Complement-mediated Cytotoxicity Research Articles

Stereoselective Synthesis of Sialyl Lewisa Antigen and the Effective Anticancer Activity of Its Bacteriophage Qβ Conjugate as an Anticancer Vaccine.

Sialyl Lewisa (sLea ), also known as cancer antigen 19-9 (CA19-9), is a tumor-associated carbohydrate antigen. The overexpression of sLea on the surface of a variety of cancer cells makes it an attractive target for anticancer immunotherapy. However, sLea -based anticancer vaccines have been under-explored. To develop a new vaccine, efficient stereoselective synthesis of sLea with an amine-bearing linker was achieved, which was subsequently conjugated with a powerful carrier bacteriophage, Qβ. Mouse immunization with the Qβ-sLea conjugate generated strong and long-lasting anti-sLea IgG antibody responses, which were superior to those induced by the corresponding conjugate of sLea with the benchmark carrier keyhole limpet hemocyanin. Antibodies elicited by Qβ-sLea were highly selective toward the sLea structure, could bind strongly with sLea -expressing cancer cells and human pancreatic cancer tissues, and kill tumor cells through complement-mediated cytotoxicity. Furthermore, vaccination with Qβ-sLea significantly reduced tumor development in a metastatic cancer model in mice, demonstrating tumor protection for the first time by a sLea -based vaccine, thus highlighting the significant potential of sLea as a promising cancer antigen.

Development of C1q Affinity Chromatography for the Study of C1q-IgG Interactions.

The classical complement system represents a central effector mechanism of Abs initiated by the binding of C1q to target bound IgG. Human C1q contains six heterotrimeric globular head groups that mediate IgG interaction, resulting in an avidity-driven binding event involving multiple IgG molecules binding a single C1q. Accordingly, surface bound IgG molecules are thought to assemble into noncovalent hexameric rings for optimal binding to the six-headed C1q. To study the C1q-Fc interaction of various Abs and screen for altered C1q binding mutants, we developed, to our knowledge, a novel HPLC-based method. Employing a single-chain form of C1q representing one C1q head group, our HPLC methodology was able to detect the interaction between the single-chain monomeric form of C1q and various ligands. We show that, despite a narrow window of specific binding owing to the low affinity of the monomeric C1q-IgG interaction, this approach clearly distinguished between IgG subclasses with established C1q binding properties. IgG3 displayed the strongest binding, followed by IgG1, with IgG2 and IgG4 showing the weakest binding. Fc mutants known to have increased C1q binding through oligomerization or enhanced C1q interaction showed greatly increased column retention, and IgG glycovariants displayed a consistent trend of increasing retention upon increasing galactosylation and sialylation. Furthermore, the column retention of IgG isotypes and glycovariants matches both the cell surface recruitment of C1q and complement-mediated cytotoxicity induced by each variant on an anti-CD20 Ab backbone. This methodology therefore provides a valuable tool for testing IgG Ab (glyco)variants for C1q binding, with clear relevance for therapeutic Ab development.

Incidence and Risk Factors of Infections Among Diffuse Large B-cell Lymphoma and Classical Hodgkin's Lymphoma Patients in a Tertiary Care Center in Saudi Arabia: A Retrospective Cohort Study.

