The vedotin antibody-drug conjugate payload drives platform-based nonclinical safety and pharmacokinetic profiles.

Nonclinical safety and pharmacokinetic data for MMAE and 14 vedotin ADCs were evaluated to determine patterns of toxicity, consistency of pharmacokinetic results, and species differences between rats and monkeys. Most nonclinical toxicities were antigen-independent, common across ADCs, and included hematologic, lymphoid, and reproductive toxicity related to MMAE pharmacology. Hematologic toxicity was the dose-limiting or predominant toxicity for the majority of vedotin ADCs in both species. Tissue expression of the targeted antigen of an ADC rarely correlated with dose-limiting toxicity (DLT); only two ADCs had antigen-dependent skin DLTs. For two additional ADCs, antigen-dependent delivery of MMAE in the bone marrow may have exacerbated the antigen-independent hematologic DLT. The highest tolerated doses and pharmacokinetics were similar within a given species, with rats tolerating higher doses than monkeys. Studies longer than one month in duration detected the same or fewer toxicities than one-month studies and had no additional findings that affected the human risk assessment. These data support opportunities to streamline ADC toxicity assessments without compromising human starting dose selection or target organ identification.

Preclinical characterization of SGN-70, a humanized antibody directed against CD70.

PURPOSE: CD70 (CD27L) is a member of the tumor necrosis factor family aberrantly expressed on a number of hematologic malignancies and some carcinomas. CD70 expression on malignant cells coupled with its highly restricted expression on normal cells makes CD70 an attractive target for monoclonal antibody (mAb)-based therapies. We developed a humanized anti-CD70 antibody, SGN-70, and herein describe the antitumor activities of this mAb. EXPERIMENTAL DESIGN: CD70 expression on primary tumors was evaluated by immunohistochemical staining of Hodgkin lymphoma, non-Hodgkin lymphoma, multiple myeloma, and renal cell carcinoma tissue microarrays. The CD70-binding and cytotoxic activities of SGN-70 were tested in vitro using a number of cell-based assays. The in vivo antitumor properties of SGN-70 were tested in severe combined immunodeficient mice bearing disseminated lymphoma and multiple myeloma xenografts. Mechanism-of-action studies were conducted using SGN-70v, a variant mAb with equivalent target-binding activity but impaired Fcgamma receptor binding compared with SGN-70. RESULTS: Immunohistochemical analysis identified CD70 expression on approximately 40% of multiple myeloma isolates and confirmed CD70 expression on a high percentage of Hodgkin lymphoma Reed-Sternberg cells, non-Hodgkin lymphoma, and renal cell carcinoma tumors. SGN-70 lysed CD70+ tumor cells via Fc-dependent functions, including antibody-dependent cellular cytotoxicity and phagocytosis and complement fixation. In vivo, SGN-70 treatment significantly decreased tumor burden and prolonged survival of tumor-bearing mice. CONCLUSIONS: SGN-70 is a novel humanized IgG1 mAb undergoing clinical development for the treatment of CD70+ cancers. SGN-70 possesses Fc-dependent antibody effector functions and mediates antitumor activity in vivo.

The Discovery and Preclinical Development of ASG-5ME, an Antibody-Drug Conjugate Targeting SLC44A4-Positive Epithelial Tumors Including Pancreatic and Prostate Cancer.

Here, we report the development of an antibody-drug conjugate, ASG-5ME, which targets the solute carrier receptor SLC44A4. SLC44A4 is a member of a family of putative choline transporters that we show to be markedly upregulated in a variety of epithelial tumors, most notably prostate and pancreatic cancer. SLC44A4 is normally expressed on the apical surface of secretory epithelial cells, but in cancer we show expression is not restricted to the luminal surface in advanced and undifferentiated tumors. ASG-5ME consists of a human IgG2 anti-SLC44A4 antibody conjugated through a cleavable linker to the microtubule-disrupting agent monomethylauristatin E. It has potent antitumor activity in both cell line - and patient-derived xenograft models of pancreatic and prostate cancers. Combination studies with ASG-5ME and nab-paclitaxel demonstrated combination effect in both pancreatic and prostate tumor models. Altogether, the data presented here suggest that ASG-5ME may have the potential to offer a new therapeutic option for the treatment of pancreatic and prostate cancers. Mol Cancer Ther; 15(11); 2679-87. ©2016 AACR.

Development of a Guinea pig model for low-dose, long-term exposure to organophosphorus nerve agents.

