PD-L1 (Programmed Death Ligand 1) Regulates T-Cell Differentiation to Control Adaptive Venous Remodeling.

OBJECTIVE: Patients with end-stage renal disease depend on hemodialysis for survival. Although arteriovenous fistulae (AVF) are the preferred vascular access for hemodialysis, the primary success rate of AVF is only 30% to 50% within 6 months, showing an urgent need for improvement. PD-L1 (programmed death ligand 1) is a ligand that regulates T-cell activity. Since T cells have an important role during AVF maturation, we hypothesized that PD-L1 regulates T cells to control venous remodeling that occurs during AVF maturation. Approach and results: In the mouse aortocaval fistula model, anti-PD-L1 antibody (200 mg, 3×/wk intraperitoneal) was given to inhibit PD-L1 activity during AVF maturation. Inhibition of PD-L1 increased T-helper type 1 cells and T-helper type 2 cells but reduced regulatory T cells to increase M1-type macrophages and reduce M2-type macrophages; these changes were associated with reduced vascular wall thickening and reduced AVF patency. Inhibition of PD-L1 also inhibited smooth muscle cell proliferation and increased endothelial dysfunction. The effects of anti-PD-L1 antibody on adaptive venous remodeling were diminished in nude mice; however, they were restored after T-cell transfer into nude mice, indicating the effects of anti-PD-L1 antibody on venous remodeling were dependent on T cells. CONCLUSIONS: Regulation of PD-L1 activity may be a potential therapeutic target for clinical translation to improve AVF maturation.

A functional mammalian display screen identifies rare antibodies that stimulate NK cell-mediated cytotoxicity.

Therapies that boost the antitumor immune response have shown a great deal of success. Although most of these therapies have focused on enhancing T cell functions, there is a growing interest in developing therapies that can target other immune cell subsets. Like T cells, natural killer (NK) cells are cytotoxic effector cells that play a key role in the antitumor response. To advance the development of NK-based therapies, we developed a functional screen to rapidly identify antibodies that can activate NK cells. We displayed antibodies on a mammalian target cell line and probed their ability to stimulate NK cell-mediated cytotoxicity. From this screen, we identified five antibodies that bound with high affinity to NK cells and stimulated NK cell-mediated cytotoxicity and interferon-γ (IFN-γ) secretion. We demonstrate that these antibodies can be further developed into bispecific antibodies to redirect NK cell-mediated cytotoxicity toward CD20+ B cell lymphoma cells and HER2+ breast cancer cells. While antibodies to two of the receptors, CD16 and NCR1, have previously been targeted as bispecific antibodies to redirect NK cell-mediated cytotoxicity, we demonstrate that bispecific antibodies targeting NCR3 can also potently activate NK cells. These results show that this screen can be used to directly identify antibodies that can enhance antitumor immune responses.

The nine lives of epitope-based antibody patents.

Epitope-based antibody claims have emerged to become one of the most important claim species in antibody patents. At the same time, they are heavily disputed. This article strives to give an overview about the as is situation both in Europe and the United States.

Characterization of antisperm antibody binding patterns in relation to sperm phenotypic attributes and field fertility in dairy bulls.

To test our hypothesis that antisperm antibodies (ASA) might alter sperm phenotypic attributes thus leading to sub-fertility/infertility in bulls, ASA were generated in crossbred male calves by immunizing with sperm two times. Cryopreserved spermatozoa from crossbred bulls (n = 24) with different field fertility ratings were incubated with ASA and different patterns of ASA immunolocalization were studied. In addition, sperm membrane integrity, acrosomal integrity and cryo-capacitation status were also assessed. Immunolocalization of sperm antigens using antisperm antibody revealed three major patterns (Acrosomal-AR, apical-AP and, acrosome and tail-AT). The proportion of ASA reactive spermatozoa was significantly (P < 0.05) higher in low- and medium-fertile bulls compared to high-fertile bulls. Among the three patterns, the proportion of spermatozoa with AR pattern was significantly (P < 0.05) higher in low- and medium-fertile bulls compared to high-fertile bulls. The proportion of membrane and acrosome intact spermatozoa was significantly (P < 0.05) higher in high-fertile bulls compared to medium- and low-fertile bulls. There were no significant differences in the proportion of cryo-capacitated spermatozoa among high-, medium- and low-fertile bulls. The relationship between ASA reactive spermatozoa and conception rates (CR) of bulls was highly (P < 0.01) significant and negative. Similarly, AR and AT pattern were also significantly (P < 0.01) and negatively related to CR of bulls. The reactivity of spermatozoa with ASA was also significantly (P < 0.01) and negatively related to the membrane and acrosome integrity of spermatozoa. It was concluded that the proportion of spermatozoa responding to ASA was higher in low-compared to high-fertile bulls and ASA localization in sperm acrosomal area was negatively related to sperm membrane and acrosomal integrity and bull fertility.

