Daratumumab is a CD38‐targeted human monoclonal antibody with direct anti‐myeloma cell mechanisms of action. Flow cytometry in relapsed and/or refractory multiple myeloma (RRMM) patients treated with daratumumab revealed cytotoxic T‐cell expansion and reduction of immune‐suppressive populations, suggesting immune modulation as an additional mechanism of action. Here, we performed an in‐depth analysis of the effects of daratumumab on immune‐cell subpopulations using high‐dimensional mass cytometry. Whole‐blood and bone‐marrow baseline and on‐treatment samples from RRMM patients who participated in daratumumab monotherapy studies (SIRIUS and GEN501) were evaluated with high‐throughput immunophenotyping. In daratumumab‐treated patients, the intensity of CD38 marker expression decreased on many immune cells in SIRIUS whole‐blood samples. Natural killer (NK) cells were depleted with daratumumab, with remaining NK cells showing increased CD69 and CD127, decreased CD45RA, and trends for increased CD25, CD27, and CD137 and decreased granzyme B. Immune‐suppressive population depletion paralleled previous findings, and a newly observed reduction in CD38 + basophils was seen in patients who received monotherapy. After 2 months of daratumumab, the T‐cell population in whole‐blood samples from responders shifted to a CD8 prevalence with higher granzyme B positivity ( P = 0.017), suggesting increased killing capacity and supporting monotherapy‐induced CD8 + T‐cell activation. High‐throughput cytometry immune profiling confirms and builds upon previous flow cytometry data, including comparable CD38 marker intensity on plasma cells, NK cells, monocytes, and B/T cells. Interestingly, a shift toward cytolytic granzyme B + T cells was also observed and supports adaptive responses in patients that may contribute to depth of response. © 2018 The Authors. Cytometry Part A published by Wiley Periodicals, Inc. on behalf of International Society for Advancement of Cytometry.
Prolyl oligopeptidase (EC 3.4.21.26) activity was measured in human tissue homogenates and body fluids. The enzyme was ubiquitously present, revealing high activity in renal cortex, epithelial cells, fibroblasts, testis, lymphocytes and thrombocytes. The activity in the body fluids was low. Prolyl oligopeptidase activity was significant higher in tumours of prostate, lung and sigmoid, than in the healthy tissues. Sera of individuals suffering from HIV infection, malaria, prostate cancer or benign prostate hypertrophy contained lowered activity. Interestingly, the low serum activity during prostate carcinoma increased upon medical treatment with anti-androgens. This suggests hormonal control of the gene transcript. A positive correlation with angiotensin converting enzyme activity in hypertensive patients was demonstrated and this further supports the possible involvement of prolyl oligopeptidase in the renin-angiotensin system and in the pathogenesis of hypertension.
CD38-targeted antibody, daratumumab, is approved for the treatment of multiple myeloma (MM). Phase 1/2 studies GEN501/SIRIUS revealed a novel immunomodulatory mechanism of action (MOA) of daratumumab that enhanced the immune response, reducing natural killer (NK) cells without affecting efficacy or safety. We further evaluated daratumumab’s effects on immune cells in whole blood samples of relapsed/refractory MM patients from both treatment arms of the phase 3 POLLUX study (lenalidomide/dexamethasone [Rd] or daratumumab plus Rd [D-Rd]) at baseline (D-Rd, 40; Rd, 45) and after 2 months on treatment (D-Rd, 31; Rd, 33) using cytometry by time-of-flight. We confirmed previous reports of NK cell reduction with D-Rd. Persisting NK cells were phenotypically distinct, with increased expression of HLA-DR, CD69, CD127, and CD27. The proportion of T cells increased preferentially in deep responders to D-Rd, with a higher proportion of CD8+ versus CD4+ T cells. The expansion of CD8+ T cells correlated with clonality, indicating generation of adaptive immune response with D-Rd. D-Rd resulted in a higher proportion of effector memory T cells versus Rd. D-Rd reduced immunosuppressive CD38+ regulatory T cells. This study confirms daratumumab’s immunomodulatory MOA in combination with immunomodulatory drugs and provides further insight into immune cell changes and activation status following daratumumab-based therapy.