An animal dosing model and related maximum tolerated dose (MTD) were developed for repeated exposures in guinea pigs to three organophosphorus chemical warfare nerve agents (CWNA). Male animals were injected subcutaneously with sarin (GB), soman (GD) or VX once a day (Monday through Friday) for 2-, 4-, or 13-weeks. An initial 13-week study for each CWNA employed doses of vehicle (normal saline), 0.2x, 0.4x, 0.6x, and 0.8x the previously established acute LD(50). A 2-week and 4-week exposure were also performed for each agent at doses less than the 13-week MTD to verify lack of toxicity. Animals dosed daily for 13 weeks with 0.4x LD(50) of GB or GD or with 0.2x LD(50) of VX did not display signs of acute cholinergic toxicity. In animals dosed daily for either 2- or 4-weeks, the MTDs were 0.4x the acute LD(50) for all three CWNA. There were no differences among these groups and their respective vehicle controls for weight gains, body temperature, complete blood cell counts, blood chemistries, nor by histopathology. At the MTD in all groups, red blood cell cholinesterase activity one hour after the last exposure was inhibited up to 90% compared with controls. The toxicity observed with repeated doses above the MTD for up to chronic exposure durations was not significantly different from symptoms observed after acute exposure. For all three nerve agents the MTDs for subacute exposure durations can be expressed by the same constant fraction of the acute LD(50), despite differences in the absolute amount of nerve agent administered.

Self-hydrolyzing maleimides improve the stability and pharmacological properties of antibody-drug conjugates.

Many antibody-drug conjugates (ADCs) are unstable in vivo because they are formed from maleimide-containing components conjugated to reactive thiols. These thiosuccinimide linkages undergo two competing reactions in plasma: elimination of the maleimide through a retro-Michael reaction, which results in loss of drug-linker from the ADC, and hydrolysis of the thiosuccinimide ring, which results in a derivative that is resistant to the elimination reaction. In an effort to create linker technologies with improved stability characteristics, we used diaminopropionic acid (DPR) to prepare a drug-linker incorporating a basic amino group adjacent to the maleimide, positioned to provide intramolecular catalysis of thiosuccinimide ring hydrolysis. This basic group induces the thiosuccinimide to undergo rapid hydrolysis at neutral pH and room temperature. Once hydrolyzed, the drug-linker is no longer subject to maleimide elimination reactions, preventing nonspecific deconjugation. In vivo studies demonstrate that the increased stability characteristics can lead to improved ADC antitumor activity and reduced neutropenia.

Toxicity in rhesus monkeys following administration of the 8-aminoquinoline 8-[(4-amino-l-methylbutyl)amino]- 5-(l-hexyloxy)-6-methoxy-4-methylquinoline (WR242511).

INTRODUCTION: Many substances that form methemoglobin (MHb) effectively counter cyanide (CN) toxicity. Although MHb formers are generally applied as treatments for CN poisoning, it has been proposed that a stable, long-acting MHb former could serve as a CN pretreatment. Using this rationale, the 8-aminoquinoline WR242511, a potent long-lasting MHb former in rodents and beagle dogs, was studied in the rhesus monkey for advanced development as a potential CN pretreatment. METHODS: In this study, WR242511 was administered intravenously (IV) in 2 female and 4 male rhesus monkeys in doses of 3.5 and/or 7.0 mg/kg; a single male also received WR242511 orally (PO) at 7.0 mg/kg. Health status and MHb levels were monitored following exposure. RESULTS: The selected doses of WR242511, which produced significant methemoglobinemia in beagle dogs in earlier studies conducted elsewhere, produced very little MHb (mean < 2.0%) in the rhesus monkey. Furthermore, transient hemoglobinuria was noted approximately 60 minutes postinjection of WR242511 (3.5 or 7.0 mg/kg), and 2 lethalities occurred (one IV and one PO) following the 7.0 mg/kg dose. Myoglobinuria was also observed following the 7.0 mg/kg dose. Histopathology analyses in the 2 animals that died revealed liver and kidney toxicity, with greater severity in the orally-treated animal. CONCLUSIONS: These data demonstrate direct and/or indirect drug-induced toxicity. It is concluded that WR242511 should not be pursued as a pretreatment for CN poisoning unless the anti-CN characteristics of this compound can be successfully dissociated from those producing undesirable toxicity.

Phase I Trial of sequential administration of recombinant DNA and adenovirus expressing L523S protein in early stage non-small-cell lung cancer.

L523S is an immunogenic lung cancer antigen that has demonstrated preclinical safety when the gene is injected intramuscularly as an expressive plasmid (pVAX/L523S) and when delivered following incorporation into an E1B-deleted adenovirus (Ad/L523S). We performed a phase I clinical trial in 13 stage IB, IIA, and IIB non-small-cell lung cancer patients. pVAX/L523S (8 mg on days 0 and 14 in all cohorts) and Ad/L523S (1, 20, 400 x 10(9) vp on days 28 and 56, cohorts 1, 2, and 3, respectively) were administered to 3 patients in each of three cohorts. No significant toxic effect was identified. All but 1 patient demonstrated greater than or equal to twofold elevation in anti-adenovirus antibodies. One of 10 evaluable patients demonstrated L523S-specific antibody by direct IgG ELISA. Two patients developed disease recurrence and all remain alive after a median of 290 days follow-up. Results suggest a high level of safety but evidence of L523S-directed immune activation was limited, suggesting a need for modification of dose, schedule, and site of vaccination (i.e., intradermal) with further clinical testing.

Control of nerve agent-induced seizures is critical for neuroprotection and survival.

