ABBV-319: a CD19-targeting glucocorticoid receptor modulator antibody-drug conjugate therapy for B-cell malignancies.

ABSTRACT: Glucocorticoids are key components of the standard-of-care treatment regimens for B-cell malignancy. However, systemic glucocorticoid treatment is associated with several adverse events. ABBV-319 is a CD19-targeting antibody-drug conjugate engineered to reduce glucocorticoid-associated toxicities while possessing 3 distinct mechanisms of action (MOA) to increase therapeutic efficacy: (1) antibody-mediated delivery of a glucocorticoid receptor modulator (GRM) payload to activate apoptosis, (2) inhibition of CD19 signaling, and (3) enhanced fragment crystallizable (Fc)-mediated effector function via afucosylation of the antibody backbone. ABBV-319 elicited potent GRM-driven antitumor activity against multiple malignant B-cell lines in vitro, as well as in cell line-derived xenografts and patient-derived xenografts (PDXs) in vivo. Remarkably, a single dose of ABBV-319 induced sustained tumor regression and enhanced antitumor activity compared with repeated dosing of systemic prednisolone at the maximum tolerated dose in mice. The unconjugated CD19 monoclonal antibody (mAb) also displayed antiproliferative activity in a subset of B-cell lymphoma cell lines through the inhibition of phosphoinositide 3-kinase signaling. Moreover, afucosylation of CD19 mAb enhanced Fc-mediated antibody-dependent cellular cytotoxicity. Notably, ABBV-319 displayed superior efficacy compared with afucosylated CD19 mAb in human CD34+ peripheral blood mononuclear cell-engrafted NSG-Tg(Hu-IL15) transgenic mice, demonstrating enhanced antitumor activity when multiple MOAs are enabled. ABBV-319 also showed durable antitumor activity across multiple B-cell lymphoma PDX models, including nongerminal center B-cell diffuse large B-cell lymphoma and relapsed lymphoma after R-CHOP treatment. Collectively, these data support the ongoing evaluation of ABBV-319 in a phase 1 clinical trial.

Perception of #TheDress in childhood is influenced by age and green-leaf preference.

The perception of the ambiguous image of #TheDress may be influenced by optical factors, such as macular pigments. Their accumulation during childhood could increase with age and the ingestion of carotenoid-containing foods. The purpose of this study was to investigate whether the visual perception of the dress in children would differ based on age and carotenoid preference. This was a cross-sectional, observational, and comparative study. A poll was administered to children aged 2 to 10 years. Parents were instructed to inquire about the color of #TheDress from their children. A carotenoid preference survey was also completed. A total of 413 poll responses were analyzed. Responses were categorized based on the perceived color of the dress: blue/black (BB) (n = 204) and white/gold (WG) (n = 209). The mean and median age of the WG group was higher than the BB group (mean 6.1, median 6.0 years, standard deviation [SD] 2.2; mean 5.5, median 5.0 years, SD 2.3; p = 0.007). Spearman correlation between age and group was 0.133 (p = 0.007). Green-leaf preference (GLP) showed a statistically significant difference between groups (Mann-Whitney U: p = 0.038). Spearman correlation between GLP and group was 0.102 (p = 0.037). Logistic regression for the perception of the dress as WG indicated that age and GLP were significant predictors (age: B weight 0.109, p = 0.012, odds ratio: 1.115; GLP: B weight 0.317, p = 0.033, odds ratio: 1.373). Older children and those with a higher GLP were more likely to perceive #TheDress as WG. These results suggest a potential relationship with the gradual accumulation of macular pigments throughout a child's lifetime.

Self-Immolative Carbamate Linkers for CD19-Budesonide Antibody-Drug Conjugates.

