A Novel Affinity Engineered Anti-CD47 Antibody With Improved Therapeutic Index That Preserves Erythrocytes and Normal Immune Cells.

Therapeutic blockade of the CD47/SIRPα axis by small molecules or monoclonal antibodies (mAbs) is a proven strategy to enhance macrophages-mediated anti-tumor activity. However, this strategy has been hampered by elevated on-target toxicities and rapid clearance due to the extensive CD47 expression on normal cells ("antigen sink") such as red blood cells (RBCs). To address these hurdles, we report on the development of STI-6643, an affinity-engineered fully human anti-CD47 IgG4 antibody with negligible binding to normal cells. STI-6643 exhibited no hemagglutination activity on human RBCs at concentrations up to 300 µg/mL yet specifically blocked the CD47/SIPRα interaction. Of particular interest, STI-6643 preserved T cell functionality in vitro and showed significantly lower immune cell depletion in vivo in contrast to three previously published competitor reference anti-CD47 clones Hu5F9, AO-176 and 13H3. In cynomolgus monkeys, STI-6643 was well-tolerated at the highest dose tested (300 mg/kg/week) and provided favorable clinical safety margins. Finally, STI-6643 displayed comparable anti-tumor activity to the high-affinity reference clone Hu5F9 in a RAJI-Fluc xenograft tumor model as monotherapy or in combination with anti-CD20 (rituximab) or anti-CD38 (daratumumab) mAbs. These data suggest that STI-6643 possesses the characteristics of an effective therapeutic candidate given its potent anti-tumor activity and low toxicity profile.

Distinct functions of activated protein C differentially attenuate acute kidney injury.

Administration of activated protein C (APC) protects from renal dysfunction, but the underlying mechanism is unknown. APC exerts both antithrombotic and cytoprotective properties, the latter via modulation of protease-activated receptor-1 (PAR-1) signaling. We generated APC variants to study the relative importance of the two functions of APC in a model of LPS-induced renal microvascular dysfunction. Compared with wild-type APC, the K193E variant exhibited impaired anticoagulant activity but retained the ability to mediate PAR-1-dependent signaling. In contrast, the L8W variant retained anticoagulant activity but lost its ability to modulate PAR-1. By administering wild-type APC or these mutants in a rat model of LPS-induced injury, we found that the PAR-1 agonism, but not the anticoagulant function of APC, reversed LPS-induced systemic hypotension. In contrast, both functions of APC played a role in reversing LPS-induced decreases in renal blood flow and volume, although the effects on PAR-1-dependent signaling were more potent. Regarding potential mechanisms for these findings, APC-mediated PAR-1 agonism suppressed LPS-induced increases in the vasoactive peptide adrenomedullin and infiltration of iNOS-positive leukocytes into renal tissue. However, the anticoagulant function of APC was responsible for suppressing LPS-induced stimulation of the proinflammatory mediators ACE-1, IL-6, and IL-18, perhaps accounting for its ability to modulate renal hemodynamics. Both variants reduced active caspase-3 and abrogated LPS-induced renal dysfunction and pathology. We conclude that although PAR-1 agonism is solely responsible for APC-mediated improvement in systemic hemodynamics, both functions of APC play distinct roles in attenuating the response to injury in the kidney.

Parent Psychological and Physical Health Outcomes in Pediatric Hematopoietic Stem Cell Transplantation.

BACKGROUND: Parents of children undergoing hematopoietic stem cell transplantation (HSCT) are at risk of adverse health outcomes due to their intense caregiver demands. OBJECTIVE: The aim of this study was to describe adverse health outcomes in parents of children who survived an allogeneic HSCT done within the past 1 to 10 years. METHODS: This cross-sectional study, conducted at a children's hospital in the western United States, enrolled English- and Spanish-speaking parents of children who survived allogeneic HSCT between 2005 and 2015. Outcome measures included Beck Anxiety and Depression Inventories, Perceived Stress and Parent Stress Scales, Physical Symptom Inventory, and Short-Form 36 version 2. Parent scores were compared with normative means. Subsequently, the parent sample was stratified by the amount of time since their child's HSCT for comparison between groups. RESULTS: Fifty-four mothers and 7 fathers (n = 61) were enrolled. Global mental health scores were lower for parents in the sample compared with norms (P = .003). Parents in the sample reported moderate anxiety and depression (20% and 23%, respectively), yet reported less parenting stress and superior health outcomes compared with norms (P < .001). Social functioning and general health scores were lower for parents whose children survived an allogeneic HSCT done within the past 1 to 4.99 years (P = .012). CONCLUSION: Parents of survivors of allogeneic HSCT may concurrently experience posttraumatic growth and stress following their child's HSCT. IMPLICATIONS FOR PRACTICE: Health screening and psychological support for parents of children post-HSCT may help to identify parents at risk of adverse outcomes and allow for early, targeted interventions.

Hospitalization Among HIV-Infected U.S. Marshals Service Prisoners.

