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Objective To determine the X-ray structure and biophysical properties of a Camelid V H H isolated from a naïve phage display library. Results Single domain antibodies (V H H) derived from the unique immune system of the Camelidae family have gained traction as useful tools for biotechnology as well as a source of potentially novel therapeutics. Here we report the structure and biophysical characterization of a V H H originally isolated from a naïve camelid phage display library. V H H R419 has a melting temperate of 66 °C and was found to be a monomer in solution. The protein crystallized in space group P 6 5 22 and the structure was solved by molecular replacement to a resolution of 1.5 Å. The structure revealed a flat paratope with CDR loops that could be classified into existing canonical loop structures. A combination of high expression yield, stability and rapid crystallization might make R419 into a candidate scaffold for CDR grafting and homology modeling.

Aberrant glycosylation and the overexpression of certain carbohydrate moieties is a consistent feature of cancers, and tumor-associated oligosaccharides are actively investigated as targets for immunotherapy. One of the most common aberrations in glycosylation patterns is the presentation of a single O-linked N-acetylgalactosamine on a threonine or serine residue known as the \"Tn antigen.\" Whereas the ubiquitous nature of Tn antigens on cancers has made them a natural focus of vaccine research, such carbohydrate moieties are not always tumor-specific and have been observed on embryonic and nonmalignant adult tissue. Here we report the structural basis of binding of a complex of a monoclonal antibody (237mAb) with a truly tumor-specific glycopeptide containing the Tn antigen. In contrast to glycopeptide-specific antibodies in complex with simple peptides, 237mAb does not recognize a conformational epitope induced in the peptide by sugar substitution. Instead, 237mAb uses a pocket coded by germ-line genes to completely envelope the carbohydrate moiety itself while interacting with the peptide moiety in a shallow groove. Thus, 237mAb achieves its striking tumor specificity, with no observed physiological cross-reactivity to the unglycosylated peptide or the free glycan, by a combination of multiple weak but specific interactions to both the peptide and to the glycan portions of the antigen.

Escherichia coli infections, a leading cause of septic shock, remain a major threat to human health because of the fatal action to endotoxin (LPS). Therapeutic attempts to neutralize endotoxin currently focus on inhibiting the interaction of the toxic component lipid A with myeloid differentiating factor 2, which forms a trimeric complex together with Toll-like receptor 4 to induce immune cell activation. The 1.73-Å resolution structure of the unique endotoxin-neutralizing protective antibody WN1 222-5 in complex with the core region shows that it recognizes LPS of all E. coli serovars in a manner similar to Toll-like receptor 4, revealing that protection can be achieved by targeting the inner core of LPS and that recognition of lipid A is not required. Such interference with Toll-like receptor complex formation opens new paths for antibody sepsis therapy independent of lipid A antagonists.

Background/Objectives: Pancreatic ductal adenocarcinoma (PDAC) is diagnosed at a late stage with distant metastasis in an overwhelming 50% of cases, and the prognosis is poor. Treating this extremely aggressive disease with standard-of-care therapies has led to modest benefits in overall survival, mainly due to a lack of targeted early treatment modalities, as early detection has not yet been possible. Mucin-16 (MUC16) is a glycoprotein overexpressed in more than 60% of patients with PDAC and is a tumor-specific biomarker. Methods: In this study, a magnetic resonance imaging (MRI) probe to facilitate the detection of early and late lesions of PDAC is developed by conjugating a MUC16-targeted humanized antibody (huAR9.6) with gadolinium. Results: In preclinical mouse models, this MUC16-targeted MRI probe demonstrates effective contrast enhancement in early lesions of PDAC in the subcutaneous setting and allows for the detection of late-stage pancreatic cancer tumors in an orthotopic model. The probe did not induce any toxicity in vital organs at the administered doses. Conclusions: This study establishes that synthesizing a MUC16-targeted MRI probe is feasible and allows for the better high-resolution contrast enhancement of MUC16+ PDAC lesions to facilitate detection and possibly better treatment strategies.

We used the protein kinase A (PKA) specific activator Sp‐8‐Br‐cAMPS and type I inhibitor Rp‐8‐Br‐cAMPS alone and in combination to define the role of PKA in the non‐self responses of larval Galleria mellonella haemocytes in vitro and in vivo. Active PKA depressed haemocyte responses whereas PKA inhibition enhanced activities, including bacterial phagocytosis, the number of haemocytes with adherent bacteria, bacterial‐induced haemocytic protein release and haemocyte adhesion to slides in vitro, as well as in vivo bacterial removal from the haemolymph. Non‐attached haemocytes had more PKA activity than attached haemocytes; therefore, active PKA limited haemocyte response to foreign materials. We found that (i) PKA inhibitor alone induced non‐self responses, including haemocyte protein discharge and lowered haemocyte counts in vivo, and induced nodulation; (ii) the enzyme activator produced effects opposite to those of the inhibitor; and (iii) together, the modulators offset each others’ effects and influenced haemocyte lysate PKA activity. These findings establish PKA as a mediator of haemocytic non‐self responses.