Antibody-drug conjugates consist of potent small-molecule payloads linked to a targeting antibody. Payloads must possess a viable functional group by which a linker for conjugation can be attached. Linker-attachment options remain limited for the connection to payloads via hydroxyl groups. A releasing group based on 2-aminopyridine was developed to enable stable attachment of para-aminobenzyl carbamate (PABC) linkers to the C21-hydroxyl group of budesonide, a glucocorticoid receptor agonist. Payload release involves a cascade of two self-immolative events that are initiated by the protease-mediated cleavage of the dipeptide-PABC bond. Budesonide release rates were determined for a series of payload-linker intermediates in buffered solution at pH 7.4 and 5.4, leading to the identification of 2-aminopyridine as the preferred releasing group. Addition of a poly(ethylene glycol) group improved linker hydrophilicity, thereby providing CD19-budesonide ADCs with suitable properties. ADC23 demonstrated targeted delivery of budesonide to CD19-expressing cells and inhibited B-cell activation in mice.

Genomic phylogeography illuminates deep cyto-nuclear discordances in midwife toads (Alytes).

The advent of genomic methods allows us to revisit the evolutionary history of organismal groups for which robust phylogenies are still lacking, particularly in species complexes that frequently hybridize. In this study, we conduct RAD-sequencing (RAD-seq) analyses of midwife toads (genus Alytes), an iconic group of western Mediterranean amphibians famous for their parental care behavior, but equally infamous for the difficulties to reconstruct their evolutionary history. Through admixture and phylogenetic analyses of thousands of loci, we provide a comprehensive phylogeographic framework for the A. obstetricans complex, as well as a fully resolved phylogeny for the entire genus. As part of this effort, we carefully explore the influence of different sampling schemes and data filtering thresholds on tree reconstruction, showing that several, slightly different, yet robust topologies may be retrieved with small datasets obtained by stringent SNP calling parameters, especially when admixed individuals are included. In contrast, analyses of incomplete but larger datasets converged on the same phylogeny, irrespective of the reconstruction method used or the proportion of missing data. The Alytes tree features three Miocene-diverged clades corresponding to the proposed subgenera Ammoryctis (A. cisternasii), Baleaphryne (A. maurus, A. dickhilleni and A. muletensis), and Alytes (A. obstetricans complex). The latter consists of six evolutionary lineages, grouped into three clades of Pliocene origin, and currently delimited as two species: (1) A. almogavarii almogavarii and A. a. inigoi; (2) A. obstetricans obstetricans and A. o. pertinax; (3) A. o. boscai and an undescribed taxon (A. o. cf. boscai). These results contradict the mitochondrial tree, due to past mitochondrial captures in A. a. almogavarii (central Pyrenees) and A. o. boscai (central Iberia) by A. obstetricans ancestors during the Pleistocene. Patterns of admixture between subspecies appear far more extensive than previously assumed from microsatellites, causing nomenclatural uncertainties, and even underlying the reticulate evolution of one taxon (A. o. pertinax). All Ammoryctis and Baleaphryne species form shallow clades, so their taxonomy should remain stable. Amid the prevalence of cyto-nuclear discordance among terrestrial vertebrates and the usual lack of resolution of conventional nuclear markers, our study advocates for phylogeography based on next-generation sequencing, but also encourages properly exploring parameter space and sampling schemes when building and analyzing genomic datasets.

Impact of dipeptide on ADC physicochemical properties and efficacy identifies Ala-Ala as the optimal dipeptide.

Side chains of natural occurring amino acids vary greatly in terms of charge state, polarity, size and hydrophobicity. Using a linear synthetic route, two amino acids were sequentially coupled to a potent glucocorticoid receptor modulator (GRM) to afford a library of dipeptide-GRM linker payloads with a range of in silico properties. The linker payloads were conjugated to a mouse anti-TNF antibody through interchain disulfide Cys. Impact of various dipeptide linkers on ADC physical properties, including solubility, hydrophobicity, and aggregation were evaluated and the in silico properties pI, Log P and tPSA of the linker drugs used to correlate with these properties. ADCs were screened in a GRE luciferase reporter assay to compare their in vitro efficacy. Data identified Ala-Ala as a superior dipeptide linker that allowed a maximum drug load of 10 while affording ADCs with low aggregation.

