Antibody-drug conjugates (ADCs) are emerging therapeutic modalities that utilize the specificity of antibodies to deliver chemotherapeutic drugs directly to tumors with the promise of maintaining antitumor efficacy while minimizing nontumor toxicities. This chapter discusses the cellular biology that impacts ADC efficacy and design a strategy as well as propose the lysosome as an antitumor target for novel ADC development. First, it describes the mechanisms that mediate internalization of plasma-bound antigens. The internalization of cell surface receptors provides ADCs with a target-specific entry point. The chapter then outlines the strategic design of ADCs and the mechanisms that drive receptor-ADC internalization. The efficacy and duration of any chemotherapeutic regimen is dictated by the innate resistance of cancer cells to a therapy or the acquisition of resistance following drug exposure and resistant cell population selection.
(R,S)-Ketamine (ketamine) is a dissociative anesthetic that also possesses analgesic and antidepressant activity. Undesirable dissociative side effects and misuse potential limit expanded use of ketamine in several mental health disorders despite promising clinical activity and intensifying medical need. (2R,6R)-Hydroxynorketamine (RR-HNK) is a metabolite of ketamine that lacks anesthetic and dissociative activity but maintains antidepressant and analgesic activity in multiple preclinical models. To enable future assessments in selected human indications, we report the safety, tolerability, pharmacokinetics (PK), and pharmacodynamics (PD) of RR-HNK in a Phase 1 study in healthy volunteers (NCT04711005). A six-level single-ascending dose (SAD) (0.1-4 mg/kg) and a two-level multiple ascending dose (MAD) (1 and 2 mg/kg) study was performed using a 40-minute IV administration emulating the common practice for ketamine administration for depression. Safety assessments showed RR-HNK possessed a minimal adverse event profile and no serious adverse events at all doses examined. Evaluations of dissociation and sedation demonstrated that RR-HNK did not possess anesthetic or dissociative characteristics in the doses examined. RR-HNK PK parameters were measured in both the SAD and MAD studies and exhibited dose-proportional increases in exposure. Quantitative electroencephalography (EEG) measurements collected as a PD parameter based on preclinical findings and ketamine's established effect on gamma-power oscillations demonstrated increases of gamma power in some participants at the lower/mid-range doses examined. Cerebrospinal fluid examination confirmed RR-HNK exposure within the central nervous system (CNS). Collectively, these data demonstrate RR-HNK is well tolerated with an acceptable PK profile and promising PD outcomes to support the progression into Phase 2.
Abstract Targeted treatment with antibody-drug conjugates (ADCs) can lead to dramatic regressions of solid tumors; combination therapies with the ADCs that could potentially help achieve long-term disease control remain largely unexplored. The aim of the study is to investigate most promising combination regimens of auristatin-based conjugates in preclinical models of cancer. A tool ADC, an auristatin-based anti-5T4 antibody conjugate (5T4-ADC) and permeable auristatin analogs were combined with dual PI3K/mTOR catalytic site inhibitor PF-05212384 (PF-384) or taxanes in a panel of common cancer cell lines of breast, lung or ovarian origin. Drug combinations were tested for their effects in vitro on cell viability, apoptosis, cell cycle or modulation of PI3K/mTOR pathway markers. Anti-tumor efficacy of selected combinations was also evaluated in a 5T4-positive human breast or lung tumor xenografts in vivo. Remarkably, ADC and auristatin analogs resulted in synergistic or additive effects on cell cytotoxicity when combined with PF-384 or with taxanes in vitro. ADC/PF-384, auristatin/PF-384 and ADC/Paclitaxel (PTX), auristatin/PTX combinations showed enhanced induction of apoptosis and selective attenuation of the downstream phospho-markers of the PI3K/mTOR pathway. Quantitative proteomic analysis of cells treated with auristatin-based agents and PF-384 alone or in combination revealed novel and unique effects on key components of mRNA translation, including translation initiation or elongation factors. In human lung or breast cancer xenograft models, dual targeting with 5T4-ADC/PF-384 or 5T4-ADC/PTX produced substantially greater antitumor effects with longer average survival as compared to monotherapy treatments. Our results provide strong rationale for combining 5T4-ADC with PI3K/mTOR pathway inhibitors or taxanes. New biological insights into molecular mechanisms underlying synergistic effects of ADC - drug combinations suggest/reveal that synergies are mediated by payload's mode of action. These findings can be applicable to several ADCs employing microtubule inhibitors that are undergoing clinical evaluation. Citation Format: Puja Sapra, Boris Shor, Maureen Dougher, Jennifer Kahler, Michelle Mack, Jane Xu, Shuyan Lu, Eugene Melamud, Fang Wang, Edward Rosfjord. Enhanced anti-tumor activity of an Auristatin-based antibody-drug conjugate in combination with PI3K/mTOR inhibitors or taxanes: Translational implications and mechanistic insights. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 2463. doi:10.1158/1538-7445.AM2015-2463