Non-Hodgkin's lymphoma (NHL) ranked fourth among all cancer types in Saudi Arabia, as reported by the Saudi Health Council in 2015. Diffuse large B-cell lymphoma (DLBCL) is the most common histological type of NHL. On the other hand, classical Hodgkin's lymphoma (cHL) ranked sixth and had a modest tendency to affect young men more frequently.Over recent decades, DLBCL patients were treated with cyclophosphamide, doxorubicin hydrochloride, oncovin, and prednisolone (CHOP) alone. Adding rituximab (R) to the standard regimen (CHOP) shows significant improvement in overall survival. However, it also has a considerable effect on the immune system, impacting complement-mediated and antibody-dependent cellular cytotoxicity and causing an immunosuppressive state through modulating T-cell immunity via neutropenia, which can let the infection spread. This study aims to evaluate the incidence and risk factors associated with infections in DLBCL patients in comparison to patients with cHLtreated with doxorubicin hydrochloride (Adriamycin), bleomycin sulfate, vinblastine sulfate, and dacarbazine (ABVD). This study is a retrospective case-control study that included 201 patients acquired between January 1st, 2010, and January 1st, 2020. Sixty-seven patients had a diagnosis ofcHL and had received ABVD, and 134 had DLBCL and had received rituximab. Clinical data were obtained from the medical records. During the study period, we enrolled 201 patients, of whom 67 had cHL, and 134 had DLBCL. DLBCL patients had a higher serum lactate dehydrogenase upon diagnosis than cHL (p = 0.005). Both groups have similar response rates with complete remission/partial remission. Compared to cHL, patients with DLBCL were more likely to have advanced disease when they first presented (stage III/IV, DLBCL: 67.3 vs. cHL: 56.5; p = 0.005). DLBCL patients had an increased risk of infection as compared to cHL patients (DLBCL: 32.1 % vs. 16.4%; p = 0.02). However, patients with a poor response to treatment had an increased risk of infection compared to patients with a favorable response regardless of the type of disease (odds ratio: 4.6; p = <0.001).When using multivariate analysis, it is revealed that unfavorable therapeutic response continues to be the only predictor raising the probability of infection in the population (odds ratio: 4.2; p = 0.003). Our study explored all potential risk factors for the occurrence of infection in DLBCL patients who received R-CHOP versus cHL. The most reliable predictor of an increased risk of infection during the follow-up period was having an unfavorable response to medication. To assess these results, additional prospective research is required.

Pseudomonas aeruginosa LecB suppresses immune responses by inhibiting transendothelial migration.

Pseudomonas aeruginosa is a Gram-negative bacterium causing morbidity and mortality in immuno-compromised humans. It produces a lectin, LecB, that is considered a major virulence factor, however, its impact on the immune system remains incompletely understood. Here we show that LecB binds to endothelial cells in human skin and mice and disrupts the transendothelial passage of leukocytes in vitro. It impairs the migration of dendritic cells into the paracortex of lymph nodes leading to a reduced antigen-specific T cell response. Under the effect of the lectin, endothelial cells undergo profound cellular changes resulting in endocytosis and degradation of the junctional protein VE-cadherin, formation of an actin rim, and arrested cell motility. This likely negatively impacts the capacity of endothelial cells to respond to extracellular stimuli and to generate the intercellular gaps for allowing leukocyte diapedesis. A LecB inhibitor can restore dendritic cell migration and T cell activation, underlining the importance of LecB antagonism to reactivate the immune response against P.aeruginosa infection.

LIS1, a glyco-humanized swine polyclonal anti-lymphocyte globulin, as a novel induction treatment in solid organ transplantation.

Anti-thymocyte or anti-lymphocyte globulins (ATGs/ALGs) are immunosuppressive drugs used in induction therapies to prevent acute rejection in solid organ transplantation. Because animal-derived, ATGs/ALGs contain highly immunogenic carbohydrate xenoantigens eliciting antibodies that are associated with subclinical inflammatory events, possibly impacting long-term graft survival. Their strong and long-lasting lymphodepleting activity also increases the risk for infections. We investigated here the in vitro and in vivo activity of LIS1, a glyco-humanized ALG (GH-ALG) produced in pigs knocked out for the two major xeno-antigens αGal and Neu5Gc. It differs from other ATGs/ALGs by its mechanism of action excluding antibody-dependent cell-mediated cytotoxicity and being restricted to complement-mediated cytotoxicity, phagocyte-mediated cytotoxicity, apoptosis and antigen masking, resulting in profound inhibition of T-cell alloreactivity in mixed leucocyte reactions. Preclinical evaluation in non-human primates showed that GH-ALG dramatically reduced CD4+ (p=0.0005,***), CD8+ effector T cells (p=0.0002,***) or myeloid cells (p=0.0007,***) but not T-reg (p=0.65, ns) or B cells (p=0.65, ns). Compared with rabbit ATG, GH-ALG induced transient depletion (less than one week) of target T cells in the peripheral blood (<100 lymphocytes/L) but was equivalent in preventing allograft rejection in a skin allograft model. The novel therapeutic modality of GH-ALG might present advantages in induction treatment during organ transplantation by shortening the T-cell depletion period while maintaining adequate immunosuppression and reducing immunogenicity.