Linker substitution influences succinimide ring hydrolysis equilibrium impacting the stability of attachment to antibody-drug conjugates.

Maleimide chemistry is widely used in antibody-drug conjugate (ADC) generation to connect drugs to antibodies through a succinimide linker. The resulting ADC is prone to payload loss via a reverse Michael reaction, leading to premature drug release in vivo. Complete succinimide hydrolysis is an effective strategy to overcome the instability of ADC. However, we discovered through previous work that hydrolysed succinimide rings can close again in a liquid formulation during storage and under thermal stress conditions. In this work, a set of maleimide linkers with hydrolysis-prone groups were designed. The corresponding ADCs were prepared and subjected to thermal stress conditions. The extent of succinimide hydrolysis and drug release was measured, and ADC properties such as SEC, DAR, pI and clog P of linkers were calculated. Our results demonstrated that even though all these groups increased the hydrolysis rate, they have different impacts on maintaining the hydrolysed succinimide ring in an open conformation and ADC stability in a liquid formulation.

Anti-TNF Thioester Glucocorticoid Antibody-Drug Conjugate Fully Inhibits Inflammation with Minimal Effect on Systemic Corticosterone Levels in a Mouse Arthritis Model.

We describe the discovery of a thioester-containing glucocorticoid receptor modulator (GRM) payload and the corresponding antibody-drug conjugate (ADC). Payload 6 was designed for rapid hepatic inactivation to minimize systemic exposure of nonconjugated GRM. Mouse PK indicated that 6 is cleared 10-fold more rapidly than a first-generation GRM payload, resulting in 10-fold lower exposure and 3-fold decrease in Cmax. The anti-mTNF conjugate ADC5 fully inhibited inflammation in mouse contact hypersensitivity with minimal effects on corticosterone, a biomarker for systemic GRM effects, at doses up to and including 100 mg/kg. Concomitant inhibition of P1NP suggests potential delivery to cells involved in the remodeling of bone, which may be a consequence of TNF-targeting or bystander payload effects. Furthermore, ADC5 fully suppressed inflammation in collagen-induced arthritis mouse model after one 10 mg/kg dose for 21 days. The properties of the anti-hTNF conjugate were suitable for liquid formulation and may enable subcutaneous dosing.

An anti-TNF-glucocorticoid receptor modulator antibody-drug conjugate is efficacious against immune-mediated inflammatory diseases.

Glucocorticoids (GCs) are efficacious drugs used for treating many inflammatory diseases, but the dose and duration of administration are limited because of severe side effects. We therefore sought to identify an approach to selectively target GCs to inflamed tissue. Previous work identified that anti-tumor necrosis factor (TNF) antibodies that bind to transmembrane TNF undergo internalization; therefore, an anti-TNF antibody-drug conjugate (ADC) would be mechanistically similar, where lysosomal catabolism could release a GC receptor modulator (GRM) payload to dampen immune cell activity. Consequently, we have generated an anti-TNF-GRM ADC with the aim of inhibiting pro-inflammatory cytokine production from stimulated human immune cells. In an acute mouse model of contact hypersensitivity, a murine surrogate anti-TNF-GRM ADC inhibited inflammatory responses with minimal effect on systemic GC biomarkers. In addition, in a mouse model of collagen-induced arthritis, single-dose administration of the ADC, delivered at disease onset, was able to completely inhibit arthritis for greater than 30 days, whereas an anti-TNF monoclonal antibody only partially inhibited disease. ADC treatment at the peak of disease was also able to attenuate the arthritic phenotype. Clinical data for a human anti-TNF-GRM ADC (ABBV-3373) from a single ascending dose phase 1 study in healthy volunteers demonstrated antibody-like pharmacokinetic profiles and a lack of impact on serum cortisol concentrations at predicted therapeutic doses. These data suggest that an anti-TNF-GRM ADC may provide improved efficacy beyond anti-TNF alone in immune mediated diseases while minimizing systemic side effects associated with standard GC treatment.