Targeted treatment of solid or liquid tumors with antibody-drug conjugates (ADCs) can lead to promising clinical benefit. The aim of the study is to investigate combination regimens of auristatin-based ADCs in preclinical models of cancer.An auristatin-based anti-5T4 antibody conjugate (5T4-ADC) and auristatin payloads were combined with the dual PI3K/mTOR catalytic site inhibitor PF-05212384 (PF-384) or taxanes in a panel of tumor cell lines. Drug interactions in vitro were evaluated using cell viability assays, apoptosis induction, immunofluorescence, mitotic index, and immunoblotting. Breast cancer cells treated with auristatin analogue or 5T4-ADC were profiled by total- and phospho-proteomics. Antitumor efficacy of selected combinations was evaluated in 5T4-positive human breast or lung tumor xenografts in vivo.In vitro, auristatin-based agents displayed strong synergistic or additive activity when combined with PF-384 or taxanes, respectively. Further, treatment of 5T4-ADC plus PF-384 resulted in stronger induction of apoptosis and cell line-specific attenuation of pAKT and pGSK. Interestingly, proteomic analysis revealed unique effects of auristatins on multiple components of mRNA translation. Addition of PF-384 further amplified effects of 5T4-ADC on translational components, providing a potential mechanism of synergy between these drugs. In human tumor xenografts, dual targeting with 5T4-ADC/PF-384 or 5T4-ADC/paclitaxel produced substantially greater antitumor effects with longer average survival as compared with monotherapy treatments.Our results provide a biologic rationale for combining 5T4-ADC with either PI3K/mTOR pathway inhibitors or taxanes and suggest that mechanisms underlying the synergy may be attributed to cellular effects of the auristatin payload.
Ethanol (EtOH) exerts its effects through various protein targets, including transient receptor potential melastatin 7 (TRPM7) channels, which play an essential role in cellular homeostasis. We demonstrated that TRPM7 is expressed in rat brain microvascular endothelial cells (rBMVECs), the major cellular component of the blood–brain barrier (BBB). Heavy alcohol drinking is often associated with HIV infection, however mechanisms underlying alcohol-induced BBB damage and HIV proteins, are not fully understood. We utilized the HIV-1 transgenic (HIV-1Tg) rat to mimic HIV-1 patients on combination anti-retroviral therapy (cART) and demonstrated TRPM7 expression in rBMVECs wass lower in adolescent HIV-1Tg rats compared to control animals, however control and HIV-1Tg rats expressed similar levels at 9 weeks, indicating persistent presence of HIV-1 proteins delayed TRPM7 expression. Binge exposure to EtOH (binge EtOH) decreased TRPM7 expression in control rBMVECs in a concentration-dependent manner, and abolished TRPM7 expression in HIV-1Tg rats. In human BMVECs (hBMVECs), TRPM7 expression was downregulated after treatment with EtOH, HIV-1 proteins, and in combination. Next, we constructed in vitro BBB models using BMVECs and found TRPM7 antagonists enhanced EtOH-mediated BBB integrity changes. Our study demonstrated alcohol decreased TRPM7 expression, whereby TRPM7 could be involved in the mechanisms underlying BBB alcohol-induced damage in HIV-1 patients on cART.
SummaryMore than 97% of achondroplasia cases are caused by one of two mutations (G1138A and G1138C) in the fibroblast growth factor receptor 3 (FGFR3) gene, which results in a specific amino acid substitution, G380R. Sporadic cases of achondroplasia have been associated with advanced paternal age, suggesting that these mutations occur preferentially during spermatogenesis. We have determined the parental origin of the achondroplasia mutation in 40 sporadic cases. Three distinct 1-bp polymorphisms were identified in the FGFR3 gene, within close proximity to the achondroplasia mutation site. Ninety-nine families, each with a sporadic case of achondroplasia in a child, were analyzed in this study. In this population, the achondroplasia mutation occurred on the paternal chromosome in all 40 cases in which parental origin was unambiguous. This observation is consistent with the clinical observation of advanced paternal age resulting in new cases of achondroplasia and suggests that factors influencing DNA replication or repair during spermatogenesis, but not during oogenesis, may predispose to the occurrence of the G1138 FGFR3 mutations. More than 97% of achondroplasia cases are caused by one of two mutations (G1138A and G1138C) in the fibroblast growth factor receptor 3 (FGFR3) gene, which results in a specific amino acid substitution, G380R. Sporadic cases of achondroplasia have been associated with advanced paternal age, suggesting that these mutations occur preferentially during spermatogenesis. We have determined the parental origin of the achondroplasia mutation in 40 sporadic cases. Three distinct 1-bp polymorphisms were identified in the FGFR3 gene, within close proximity to the achondroplasia mutation site. Ninety-nine families, each with a sporadic case of achondroplasia in a child, were analyzed in this study. In this population, the achondroplasia mutation occurred on the paternal chromosome in all 40 cases in which parental origin was unambiguous. This observation is consistent with the clinical observation of advanced paternal age resulting in new cases of achondroplasia and suggests that factors influencing DNA replication or repair during spermatogenesis, but not during oogenesis, may predispose to the occurrence of the G1138 FGFR3 mutations.
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