Complement-dependent cytotoxicity of human autoantibodies against myelin oligodendrocyte glycoprotein.

The autoantibody to myelin oligodendrocyte glycoprotein (MOG), a component of the central nervous system myelin, has been identified in a subset of demyelinating diseases. However, there is no convincing evidence to support the direct pathogenic contribution of this autoantibody. To elucidate the role of anti-MOG autoantibodies in human demyelinating disorders, we assessed the effect of autoantibodies on MOG-expressing cells. Mammalian cells expressing the human MOG protein reacted with human anti-MOG autoantibodies in the presence or absence of complement. Sera from 86 patients and 11 healthy sera were used. We analyzed anti-MOG antibody titers, IgG subclass, and their cytotoxic ability in sera from patients with various neurological diseases. Membrane attack complex (MAC) formation was examined by detection of complement C9 or C9neo with western blot or flow cytometry. Among 86 patients, 40 were determined to be MOG-IgG-positive and 46 were negative. Anti-MOG-positive sera, but not -negative sera, caused cell death in MOG-expressing cells. This cytotoxic effect was disappeared after heat inactivation of sera. Importantly, anti-MOG IgG and externally added complement were necessary for sufficient cytotoxic effects. Anti-MOG autoantibodies were histologically colocalized with complement and formed a membrane attack complex consisting of anti-MOG IgG and complement factors. The human MOG antibody specifically killed MOG-expressing cells in vitro in the presence of externally added complement. Membrane attack complexes were formed on the cells, indicating that this autoantibody activated complement-mediated cytotoxicity. Further studies in larger numbers of patients are needed to characterize the role of complement in MOGAD.

New approach to desensitization in solid organ transplantation-imlifidase

The IgG-degrading enzyme derived from Streptococcus pyogenes is a recombinant cysteine protease of S. pyogenes produced in Escherichia coli that cleaves all four human subclasses of IgG with strict specificity. The proteolytic activity on IgG molecules prevents the occurrence of IgG-mediated antibody-dependent, cellular cytotoxicity and complement-mediated cytotoxicity, two processes that are critical for antibody rejection. The results from phase II studies demonstrated that desensitization with imlifidase represents a therapeutic strategy that can operationalize desensitization, allowing life-saving transplants from deceased donors (DD) and living donors (LD) to proceed in highly sensitized kidney transplant candidates with low risk of hyperacute rejection. Its action onset is rapid, allowing kidney transplantation from a deceased donor. Disadvantages of imlifidase include a quick reappearance of DSAs, which poses a risk of antibody-mediated rejection, the quick development of anti-Ides antibodies, which rules out repeated use of imlifidase and its IgG-degrading potential, limiting the use of therapeutic antibodies. Imlifdase received conditional approval on 26 August 2020 in the EU for desensitization treatment of highly sensitized adult kidney transplant patients with positive crossmatch against an available deceased donor.

Autoantibody panel on small extracellular vesicles for the early detection of lung cancer

This study evaluated a different form of autoantibody, which is on small extracellular vesicles (sEVs), as a biomarker for early detection of lung cancer. The sEVs were isolated from plasma by ultracentrifugation and validated with morphology and typical markers. The autoantibody levels were quantified by enzyme-linked immunosorbent assay, and further analysis indicated that the autoantibody panel on sEVs was better than that from serum to distinguish benign lung disease (n = 32) from lung cancer (n = 90). In the prospective study, autoantibody on sEVs performed better in identification of patients with a higher risk of lung cancer. Furthermore, with immunogold labeling transmission electron microscopy, Nanoflow cytometry and binding tests, we illustrated that the autoantibodies could bind to the antigens on sEVs, which may explain the detected autoantibodies on sEVs. Besides, the binding resulted in the attenuation of complement-mediated cytotoxicity, which may contribute to the immune escape of lung cancer.

Development and Validation of an Efficient and Highly Sensitive Enzyme-Linked Immunosorbent Assay for Alemtuzumab Quantification in Human Serum and Plasma.