This study evaluated the potency and rapidity of some anticholinergics (atropine, biperiden, and trihexyphenidyl) and benzodiazepines (diazepam and midazolam) as an anticonvulsant treatment against seizures induced by six nerve agents (tabun, sarin, soman, cyclosarin, VR, and VX) and summarized the relationship between anticonvulsant activity and nerve agent-induced lethality and neuropathology. Guinea pigs, previously implanted with cortical electrodes for EEG recording, were pretreated with pyridostigmine bromide (0.026 mg/kg im) 30 min prior to challenge with 2x LD50 dose (sc) of a given nerve agent; in a separate experiment, animals were challenged with 5x LD50 sc of soman. One minute after agent challenge the animals were treated im with 2 mg/kg atropine SO(4) admixed with 25 mg/kg 2-PAM Cl. Five minutes after the start of EEG seizures, animals were treated im with different doses of anticholinergics or benzodiazepines and observed for seizure termination. The time to seizure onset, the time to seizure termination, and 24-h lethality were recorded. The anticonvulsant ED50 of each drug for termination of seizures induced by each agent was calculated and compared. Brain tissue from animals that survived 24 h was examined for pathology. All drugs were capable of terminating seizure activity, with midazolam and trihexyphenidyl being significantly more potent than the other drugs, and midazolam being more rapid in controlling seizure than atropine, trihexyphenidyl, or diazepam against each agent. Seizures induced by sarin or VX required lower doses of all the test anticonvulsants. The dose of a given drug that was an effective anticonvulsant against a 2x LD50 challenge of soman was equally effective against seizures induced by a 5x LD50 challenge. All nerve agents were capable of producing neuropathology. Seizure control was strongly associated with protection against acute lethality and brain pathology.

Impact of inhalation exposure modality and particle size on the respiratory deposition of ricin in BALB/c mice.

Ricin is a toxic lectin derived from the seed of Ricinus communis (castor plant). It is lethal in small quantities when disseminated as an aerosol. We determined the impact of using two types of exposure chambers and different particle sizes on the deposition of ricin aerosols in mice. Initially, two types of inhalation exposure chambers (whole-body [WB] or nose-only [NO]) were compared using the same size aerosol (1 micro m) to determine the potential impact upon respiratory deposition and presented dose. We then assessed the role of particle size on deposition by using aerosols with two distinctly sized particle distributions. Selected organs were collected at four time points after exposure and were analyzed by quantitative enyzme-linked immunosorbent assay (ELISA) and epifluorescence microscopy. Results of the exposure chamber comparison, using 1- micro m particles only, indicated approximately 50% of the total ricin in the 4 organs was detected in the lung tissue 1 h after exposure. The trachea and nasopharyngeal region of the animals exposed using the WB chamber contained significantly more ricin than those of animals exposed in the NO chamber. Histopathology indicated an accumulation of ricin in both the tracheobronchial and pulmonary regions with pronounced bronchiolar degradation 48 h postexposure. When particles larger than 3 micro m were used, results indicated a considerable amount of ricin initially detected in the trachea, although this finding was discounted due to the heterodispersity of the particles generated. Interestingly, no animals died as a result of exposure to the equivalent of 4 LD50s (as determined using a 1- micro m particle) when exposed to the larger size distribution of particles. This result indicates a differential lethality that is contingent upon aerosol size.

Acute changes in lung histopathology and bronchoalveolar lavage parameters in mice exposed to the choking agent gas phosgene.

Phosgene (CG) is a highly irritant gas widely used industrially as a chemical intermediate for the production of dyes, pesticides, and plastics, and can cause life-threatening pulmonary edema within 24 hours of exposure. This study was designed to investigate acute changes in lung tissue histopathology and selected bronchoalveolar lavage fluid (BALF) factors over time to determine early diagnostic indicators of exposure. Three groups of 40 male mice each were exposed to 32 mg/m3 (8 ppm) CG for 20 minutes, and 3 groups of 40 control male mice were exposed to filtered room air for 20 minutes, both exposures were followed by room air washout for 5 minutes. At 1, 4.8, 12, 24, 48, and 72 hours after exposure each group of mice was euthanized and processed for histopathology, bronchoalveolar lavage or gravimetric measurements, respectively. Over time, the histopathological lesions were characterized by acute changes consisting of alveolar and interstitial edema, fibrin and hemorrhage, followed by significant alveolar and interstitial flooding with inflammatory cell infiltrates and scattered bronchiolar and terminal airway epithelial degeneration and necrosis. From 48 to 72 hours, there was partial resolution of the edema and degenerative changes, followed by epithelial and fibroblastic regeneration centered on the terminal bronchiolar areas. Bronchoalveolar lavage was processed for cell differential counts, LDH, and protein determination. Comparative analysis revealed significant increases in both postexposure lung wet/dry weight ratios, and early elevations of BALF LDH and protein, and later elevations in leukocytes. This article describes the use of histopathology to chronicle the temporal pulmonary changes subsequent to whole body exposure to phosgene, and correlate these changes with BALF ingredients and postexposure lung wet weights in an effort to characterize toxic gas-induced acute lung injury and identify early markers of phosgene exposure.