Rubella seroprevalence using residual samples from the South African measles surveillance program: a cross-sectional analytic study.

: South Africa is yet to introduce rubella-containing vaccines (RCV) into its routine immunization schedule. Selecting the target population when introducing RCV should take into account the ages of susceptible individuals in the population. We aimed to determine the seroprevalence of antibodies to rubella and characterize immunity gaps among individuals of all ages in South Africa.

Circulating T Cell Subpopulations Correlate With Immune Responses at the Tumor Site and Clinical Response to PD1 Inhibition in Non-Small Cell Lung Cancer.

Agents targeting the PD1-PDL1 axis have transformed cancer therapy. Factors that influence clinical response to PD1-PDL1 inhibitors include tumor mutational burden, immune infiltration of the tumor, and local PDL1 expression. To identify peripheral correlates of the anti-tumor immune response in the absence of checkpoint blockade, we performed a retrospective study of circulating T cell subpopulations and matched tumor gene expression in melanoma and non-small cell lung cancer (NSCLC) patients.

Antibody Drug-Target Engagement Measurement in Tissue Using Quantitative Affinity Extraction Liquid Chromatography-Mass Spectrometry: Method Development and Qualification.

We demonstrate a novel strategy using affinity extraction (AE) LC-MS to directly measure drug exposure and target engagement, two critical pharmacological questions, with a single assay. The assay measures total drug and target concentration at the site of therapeutic action, as well as the amount of target bound to drug. The case study presented applies the strategy to measure drug engagement of a membrane bound receptor (CD40) that is critical to immune regulation in colon biopsies collected from monkey dosed with an anti-CD40 antibody.

Fc-Silent Anti-CD154 Domain Antibody Effectively Prevents Nonhuman Primate Renal Allograft Rejection.

The advent of costimulation blockade provides the prospect for targeted therapy with improved graft survival in transplant patients. Perhaps the most effective costimulation blockade in experimental models is the use of reagents to block the CD40/CD154 pathway. Unfortunately, successful clinical translation of anti-CD154 therapy has not been achieved.

B cells from African American lupus patients exhibit an activated phenotype.

Systemic lupus erythematosus (SLE) is a complex systemic autoimmune disease driven by both innate and adaptive immune cells. African Americans tend to present with more severe disease at an earlier age compared with patients of European ancestry. In order to better understand the immunological differences between African American and European American patients, we analyzed the frequencies of B cell subsets and the expression of B cell activation markers from a total of 68 SLE patients and 69 normal healthy volunteers.

Functional Antagonism of Human CD40 Achieved by Targeting a Unique Species-Specific Epitope.

Current clinical anti-CD40 biologic agents include both antagonist molecules for the treatment of autoimmune diseases and agonist molecules for immuno-oncology, yet the relationship between CD40 epitope and these opposing biological outcomes is not well defined. This report describes the identification of potent antagonist domain antibodies (dAbs) that bind to a novel human CD40-specific epitope that is divergent in the CD40 of nonhuman primates. A similarly selected anti-cynomolgus CD40 dAb recognizing the homologous epitope is also a potent antagonist.

Identification and synthesis of potent and selective pyridyl-isoxazole based agonists of sphingosine-1-phosphate 1 (S1P1).

The synthesis and structure-activity relationship (SAR) of a series of pyridyl-isoxazole based agonists of S1P1 are discussed. Compound 5b provided potent in vitro activity with selectivity, had an acceptable pharmacokinetic profile, and demonstrated efficacy in a dose dependent manner when administered orally in a rodent model of arthritis.

Potent and Selective Agonists of Sphingosine 1-Phosphate 1 (S1P1): Discovery and SAR of a Novel Isoxazole Based Series.

Sphingosine 1-phosphate (S1P) is the endogenous ligand for the sphingosine 1-phosphate receptors (S1P1-5) and evokes a variety of cellular responses through their stimulation. The interaction of S1P with the S1P receptors plays a fundamental physiological role in a number of processes including vascular development and stabilization, lymphocyte migration, and proliferation. Agonism of S1P1, in particular, has been shown to play a significant role in lymphocyte trafficking from the thymus and secondary lymphoid organs, resulting in immunosuppression.

Integrated Pharmacokinetic/Pharmacodynamic Analysis for Determining the Minimal Anticipated Biological Effect Level of a Novel Anti-CD28 Receptor Antagonist BMS-931699.

BMS-931699 (lulizumab pegol), a domain antibody (dAb) conjugated with 40-kDa branched polyethylene glycol, is a human anti-CD28 receptor antagonist under development for the treatment of inflammatory and autoimmune diseases. In the present work, the minimal anticipated biologic effect level (MABEL) was determined for BMS-931699 by integrating all the available preclinical data. The relevance of the in vitro mixed lymphocyte reaction (MLR) assay to a whole blood CD28 receptor occupancy (RO) assessment, as well as the relationship between the CD28 RO and the inhibition of T-cell-dependent antibody response to keyhole limpet hemocyanin in vivo, was demonstrated through an integrated pharmacokinetic/pharmacodynamic analysis using anti-hCD28 dAb-001 (differing from BMS-931699 by two additional amino acids at the N-terminus) and a mouse surrogate.

