Bronchioalveolar organoids: A preclinical tool to screen toxicity associated with antibody-drug conjugates.

Despite extensive preclinical testing, cancer therapeutics can result in unanticipated toxicity to non-tumor tissue in patients. These toxicities may pass undetected in preclinical experiments due to modeling limitations involving poor biomimicry of 2-dimensional in vitro cell cultures and due to lack of interspecies translatability in in vivo studies. Instead, primary cells can be grown into miniature 3-dimensional structures that recapitulate morphological and functional aspects of native tissue, termed "organoids." Here, human bronchioalveolar organoids grown from primary alveolar epithelial cells were employed to model lung epithelium and investigate off-target toxicities associated with antibody-drug conjugates (ADCs). ADCs with three different linker-payload combinations (mafodotin, vedotin, and deruxtecan) were tested in bronchioalveolar organoids generated from human, rat, and nonhuman primate lung cells. Organoids demonstrated antibody uptake and changes in viability in response to ADC exposure that model in vivo drug sensitivity. RNA sequencing identified inflammatory activation in bronchioalveolar cells in response to deruxtecan. Future studies will explore specific cell populations involved in interstitial lung disease and incorporate immune cells to the culture.

Elimination of tucatinib, a small molecule kinase inhibitor of HER2, is primarily governed by CYP2C8 enantioselective oxidation of gem-dimethyl.

PURPOSE: Tucatinib, a small molecule for the treatment of metastatic HER2-positive breast cancer, was extensively metabolized in humans to multiple oxidative metabolites. To fully understand the elimination and biotransformation pathways of tucatinib, we investigated the in vitro and in vivo metabolism of tucatinib, and also conducted a Phase I trial using [14C]tucatinib. METHODS: To identify the responsible enzymes for tucatinib clearance, we investigated the in vitro metabolism of tucatinib including enzyme phenotyping, which facilitated the discovery of several metabolites in human and monkey plasma and excreta, in particular M1 (ONT-993, an aliphatic hydroxylated metabolite). Stereoselective formation of M1 was further investigated in vitro, in vivo, and in silico. RESULTS: In humans, approximately 86% of the total radiolabeled dose was recovered in feces and 4% in urine; in plasma, approximately 76% of radioactivity circulated as parent drug, with 19% attributed to multiple metabolites. The primary isoforms responsible for the elimination of tucatinib were CYP2C8 and CYP3A4/5. CYP2C8 was shown to possess sole catalytic activity for the formation of M1, whereas CYP3A4/5 and aldehyde oxidase catalyzed the formation of the remaining metabolites. Subsequent investigation revealed that M1 was formed in a stereoselective manner. Examination of the enantiomeric ratio of M1 stereoisomers observed in humans relative to cynomolgus monkeys revealed comparable results, suggesting that the enantiomers that comprise M1 were not considered to be unique or disproportionately high in human. CONCLUSION: CYP2C8 and CYP3A4/5 are the primary drug-metabolizing enzymes involved in the in vitro metabolism of tucatinib, which provided the basis to describe human disposition of tucatinib and formation of the observed metabolites.

Persistence of Trichomonas vaginalis serostatus in men over time.

PURPOSE: Previous epidemiologic studies have observed positive associations between Trichomonas vaginalis (Tv) serostatus and both prostate cancer (PCa) risk and mortality. However, only a few small older studies have examined Tv antibody persistence over time, all of which were composed mainly of female patients. Therefore, we examined Tv antibody persistence over time, as well as intra-individual variability, among middle- to older-aged men in the Southern Community Cohort Study (SCCS). METHODS: We tested baseline and repeat plasma specimens (collected 1-3 years later) from 248 male participants for Tv antibodies. We used the same enzyme-linked immunosorbent assay as in previous studies of Tv serostatus and PCa. RESULTS: At baseline, 46 (18.5 %) participants were seropositive for Tv infection. Seventy-six percent of these men were still seropositive 1-3 years later. A similar proportion of men "seroconverted" (4.0 %) as "seroreverted" (4.4 %), all of whom had absorbance values near the cutoff point for seropositivity. Overall, substantial agreement was observed between baseline and repeat serostatus (κ = 0.72, 95 % confidence interval 0.60-0.83). CONCLUSION: Tv seropositivity was largely persistent between plasma specimens collected 1-3 years apart from middle- to older-aged men. These high levels of persistence are similar to those observed for other sexually transmitted infections frequently investigated in relation to PCa.

