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ABSTRACT As a principal metabolite of ginsenosides, compound K (CK) exhibits remarkable antioxidant, anti‐inflammatory, antidiabetic, and anticancer bioactivities. However, CK is naturally scarce in ginseng and its industrial application is limited by the lack of efficient deglycosylating enzymes for large‐scale CK bioproduction. Here, we report a synthetic biology‐driven approach for high‐yield CK production via recombinant β‐glycosidase‐mediated bioconversion of the polar ginsenoside Rb1. The β‑glycosidase‐encoding gene was introduced into Escherichia coli BL21 (DE3), and the resulting engineered strain produced 2.1 g/L β‑glycosidase in shake flasks and 10.1 g/L in a 5‐L fermentor. The crude β‐glycosidase exhibited excellent enzymatic activity; at a concentration of 1.5 g/L, it converted 95% of the Rb1 substrate (initial concentration 10 g/L) to CK within 4 h. In vitro studies demonstrated CK's high cytocompatibility and potent anti‐aging effects, including suppression of reactive oxygen species ( via upregulation of superoxide dismutase, catalase, and glutathione peroxidase), enhanced collagen I, Ⅲ and elastin synthesis, and matrix metalloproteinase‐1 inhibition. These findings highlight CK as a promising candidate for anti‐wrinkle formulations, supported by the feasibility of scalable bioproduction.

Introduction Despite improvements in outcomes with triplet and quadruplet combination regimens in front-line (1L) multiple myeloma (MM) treatment (Tx), pts with disease that relapses early after 1L Tx or responds inadequately to 1L autologous stem cell transplantation (ASCT) have poor prognosis. Therefore, Txs with novel mechanisms of action are needed. Ide-cel, a BCMA-directed CAR T cell therapy, resulted in deep and durable responses in pts with functional high-risk MM. KarMMa-2 (NCT03601078) is a phase 2, multicenter, multicohort trial of ide-cel: cohort 2a includes pts who progressed ≤ 18 mo of starting 1L therapy with ASCT, cohort 2b includes pts who progressed ≤ 18 mo of starting 1L therapy without ASCT, and cohort 2c includes pts who achieved less than very good partial response ~100 d after 1L ASCT. With a median follow-up of 21.5, 30.1, and 39.4 mo, respectively, median PFS (95% CI) for cohorts 2a, 2b, and 2c was 11.4 mo (5.6-19.6), not reached (NR; 12.2-NR), and NR (38.0-NR). Previous correlative analyses of ide-cel in relapsed and refractory MM have shown that pts with lower pre-Tx tumor burden and lower pre-Tx indicators of basal inflammation had better responses. This cross-cohort analysis evaluated tumor burden, baseline inflammatory status, and T-cell quality differences to develop hypotheses for biologic features underpinning robust PFS previously observed with ide-cel. Methods Levels of soluble BCMA (sBCMA) and 25 exploratory cytokines at infusion and post-Tx were measured by immunoassay. Apheresis and drug product material were characterized by high-parameter flow cytometry and immunoassay. Pharmacokinetics (PK) were evaluated by droplet digital polymerase chain reaction. These analyses were exploratory and post hoc in nature; statistical tests were utilized to identify relationships of interest to develop working hypotheses of the underlying biology. Results Baseline tumor burden, evaluated by median sBCMA (interquartile range [IQR]), was highest in cohort 2b at 156 ng/mL (83-300) compared with 2a at 91 ng/mL (41-169) and 2c at 27 ng/mL (19-48). Pts in cohort 2b had the lowest baseline levels of a subset of inflammatory cytokines, including interferon [IFN]-γ and interleukin-4. Phenotypic data from apheresis material showed that, among the 3 cohorts, pts in cohort 2b also had the lowest amount of effector memory T cells positive for CD45RA (TEMRA; terminally differentiated T cells) and CD57+ T cells (senescent T cells), and the highest amount of CCR7+CD27+ T cells (early memory T cells). Cohorts 2a and 2c had similar phenotypic profiles in the apheresis material. Median time from ASCT to infusion (IQR) was 12.1 mo (9.7-14.3) in cohort 2a and 5.8 mo (5.2-6.2) in cohort 2c. Pts in cohort 2b also had a higher CD4:CD8 ratio in the apheresis material vs the other 2 cohorts. Profiling the drug products from pts in cohort 2b showed higher CAR+ T cell frequency and CAR-specific IFN-γ, and tumor necrosis factor-α and granzyme secretion upon stimulation vs 2a and 2c, with a consistent and comparable potency profile among all cohorts. While sBCMA level at infusion was lower in cohort 2c vs 2a, the similarity in inflammatory state and T-cell quality may reflect impacts from recent melphalan use before ASCT and bone marrow recovery after ASCT, as most pts in both cohorts were < 18 mo from ASCT. Robust cell expansion was observed across cohorts. Conclusions The encouraging median PFS outcome previously observed in cohort 2b relative to existing therapies in this high-risk pt population may be driven by the higher T-cell quality and better inflammatory state pre-Tx despite high baseline tumor burden (sBCMA). Pts in cohorts 2c and 2a had comparatively less optimal baseline immune status vs cohort 2b. The promising PFS observed in cohort 2c may be reflective of sufficient T-cell quality to drive optimal responses in the context of relatively low tumor burden at study entry. The median PFS in cohort 2a may reflect the combination of high tumor burden and less optimal baseline inflammatory status. These observations suggest a working model in which ide-cel may demonstrate robust PFS in pts with early-line, high-risk MM and high tumor burden with good baseline immune status and T-cell quality, and in pts with less favorable baseline immune status and T-cell quality when tumor burden is relatively low and stable.