Alemtuzumab is a humanized monoclonal antibody that targets the CD52 glycoprotein expressed on most lymphocytes, subsequently inducing complement-mediated and antibody-mediated cytotoxicity. Owing to its ability to induce profound immune depletion, alemtuzumab is frequently used in patients before allogeneic hematopoietic stem cell transplantation to prevent graft rejection and acute graft-versus-host disease. In this clinical context, a stable immunoassay with high sensitivity and specificity to determine alemtuzumab levels is essential for performing pharmacokinetic and pharmacodynamic analyses; however, the available methods have several limitations. Here, we report the successful development and validation of an efficient and highly sensitive enzyme-linked immunosorbent assay technique based on commercially available reagents to quantify alemtuzumab in human serum or plasma. This enzyme-linked immunosorbent assay technique was developed and validated in accordance with the European Medicines Agency guidelines on bioanalytical method validation. The assay sensitivity (lower limit of quantification) is 0.5 ng·mL -1 , and the dynamic range is 0.78-25 ng·mL -1 . To accommodate quantification of peak concentration and concentrations below the lympholytic level (<0.1 mcg·mL -1 ), patients' serum samples were prediluted 20-400 times according to the expected alemtuzumab concentration. The overall within-run accuracy was between 96% and 105%, whereas overall within-run precision (coefficient of variation) was between 3% and 9%. The between-run assessment provided an overall accuracy between 86% and 95% and an overall coefficient of variation between 5% and 14%. The developed assay provides accurate insight into alemtuzumab exposure and its effects on the clinical response to treatment, which is key to optimizing treatment strategies.

Antibody-mediated cell depletion therapies in multiple sclerosis.

Development of disease-modifying therapies including monoclonal antibody (mAb)-based therapeutics for the treatment of multiple sclerosis (MS) has been extremely successful over the past decades. Most of the mAb-based therapies approved for MS deplete immune cell subsets and act through activation of cellular Fc-gamma receptors expressed by cytotoxic lymphocytes and phagocytes, resulting in antibody-dependent cellular cytotoxicity or by initiation of complement-mediated cytotoxicity. The therapeutic goal is to eliminate pathogenic immune cell components and to potentially foster the reconstitution of a new and healthy immune system. Ab-mediated immune cell depletion therapies include the CD52-targeting mAb alemtuzumab, CD20-specific therapeutics, and new Ab-based treatments which are currently being developed and tested in clinical trials. Here, we review recent developments in effector mechanisms and clinical applications of Ab-based cell depletion therapies, compare their immunological and clinical effects with the prototypic immune reconstitution treatment strategy, autologous hematopoietic stem cell transplantation, and discuss their potential to restore immunological tolerance and to achieve durable remission in people with MS.

A desirable transgenic strategy using GGTA1 endogenous promoter-mediated knock-in for xenotransplantation model