Epitopes of the highly immunogenic Trichomonas vaginalis α-actinin are serodiagnostic targets for both women and men.

There is a need for a point-of-care serodiagnostic test for women and men for sexually transmitted infections (STIs) caused by Trichomonas vaginalis. Sera from women with this STI and sera from men that were analyzed in studies showing a relationship between serostatus and prostate cancer are highly seropositive in response to trichomonad α-actinin and its truncated protein (ACT-P2) (positive control sera). Epitope mapping experiments showed that positive control sera from women had antibodies to 13 distinct epitopes, 5 of which were detected by positive control sera from men. Sera from women and men that were unreactive with α-actinin (negative control sera) failed to detect any of the epitopes or other α-actinin amino acid sequences. The T. vaginalis α-actinin amino acid sequence and the sequences of the epitopes showed little or no identity with those of other proteins of microbial pathogens or the human α-actinin 1 (HuACTN1) homolog. Immunoassays such as dot blot, immunoblot, and enzyme-linked immunosorbent assays were used. Positive control sera did not detect HuACTN1 in immunoassays, and the range of levels of identity of α-actinin epitopes with HuACTN1 was 0% to 50%. Comparison of the T. vaginalis α-actinin epitopes with proteins in data banks, such as Tritrichomonas suis, Candida albicans, and Saccharomyces cerevisiae proteins, gave a range of identity levels of 0% to 22%. Specific 15-mer peptide epitopes of α-actinin with low to no identity with other proteins were synthesized and were reactive with positive control sera only. These findings identify epitopes of α-actinin as candidate serodiagnostic targets and suggest strongly that a highly seropositive reaction to α-actinin suggests exposure to T. vaginalis.

Identification, characterization, and synthesis of peptide epitopes and a recombinant six-epitope protein for Trichomonas vaginalis serodiagnosis.

There is a need for a rapid, accurate serodiagnostic test useful for both women and men infected by Trichomonas vaginalis, which causes the number one sexually transmitted infection (STI). Women and men exposed to T. vaginalis make serum antibody to fructose-1,6-bisphosphate aldolase (ALD), α-enolase (ENO), and glyceraldehyde-3-phosphate dehydrogenase (GAP). We identified, by epitope mapping, the common and distinct epitopes of each protein detected by the sera of women patients with trichomonosis and by the sera of men highly seropositive to the immunogenic protein α-actinin (positive control sera). We analyzed the amino acid sequences to determine the extent of identity of the epitopes of each protein with other proteins in the databanks. This approach identified epitopes unique to T. vaginalis, indicating these peptide-epitopes as possible targets for a serodiagnostic test. Individual or combinations of 15-mer peptide epitopes with low to no identity with other proteins were reactive with positive control sera from both women and men but were unreactive with negative control sera. These analyses permitted the synthesis of a recombinant His6 fusion protein of 111 amino acids with an Mr of ~13.4 kDa, which consisted of 15-mer peptides of two distinct epitopes each for ALD, ENO, and GAP. This recombinant protein was purified by affinity chromatography. This composite protein was detected by enzyme-linked immunosorbent assay (ELISA), dot blots, and immunoblots, using positive control sera from women and men. These data indicate that it is possible to identify epitopes and that either singly, in combination, or as a composite protein represent targets for a point-of-care serodiagnostic test for T. vaginalis.