Optogenetic activation of intracellular antibodies for direct modulation of endogenous proteins.

Intracellular antibodies have become powerful tools for imaging, modulating and neutralizing endogenous target proteins. Here, we describe an optogenetically activated intracellular antibody (optobody) consisting of split antibody fragments and blue-light inducible heterodimerization domains. We expanded this optobody platform by generating several optobodies from previously developed intracellular antibodies, and demonstrated that photoactivation of gelsolin and ß2-adrenergic receptor (ß2AR) optobodies suppressed endogenous gelsolin activity and ß2AR signaling, respectively.

Sclerostin Antibody-Induced Changes in Bone Mass Are Site Specific in Developing Crania.

Sclerostin antibody (Scl-Ab) is an anabolic bone agent that has been shown to increase bone mass in clinical trials of adult diseases of low bone mass, such as osteoporosis and osteogenesis imperfecta (OI). Its use to decrease bone fragility in pediatric OI has shown efficacy in several growing mouse models, suggesting translational potential to pediatric disorders of low bone mass. However, the effects of pharmacologic inhibition of sclerostin during periods of rapid growth and development have not yet been described with respect to the cranium, where lifelong deficiency of functioning sclerostin leads to patterns of excessive bone growth, cranial compression, and facial palsy. In the present study, we undertook dimensional and volumetric measurements in the skulls of growing Brtl/+ OI mice treated with Scl-Ab to examine whether therapy-induced phenotypic changes were similar to those observed clinically in patients with sclerosteosis or Van Buchem disorder. Mice treated between 3 and 14 weeks of age with high doses of Scl-Ab show significant calvarial thickening capable of rescuing OI-induced deficiencies in skull thickness. Other changes in cranial morphology, such as lengths and distances between anatomic landmarks, intracranial volume, and suture interdigitation, showed minimal effects of Scl-Ab when compared with growth-induced differences over the treatment duration. Treatment-induced narrowing of foramina was limited to sites of vascular but not neural passage, suggesting patterns of local regulation. Together, these findings reveal a site specificity of Scl-Ab action in the calvaria with no measurable cranial nerve impingement or brainstem compression. This differentiation from the observed outcomes of lifelong sclerostin deficiency complements reports of Scl-Ab treatment efficacy at other skeletal sites with the prospect of minimal cranial secondary complications. © 2019 American Society for Bone and Mineral Research. © 2019 American Society for Bone and Mineral Research.

Antibody-based nanotechnology.

Antibodies are considered the hallmark of the adaptive immune system in that they mediate various key biological functions, such as direct neutralization and recruitment of effector immune cells to eliminate invading pathogens. Antibodies exhibit several unique properties, including high diversity (enabling binding to a wide range of targets), high specificity and structural integrity. These properties and the understanding that antibodies can be utilized in a wide range of applications have motivated the scientific community to develop new approaches for antibody repertoire analysis and rapid monoclonal antibody discovery. Today, antibodies are key modules in the pharmaceutical and diagnostic industries. By virtue of their high affinity and specificity to their targets and the availability of technologies to engineer different antibodies to a wide range of targets, antibodies have become the most promising natural biological molecules in a range of biotechnological applications, such as: highly specific and sensitive nanobiosensors for the diagnostics of different biomarkers; nanoparticle-based targeted drug delivery systems to certain cells or tissues; and nanomachines, which are nanoscale mechanical devices that enable energy conversion into precise mechanical motions in response to specific molecular inputs. In this review, we start by describing the unique properties of antibodies, how antibody diversity is generated, and the available technologies for antibody repertoire analysis and antibody discovery. Thereafter, we provide an overview of some antibody-based nanotechnologies and discuss novel and promising approaches for the application of antibodies in the nanotechnology field. Overall, we aim to bridge the knowledge gap between the nanotechnology and antibody engineering disciplines by demonstrating how technological advances in the antibody field can be leveraged to develop and/or enhance new technological approaches in the nanotechnology field.

Rituximab, plasma exchange and immunoglobulins: an ineffective treatment for chronic active antibody-mediated rejection.