Converting the complete genome sequence of Candida albicans into meaningful biological information will require comprehensive screens for identifying functional classes of genes. Most systems described so far are not applicable to C. albicans because of its difficulty with mating, its diploid nature, and the lack of functional random insertional mutagenesis methods. We examined artificial gene suppression as a means to identify gene products critical for growth of this pathogen; these represent new antifungal drug targets. To achieve gene suppression we combined antisense RNA inhibition and promoter interference. After cloning antisense complementary DNA (cDNA) fragments under control of an inducible GAL1 promoter, we transferred the resulting libraries to C. albicans. Over 2,000 transformant colonies were screened for a promoter-induced diminished-growth phenotype. After recovery of the plasmids, sequence determination of their inserts revealed the messenger RNA (mRNA) they inhibited or the gene they disrupted. Eighty-six genes critical for growth were identified, 45 with unknown function. When used in high-throughput screening for antifungals, the crippled C. albicans strains generated in this study showed enhanced sensitivity to specific drugs.
Structure-guided design led to the identification of the novel, potent, and selective phosphodiesterase 2 (PDE2) inhibitor 12. Compound 12 demonstrated a >210-fold selectivity versus PDE10 and PDE11 and was inactive against all other PDE family members up to 10 μM. In vivo evaluation of 12 provided evidence that it is able to engage the target and to increase cGMP levels in relevant brain regions. Hence, 12 is a valuable tool compound for the better understanding of the role of PDE2 in cognitive impairment and other central nervous system related disorders.
The relationship between CD26/dipeptidyl peptidase IV, an ectopeptidase involved in T cell activation, and the binding protein for adenosine deaminase (ADAbp) was studied. Monoclonal antibodies (mAb) against CD26 and ADAbp, respectively, showed a similar binding profile on various lymphocyte subsets from the peripheral blood. The adenosine deaminase (ADA) itself blocked the binding of a specific set of anti-CD26 mAb (among these the anti-TA5.9 mAb) on lymphocytic CD26; ADA also hindered the binding of soluble CD26 to the same immobilized anti-CD26 mAb. In addition, the interaction between immobilized ADA and purified CD26/DPP IV was inhibited by the anti-TA5.9 mAb. ADA did not inhibit the specific peptidase activity of CD26. Neither soluble nor immobilized ADA was able to down-modulate CD26 on the lymphocyte surface. Our data thus confirm the identity between ADAbp and CD26 and identify some epitopes, crucial in the binding of ADA to CD26.
A novel series of pyrido [4,3-e][1,2,4]triazolo- [4,3-a]pyrazines is reported as potent PDE2/PDE10 inhibitors with drug-like properties. Selectivity for PDE2 was obtained by introducing a linear, lipophilic moiety on the meta-position of the phenyl ring pending from the triazole. The SAR and protein flexibility were explored with free energy perturbation calculations. Rat pharmacokinetic data and in vivo receptor occupancy data are given for two representative compounds 6 and 12.
The proline-specific peptidases, aminopeptidase P (EC 3.4.11.9) and dipeptidyl peptidase IV (EC 3.4.14.5), were measured in human tissue homogenates and physiological fluids. All tissues examined contained measurable aminopeptidase P and dipeptidyl peptidase IV activities. High specific activities for both enzymes under study were found in benign prostatic hypertrophy. Normal prostate and prostatic adenocarcinoma had a much lower activity. This difference, however, is not reflected in the serum values of the patients. The most striking finding is the extremely high activity of dipeptidyl peptidase IV in prostatosomes, prostate-derived organelles, which occur freely in human seminal plasma, and which are important for enhancement of sperm forward motility.
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