Pig-to-human organ transplantation is a feasible solution to resolve the shortage of organ donors for patients that wait for transplantation. To overcome immunological rejection, which is the main hurdle in pig-to-human xenotransplantation, various engineered transgenic pigs have been developed. Ablation of xeno-reactive antigens, especially the 1,3-Gal epitope (GalT), which causes hyperacute rejection, and insertion of complement regulatory protein genes, such as hCD46, hCD55, and hCD59, and genes to regulate the coagulation pathway or immune cell-mediated rejection may be required for an ideal xenotransplantation model. However, the technique for stable and efficient expression of multi-transgenes has not yet been settled to develop a suitable xenotransplantation model. To develop a stable and efficient transgenic system, we knocked-in internal ribosome entry sites (IRES)-mediated transgenes into the α 1,3-galactosyltransferase (GGTA1) locus so that expression of these transgenes would be controlled by the GGTA1 endogenous promoter. We constructed an IRES-based polycistronic hCD55/hCD39 knock-in vector to target exon4 of the GGTA1 gene. The hCD55/hCD39 knock-in vector and CRISPR/Cas9 to target exon4 of the GGTA1 gene were co-transfected into white yucatan miniature pig fibroblasts. After transfection, hCD39 expressed cells were sorted by FACS. Targeted colonies were verified using targeting PCR and FACS analysis, and used as donors for somatic cell nuclear transfer. Expression of GalT, hCD55, and hCD39 was analyzed by FACS and western blotting. Human complement-mediated cytotoxicity and human antibody binding assays were conducted on peripheral blood mononuclear cells (PBMCs) and red blood cells (RBCs), and deposition of C3 by incubation with human complement serum and platelet aggregation were analyzed in GGTA1 knock-out (GTKO)/CD55/CD39 pig cells. We obtained six targeted colonies with high efficiency of targeting (42.8% of efficiency). Selected colony and transgenic pigs showed abundant expression of targeted genes (hCD55 and hCD39). Knocked-in transgenes were expressed in various cell types under the control of the GGTA1 endogenous promoter in GTKO/CD55/CD39 pig and IRES was sufficient to express downstream expression of the transgene. Human IgG and IgM binding decreased in GTKO/CD55/CD39 pig and GTKO compared to wild-type pig PBMCs and RBCs. The human complement-mediated cytotoxicity of RBCs and PBMCs decreased in GTKO/CD55/CD39 pig compared to cells from GTKO pig. C3 was also deposited less in GTKO/CD55/CD39 pig cells than wild-type pig cells. The platelet aggregation was delayed by hCD39 expression in GTKO/CD55/CD39 pig. In the current study, knock-in into the GGTA1 locus and GGTA1 endogenous promoter-mediated expression of transgenes are an appropriable strategy for effective and stable expression of multi-transgenes. The IRES-based polycistronic transgene vector system also caused sufficient expression of both hCD55 and hCD39. Furthermore, co-transfection of CRISPR/Cas9 and the knock-in vector not only increased the knock-in efficiency but also induced null for GalT by CRISPR/Cas9-mediated double-stranded break of the target site. As shown in human complement-mediated lysis and human antibody binding to GTKO/CD55/CD39 transgenic pig cells, expression of hCD55 and hCD39 with ablation of GalT prevents an effective immunological reaction in vitro. As a consequence, our technique to produce multi-transgenic pigs could improve the development of a suitable xenotransplantation model, and the GTKO/CD55/CD39 pig developed could prolong the survival of pig-to-primate xenotransplant recipients.

Fc-Mediated Functions of Porcine IgG Subclasses.

The pig is an important agricultural species and powerful biomedical model. We have established the pig, a large natural host animal for influenza with many physiological similarities to humans, as a robust model for testing the therapeutic potential of monoclonal antibodies. Antibodies provide protection through neutralization and recruitment of innate effector functions through the Fc domain. However very little is known about the Fc-mediated functions of porcine IgG subclasses. We have generated 8 subclasses of two porcine monoclonal anti influenza hemagglutinin antibodies. We characterized their ability to activate complement, trigger cytotoxicity and phagocytosis by immune cells and assayed their binding to monocytes, macrophages, and natural killer cells. We show that IgG1, IgG2a, IgG2b, IgG2c and IgG4 bind well to targeted cell types and mediate complement mediated cellular cytotoxicity (CDCC), antibody dependent cellular cytotoxicity (ADCC) and antibody mediated cell phagocytosis (ADCP). IgG5b and IgG5c exhibited weak binding and variable and poor functional activity. Immune complexes of porcine IgG3 did not show any Fc-mediated functions except for binding to monocytes and macrophages and weak binding to NK cells. Interestingly, functionally similar porcine IgG subclasses clustered together in the genome. These novel findings will enhance the utility of the pig model for investigation of therapeutic antibodies.

Pathomechanisms in demyelination and astrocytopathy: autoantibodies to AQP4, MOG, GFAP, GRP78 and beyond

The purpose of this review is to highlight the recently emerging pathomechanisms of diseases associated with autoantibodies to AQP4, MOG, GFAP, GRP78 and further novel targets. We discuss novel biomarkers and therapeutic approaches. Although complement-mediated cytotoxicity (CDC) is regarded as the major effector mechanism for AQP4-IgG in neuromyelitis optica spectrum disorders (NMOSD), recent studies helped to understand the relevance of complement-independent effector mechanisms. For MOG-IgG mediated diseases the role of CDC is less clear. MOG-IgG may trigger a tightly controlled FcR and BTK-driven microglia proliferative response in MOG-antibody-associated diseases. Differences of antibody-mediated tissue damage may reflect differential response to therapy. In addition, antibodies to GFAP, GRP78 and further novel targets have been implicated in demyelination and astrocytopathy. Elucidating the whole spectrum of effector functions in diseases mediated by AQP4-IgG and MOG-IgG and understanding the role of additional novel autoantibodies involved in demyelination and astrocytopathy may guide further novel treatment decisions.

