Pretargeted alpha therapy in MUC16-positive high-grade serous ovarian cancer.

Background: Peritoneal metastasis with micrometastatic cell clusters is a common feature of advanced ovarian cancer. Targeted alpha therapy (TAT) is an attractive approach for treating micrometastatic diseases as alpha particles release enormous amounts of energy within a short distance. A pretargeting approach - leveraging the inverse-electron-demand Diels-Alder reaction between tetrazines (Tz) and trans-cyclooctene (TCO) - can minimize off-target toxicity related to TAT, often associated with full-length antibodies.

Interrogating the Theranostic Capacity of a MUC16-Targeted Antibody for Ovarian Cancer.

Aberrantly expressed glycans on mucins such as mucin-16 (MUC16) are implicated in the biology that promotes ovarian cancer (OC) malignancy. Here, we investigated the theranostic potential of a humanized antibody, huAR9.6, targeting fully glycosylated and hypoglycosylated MUC16 isoforms.

Structural Basis for Multivalent MUC16 Recognition and Robust Anti-Pancreatic Cancer Activity of Humanized Antibody AR9.6.

Mucin-16 (MUC16) is a target for antibody-mediated immunotherapy in pancreatic ductal adenocarcinoma (PDAC) among other malignancies. The MUC16-specific monoclonal antibody AR9.6 has shown promise for PDAC immunotherapy and imaging.

2.1 Å crystal structure of the Mycobacterium tuberculosis serine hydrolase, Hip1, in its anhydro-form (Anhydrohip1).

The 2.6 Å crystal structure of the apo form of Hip1 (hydrolase important for pathogenesis) has been previously reported. However, very little is known about the active site architecture of this M.

Preclinical Evaluation of a Humanized, Near-Infrared Fluorescent Antibody for Fluorescence-Guided Surgery of MUC16-Expressing Pancreatic Cancer.

Surgery remains the only potentially curative treatment option for pancreatic cancer, but resections are made more difficult by infiltrative disease, proximity of critical vasculature, peritumoral inflammation, and dense stroma. Surgeons are limited to tactile and visual cues to differentiate cancerous tissue from normal tissue. Furthermore, translating preoperative images to the intraoperative setting poses additional challenges for tumor detection, and can result in undetected and unresected lesions.

X-ray Crystal Structure Analysis of VHH-Protein Antigen Complexes.

VHHs are antigen-binding domains cloned from heavy-chain antibodies found in camelids. These proteins have generated considerable interest in a variety of applications as research reagents, crystallization chaperones, and therapeutics. The evolutionary adaptations of VHHs have resulted in biophysical properties and antigen-binding modalities which are distinct from those of conventional antibodies.

Crystal structure of a human MUC16 SEA domain reveals insight into the nature of the CA125 tumor marker.

MUC16 is a membrane bound glycoprotein involved in the progression and metastasis of pancreatic and ovarian cancer. The protein is shed into the serum and the resulting cancer antigen 125 (CA125) can be detected by immunoassays. The CA125 epitope is used for monitoring ovarian cancer treatment progression, and has emerged as a potential target for antibody mediated immunotherapy.

ImmunoPET of Ovarian and Pancreatic Cancer with AR9.6, a Novel MUC16-Targeted Therapeutic Antibody.

Purpose: Advances in our understanding of the contribution of aberrant glycosylation to the pro-oncogenic signaling and metastasis of tumor cells have reinvigorated the development of mucin-targeted therapies. Here, we validate the tumor-targeting ability of a novel monoclonal antibody (mAb), AR9.6, that binds MUC16 and abrogates downstream oncogenic signaling to confer a therapeutic response.

Expression and Purification of Human Mitochondrial Intramembrane Protease PARL.

Rhomboid proteases are a ubiquitous superfamily of serine intramembrane peptidases that play a role in a wide variety of cellular processes. The mammalian mitochondrial rhomboid protease, Presenilin-Associated Rhomboid Like (PARL), is a critical regulator of mitochondrial homeostasis through the cleavage of its substrates, which have roles in mitochondrial quality control and apoptosis. However, neither structural nor functional information for this important protease is available, because the expression of eukaryotic membrane proteins to sufficient levels in an active form often represents a major bottleneck for in vitro studies.

Role of a noncanonical disulfide bond in the stability, affinity, and flexibility of a VHH specific for the Listeria virulence factor InlB.

A distinguishing feature of camel (Camelus dromedarius) VHH domains are noncanonical disulfide bonds between CDR1 and CDR3. The disulfide bond may provide an evolutionary advantage, as one of the cysteines in the bond is germline encoded. It has been hypothesized that this additional disulfide bond may play a role in binding affinity by reducing the entropic penalty associated with immobilization of a long CDR3 loop upon antigen binding.

Role of glycosylation on the ensemble of conformations in the MUC1 immunodominant epitope.

MUC1 is a membrane glycoprotein, which in adenocarninomas is overexpressed and exhibits truncated O-glycosylation. Overexpression and altered glycosylation make MUC1 into a candidate for immunotherapy. Monoclonal antibodies directed against MUC1 frequently bind an immunodominant epitope that contains a single site for O-glycosylation.

Structure of a VH isolated from a naïve phage display library.

Objective: To determine the X-ray structure and biophysical properties of a Camelid VH isolated from a naïve phage display library.