BACKGROUND: Chronic active antibody-mediated rejection (c-aABMR) is an important cause of allograft failure and graft loss in long-term kidney transplants. METHODS: To determine the efficacy and safety of combined therapy with rituximab, plasma exchange (PE) and intravenous immunoglobulins (IVIG), a cohort of patients with transplant glomerulopathy (TG) that met criteria of active cABMR, according to BANFF'17 classification, was identified. RESULTS: We identified 62 patients with active c-aABMR and TG (cg ≥ 1). Twenty-three patients were treated with the combination therapy and, 39 patients did not receive treatment and were considered the control group. There were no significant differences in the graft survival between the two groups. The number of graft losses at 12 and 24 months and the decline of eGFR were not different and independent of the treatment. A decrease of eGFR≥13 ml/min between 6 months before and c-aABMR diagnosis, was an independent risk factor for graft loss at 24 months (OR = 5; P = 0.01). Infections that required hospitalization during the first year after c-aABMR diagnosis were significantly more frequent in treated patients (OR = 4.22; P = 0.013), with a ratio infection/patient-year of 0.65 and 0.20 respectively. CONCLUSIONS: Treatment with rituximab, PE, and IVIG in kidney transplants with c-aABMR did not improve graft survival and was associated with a significant increase in severe infectious complications. TRIAL REGISTRATION: Agencia Española de Medicametos y Productos Sanitarios (AEMPS): 14566/RG 24161. Study code: UTR-INM-2017-01.

Heterogeneity and longevity of antibody memory to viruses and vaccines.

Determining the duration of protective immunity requires quantifying the magnitude and rate of loss of antibodies to different virus and vaccine antigens. A key complication is heterogeneity in both the magnitude and decay rate of responses of different individuals to a given vaccine, as well as of a given individual to different vaccines. We analyzed longitudinal data on antibody titers in 45 individuals to characterize the extent of this heterogeneity and used models to determine how it affected the longevity of protective immunity to measles, rubella, vaccinia, tetanus, and diphtheria. Our analysis showed that the magnitude of responses in different individuals varied between 12- and 200-fold (95% coverage) depending on the antigen. Heterogeneity in the magnitude and decay rate contribute comparably to variation in the longevity of protective immunity between different individuals. We found that some individuals have, on average, slightly longer-lasting memory than others-on average, they have higher antibody levels with slower decay rates. We identified different patterns for the loss of protective levels of antibodies to different vaccine and virus antigens. Specifically, we found that for the first 25 to 50 years, virtually all individuals have protective antibody titers against diphtheria and tetanus, respectively, but about 10% of the population subsequently lose protective immunity per decade. In contrast, at the outset, not all individuals had protective titers against measles, rubella, and vaccinia. However, these antibody titers wane much more slowly, with a loss of protective immunity in only 1% to 3% of the population per decade. Our results highlight the importance of long-term longitudinal studies for estimating the duration of protective immunity and suggest both how vaccines might be improved and how boosting schedules might be reevaluated.

Dissecting FcγR Regulation through a Multivalent Binding Model.

Many immune receptors transduce activation across the plasma membrane through their clustering. With Fcγ receptors (FcγRs), this clustering is driven by binding to antibodies of differing affinities that are in turn bound to multivalent antigen. As a consequence of this activation mechanism, accounting for and rationally manipulating immunoglobulin (Ig)G effector function is complicated by, among other factors, differing affinities between FcγR species and changes in the valency of antigen binding. In this study, we show that a model of multivalent receptor-ligand binding can effectively account for the contribution of IgG-FcγR affinity and immune complex valency. This model in turn enables us to make specific predictions about the effect of immune complexes of defined composition. In total, these results enable both rational immune complex design for a desired IgG effector function and the deconvolution of effector function by immune complexes.

The clinical impact of donor-specific antibodies in heart transplantation.

Donor-specific antibodies (DSA) are integral to the development of antibody-mediated rejection (AMR). Chronic AMR is associated with high mortality and an increased risk for cardiac allograft vasculopathy (CAV). Anti-donor HLA antibodies are present in 3-11% of patients at the time of heart transplantation (HTx), with de novo DSA (predominantly anti-HLA class II) developing post-transplant in 10-30% of patients. DSA are associated with lower graft and patient survival after HTx, with one study suggesting a three-fold increase in mortality in patients who develop de novo DSA (dnDSA). DSA against anti-HLA class II, notably DQ, are at particularly high risk for graft loss. Although detection of DSA is not a criterion for pathologic diagnosis of AMR, circulating DSA are found in almost all cases of AMR. MFI thresholds of ~5000 for DSA against class I antibodies, 2000 against class II antibodies, or an overall cut-off of 5-6000 for any DSA, have been suggested as being predictive for AMR. There is no firm consensus on pre-transplant strategies to treat HLA antibodies, or for the elimination of antibodies after diagnosis of AMR. Minimizing the risk of dnDSA is rational but data on risk factors in HTx are limited. The effect of different immunosuppressive regimens is largely unexplored in HTx, but studies in kidney transplantation emphasize the importance of adherence and maintaining adequate immunosuppression. One study has suggested a reduced risk for dnDSA with rabbit antithymocyte globulin induction. Management of DSA pre- and post-HTx varies but typically most centers rely on a plasmapheresis or immunoadsorption, with or without rituximab and/or intravenous immunoglobulin. Based on the literature and a multi-center survey, an algorithm for a suggested surveillance and therapeutic strategy is provided.