Optimization of Drug-Linker to Enable Long-term Storage of Antibody-Drug Conjugate for Subcutaneous Dosing.

To facilitate subcutaneous dosing, biotherapeutics need to exhibit properties that enable high-concentration formulation and long-term stability in the formulation buffer. For antibody-drug conjugates (ADCs), the introduction of drug-linkers can lead to increased hydrophobicity and higher levels of aggregation, which are both detrimental to the properties required for subcutaneous dosing. Herein we show how the physicochemical properties of ADCs could be controlled through the drug-linker chemistry in combination with prodrug chemistry of the payload, and how optimization of these combinations could afford ADCs with significantly improved solution stability. Key to achieving this optimization is the use of an accelerated stress test performed in a minimal formulation buffer.

Discovery of ABBV-154, an anti-TNF Glucocorticoid Receptor Modulator Immunology Antibody-Drug Conjugate (iADC).

Stable attachment of drug-linkers to the antibody is a critical requirement, and for maleimide conjugation to cysteine, it is achieved by ring hydrolysis of the succinimide ring. During ADC profiling in our in-house property screening funnel, we discovered that the succinimide ring open form is in equilibrium with the ring closed succinimide. Bromoacetamide (BrAc) was identified as the optimal replacement, as it affords stable attachment of the drug-linker to the antibody while completely removing the undesired ring open-closed equilibrium. Additionally, BrAc also offers multiple benefits over maleimide, especially with respect to homogeneity of the ADC structure. In combination with a short, hydrophilic linker and phosphate prodrug on the payload, this afforded a stable ADC (ABBV-154) with the desired properties to enable long-term stability to facilitate subcutaneous self-administration.

Vegetation cover and occurrence of salamanders in the western Mediterranean.

The Mediterranean region possesses a rich diversity of salamanders, which also exhibit a high degree of ecological diversification. It is assumed that the presence of salamanders is dependent on the level of vegetation cover in terrestrial habitats, but the strength of this association is likely to vary among species. In this study, we investigated the patterns of habitat utilization for Mediterranean salamanders based on 589 records of 33 species. We tested the hypothesis that the association between salamander presence and vegetation density varies among genera or reproductive modes (i.e. terrestrial vs aquatic). The results show that vegetation cover has similar influences on terrestrial and aquatic groups but important differences are identified for lotic and lentic aquatic species. Our findings also indicate that the aquatic lotic, terrestrial, and small-body species deviate significantly from that expected from the background range of variation.

Design and Development of Glucocorticoid Receptor Modulators as Immunology Antibody-Drug Conjugate Payloads.

Glucocorticoid receptor modulators (GRM) are the first-line treatment for many immune diseases, but unwanted side effects restrict chronic dosing. However, targeted delivery of a GRM payload via an immunology antibody-drug conjugate (iADC) may deliver significant efficacy at doses that do not lead to unwanted side effects. We initiated our α-TNF-GRM ADC project focusing on identifying the optimal payload and a linker that afforded stable attachment to both the payload and antibody, resulting in the identification of the synthetically accessible maleimide-Gly-Ala-Ala linker. DAR 4 purified ADCs were shown to be more efficacious in a mouse contact hypersensitivity model than the parent α-TNF antibody. Analysis of P1NP and corticosterone biomarkers showed there was a sufficient therapeutic window between efficacy and unwanted effects. In a chronic mouse arthritis model, α-TNF-GRM ADCs were more efficacious than both the parent α-TNF mAb and an isotype control bearing the same GRM payload.

Discovery of ABBV-3373, an Anti-TNF Glucocorticoid Receptor Modulator Immunology Antibody Drug Conjugate.

