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Despite the use of rabbits in biomedical research, including regulatory toxicology and cardiovascular studies, little data exist on heart findings in this species. This study was designed to document myocardial findings in female rabbits and the impact of study-related procedures typical for vaccine toxicology studies. One hundred and forty 6- to 8-month-old female New Zealand White rabbits were divided equally into 2 groups, high and low study procedure groups (group 1 and group 2, respectively). All animals received intramuscular (IM) injections of sterile saline every 2 weeks for 5 times and were necropsied 2 days after the final IM injection. Clinical chemistry, hematology, and urinalysis were evaluated. Blood for stress biomarkers (norepinephrine, epinephrine, cortisol, and corticosterone), C-reactive protein, cardiac troponin I, and creatine kinase were collected at time 0 (just before dose administration) and then at 4, 24, and 48 hr after dose administration in group 1 only. Hearts were assessed histologically. Focal to multifocal minimal inflammatory cell infiltrates were common (∼80%), particularly in the left ventricle and interventricular septum, and were similar to the types of infiltrates identified in other laboratory animal species. Additionally, study-related procedures elevated serum stress biomarkers and exacerbated the frequency and severity of myocardial inflammatory cell infiltrates.

The Staphylococcus aureus virulence factor clumping factor A (ClfA) is a component of an investigational S. aureus prophylactic vaccine. ClfA enables S. aureus to bind to fibrinogen and platelets during the initial stages of invasive disease. Here we demonstrate that ectopic expression of ClfA is sufficient to render nonpathogenic Lactococcus lactis lethal in a murine model of systemic infection. In contrast, L. lactis expressing ClfAY338A, which cannot bind fibrinogen, did not cause death in the mice. Pathogenicity was also prevented by immunization with ClfA. This model was then used to define a preclinical correlate of protection by measuring functional antibody in a S. aureus fibrinogen binding inhibition assay (FBI) and correlating that titer with protective outcomes. Although many humans have pre-existing antibodies that bind to ClfA, only sera with a threshold functional titer in the FBI were protective in this preclinical model. This confirms that fibrinogen binding is critical for ClfA-mediated pathogenesis and demonstrates that functional antibodies against ClfA are sufficient to protect against ClfA-mediated pathogenesis in vivo, enabling the definition of a preclinical correlate of protection for ClfA-containing vaccines based on FBI titer.

In present study, the potential drug delivery of nanoformulations was validated via the comparison of cellular uptake of nanoparticles in various cell lines and in vivo pulmonary cellular uptake in intratracheally (IT) dosed rat model. Nanoparticles were prepared by a bench scale wet milling device and incubated with a series of cell lines, including Caco-2, RAW, MDCK and MDCK transfected MDR1 cells. IT dosed rats were examined for the pulmonary cellular uptake of nanoparticles. The processes of nanoparticle preparation did not alter the crystalline state of the material. The uptake of nanoparticles was observed most extensively in RAW cells and the least in Caco-2 cells. Efflux transporter P-gp did not prevent cell from nanoparticles uptake. The cellular uptake of nanoparticles was also confirmed in bronchoalveolar lavage (BAL) fluid cells and in bronchiolar epithelial cells, type II alveolar epithelial cells in the intratracheally administrated rats. The nanoparticles uptake in MDCK, RAW cells and in vivo lung epithelial cells indicated the potential applications of nanoformulation for poorly soluble compounds. The observed limited direct uptake of nanoparticles in Caco-2 cells suggests that the improvement in oral bioavailability by particle size reduction is via increased dissolution rate rather than direct uptake.

The use of predictive preclinical models in drug discovery is critical for compound selection, optimization, preclinical to clinical translation, and strategic decision-making. Trophoblast glycoprotein (TPBG), also known as 5T4, is the therapeutic target of several anticancer agents currently in clinical development, largely due to its high expression in tumors and low expression in normal adult tissues. In this study, mice were engineered to express human TPBG under endogenous regulatory sequences by replacement of the murine Tpbg coding sequence. The gene replacement was considered functional since the hTPBG knockin (hTPBG-KI) mice did not exhibit clinical observations or histopathological phenotypes that are associated with Tpbg gene deletion, except in rare instances. The expression of hTPBG in certain epithelial cell types and in different microregions of the brain and spinal cord was consistent with previously reported phenotypes and expression patterns. In pharmacokinetic studies, the exposure of a clinical-stage anti-TPBG antibody-drug conjugate (ADC), A1mcMMAF, was lower in hTPBG-KI versus wild-type animals, which was evidence of target-related increased clearance in hTPBG-KI mice. Thus, the hTPBG-KI mice constitute an improved system for pharmacology studies with current and future TPBG-targeted therapies and can generate more precise pharmacokinetic and pharmacodynamic data. In general the strategy of employing gene replacement to improve pharmacokinetic assessments should be broadly applicable to the discovery and development of ADCs and other biotherapeutics.

Antibody–drug conjugates (ADC) represent a promising therapeutic modality for the clinical management of cancer. We sought to develop a novel ADC that targets 5T4, an oncofetal antigen expressed on tumor-initiating cells (TIC), which comprise the most aggressive cell population in the tumor. We optimized an anti-5T4 ADC (A1mcMMAF) by sulfydryl-based conjugation of the humanized A1 antibody to the tubulin inhibitor monomethylauristatin F (MMAF) via a maleimidocaproyl linker. A1mcMMAF exhibited potent in vivo antitumor activity in a variety of tumor models and induced long-term regressions for up to 100 days after the last dose. Strikingly, animals showed pathologic complete response in each model with doses as low as 3 mg antibody/kg dosed every 4 days. In a non–small cell lung cancer patient-derived xenograft model, in which 5T4 is preferentially expressed on the less differentiated tumor cells, A1mcMMAF treatment resulted in sustained tumor regressions and reduced TIC frequency. These results highlight the potential of ADCs that target the most aggressive cell populations within tumors, such as TICs. In exploratory safety studies, A1mcMMAF exhibited no overt toxicities when administered to cynomolgus monkeys at doses up to 10 mg antibody/kg/cycle × 2 and displayed a half-life of 5 days. The preclinical efficacy and safety data established a promising therapeutic index that supports clinical testing of A1mcMMAF. Mol Cancer Ther; 12(1); 38–47. ©2012 AACR.