Migration-based selections of antibodies that convert bone marrow into trafficking microglia-like cells that reduce brain amyloid ß.

One goal of regenerative medicine is to repair damaged tissue. This requires not only generating new cells of the proper phenotype, but also selecting for those that properly integrate into sites of injury. In our laboratory we are using a cell-migration-based in vivo selection system to generate antibodies that induce cells to both differentiate and selectively localize to different tissues. Here we describe an antibody that induces bone marrow stem cells to differentiate into microglia-like cells that traffic to the brain where they organize into typical networks. Interestingly, in the APP/PS1 Alzheimer's disease mouse model, these induced microglia-like cells are found at sites of plaque formation and significantly reduce their number. These results raise the intriguing question as to whether one can use such antibody-induced differentiation of stem cells to essentially recapitulate embryogenesis in adults to discover cells that can regenerate damaged organ systems.

Thalamic Reticular Nucleus in Caiman crocodilus: Immunohistochemical Staining.

The thalamic reticular nucleus in reptiles, Caiman crocodilus, shares a number of morphological similarities with its counterpart in mammals. In view of the immunohistochemical properties of this nucleus in mammals and the more recently identified complexity of this neuronal aggregate in Caiman, this nucleus was investigated using a number of antibodies. These results were compared with findings described for other amniotes. The following antibodies gave consistent and reproducible results: polyclonal sheep anti-parvalbumin (PV), monoclonal mouse anti-PV, and polyclonal sheep anti-glutamic acid decarboxylase (GAD). In the transverse plane, this nucleus is divided into two. In each part, a compact group of cells sits on top of the fibers of the forebrain bundle with scattered cells among these fibers. In the lateral forebrain bundle, this neuronal aggregate is represented by the dorsal peduncular nucleus and the perireticular nucleus while, in the medial forebrain bundle, these parts are the interstitial nucleus and the scattered cells in this fiber tract. The results of this study are the following. First, the thalamic reticular nucleus of Caiman contains GAD(+) and PV(+) neurons, which is similar to what has been described in other amniotes. Second, the morphology and distribution of many GAD(+) and PV(+) neurons in the dorsal peduncular and perireticular nuclei are similar and suggest that these neurons colocalize these markers. Third, neurons in the interstitial nucleus and in the medial forebrain bundle are GAD(+) and PV(+). At the caudal pole of the thalamic reticular nucleus, PV immunoreactive cells predominated and avoided the central portion of this nucleus where GAD(+) cells were preferentially located. However, GAD(+) cells were sparse when compared with PV(+) cells. This immunohistochemically different area in the caudal pole is considered to be an area separate from the thalamic reticular nucleus.

Structure-function characterization of three human antibodies targeting the vaccinia virus adhesion molecule D8.

Vaccinia virus (VACV) envelope protein D8 is one of three glycosaminoglycan adhesion molecules and binds to the linear polysaccharide chondroitin sulfate (CS). D8 is also a target for neutralizing antibody responses that are elicited by the smallpox vaccine, which has enabled the first eradication of a human viral pathogen and is a useful model for studying antibody responses. However, to date, VACV epitopes targeted by human antibodies have not been characterized at atomic resolution. Here, we characterized the binding properties of several human anti-D8 antibodies and determined the crystal structures of three VACV-mAb variants, VACV-66, VACV-138, and VACV-304, separately bound to D8. Although all these antibodies bound D8 with high affinity and were moderately neutralizing in the presence of complement, VACV-138 and VACV-304 also fully blocked D8 binding to CS-A, the low affinity ligand for D8. VACV-138 also abrogated D8 binding to the high-affinity ligand CS-E, but we observed residual CS-E binding was observed in the presence of VACV-304. Analysis of the VACV-138- and VACV-304-binding sites along the CS-binding crevice of D8, combined with different efficiencies of blocking D8 adhesion to CS-A and CS-E allowed us to propose that D8 has a high- and low-affinity CS-binding region within its central crevice. The crevice is amenable to protein engineering to further enhance both specificity and affinity of binding to CS-E. Finally, a wild-type D8 tetramer specifically bound to structures within the developing glomeruli of the kidney, which express CS-E. We propose that through structure-based protein engineering, an improved D8 tetramer could be used as a potential diagnostic tool to detect expression of CS-E, which is a possible biomarker for ovarian cancer.