Newborn screening for congenital hypothyroidism remains challenging decades after broad implementation worldwide. Testing protocols are not uniform in terms of targets (TSH and/or T4) and protocols (parallel vs. sequential testing; one or two specimen collection times), and specificity (with or without collection of a second specimen) is overall poor. The purpose of this retrospective study is to investigate the potential impact of multivariate pattern recognition software (CLIR) to improve the post-analytical interpretation of screening results. Seven programs contributed reference data (N = 1,970,536) and two sets of true (TP, N = 1369 combined) and false (FP, N = 15,201) positive cases for validation and verification purposes, respectively. Data were adjusted for age at collection, birth weight, and location using polynomial regression models of the fifth degree to create three-dimensional regression surfaces. Customized Single Condition Tools and Dual Scatter Plots were created using CLIR to optimize the differential diagnosis between TP and FP cases in the validation set. Verification testing correctly identified 446/454 (98%) of the TP cases, and could have prevented 1931/5447 (35%) of the FP cases, with variable impact among locations (range 4% to 50%). CLIR tools either as made here or preferably standardized to the recommended uniform screening panel could improve performance of newborn screening for congenital hypothyroidism.

Abstract Nivolumab, an anti–programmed death-1 (PD-1) monoclonal antibody, showed promising activity in relapsed or refractory (R/R) follicular lymphoma (FL) in a phase 1 study. We conducted a phase 2 trial to further evaluate its efficacy and safety in patients with R/R FL and to explore biomarkers of response. Patients with R/R FL and at least 2 prior lines of therapy, each containing a CD20 antibody or an alkylating agent, were treated with nivolumab 3 mg/kg every 2 weeks. The primary end point was objective response rate (ORR) assessed by an independent radiologic review committee. Biomarker analyses included gene expression profiling and multiplex immunofluorescence studies of pretreatment tumor samples. A total of 92 patients were treated. After a minimum follow-up of 12 months, ORR was 4% (4 of 92 patients). Median progression-free survival (PFS) was 2.2 months (95% confidence interval [CI], 1.9-3.6 months). Median duration of response was 11 months (95% CI, 8-14 months). Exploratory analyses suggested that responders had significantly higher proportion of CD3+ T cells in the tumor microenvironment than nonresponders, but no significant differences in PD-1 or programmed death-ligand 1 expression were observed. High expression of a set of tumor-associated macrophage genes was associated with reduced PFS (hazard ratio, 3.28; 95% CI, 1.76-6.11; P = .001). The safety profile was consistent with previous reports of nivolumab. In conclusion, nivolumab monotherapy was associated with very limited activity in patients with R/R FL. Better understanding of the immune biology of this disease may facilitate the development of effective checkpoint-based strategies. This trial was registered at www.clinicaltrials.gov as #NCT02038946.