MUC1 Specific Immune Responses Enhanced by Coadministration of Liposomal DDA/MPLA and Lipoglycopeptide.

Mucin 1 (MUC1), a well-known tumor-associated antigen and attractive target for tumor immunotherapy, is overexpressed in most human epithelial adenomas with aberrant glycosylation. However, its low immunogenicity impedes the development of MUC1-targeted antitumor vaccines. In this study, we investigated three liposomal adjuvant systems containing toll-like receptor 4 (TLR4) agonist monophosphoryl lipid A (MPLA) and auxiliary lipids of different charges: cationic lipid dimethyldioctadecylammonium (DDA), neutral lipid distearoylglycerophosphocholine (DSPC) or anionic lipid dioleoylphosphatidylglycerol (DOPG), respectively. ELISA assay evidenced that the positively charged DDA/MPLA liposomes are potent immune activators, which induced remarkable levels of anti-MUC1 antibodies and exhibited robust Th1-biased immune responses. Importantly, the antibodies induced by DDA/MPLA liposomes efficiently recognized and killed MUC1-positive tumor cells through complement-mediated cytotoxicity. In addition, antibody titers in mice immunized with P2-MUC1 vaccine were significantly higher than those from mice immunized with P1-MUC1 or MUC1 vaccine, which indicated that the lipid conjugated on MUC1 antigen also played important role for immunomodulation. This study suggested that the liposomal DDA/MPLA with lipid-MUC1 is a promising antitumor vaccine, which can be used for the immunotherapy of various epithelial carcinomas represented by breast cancer.

Preclinical Immunopharmacologic Assessment of KPL-404, a Novel, Humanized, Non-Depleting Antagonistic Anti-CD40 Monoclonal Antibody

The CD40/CD40L pathway plays a major role in multiple inflammatory processes involving different immune and stromal cells. Abnormal activation of this pathway has been implicated in pathogenesis of complex autoimmune diseases including systemic lupus erythematosus, rheumatoid arthritis, Graves' disease, and Sjogren's Syndrome. We completed in vitro and in vivo preclinical characterization of KPL-404, a novel humanized anti-CD40 IgG4 monoclonal antibody, to demonstrate its potency, efficacy, and pharmacokinetic profile; safety was also assessed. In vitro, KPL-404 bound recombinant human and cynomolgus monkey CD40 with comparable affinity in the nanomolar range. KPL-404 binding to cell surface CD40 did not induce antibody- or complement-mediated cytotoxicity of CD40-expressing cells. Pharmacological antagonistic activity of KPL-404 was demonstrated in vitro by inhibition of CD40-mediated downstream NF-kB activation. In the in vivo study with cynomolgus monkeys, KPL-404, administered intravenously as a single dose (10 mg/kg) or two monthly doses of 1 or 5 mg/kg, did not elicit observable safety findings, including thrombocytopenia over 8 weeks. KPL-404 engaged CD40 expressed on peripheral B cells for 2 and 4 weeks after a single administration of 5 or 10 mg/kg IV, respectively, without depletion of peripheral B cells. At 5 mg/kg IV, KPL-404 blocked both primary and secondary responses to T-cell dependent antibody responses to test antigens, KLH, and tetanus toxoid. These data illustrated the relationship between KPL-404 serum concentration and pharmacodynamic effects of CD40-targeting in circulation and in lymphoid tissues. These data support clinical development of KPL-404 in autoimmune diseases. SIGNIFICANCE STATEMENT: We aimed to develop a potent and efficacious CD40 antagonist. In vitro and in vivo findings show that KPL-404 blocks the anti-CD40 antibody that potently inhibits primary and secondary antibody responses at pharmacologically relevant concentrations, has a favorable pharmacokinetic profile, and does not deplete B cells by antibody-dependent cellular cytotoxicity or apoptosis ("nondepleting"). These findings support clinical development of KPL-404 as a potential therapeutic in autoimmune diseases.

