Afucosylation of HLA-specific IgG1 as a potential predictor of antibody pathogenicity in kidney transplantation.

Antibody-mediated rejection (AMR) is the leading cause of graft failure. While donor-specific antibodies (DSAs) are associated with a higher risk of AMR, not all patients with DSAs develop rejection, suggesting that the characteristics of alloantibodies determining their pathogenicity remain undefined. Using human leukocyte antigen (HLA)-A2-specific antibodies as a model, we apply systems serology tools to investigate qualitative features of immunoglobulin G (IgG) alloantibodies including Fc-glycosylation patterns and FcγR-binding properties. Levels of afucosylated anti-A2 antibodies are elevated in seropositive patients, especially those with AMR, suggesting potential cytotoxicity via FcγRIII-mediated mechanisms. Afucosylation of both glycoengineered monoclonal and naturally glycovariant polyclonal serum IgG specific to HLA-A2 drives potentiated binding to, slower dissociation from, and enhanced signaling through FcγRIII, a receptor widely expressed on innate effector cells, and greater cytotoxicity against HLA-A2+ cells mediated by natural killer (NK) cells. Collectively, these results suggest that afucosylated DSA may be a biomarker of AMR and contribute to pathogenesis.

Glycosylation of Antigen-Specific Antibodies: Perspectives on Immunoglobulin G Glycosylation in Vaccination and Immunotherapy.

Exciting developments have been made in understanding antibody-mediated immunity, deepening understanding of antibody effector functions increasingly recognized as critical mechanisms of action beyond antigen recognition, and significantly broadening the evidence base for the importance of these effector mechanisms across diverse infectious and autoimmune diseases. Because these activities critically depend on the specific glycoforms present on a conserved site of the IgG Fc domain, relationships between the Fc glycosylation profiles of antigen-specific antibody pools and outcomes in infectious and autoimmune disease have begun to be defined, pointing to the key role of this posttranslational modification as a biomarker and mechanistic modifier of antibody-mediated immunity. Here we summarize studies evaluating the profiles and activities of antigen-specific antibodies elicited by infection and vaccination as well as within the context of allo- and autoimmunity, and consider current approaches to rational modification of Fc glycans in vivo.

Implementation of a three-tiered approach to identify and characterize anti-drug antibodies raised against HIV-specific broadly neutralizing antibodies.

The ability to detect, quantify, and interrogate the properties of immune responses raised against biological therapeutics is not only important to our understanding of these molecules, but also to their success in the clinic. A tiered assay approach to identify the presence, specificity, and titer of anti-drug antibody (ADA) responses has been adopted as a gold standard by industry leaders, the FDA, and the EMA. In order to support pre-clinical and clinical trials, these assays must be standardized, and their performance sufficiently characterized to ensure the accuracy and reproducibility of results under relevant testing conditions. Here we present implementation of electrochemiluminiscence assays that fit into the tiered paradigm of ADA testing for five HIV broadly neutralizing antibodies (3BNC117, 3BNC117-LS, 10-1074, PGT121, and PGDM1400) in compliance with Good Clinical Laboratory practices. Assay sensitivities and matrix effects were evaluated and used to inform the development of positivity cut points. Once cut points were established, assay precision, specificity, free-drug tolerance, and robustness were defined. In all cases, assay characteristics met or surpassed recommendations set forth by the FDA. To further evaluate the performance of these assays and the tiered approach, samples from non-human primates that had received a subset of the five therapeutics were evaluated. In sum, this study reports qualification of a set of ADA assays available to the scientific community as pre-clinical and clinical trials of broadly HIV-neutralizing antibodies proceed, and a framework that is easily adapted as new drug products are advanced in the clinic.

Biophysical and Functional Characterization of Rhesus Macaque IgG Subclasses.