Using a convergent synthetic route to enable multiple points of diversity, a series of glucocorticoid receptor modulators (GRM) were profiled for potency, selectivity, and drug-like properties in vitro. Despite covering a large range of diversity, profiling the nonconjugated small molecule was suboptimal and they were conjugated to a mouse antitumor necrosis factor (TNF) antibody using the MP-Ala-Ala linker. Screening of the resulting antibody drug conjugates (ADCs) provided a better assessment of efficacy and physical properties, reinforcing the need to conduct structure-activity relationship studies on the complete ADC. DAR4 ADCs were screened in an acute mouse contact hypersensitivity model measuring biomarkers to ensure a sufficient therapeutic window. In a chronic mouse arthritis model, mouse anti-TNF GRM ADCs were efficacious after a single dose of 10 mg/kg i.p. for over 30 days. Data on the unconjugated payloads and mouse surrogate anti-TNF ADCs identified payload 17 which was conjugated to a human anti-TNF antibody and advanced to the clinic as ABBV-3373.

A new species of the genus Tylototriton (Urodela, Salamandridae) from western Thailand.

We describe a new species of the newt genus Tylototriton from Umphang Wildlife Sanctuary, Tak Province, western Thailand based on molecular and morphological evidence and named here as Tylototritonumphangensis sp. nov. The new species is assigned to the subgenus Tylototriton and differs from other species in having dark-brown to blackish-brown body and limbs, truncate snout, prominent antero-medial ends of the expansion of the dentary bones, laterally protruding quadrate regions, indistinct and small rib nodules, a well-segmented vertebral ridge, and rough dorsolateral bony ridges, which are steeper anterior, and curved medially at the posterior ends. The molecular data show that Tylototritonumphangensis sp. nov. differs from T.uyenoi sensu stricto by a 5% genetic sequence divergence of the mitochondrial NADH dehydrogenase subunit 2 region gene. The new species and T.uyenoi are both endemic to Thailand, distributed along the Northwest Thai (Dawna) Uplands of Indochina. To clarify the species boundary between Tylototritonumphangensis sp. nov. and T.uyenoi, additional field research is needed in adjacent areas. Tylototritonumphangensis sp. nov. is restricted to evergreen hill forests in Umphang Wildlife Sanctuary. We suggest that the new species should be classified as Endangered (EN) in the IUCN Red List.

l-Proline as a Valuable Scaffold for the Synthesis of Novel Enantiopure Neonicotinoids Analogs.

In this research, six neonicotinoid analogs derived from l-proline were synthesized, characterized, and evaluated as insecticides against Xyleborus affinis. Most of the target compounds showed good to excellent insecticidal activity. To the best of our knowledge, this is the first report dealing with the use of enantiopure l-proline to get neonicotinoids. These results highlighted the compound 9 as an excellent candidate used as the lead chiral insecticide for future development. Additionally, molecular docking with the receptor and compound 9 was carried out to gain insight into its high activity when compared to dinotefuran. Finally, the neurotoxic evaluation of compound 9 showed lower toxicity than the classic neonicotinoid dinotefuran.

A new subspecies of African fire salamander Salamandra algira (Urodela, Salamandridae) from the Middle Atlas Mountains, Morocco.

A new subspecies within the Salamandra algira species complex from north-western Africa is described. Previous molecular analysis showed that the populations of S. algira splendens from north-western Morocco consisted of two well supported clades: clade 1 distributed in the Rif Mountains, from Chefchaouen (type locality) to Al Hoceima, and clade 2, located southern from clade 1 being isolated in the northern and central Middle Atlas Mountains. Clade 2 is herein described as a distinct subspecies: Salamandra algira atlantica ssp. nov. based on morphological data, allopatric range and molecular divergence. This new subspecies shows an uncorrected pairwise distance of 0.0265 from clade 1 based on cytochrome b DNA sequences. Salamandra algira atlantica ssp. nov. is a slender and large sized salamander with a highly variable colouration pattern. It can be distinguished from S. algira splendens by the greater proportion of coral red in the background colouration, being the only known subspecies of S. algira in which coral red can exceed the proportion of black. Variable number (0-5) of yellow to golden yellow dorsal blotches, but usually in lower numbers than the nominotypical subspecies. Salamandra algira atlantica ssp. nov. inhabits subhumid to humid forests and karstic systems at mid to high elevations. We briefly discuss the phylogenetic and taxonomic issues among the genus Salamandra which encompasses more valid species than currently recognised.