Immune response to allogeneic equine mesenchymal stromal cells

BackgroundMesenchymal stromal cells (MSCs) are believed to be hypoimmunogeneic with potential use for allogeneic administration.MethodsBone marrow was harvested from Connemara (n = 1), Standardbred (n = 6), and Thoroughbred (n = 3) horses. MSCs were grouped by their level of expression of major histocompatibility factor II (MHC II). MSCs were then sub-grouped by those MSCs derived from universal blood donor horses. MSCs were isolated and cultured using media containing fetal bovine serum until adequate numbers were acquired. The MSCs were cultured in xenogen-free media for 48 h prior to use and during all assays. Autologous and allogeneic MSCs were then directly co-cultured with responder leukocytes from the Connemara horse in varying concentrations of MSCs to leukocytes (1:1, 1:10, and 1:100). MSCs were also cultured with complement present and heat-inactivated complement to determine whether complement alone would decrease MSC viability. MSCs underwent haplotyping of their equine leukocyte antigen (ELA) to determine whether the MHC factors were matched or mismatched between the donor MSCs and the responder leukocytes.ResultsAll allogeneic MSCs were found to be ELA mismatched with the responder leukocytes. MHC II-low and universal blood donor MSCs caused no peripheral blood mononuclear cell (PBMC) proliferation, no increase in B cells, and no activation of CD8 lymphocytes. Universal blood donor MSCs stimulated a significant increase in the number of T regulatory cells. Neutrophil interaction with MSCs showed that universal blood donor and MHC II-high allogeneic MSCs at the 6 h time point in co-culture caused greater neutrophil activation than the other co-culture groups. Complement-mediated cytotoxicity did not consistently cause MSC death in cultures with active complement as compared to those with inactivated complement. Gene expression assays revealed that the universal blood donor group and the MHC II-low MSCs were more metabolically active both in the anabolic and catabolic gene categories when cultured with allogeneic lymphocytes as compared to the other co-cultures. These upregulated genes included CD59, FGF-2, HGF, IDO, IL-10, IL-RA, IL-2, SOX2, TGF-β1, ADAMSTS-4, ADAMSTS-5, CCL2, CXCLB/IL-8, IFNγ, IL-1β, and TNFα.ConclusionsMHC II-low MSCs are the most appropriate type of allogeneic MSC to prevent activation of the innate and cell-mediated component of the adaptive immune systems and have increased gene expression as compared to other allogeneic MSCs.

Hyper-CVAD Plus Ofatumumab As Frontline Therapy in CD20+ Acute Lymphoblastic Leukemia: Updated Results of a Phase II Trial

Hyper-CVAD Plus Ofatumumab As Frontline Therapy in CD20+ Acute Lymphoblastic Leukemia: Updated Results of a Phase II Trial

Sialylation of CD55 by ST3GAL1 Facilitates Immune Evasion in Cancer.

Altered glycosylations, which are associated with expression and activities of glycosyltransferases, can dramatically affect the function of glycoproteins and modify the behavior of tumor cells. ST3GAL1 is a sialyltransferase that adds sialic acid to core 1 glycans, thereby terminating glycan chain extension. In breast carcinomas, overexpression of ST3GAL1 promotes tumorigenesis and correlates with increased tumor grade. In pursuing the role of ST3GAL1 in breast cancer using ST3GAL1-siRNA to knockdown ST3GAL1, we identified CD55 to be one of the potential target proteins of ST3GAL1. CD55 is an important complement regulatory protein, preventing cells from complement-mediated cytotoxicity. CD55 had one N-linked glycosylation site in addition to a Ser/Thr-rich domain, which was expected to be heavily O-glycosylated. Detailed analyses of N- and O-linked oligosaccharides of CD55 released from scramble or ST3GAL1 siRNA-treated breast cancer cells by tandem mass spectrometry revealed that the N-glycan profile was not affected by ST3GAL1 silencing. The O-glycan profile of CD55 demonstrated a shift in abundance to nonsialylated core 1 and monosialylated core 2 at the expense of the disialylated core 2 structure after ST3GAL1 silencing. We also demonstrated that O-linked desialylation of CD55 by ST3GAL1 silencing resulted in increased C3 deposition and complement-mediated lysis of breast cancer cells and enhanced sensitivity to antibody-dependent cell-mediated cytotoxicity. These data demonstrated that ST3GAL1-mediated O-linked sialylation of CD55 acts like an immune checkpoint molecule for cancer cells to evade immune attack and that inhibition of ST3GAL1 is a potential strategy to block CD55-mediated immune evasion.