Inhibition of Donor-Reactive CD8+ T Cell Responses by Selective CD28 Blockade Is Independent of Reduced ICOS Expression.

Programmed T cell differentiation is critically influenced by the complement of costimulatory and coinhibitory signals transmitted during initial antigen encounter. We previously showed that selective CD28 blockade with novel domain antibodies that leave CTLA-4-mediated coinhibitory signaling intact resulted in more profound attenuation of donor-reactive T cell responses and improved graft survival in a murine transplant model. Selective CD28 blockade was also associated with decreased ICOS expression on donor-reactive CD8+ T cell responses as compared to CTLA-4 Ig, but the functional importance of this reduced ICOS expression was not known.

Engineering of a novel anti-CD40L domain antibody for treatment of autoimmune diseases.

CD40-CD40L interactions play a critical role in regulating immune responses. Blockade of CD40L by Abs, such as the anti-CD40L Ab 5c8, demonstrated positive clinical effects in patients with autoimmune diseases; however, incidents of thromboembolism (TE) precluded further development of these molecules. In this study, we examined the role of the Fc domain interaction with FcγRs in modulating platelet activation and potential for TE.

2B4 (CD244) induced by selective CD28 blockade functionally regulates allograft-specific CD8+ T cell responses.

Mounting evidence in models of both autoimmunity and chronic viral infection suggests that the outcome of T cell activation is critically impacted by the constellation of co-stimulatory and co-inhibitory receptors expressed on the cell surface. Here, we identified a critical role for the co-inhibitory SLAM family member 2B4 (CD244) in attenuating primary antigen-specific CD8(+) T cell responses in the presence of immune modulation with selective CD28 blockade. Our results reveal a specific up-regulation of 2B4 on antigen-specific CD8(+) T cells in animals in which CD28 signaling was blocked.

A monovalent anti-human CD28 domain antibody antagonist: preclinical efficacy and safety.

Targeting the CD28-CD80/86 pathway with an anti-CD28 antagonist is a promising alternative to current therapies for autoimmunity. However, attempts at generating conventional anti-CD28 mAbs lacking stimulatory activity has been challenging. In this study, we describe anti-human CD28 receptor antagonist domain Abs (dAbs) that are specific for human CD28.

Pellet digestion: a simple and efficient sample preparation technique for LC-MS/MS quantification of large therapeutic proteins in plasma.

Background: There is a need for a simple and efficient sample preparation technique for LC-MS/MS quantification of large therapeutic proteins in plasma.

Results: The sample preparation technique presented here is based upon trypsin digestion of the pellet obtained following precipitation of the protein analyte from plasma. The pellet digestion technique was shown to facilitate efficient digestion of large therapeutic proteins, with concomitant removal of a substantial amount of potentially problematic plasma phospholipids.

Small molecule antagonist of leukocyte function associated antigen-1 (LFA-1): structure-activity relationships leading to the identification of 6-((5S,9R)-9-(4-cyanophenyl)-3-(3,5-dichlorophenyl)-1-methyl-2,4-dioxo-1,3,7-triazaspiro[4.4]nonan-7-yl)nicotinic acid (BMS-688521).

Leukocyte function-associated antigen-1 (LFA-1), also known as CD11a/CD18 or alpha(L)beta(2), belongs to the beta(2) integrin subfamily and is constitutively expressed on all leukocytes. The major ligands of LFA-1 include three intercellular adhesion molecules 1, 2, and 3 (ICAM 1, 2, and 3). The interactions between LFA-1 and the ICAMs are critical for cell adhesion, and preclinical animal studies and clinical data from the humanized anti-LFA-1 antibody efalizumab have provided proof-of-concept for LFA-1 as an immunological target.

An LFA-1 (alphaLbeta2) small-molecule antagonist reduces inflammation and joint destruction in murine models of arthritis.

LFA-1 appears to play a central role in normal immune responses to foreign Ags. In autoimmune or inflammatory diseases, there is increased expression of LFA-1 and/or its counterligand, ICAM-1. Others have demonstrated that the targeted disruption of LFA-1:ICAM interactions, either by gene deletion or Ab treatment in mice, results in reduced leukocyte trafficking, inflammatory responses, and inhibition of inflammatory arthritis in the K/BxN serum transfer model.

Assessment of belatacept-mediated costimulation blockade through evaluation of CD80/86-receptor saturation.