Results: Single domain antibodies (VH) 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 VH originally isolated from a naïve camelid phage display library.

Subtle Changes in the Combining Site of the Chlamydiaceae-Specific mAb S25-23 Increase the Antibody-Carbohydrate Binding Affinity by an Order of Magnitude.

Murine antibodies S25-23, S25-26, and S25-5 derive from a common germ-line origin, and all bind the Chlamydiaceae family-specific epitope αKdo(2→8)αKdo(2→4)αKdo (where Kdo is 3-deoxy-α-d- manno-oct-2-ulosonic acid) with high affinity and specificity. These antibodies recognize the entire trisaccharide antigen in a linkage-dependent manner via a groove composed largely of germ-line residues. Despite sharing identical heavy and light chain genes, S25-23 binds the family-specific epitope with nanomolar affinity, which is an order of magnitude higher than that of S25-26, while S25-5 displays an affinity between those of S25-23 and S25-26.

Immunological Functions and Evolutionary Emergence of Heavy-Chain Antibodies.

Homodimeric antibodies devoid of light chains have evolved multiple times through convergent evolution, yet their specific immunological functions remain poorly understood. We survey the molecular and structural features of these antibodies, their immunological functions in host defense, and reflect on the long-standing question of the evolutionary forces driving their emergence.

Structural basis of VH-mediated neutralization of the food-borne pathogen .

causes listeriosis, a potentially fatal food-borne disease. The condition is especially harmful to pregnant women. outbreaks can originate from diverse foods, highlighting the need for novel strategies to improve food safety.

Epitope Mapping of Antibody-Antigen Interactions with X-Ray Crystallography.

Therapeutic antibodies constitute one of the fastest areas of growth in the field of biologic drugs. A molecular understanding of how antibodies interact with their target antigens is known as epitope mapping. The data provided by epitope mapping is extremely valuable in the process of antibody humanization, as well as in vaccine design.

Glycosylation of MUC1 influences the binding of a therapeutic antibody by altering the conformational equilibrium of the antigen.

In cancer cells, the glycoprotein Mucin 1 (MUC1) undergoes abnormal, truncated glycosylation. The truncated glycosylation exposes cryptic peptide epitopes that can be recognized by antibodies. Since these immunogenic regions are cancer specific, they represent ideal targets for therapeutic antibodies.

Molecular basis of antibody binding to mucin glycopeptides in lung cancer.

Glycopeptides bearing Tn epitopes are emerging targets for cancer diagnosis and immunotherapy. In this study, we analyzed membrane proteins containing O-glycosylated tandem repeat (TR) sequences in lung cancer patients of different types and stages, using gene microarray data in public domain. The expression of Tn and glycopeptide epitopes on the surface of lung cancer cell lines were studied by monoclonal IgG antibodies 14A, 16A, and B72.

Single-chain antibody-fragment M6P-1 possesses a mannose 6-phosphate monosaccharide-specific binding pocket that distinguishes N-glycan phosphorylation in a branch-specific manner†.

The acquisition of mannose 6-phosphate (Man6P) on N-linked glycans of lysosomal enzymes is a structural requirement for their transport from the Golgi apparatus to lysosomes mediated by the mannose 6-phosphate receptors, 300 kDa cation-independent mannose 6-phosphate receptor (MPR300) and 46 kDa cation-dependent mannose 6-phosphate receptor (MPR46). Here we report that the single-chain variable domain (scFv) M6P-1 is a unique antibody fragment with specificity for Man6P monosaccharide that, through an array-screening approach against a number of phosphorylated N-glycans, is shown to bind mono- and diphosphorylated Man6 and Man7 glycans that contain terminal αMan6P(1 → 2)αMan(1 → 3)αMan. In contrast to MPR300, scFv M6P-1 does not bind phosphodiesters, monophosphorylated Man8 or mono- or diphosphorylated Man9 structures.

Directed evolution of the Escherichia coli cAMP receptor protein at the cAMP pocket.

The Escherichia coli cAMP receptor protein (CRP) requires cAMP binding to undergo a conformational change for DNA binding and transcriptional regulation. Two CRP residues, Thr(127) and Ser(128), are known to play important roles in cAMP binding through hydrogen bonding and in the cAMP-induced conformational change, but the connection between the two is not completely clear. Here, we simultaneously randomized the codons for these two residues and selected CRP mutants displaying high CRP activity in a cAMP-producing E.

In situ proteolysis, crystallization and preliminary X-ray diffraction analysis of a VHH that binds listeria internalin B.

The variable region of camelid heavy-chain antibodies produces the smallest known antibody fragment with antigen-binding capability (a VHH). The VHH R303 binds internalin B (InlB), a virulence factor expressed by the pathogen Listeria monocytogenes. InlB is critical for initiation of Listeria infection, as it binds a receptor (c-Met) on epithelial cells, triggering the entry of bacteria into host cells.

The structure of lactoferrin-binding protein B from Neisseria meningitidis suggests roles in iron acquisition and neutralization of host defences.

Pathogens have evolved a range of mechanisms to acquire iron from the host during infection. Several Gram-negative pathogens including members of the genera Neisseria and Moraxella have evolved two-component systems that can extract iron from the host glycoproteins lactoferrin and transferrin. The homologous iron-transport systems consist of a membrane-bound transporter and an accessory lipoprotein.