Since the start of X-linked adrenoleukodystrophy (ALD) newborn screening in California, more than half of the diagnosed cases were found to have an ATP binding cassette subfamily D member 1 (ABCD1) gene variant of uncertain significance (VUS). To determine retrospectively the likelihood that these were true positive cases, we used a web-based post-analytical tool in Collaborative Laboratory Integrated Reports (CLIR). Confirmatory plasma very long-chain fatty-acids (VLCFA) profiles for ALD screen positive infant boys were run through the CLIR ALD tool. We compared the distribution by ABCD1 variant classification (pathogenic, likely pathogenic, VUS, and no variant) with the CLIR tool score interpretation (non-informative, possibly ALD, likely ALD, and very likely ALD) and the current case diagnosis. The study showed that CLIR tool positive interpretations were consistent with 100% of the pathogenic and likely pathogenic variants on the ABCD1 gene if a more conservative guideline was used. The tool interpretations were also consistent with screened cases that were determined to not have disease (our no-disorder group). The CLIR tool identified 19 diagnosed ALD cases with VUS to be potential false positives, representing a 40% reduction among all diagnosed ALD cases with VUS. The reduction could be extended to 65% if a more aggressive threshold was used. Identifying such preventable false positives could alleviate the follow-up burden for patients, their families, and California Special Care Centers.

Newborn screening for one or more lysosomal disorders has been implemented in several US states, Japan and Taiwan by multiplexed enzyme assays using either tandem mass spectrometry or digital microfluidics. Another multiplex assay making use of immunocapture technology has also been proposed. To investigate the potential variability in performance of these analytical approaches, we implemented three high-throughput screening assays for the simultaneous screening for four lysosomal disorders: Fabry disease, Gaucher disease, mucopolysaccharidosis type I, and Pompe disease. These assays were tested in a prospective comparative effectiveness study using nearly 100,000 residual newborn dried blood spot specimens. In addition, 2nd tier enzyme assays and confirmatory molecular genetic testing were employed. Post-analytical interpretive tools were created using the software Collaborative Laboratory Integrated Reports (CLIR) to determine its ability to improve the performance of each assay vs. the traditional result interpretation based on analyte-specific reference ranges and cutoffs. This study showed that all three platforms have high sensitivity, and the application of CLIR tools markedly improves the performance of each platform while reducing the need for 2nd tier testing by 66% to 95%. Moreover, the addition of disease-specific biochemical 2nd tier tests ensures the lowest false positive rates and the highest positive predictive values for any platform.

Short-chain acyl-CoA dehydrogenase deficiency (SCADD) is a rare autosomal recessive disorder of β-oxidation caused by pathogenic variants in the ACADS gene. Analyte testing for SCADD in blood and urine, including newborn screening (NBS) using tandem mass spectrometry (MS/MS) on dried blood spots (DBSs), is complicated by the presence of two relatively common ACADS variants (c.625G>A and c.511C>T). Individuals homozygous for these variants or compound heterozygous do not have clinical disease but do have reduced short-chain acyl-CoA dehydrogenase (SCAD) activity, resulting in elevated blood and urine metabolites. As part of a larger study of the potential role of exome sequencing in NBS in California, we reviewed ACADS sequence and MS/MS data from DBSs from a cohort of 74 patients identified to have SCADD. Of this cohort, approximately 60% had one or more of the common variants and did not have the two rare variants, and thus would need no further testing. Retrospective analysis of ethylmalonic acid, glutaric acid, 2-hydroxyglutaric acid, 3-hydroxyglutaric acid, and methylsuccinic acid demonstrated that second-tier testing applied before the release of the newborn screening result could reduce referrals by over 50% and improve the positive predictive value for SCADD to above 75%.