Antibodies raised in Indian rhesus macaques [Macaca mulatta (MM)] in many preclinical vaccine studies are often evaluated in vitro for titer, antigen-recognition breadth, neutralization potency, and/or effector function, and in vivo for potential associations with protection. However, despite reliance on this key animal model in translation of promising candidate vaccines for evaluation in first in man studies, little is known about the properties of MM immunoglobulin G (IgG) subclasses and how they may compare to human IgG subclasses. Here, we evaluate the binding of MM IgG1, IgG2, IgG3, and IgG4 to human Fc gamma receptors (FcγR) and their ability to elicit the effector functions of human FcγR-bearing cells, and unlike in humans, find a notable absence of subclasses with dramatically silent Fc regions. Biophysical, in vitro, and in vivo characterization revealed MM IgG1 exhibited the greatest effector function activity followed by IgG2 and then IgG3/4. These findings in rhesus are in contrast with the canonical understanding that IgG1 and IgG3 dominate effector function in humans, indicating that subclass-switching profiles observed in rhesus studies may not strictly recapitulate those observed in human vaccine studies.

Increased dendritic spine density and tau expression are associated with individual differences in steroidal regulation of male sexual behavior.

Male sexual behavior (MSB) is modulated by gonadal steroids, yet this relationship is highly variable across species and between individuals. A significant percentage (~30%) of B6D2F1 hybrid male mice demonstrate MSB after long-term orchidectomy (herein after referred to as "maters"), providing an opportunity to examine the mechanisms that underlie individual differences in steroidal regulation of MSB. Use of gene expression arrays comparing maters and non-maters has provided a first pass look at the genetic underpinnings of steroid-independent MSB. Surprisingly, of the ~500 genes in the medial preoptic area (MPOA) that differed between maters and non-maters, no steroid hormone or receptor genes were differentially expressed between the two groups. Interestingly, best known for their association with Alzheimer's disease, amyloid precursor protein (APP) and the microtubule-associated protein tau (MAPT) were elevated in maters. Increased levels of their protein products (APP and tau) in their non-pathological states have been implicated in cell survival, neuroprotection, and supporting synaptic integrity. Here we tested transgenic mice that overexpress tau and found facilitated mounting and intromission behavior after long-term orchidectomy relative to littermate controls. In addition, levels of synaptophysin and spinophilin, proteins generally enriched in synapses and dendritic spines respectively, were elevated in the MPOA of maters. Dendritic morphology was also assessed in Golgi-impregnated brains of orchidectomized B6D2F1 males, and hybrid maters exhibited greater dendritic spine density in MPOA neurons. In sum, we show for the first time that retention of MSB in the absence of steroids is correlated with morphological differences in neurons.

Small-molecule quinolinol inhibitor identified provides protection against BoNT/A in mice.

Botulinum neurotoxins (BoNTs), etiological agents of the life threatening neuroparalytic disease botulism, are the most toxic substances currently known. The potential for the use as bioweapon makes the development of small-molecule inhibitor against these deadly toxins is a top priority. Currently, there are no approved pharmacological treatments for BoNT intoxication. Although an effective vaccine/immunotherapy is available for immuno-prophylaxis but this cannot reverse the effects of toxin inside neurons. A small-molecule pharmacological intervention, especially one that would be effective against the light chain protease, would be highly desirable. Similarity search was carried out from ChemBridge and NSC libraries to the hit (7-(phenyl(8-quinolinylamino)methyl)-8-quinolinol; NSC 84096) to mine its analogs. Several hits obtained were screened for in silico inhibition using AutoDock 4.1 and 19 new molecules selected based on binding energy and Ki. Among these, eleven quinolinol derivatives potently inhibited in vitro endopeptidase activity of botulinum neurotoxin type A light chain (rBoNT/A-LC) on synaptosomes isolated from rat brain which simulate the in vivo system. Five of these inhibitor molecules exhibited IC(50) values ranging from 3.0 nM to 10.0 µM. NSC 84087 is the most potent inhibitor reported so far, found to be a promising lead for therapeutic development, as it exhibits no toxicity, and is able to protect animals from pre and post challenge of botulinum neurotoxin type A (BoNT/A).