To determine the X-ray structure and biophysical properties of a Camelid V H isolated from a naïve phage display library. Single domain antibodies (V H) derived from the unique immune system of the Camelidae family have gained traction as useful tools for biotechnology as well as a source of potentially novel therapeutics. Here we report the structure and biophysical characterization of a V H originally isolated from a naïve camelid phage display library. V H R419 has a melting temperate of 66 °C and was found to be a monomer in solution. The protein crystallized in space group P6 22 and the structure was solved by molecular replacement to a resolution of 1.5 Å. The structure revealed a flat paratope with CDR loops that could be classified into existing canonical loop structures. A combination of high expression yield, stability and rapid crystallization might make R419 into a candidate scaffold for CDR grafting and homology modeling.

Petrolisthes cinctipes (Randall 1840) and P. manimaculis Glassell 1945 are two conspecific anomuran crabs that live in the upper intertidal zone along the California coast. Morphometric measurements and vitellogenin (Vg) levels were recorded to understand the reproductive life histories of the two species. Using polyacrylamide gel electrophoresis (SDS-PAGE), vitellin (Vn) of both conspecifics consisted of three major subunits (MW 93 ± 2 kDa, 82 ± 2 kDa, and 65.7 ± 1.4 kDa) and two minor subunits (111 ± 2.3 kDa and 40 ± 1.3 kDa). Using fast-protein liquid chromatography (FPLC), the native molecular mass of P. cinctipes Vn was found to be 301 ± 14 kDa, and 324 ± 11 kDa for P. manimaculis. A Western blot was used to reveal that two of the major Petrolisthes Vn subunits, 93 kDa and 65.7 kDa, successfully bound with Homarus anti-Vn antisera. These enzyme-linked immunosorbant assays (ELISA) were capable of measuring Vn and hemolymph Vg of both species of Petrolisthes, with an effective range from 9–3,000 ng/ml. Mean Vg levels of P. cinctipes were elevated from August (300 ± 105 μg/ml) through February (120 ± 30 μg/ml), and the lowest levels occurred between March (30 ± 6 μg/ml) and July (10 ± 3 μg/ml). The gonadosomatic index (GSI) of P. cinctipes was elevated from September through January, decreasing from February and quiescent during late spring and summer months. Berried females were found between December and April. From these data on the GSI, the occurrence of berried females, and Vg levels of P. cinctipes, it appears that ordinary vitellogenic events increase in late August, with ovoposition beginning in December and running through February. Embryos were carried through the end of April. There was a reduced reproductive activity during the warmer summer months.

Escherichia coli infections, a leading cause of septic shock, remain a major threat to human health because of the fatal action to endotoxin (LPS). Therapeutic attempts to neutralize endotoxin currently focus on inhibiting the interaction of the toxic component lipid A with myeloid differentiating factor 2, which forms a trimeric complex together with Toll-like receptor 4 to induce immune cell activation. The 1.73-Å resolution structure of the unique endotoxin-neutralizing protective antibody WN1 222-5 in complex with the core region shows that it recognizes LPS of all E. coli serovars in a manner similar to Toll-like receptor 4, revealing that protection can be achieved by targeting the inner core of LPS and that recognition of lipid A is not required. Such interference with Toll-like receptor complex formation opens new paths for antibody sepsis therapy independent of lipid A antagonists.

We used the protein kinase A (PKA) specific activator Sp-8-Br-cAMPS and type I inhibitor Rp-8-Br-cAMPS alone and in combination to define the role of PKA in the non-self responses of larval Galleria mellonella haemocytes in vitro and in vivo. Active PKA depressed haemocyte responses whereas PKA inhibition enhanced activities, including bacterial phagocytosis, the number of haemocytes with adherent bacteria, bacterial-induced haemocytic protein release and haemocyte adhesion to slides in vitro, as well as in vivo bacterial removal from the haemolymph. Non-attached haemocytes had more PKA activity than attached haemocytes; therefore, active PKA limited haemocyte response to foreign materials. We found that (i) PKA inhibitor alone induced non-self responses, including haemocyte protein discharge and lowered haemocyte counts in vivo, and induced nodulation; (ii) the enzyme activator produced effects opposite to those of the inhibitor; and (iii) together, the modulators offset each others' effects and influenced haemocyte lysate PKA activity. These findings establish PKA as a mediator of haemocytic non-self responses.