Very low hepatitis C antibody levels predict false-positive results and avoid supplemental testing.

BACKGROUND: False-positive results for hepatitis C virus antibody (anti-HCV) occur with unacceptable frequency in low-prevalence populations. The purpose of the study was to determine whether signal-to-cutoff (S/CO) ratios of anti-HCV assay-reactive samples could be used to discriminate false-positive from true-positive anti-HCV results and avoid the need for supplemental testing. STUDY DESIGN AND METHODS: Using receiver-operating characteristic curve, the cutoff point that identifies the major proportion (>/=95%) of false-positive results, with a minor proportion (<5%) of true-positive anti-HCV results, was determined. An anti-HCV assay (VITROS, Ortho Clinical Diagnostics) was used to detect the antibodies. The third-generation recombinant immunoblot assay and HCV RNA tests were performed on all included donors. Third-generation RIBA is the gold standard for identifying false-positive antibody results. RESULTS: A total of 649 anti-HCV-positive blood donors were identified. A S/CO ratio of less than 4.5, defining very low levels in this value, was the optimal cutoff point to identify false-positive results; 315 of 322 samples with very low levels were false-positive anti-HCV results (97.8%; 95% confidence interval [CI], 95.8%-99.0%) and 7 were true-positive (2.2%; 95% CI, 1.0%-4.3%). Viremia was detected in none of them. A direct relationship was observed between positive supplemental testing and increased antibody levels in the other 327 samples. CONCLUSION: The high prediction rate of false-positive anti-HCV results using very low levels by the Ortho VITROS anti-HCV assay safely avoids the need for supplemental testing.

Acute sterile endophthalmitis following intravitreal bevacizumab: case series.

BACKGROUND: Since the ophthalmological community adopted the use of intravitreal bevacizumab as an accepted off-label treatment for neovascular diseases, the amount of knowledge regarding its effects and properties has been increasing continually. In the last few years, there have been an increasing number of reports about sterile intraocular inflammation and intraocular pressure elevations after intravitreal bevacizumab. In the following case series, we describe the clinical presentation and outcomes of ten consecutive cases of patients developing mild-to-severe sterile intraocular inflammation after intravitreal bevacizumab and their management. METHODS: This report presents a retrospective case series. We reviewed the medical records of ten consecutive patients from a group of 46, in whom repackaged bevacizumab in individual aliquots from two vials from the same batch were used. All surgical procedures were performed using standard sterile techniques in the operating room. At each follow-up visit, patients underwent a complete ophthalmological examination including visual acuity assessment, intraocular pressure, biomicroscopy, and posterior fundus examination. RESULTS: Ten patients presented sterile endophthalmitis with an onset time of 3.5±1.95 days. The clinical characteristics were mild pain, slight visual loss, conjunctival hyperemia, and various degrees of intraocular inflammation with microhypopyon. All cultures were negative. All patients were managed with topical steroids and antibiotics, except two, in whom, due to severe vitreous cells, intravitreal antibiotics were used. Three patients showed a transient elevation of intraocular pressure. Only 50% of the patients regained a visual acuity equal or better to the baseline visual acuity on file. CONCLUSION: The increasing number of intravitreal injections of bevacizumab applied every day, due to its widespread acceptance, might be one reason why the number of cases of sterile endophthalmitis is rising. Fast recognition and accurate differential diagnosis is important to avoid unnecessary treatments and long-term complications. The low incidence of this event should not preclude the use of intravitreal injections in eyes that could benefit greatly from this therapy.