The U.S. Marshals Service (USMS) prisoner population is diverse and includes immigration violators, fugitives that have evaded apprehension, perpetrators of Medicaid fraud, and parole and probation violators. Unlike state and local jails, the USMS has numerous housing options for its prisoners. Given the unique characteristics, federal prisoners' quality of care, and subsequent clinical outcomes, may differ from those of state and local inmates. However, little is known about hospitalization rates and length of stay for HIV-positive USMS prisoners. The purpose of this study is to examine hospitalizations among HIV-infected prisoners in the custody of the USMS.

Sex differences in responses to epidural steroid injection for low back pain.

UNLABELLED: Sex differences in clinical and experimental pain responses have been widely reported; however, few studies have examined sex differences in outcomes from interventional pain treatment and the predictors thereof. The aims of this study were to examine sex differences in (1) the acute pain produced by epidural steroid injections (ESIs), (2) clinical improvements in pain and pain-related psychological distress and disability after ESIs, and (3) predictors of the clinical response to ESIs. A total of 57 patients (37 menopausal women and 20 men), seen in the pain clinic of a regional medical center for ESI therapy, participated. Patients rated the painfulness of the ESI procedure itself. Also, clinical pain, depression, and disability were assessed before treatment and at 2 weeks and 2 months after the ESIs. Participants also were queried about their expectations of successful pain relief, coping strategies, and pain-related anxiety, which were examined as predictors of treatment outcome. Men reported significantly greater pain intensity and unpleasantness than women for the first injection only. All groups showed significant reductions in clinical pain, depression, and disability at 2 weeks compared to baseline, but minimal change occurred between 2 weeks and 2 months past baseline. No sex differences in the magnitude of treatment response emerged; however, specific dimensions of pain coping were associated with treatment responses in a sex-dependent manner. These findings suggest that the determinants of ESI pain and treatment outcome might differ across sex. PERSPECTIVE: Sex-related influences on pain responses have been widely reported, but few studies have explored sex-dependent predictors of treatment response. These findings indicate that pain coping was differentially associated with outcomes after ESI in women versus men.

Optimizing an artificial metabolic pathway: engineering the cofactor specificity of Corynebacterium 2,5-diketo-D-gluconic acid reductase for use in vitamin C biosynthesis.

The strict cofactor specificity of many enzymes can potentially become a liability when these enzymes are to be employed in an artificial metabolic pathway. The preference for NADPH over NADH exhibited by the Corynebacterium 2,5-diketo-D-gluconic acid (2,5-DKG) reductase may not be ideal for use in industrial scale vitamin C biosynthesis. We have previously reported making a number of site-directed mutations at five sites located in the cofactor-binding pocket that interact with the 2'-phosphate group of NADPH. These mutations conferred greater activity with NADH upon the Corynebacterium 2,5-DKG reductase [Banta, S., Swanson, B. A., Wu, S., Jarnagin, A., and Anderson, S. (2002) Protein Eng. 15, 131-140; (1)]. The best of these mutations have now been combined to see if further improvements can be obtained. In addition, several chimeric mutants have been produced that contain the same residues as are found in other members of the aldo-keto reductase superfamily that are naturally able to use NADH as a cofactor. The most active mutants obtained in this work were also combined with a previously reported substrate-binding pocket double mutant, F22Y/A272G. Mutant activity was assayed using activity-stained native polyacrylamide gels. Superior mutants were purified and subjected to a simplified kinetic analysis. The simplified kinetic analysis was extended for the most active mutants in order to obtain the kinetic parameters in the full-ordered bi bi rate equation in the absence of products, with both NADH and NADPH as cofactors. The best mutant 2,5-DKG reductase produced in this work was the F22Y/K232G/R238H/A272G quadruple mutant, which exhibits activity with NADH that is more than 2 orders of magnitude higher than that of the wild-type enzyme, and it retains a high level of activity with NADPH. This new 2,5-DKG reductase may be a valuable new catalyst for use in vitamin C biosynthesis.

Alteration of the specificity of the cofactor-binding pocket of Corynebacterium 2,5-diketo-D-gluconic acid reductase A.

The NADPH-dependent 2,5-diketo-D-gluconic acid (2,5-DKG) reductase enzyme is a required component in some novel biosynthetic vitamin C production processes. This enzyme catalyzes the conversion of 2,5-DKG to 2-keto-L-gulonic acid, which is an immediate precursor to L-ascorbic acid. Forty unique site-directed mutations were made at five residues in the cofactor-binding pocket of 2,5-DKG reductase A in an attempt to improve its ability to use NADH as a cofactor. NADH is more stable, less expensive and more prevalent in the cell than is NADPH. To the best of our knowledge, this is the first focused attempt to alter the cofactor specificity of a member of the aldo-keto reductase superfamily by engineering improved activity with NADH into the enzyme. Activity of the mutants with NADH or NADPH was assayed using activity-stained native polyacrylamide gels. Eight of the mutants at three different sites were identified as having improved activity with NADH. These mutants were purified and subjected to a kinetic characterization with NADH as a cofactor. The best mutant obtained, R238H, produced an almost 7-fold improvement in catalysis with NADH compared with the wild-type enzyme. Surprisingly, most of this catalytic improvement appeared to be due to an improvement in the apparent kcat for the reaction rather than a large improvement in the affinity of the enzyme for NADH.