Bispecific antibodies, while showing great therapeutic potential, pose formidable challenges with respect to their assembly, stability, immunogenicity, and pharmacodynamics. Here we describe a novel class of bispecific antibodies with native human immunoglobulin format. The design exploits differences in the affinities of the immunoglobulin isotypes for Protein A, allowing efficient large-scale purification. Using this format, we generated a bispecific antibody, REGN1979, targeting the B cell marker, CD20, and the CD3 component of the T cell receptor, which triggers redirected killing of B cells. In mice, this antibody prevented growth of B cell tumors and also caused regression of large established tumors. In cynomolgus monkeys, low doses of REGN1979 caused prolonged depletion of B cells in peripheral blood with a serum half-life of approximately 14 days. Further, the antibody induced a deeper depletion of B cells in lymphoid organs than rituximab. This format has broad applicability for development of clinical bispecific antibodies.
According to a report, statistics have shown that there is a decrease in the percentage of teens who have had sex and an increase in contraceptive use at first sex among teens. However, it also found that teens are inconsistent in contraceptive use. 1 in every 2 teens surveyed by the National Campaign to Prevent Teen Pregnancy cites pressure from partners as a main reason for not using contraception. Drinking and drug use also play a large role, as noted by more than 50% of the teens. The nationally representative survey questioned 515 teens aged 12-17 years. While the poll showed that nearly 9 of 10 teens believe it is important to use contraception each and every time they have sex, they are not following through in their actions. In addition, the report notes that Hispanic and very young teens are at particularly high risk of unplanned pregnancy. In view of this, it remains challenging to ensure adequate access to contraceptive care, particularly care provided in a confidential and nonjudgmental manner. The priority should always be to encourage teens to delay sexual activity and to protect their physical health, their emotional health, and their opportunities for the future.
Abstract Few biological markers of immune function have been thoroughly validated for use in epidemiologic studies that involve delayed sample processing and analysis. Here, we report our validation results for flow cytometric detection of intracellular T-helper 1/T-helper 2 (Th1/Th2) cytokines using 500 μL of whole blood obtained from children and adults. The detection of Th1/Th2 cytokine profiles by flow cytometry is a practical and mechanistically relevant assay because dysregulated cytokine production has been observed in many immune-mediated disorders, including cancer. We evaluated the intraassay and intraindividual and interindividual variability and the effects of a 24- to 72-hour delayed analysis on Th1 and Th2 end points. We compared the distributions of %CD4 lymphocytes, %Th1, and %Th2 in young children (age 1 year, n = 50) and adults (age 25–52 years, n = 16). Subjects sampled monthly for up to 1 year showed minimal variation in CD4, Th1, and Th2 end points. Delayed analysis of samples (up to 24 hours) resulted in no significant differences in the expression of CD4, Th1, and Th2; however, at 48 and 72 hours, all end points differed significantly from baseline (P < 0.01). A random effects model confirmed that interindividual variability was much greater than intraindividual variability for CD4 and Th1. Compared with adults, children had marginally higher %CD4, similar %Th2, but significantly lower %Th1 (P < 0.01). These results show that flow cytometric detection of CD4, Th1, and Th2 markers using whole blood is reproducible and that these biomarkers can be effectively used in human population studies that involve transported samples, delayed processing and analysis, and limited blood volumes.
Background— Remodeling of the arterial extracellular matrix participates importantly in atherogenesis and plaque complication. Increased expression of the elastinolytic and collagenolytic enzyme cathepsin L (Cat L) in human atherosclerotic lesions suggests its participation in these processes, a hypothesis tested here in mice. Methods and Results— We generated Cat L and low-density lipoprotein receptor (LDLr) double-deficient (LDLr −/− Cat L −/− ) mice by crossbreeding Cat L–null (Cat L −/− ) and LDLr-deficient (LDLr −/− ) mice. After 12 and 26 weeks of a Western diet, LDLr −/− Cat L −/− mice had significantly smaller atherosclerotic lesions and lipid cores compared with littermate control LDLr −/− Cat L +/− and LDLr −/− Cat L +/+ mice. In addition, lesions from the compound mutant mice showed significantly reduced levels of collagen, medial elastin degradation, CD4 + T cells, macrophages, and smooth muscle cells. Mechanistic studies showed that Cat L contributes to the degradation of extracellular matrix elastin and collagen by aortic smooth muscle cells. Smooth muscle cells from LDLr −/− Cat L −/− mice or those treated with a Cat L–selective inhibitor demonstrated significantly less degradation of elastin and collagen and delayed transmigration through elastin in vitro. Cat L deficiency also significantly impaired monocyte and T-lymphocyte transmigration through a collagen matrix in vitro, suggesting that blood-borne leukocyte penetration through the arterial basement membrane requires Cat L. Cysteine protease active site labeling demonstrated that Cat L deficiency did not affect the activity of other atherosclerosis-associated cathepsins in aortic smooth muscle cells and monocytes. Conclusions— Cat L directly participates in atherosclerosis by degrading elastin and collagen and regulates blood-borne leukocyte transmigration and lesion progression.
Benzene is an established human carcinogen, producing leukemia, hematotoxicity and perhaps lymphoma. Its carcinogenicity is most likely dependent upon its conversion to phenol and hydroquinone, the latter being oxidized to the highly toxic 1,4-benzoquinone in the bone marrow. Exposure of human lymphocytes and cell lines to hydroquinone has previously been shown to cause various forms of genetic damage, including aneusomy and the loss and gain of chromosomes. However, the target cells for leukemogenesis are the pluripotent stem cells or early progenitor cells which carry the CD34 antigen (CD34 + cells). In this study, human cord blood, which is particularly rich in CD34 + cells, was exposed to hydroquinone for 72 h in a medium that favored CD34 + cell survival and growth. CD34 + and CD34 – cells were then isolated. Fluorescence in situ hybridization was employed to determine the level of aneusomy of chromosomes 7 and 8 in both cell types. CD34 + cells were generally more susceptible to aneusomy induction by hydroquinone than CD34 – cells. Increased trisomy and monosomy of chromosomes 7 and 8 were observed in CD34 + cells ( Ptrend < 0.001), whereas in CD34 – cells only an increased level of monosomy 7 was detected ( Ptrend = 0.002). Particularly striking effects of hydroquinone were observed in CD34 + cells on monosomy 7 and trisomy 8, two common clonal aberrations found in myeloid leukemias, suggesting that these aneusomies produced by hydroquinone in CD34 + cells play a role in benzene-induced leukemogenesis.
The immune response plays an important role in the pathophysiology of numerous diseases including asthma, autoimmunity and cancer. Application of biomarkers of immunotoxicity in epidemiology studies and human clinical trials can improve our understanding of the mechanisms that underlie the associations between environmental exposures and development of these immune-mediated diseases. Immunological biomarkers currently used in environmental health studies include detection of key components of innate and adaptive immunity (e.g., complement, immunoglobulin and cell subsets) as well as functional responses and activation of key immune cells. The use of high-throughput assays, including flow cytometry, Luminex, and Multi-spot cytokine detection methods can further provide quantitative analysis of immune effects. Due to the complexity and redundancy of the immune response, an integrated assessment of several components of the immune responses is needed. The rapidly expanding field of immunoinformatics will also aid in the synthesis of the vast amount of data being generated. This review discusses and provides examples of how the identification and development of immunological biomarkers for use in studies of environmental exposures and immune-mediated disorders can be achieved.