ARGX-117, a therapeutic complement inhibiting antibody targeting C2

BackgroundActivation of the classical and lectin pathway of complement may contribute to tissue damage and organ dysfunction of antibody-mediated diseases and ischemia-reperfusion conditions. Complement factors are being considered as targets for therapeutic intervention.ObjectiveWe sought to characterize ARGX-117, a humanized inhibitory monoclonal antibody against complement C2.MethodsThe mode-of-action and binding characteristics of ARGX-117 were investigated in detail. Furthermore, its efficacy was analyzed in in vitro complement cytotoxicity assays. Finally, a pharmacokinetic/pharmacodynamic study was conducted in cynomolgus monkeys.ResultsThrough binding to the Sushi-2 domain of C2, ARGX-117 prevents the formation of the C3 proconvertase and inhibits classical and lectin pathway activation upstream of C3 activation. As ARGX-117 does not inhibit the alternative pathway, it is expected not to affect the antimicrobial activity of this complement pathway. ARGX-117 prevents complement-mediated cytotoxicity in in vitro models for autoimmune hemolytic anemia and antibody-mediated rejection of organ transplants. ARGX-117 exhibits pH- and calcium-dependent target binding and is Fc-engineered to increase affinity at acidic pH to the neonatal Fc receptor, and to reduce effector functions. In cynomolgus monkeys, ARGX-117 dose-dependently reduces free C2 levels and classical pathway activity. A 2-dose regimen of 80 and 20 mg/kg separated by a week, resulted in profound reduction of classical pathway activity lasting for at least 7 weeks.ConclusionsARGX-117 is a promising new complement inhibitor that is uniquely positioned to target both the classical and lectin pathways while leaving the alternative pathway intact.

Nanodelivery system enhances the immunogenicity of dengue-2 nonstructural protein 1, DENV-2 NS1

Nonstructural protein 1 (NS1) of dengue virus (DENV) is currently recognized as a dengue vaccine candidate. Unfortunately, most of non-replicating immunogens typically stimulate unsatisfactory immune responses, thus, the additional adjuvant is required. In this study, C-terminal truncated DENV-2 NS1 loaded in N,N,N, trimethyl chitosan nanoparticles (NS11-279TMC NPs) was prepared through the ionic gelation method. The immunogenicity of NS11-279TMC NPs was investigated using human ex vivo as well as the murine model. Through a human ex vivo model, it was demonstrated in this study that not only can TMC particles effectively deliver NS11-279 protein into monocyte-derived dendritic cells (MoDCs), but also potently stimulate those cells, resulting in increased expression of maturation marker (CD83), costimulating molecules (CD80, CD86 and HLA-DR) and markedly secreted various types of innate immune cytokines/chemokines. Moreover, mice administered with NS11-279TMC NPs strongly elicited both antibody and T cell responses, produced higher levels of IgG, IgG1, IgG2a and potently activated CD8+ T cells, as compared to mice administered with soluble NS11-279. Importantly, we further demonstrated that anti-NS11-279 antibody induced by this platform of NS11-279 effectively eliminated DENV-2 infected cells through antibody dependent complement-mediated cytotoxicity. Significantly, anti-DENV2 NS11-279 antibody exerted cross-antiviral activity against DENV-1 and −4 but not against DENV-3 infected cells. These findings demonstrate that TMC exerts a desirable adjuvant for enhancing delivery and antigenicity of NS1 based dengue vaccine.