with essential functions in cell growth and infection. Here, we report that a borrelial protease, B. burgdorferi high temperature requirement A protease (BbHtrA), cleaves BB0323 between asparagine (N) and leucine (L) at positions 236 and 237, while the replacement of these residues with alanine in the mutant protein prevents its cleavage, despite preserving its normal secondary structure. The N-terminal BB0323 protein binds BbHtrA, but its cleavage site mutant displays deficiency in such interaction. An isogenic borrelial mutant with NL-to-AA substitution in BB0323 (referred to as Bbbb0323NL) maintains normal growth yet is impaired for infection of mice or transmission from infected ticks. Notably, the BB0323 protein is still processed in Bbbb0323NL, albeit with lower levels of mature N-terminal BB0323 protein and multiple aberrantly processed polypeptides, which could result from nonspecific cleavages at other asparagine and leucine residues in the protein. The lack of infectivity of Bbbb0323NL is likely due to the impaired abundance or stoichiometry of a protein complex involving BB0238, another spirochete protein. Together, these studies highlight that a precise proteolytic event and a particular protein-protein interaction, involving multiple borrelial virulence determinants, are mutually inclusive and interconnected, playing essential roles in the infectivity of Lyme disease pathogens.

Tick gut barriers impacting tick-microbe interactions and pathogen persistence.

Ticks are regarded as one of the most ancient, unique, and highly evolved ectoparasites. They can parasitize diverse vertebrates and transmit a number of widespread infections. Once acquired from infected hosts, many tick-borne pathogens, like Borrelia burgdorferi, are confined within the tick gut lumen and are surrounded by discrete gut barriers. Such barriers include the peritrophic membrane (PM) and the dityrosine network (DTN), which are in close contact with resident microbiota and invading pathogens, influencing their survival within the vector. Herein, we review our current state of knowledge about tick-microbe interactions involving the PM and DTN structures. As a model, we will focus on Ixodes ticks, their microbiome, and the pathogen of Lyme disease. We will address the most salient findings on the structural and physiological roles of these Ixodes gut barriers on microbial interactions, with a comparison to analogous functions in other model vectors, such as mosquitoes. We will distill how this information could be leveraged towards a better understanding of the basic mechanisms of gut biology and tick-microbial interactions, which could contribute to potential therapeutic strategies in response to ticks and tick-borne infections.

A novel tick protein supports integrity of gut peritrophic matrix impacting existence of gut microbiome and Lyme disease pathogens.

The peritrophic matrix (PM) is an acellular membrane that covers the gut epithelium in arthropods and physically separates it from the lumen. The structure is thought to play an important role in tick biology. The PM is also known to impact the persistence of tick-borne pathogens like Borrelia burgdorferi, although limited information is available about its molecular constituents or their biological significance. Herein, we characterise a novel PM-associated gut protein in Ixodes scapularis ticks, annotated as Peritrophic Membrane Chitin Binding Protein (PM_CBP), for its role in the integrity and function of the matrix. The PM_CBP displays homology to the chitin deacetylase metalloenzyme, shows upregulation during tick feeding, and is localized at the luminal surface of the gut epithelium. The structural integrity of the PM was impaired both by the knock down of PM_CBP expression via RNA interference and by treatment with anti-PM_CBP antibodies, as revealed by its electron microscopic appearance. Additionally, the duration of tick engorgement on mice and the passage of experimentally-inoculated fluorescent dextran molecules across the PM are affected by the knock down of PM_CBP expression. The transfer of anti-PM_CBP antibodies into the tick gut impacted the overall composition of the resident microbiome, and also influenced B. burgdorferi acquisition in ticks and its transmission to mice. Taken together, these data highlight the biological significance of the Ixodes PM and suggest that the targeting of its molecular constituents may contribute to the development of novel interventions against tick-borne infections.

Intrinsic Differences in Backbone Dynamics between Wild Type and DNA-Contact Mutants of the p53 DNA Binding Domain Revealed by Nuclear Magnetic Resonance Spectroscopy.

Mutations in p53's DNA binding domain (p53DBD) are associated with 50% of all cancers, making it an essential system to investigate and understand the genesis and progression of cancer. In this work, we studied the changes in the structure and dynamics of wild type p53DBD in comparison with two of its "hot-spot" DNA-contact mutants, R248Q and R273H, by analysis of backbone amide chemical shift perturbations and 15N spin relaxation measurements. The results of amide chemical shift changes indicated significantly more perturbations in the R273H mutant than in wild type and R248Q p53DBD. Analysis of 15N spin relaxation rates and the resulting nuclear magnetic resonance order parameters suggests that for most parts, the R248Q mutant exhibits limited conformational flexibility and is similar to the wild type protein. In contrast, R273H showed significant backbone dynamics extending up to its ß-sandwich scaffold in addition to motions along the DNA binding interface. Furthermore, comparison of rotational correlation times between the mutants suggests that the R273H mutant, with a higher correlation time, forms an enlarged structural fold in comparison to the R248Q mutant and wild type p53DBD. Finally, we identify three regions in these proteins that show conformational flexibility to varying degrees, which suggests that the R273H mutant, in addition to being a DNA-contact mutation, exhibits properties of a conformational mutant.