SGN-CD228A Is an Investigational CD228-Directed Antibody-Drug Conjugate with Potent Antitumor Activity across a Wide Spectrum of Preclinical Solid Tumor Models.

SGN-CD228A is an investigational antibody-drug conjugate (ADC) directed to melanotransferrin (CD228, MELTF, MFI2, p97), a cell-surface protein first identified in melanoma. SGN-CD228A consists of a humanized antibody, hL49, with high specificity and affinity for CD228 that is stably conjugated to 8 molecules of the clinically validated microtubule-disrupting agent monomethyl auristatin E (MMAE) via a novel glucuronide linker. We performed comprehensive IHC studies, which corroborated published RNA sequencing data and confirmed low CD228 expression in normal tissues and high expression in several cancers, including melanoma, squamous non-small cell lung cancer (NSCLC), triple-negative breast cancer (TNBC), colorectal cancer, and pancreatic cancer. SGN-CD228A was efficiently internalized in various tumor cell types, and its cytotoxic activity was dependent on CD228 expression and internalization and intrinsic sensitivity to the MMAE payload. Compared with the valine-citrulline dipeptide linker, the novel glucuronide linker increased the cellular retention of MMAE in vitro and conferred improved antitumor activity against melanoma cell lines in vitro and in vivo. In addition, SGN-CD228A was active across melanoma, TNBC, and NSCLC cell line- and patient-derived xenograft models with heterogeneous antigen expression. In vivo, CD228 expression was important for response to SGN-CD228A but was not well correlated across all tumor types, suggesting that other factors associated with ADC activity are important. Overall, SGN-CD228A is a CD228-directed, investigational ADC that employs innovative technology and has compelling preclinical antitumor activity. SGN-CD228A is investigated in a Phase I clinical trial (NCT04042480) in patients with advanced solid tumors.

The Homeodomain Transcription Factors Vax1 and Six6 Are Required for SCN Development and Function.

The brain's primary circadian pacemaker, the suprachiasmatic nucleus (SCN), is required to translate day-length and circadian rhythms into neuronal, hormonal, and behavioral rhythms. Here, we identify the homeodomain transcription factor ventral anterior homeobox 1 (Vax1) as required for SCN development, vasoactive intestinal peptide expression, and SCN output. Previous work has shown that VAX1 is required for gonadotropin-releasing hormone (GnRH/LHRH) neuron development, a neuronal population controlling reproductive status. Surprisingly, the ectopic expression of a Gnrh-Cre allele (Gnrhcre) in the SCN confirmed the requirement of both VAX1 (Vax1flox/flox:Gnrhcre, Vax1Gnrh-cre) and sine oculis homeobox protein 6 (Six6flox/flox:Gnrhcre, Six6Gnrh-cre) in SCN function in adulthood. To dissociate the role of Vax1 and Six6 in GnRH neuron and SCN function, we used another Gnrh-cre allele that targets GnRH neurons, but not the SCN (Lhrhcre). Both Six6Lhrh-cre and Vax1Lhrh-cre were infertile, and in contrast to Vax1Gnrh-cre and Six6Gnrh-cre mice, Six6Lhrh-cre and Vax1Lhrh-cre had normal circadian behavior. Unexpectedly, ~ 1/4 of the Six6Gnrh-cre mice were unable to entrain to light, showing that ectopic expression of Gnrhcre impaired function of the retino-hypothalamic tract that relays light information to the brain. This study identifies VAX1, and confirms SIX6, as transcription factors required for SCN development and function and demonstrates the importance of understanding how ectopic CRE expression can impact the results.

Differential CRE Expression in Lhrh-cre and GnRH-cre Alleles and the Impact on Fertility in Otx2-Flox Mice.

There is an increasing trend in studies utilizing cell-specific deletion of genes through conditional gene deletion by CRE recombination. Despite numerous advantages, this strategy also has limitations such as ectopic CRE-expression and germline recombination. Two commonly used gonadotropin-releasing hormone (Gnrh)-driven CRE-expressing mice both target GnRH neurons. However, a direct comparison of the cells targeted and their phenotypic outcome have not yet been presented. To compare where recombination takes place, we crossed the Gnrh-cre and Lhrh-cre lines with the Rosa26-LacZ reporter mouse. Lhrh-cre allowed recombination of the Rosa26-LacZ gene in ∼700 cells, which is comparable to the GnRH neuronal population. Surprisingly, there were > 20 times more LacZ expressing cells in the adult Gnrh-cre:Rosa26-LacZ than the Lhrh-cre:Rosa26-LacZ brain. The greatest differences in targeting of the Gnrh-cre and Lhrh-cre lines were found in the septum, the suprachiasmatic nucleus, and the septohypothalamic area. This difference in cells targeted was present from embryonic day 12. A prior study using the Gnrh-cre to delete the transcription factor Otx2 found fewer GnRH neurons, leading to male and female subfertility. To recapitulate this study, we performed a fertility assay in Otx2:Lhrh-cre mice. We confirmed the requirement for Otx2 in GnRH neuron development, fertility and correct gonadotropin hormone release in Otx2:Lhrh-cre males, but the subfertility was more modest than in Otx2:Gnrh-cre and absent in female Otx2:Lhrh-cre. This suggests that ectopic expression of Gnrh-cre contributes to the reproductive phenotype observed. Finally, the Cre alleles caused germline recombination of the flox allele when transmitted from either parent, generating embryonic lethal knock-out offspring, producing smaller live litters.

Seasonal changes in neuronal turnover in a forebrain nucleus in adult songbirds.

Neuronal death and replacement, or neuronal turnover, in the adult brain are one of many fundamental processes of neural plasticity. The adult avian song control circuit provides an excellent model for exploring mature neuronal death and replacement by new neurons. In the song control nucleus, HVC of adult male Gambel's white-crowned sparrows (Zonotrichia leucophrys gambelli) nearly 68,000 neurons are added each breeding season and die during the subsequent nonbreeding season. To accommodate large seasonal differences in HVC neuron number, the balance between neuronal addition and death in HVC must differ between seasons. To determine whether maintenance of new HVC neurons changes within and between breeding and nonbreeding conditions, we pulse-labeled two different cohorts of new HVC neurons under both conditions and quantified their maintenance. We show that the maintenance of new HVC neurons, as well as new nonneuronal cells, was higher at the onset of breeding conditions than at the onset of nonbreeding conditions. Once a steady-state HVC volume and neuronal number were attained in either breeding or nonbreeding conditions, neuronal and nonneuronal maintenance were similarly low. We found that new neuronal number correlated with a new nonneuronal number within each cohort of new neurons. Together, these data suggest that sex steroids promote the survival of an initial population of new neurons and nonneuronal cells entering HVC. However, once HVC is fully grown or regressed, neuronal and nonneuronal cell turnover is regulated by a common mechanism likely independent of direct sex steroid signaling.