Background: The selective inhibitor of T-cell costimulation, belatacept, blocks CD28-mediated T-cell activation by binding CD80 and CD86 on antigen-presenting cells. Understanding the extent to which belatacept binds to its targets in patients may enable correlation of belatacept exposure to receptor saturation as a pharmacodynamic measure of costimulation blockade.

Methods: Flow cytometry-based receptor competition assays were developed to monitor concentration-dependent occupancy of CD80 and CD86 receptors in whole blood and dendritic cell cultures in vitro.

Abatacept does not induce direct gene expression changes in antigen-presenting cells.

Background: It has been proposed that ligation of CD80 and CD86 induces reverse signaling into antigen-presenting cells. In this study, we tested the ability of abatacept, a soluble human fusion protein comprising the extracellular domain of cytotoxic T lymphocyte antigen 4 and a fragment of the Fc domain of IgG(1), to activate antigen-presenting cells by measuring changes in global transcriptional responses.

Methods: Affymetrix chips were used to measure gene expression levels using mRNA isolated from immature and mature human dendritic cells and a B cell line following 6 h of treatment with abatacept.

Abatacept modulates human dendritic cell-stimulated T-cell proliferation and effector function independent of IDO induction.

Abatacept, the first in a new class of agents for RA, modulates CD28-mediated T-cell costimulation. Abatacept was evaluated for its ability to regulate human T-cell proliferation and cytokine production initiated by dendritic cells. Abatacept reduced T-cell proliferation by >95% at concentrations between 0.

Abatacept binds to the Fc receptor CD64 but does not mediate complement-dependent cytotoxicity or antibody-dependent cellular cytotoxicity.

Objective: To assess the ability of abatacept to mediate complement-dependent cytotoxicity (CDC) or antibody-dependent cellular cytotoxicity (ADCC) of antigen-presenting cells, and to characterize the binding of abatacept to the 3 Fc receptor classes.

Methods: CDC was measured in vitro using rabbit, baby rabbit, guinea pig, or human complement with human B cell line PM-LCL as the target. ADCC was also measured with PM-LCL target cells, but with human peripheral blood mononuclear cells from 12 healthy blood donors as effectors.

Design of LFA-1 antagonists based on a 2,3-dihydro-1H-pyrrolizin-5(7aH)-one scaffold.

A new class of lymphocyte function-associated antigen-1 (LFA-1) antagonists is described. Elaboration of the 2,3-dihydro-1H-pyrrolizin-5(7aH)-one scaffold resulted in the synthesis of potent inhibitors of the LFA-1/ICAM-1 interaction. Along with the in vitro activity, we present the X-ray crystal structure of the complex of compound 9b, in a novel binding mode to the I-domain of LFA-1.

Discovery and development of 5-[(5S,9R)-9-(4-cyanophenyl)-3-(3,5-dichlorophenyl)-1-methyl-2,4-dioxo-1,3,7-triazaspiro[4.4]non-7-yl-methyl]-3-thiophenecarboxylic acid (BMS-587101)--a small molecule antagonist of leukocyte function associated antigen-1.

LFA-1 (leukocyte function-associated antigen-1), is a member of the beta2-integrin family and is expressed on all leukocytes. This letter describes the discovery and preliminary SAR of spirocyclic hydantoin based LFA-1 antagonists that culminated in the identification of analog 8 as a clinical candidate. We also report the first example of the efficacy of a small molecule LFA-1 antagonist in combination with CTLA-4Ig in an animal model of transplant rejection.

De novo design, synthesis, and in vitro activity of LFA-1 antagonists based on a bicyclic[5.5]hydantoin scaffold.

LFA-1 (leukocyte function-associated antigen-1), is a member of the beta(2)-integrin family and is expressed on all leukocytes. The LFA-1/ICAM interaction promotes tight adhesion between activated leukocytes and the endothelium, as well as between T cells and antigen-presenting cells. Evidence from both animal models and clinical trials provides support for LFA-1 as a target in several different inflammatory diseases.

Potent reversible inhibitors of the protein tyrosine phosphatase CD45.

The cytosolic portion of CD45, a major transmembrane glycoprotein found on nucleated hematopoietic cells, contains protein tyrosine phosphatase activity and is critical for T-cell receptor-mediated T-cell activation. CD45 inhibitors could have utility in the treatment of autoimmune disorders and organ graft rejection. A number of 9,10-phenanthrenediones were identified that reversibly inhibited CD45-mediated p-nitrophenyl phosphate (pNPP) hydrolysis.

Role of p53 in the regulation of irradiation-induced apoptosis in neuroblastoma cells.

Wild-type p53 plays a crucial role in the control of apoptosis following ionizing radiation (IR); conversely, mutant p53 is associated with IR resistance. Although wild-type p53 is expressed in virtually all neuroblastoma tumors, treatment failures secondary to inadequate local control with radiotherapy are a problem in patients with advanced stage disease. This apparent paradox is the focus of our interest.