A Heterologous Model of Thrombospondin Type 1 Domain-Containing 7A-Associated Membranous Nephropathy.

Thrombospondin type 1 domain-containing 7A (THSD7A) is a target for autoimmunity in patients with membranous nephropathy (MN). Circulating autoantibodies from patients with THSD7A-associated MN have been demonstrated to cause MN in mice. However, THSD7A-associated MN is a rare disease, preventing the use of patient antibodies for larger experimental procedures. Therefore, we generated antibodies against the human and mouse orthologs of THSD7A in rabbits by coimmunization with the respective cDNAs. Injection of these anti-THSD7A antibodies into mice induced a severe nephrotic syndrome with proteinuria, weight gain, and hyperlipidemia. Immunofluorescence analyses revealed granular antigen-antibody complexes in a subepithelial location along the glomerular filtration barrier 14 days after antibody injection, and immunohistochemistry for rabbit IgG and THSD7A as well as ultrastructural analyses showed the typical characteristics of human MN. Mice injected with purified IgG from rabbit serum that was taken before immunization failed to develop any of these changes. Notably, MN developed in the absence of detectable complement activation, and disease was strain dependent. In vitro, anti-THSD7A antibodies caused cytoskeletal rearrangement and activation of focal adhesion signaling. Knockdown of the THSD7A ortholog, thsd7aa, in zebrafish larvae resulted in altered podocyte differentiation and impaired glomerular filtration barrier function, with development of pericardial edema, suggesting an important role of THSD7A in glomerular filtration barrier integrity. In summary, our study introduces a heterologous mouse model that allows further investigation of the molecular events that underlie MN.

Designing Probes for Immunodiagnostics: Structural Insights into an Epitope Targeting Burkholderia Infections.

Structure-based epitope prediction drives the design of diagnostic peptidic probes to reveal specific antibodies elicited in response to infections. We previously identified a highly immunoreactive epitope from the peptidoglycan-associated lipoprotein (Pal) antigen from Burkholderia pseudomallei, which could also diagnose Burkholderia cepacia infections. Here, considering the high phylogenetic conservation within Burkholderia species, we ask whether cross-reactivity can be reciprocally displayed by the synthetic epitope from B. cenocepacia. We perform comparative analyses of the conformational preferences and diagnostic performances of the corresponding epitopes from the two Burkholderia species when presented in the context of the full-length proteins or as isolated peptides. The effects of conformation on the diagnostic potential and cross-reactivity of Pal peptide epitopes are rationalized on the basis of the 1.8 Å crystal structure of B. cenocepacia Pal and through computational analyses. Our results are discussed in the context of designing new diagnostic molecules for the early detection of infectious diseases.

Antisperm antibodies in repeat-breeding cows: Frequency, detection and validation of threshold levels employing sperm immobilization, sperm agglutination and immunoperoxidase assay.

Antisperm antibodies have been found in repeat-breeding(RB) cows, and those causing agglutination and/or immobilization of sperm are considered to be closely related to unexplained infertility. However, a standard protocol for identifying antisperm antibodies (ASA) in cattle is not validated. Therefore, an investigation was undertaken to evaluate sperm immobilization (SIT), sperm agglutination (SAT) and immunoperoxidase (IPT)assays for detection of ASA in serum and their respective threshold levels for confirmation. Animals (heifers, normally breeding, repeat-breeding and pregnant animals) that were free from IBR, brucellosis and uterine infections (screened by clinical examination) were included in the study. Sperm agglutinating, sperm immobilizing and antisperm antibodies evaluated by respective assay were significantly higher (p < .05) in RB cows compared to other groups. The SIT assay was able to identify 61% of RB caused by ASA, more than those employing SAT and IPT. Furthermore, a dilution rate of 1:5 and 1:80 (confirms 59.0 and 57.0% RB+ve)were sufficient to diagnose ASA by SAT and IPT, respectively. Results indicate the presence of __12.6% clumped spermatozoa and __ 2.6%(cut-off value) peroxidase-positive spermatozoa at 1:5 and 1:80 dilutions diagnosed with SAT and IPT, respectively, may be considered as repeaters arising out of ASA. Furthermore, study also showed the presence of lower incidence of ASA positivity in other groups of animals (heifer