Zileuton, a redox and iron chelator 5-lipoxygenase (5-LOX) inhibitor and, leukotriene receptor antagonists are presently used clinically in the long term treatment of asthma. Recent data implicate 5-LOX pathway in pain signaling. We report 5-LOX expression in the central nervous system (CNS) and analyze the pain efficacy of a new class of non redox, non iron chelating 5-LOX inhibitor. CJ-13610, 4-(3-(4-(2-methyl-1H-imidazol-1-yl) phenylthio) phenyl)-tetrahydro-2H-pyran-4-carboxamide, demonstrated antihyperalgesic activity in inflammatory pain models including the acute carrageenan model and the chronic inflammatory model using complete Freund's adjuvant. Following complete Freund's adjuvant stimulus leukotrieneB(4) concentration in the brain was elevated (9+/-1 ng/g, mean+/-S.E.M.) by about 3 times that of the control group (3+/-0.11, mean+/-S.E.M.). Hyperalgesia and leukotrieneB(4) concentration were both reversed following CJ-13610 treatment. Furthermore, we demonstrate CJ-13610 efficacy against osteoarthritis like pain using the rat medial meniscal transection model. CJ-13610 at oral doses of 0.6, 2 and 6 mg/kg/day reversed two modalities of pain in this model; tactile allodynia and weight bearing differential. Taken together, these data suggest that 5-LOX pathway and the leukotriene products are important mediators of pain.

Antibody-drug conjugates (ADCs) represent a promising therapeutic modality for the clinical management of cancer. We sought to develop a novel ADC that targets 5T4 (TPBG), an oncofetal antigen expressed on tumor-initiating cells (TICs), which comprise the most aggressive cell population in the tumor. We optimized an anti-5T4 ADC (A1mcMMAF) by sulfydryl-based conjugation of the humanized A1 antibody to the tubulin inhibitor monomethylauristatin F (MMAF) via a maleimidocaproyl linker. A1mcMMAF exhibited potent in vivo anti-tumor activity in a variety of tumor models and induced long-term regressions for up to 100 days after the last dose. Strikingly, animals showed pathological complete response in each model. In a non-small cell lung cancer patient-derived xenograft in which 5T4 is preferentially expressed on the less differentiated tumor cells, A1mcMMAF treatment resulted in sustained tumor regressions and reduced TIC frequency. These results highlight the potential of ADCs that target the most aggressive cell populations within tumors. An optimized pharmacokinetic/pharmacodynamic (PK/PD) model of tumor growth and drug kill was used to characterize the ADC concentration response relationship in mouse. A holistic secondary parameter, tumor static concentration (TSC), was derived from model parameters to quantify efficacy and support early clinical trial design. Tumor static concentrations [80% confidence] of A1mcMMAF ranged from 1.1[0.9 -1.4] μg/ml to 11.6 [9.6 - 14.1] μg/ml across tumor models. For comparison, in the clinic T-DM1 has an average concentration of 14 μg/ml at an efficacious dose of 3.6 mg/kg Q3wk (HER+ breast cancer) (Krop et al. 2010) and Brentuximab-vedotin has an average concentration of 3.65 μg/ml at an efficacious dose of 1.8 mg/kg Q 3wk (HL/ ALCL) (Younes et al. 2010). Taken together, the preclinical data established a promising therapeutic index that supports clinical testing of A1mcMMAF. Expression analysis profiling using clinical and preclinical data indicated that lung and breast tumors demonstrated differentially high expression of 5T4 in comparison to normal tissues. An IHC assay developed in house confirmed the hypothesis that a broad range of 5T4 expression was measurable in NSCLC patient tumor samples. Additionally, we developed an assay that measures 5T4 expression on circulating tumor cells (CTCs) and used this assay to measure and characterize a broad range of 5T4 expression in CTCs obtained from the blood of NSCLC patients. We intend to deploy these co-developed immunoassays to guide A1mcMMAF clinical development. Citation Format: Puja Sapra, Marc Damelin, Kimberly Marquette, Kenneth G. Geles, Jonathon Golas, Maureen Dougher, Bitha Narayanan, Andreas Giannakou, Kiran Khandke, Russell Dushin, Elana Ernstoff, Judy Lucas, Mauricio Leal, George Hu, Alison Betts, Nahor Haddish-Berhane, Eric Powell, Steven Pirie-Shepherd, Christopher O'Donnell, Lioudmila Tchistiakova, Hans-Peter Gerber, Dena Marrinucci, Eric Tucker. Preclinical development and translational research on a novel antibody-drug conjugate that targets 5T4, an oncofetal antigen expressed on tumor-initiating cells. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 4752. doi:10.1158/1538-7445.AM2013-4752

Antibody-drug conjugates (ADCs) represent a promising therapeutic modality for the clinical management of cancer. We have developed a novel ADC that targets 5T4 (also known as TPBG), a tumor-associated antigen that rapidly internalizes. In preclinical models of NSCLC, 5T4 was identified as a marker of undifferentiated tumorigenic cells that express properties of tumor-initiating cells (TICs) and was associated with a highly proliferating (Ki67 positive) cell phenotype. Based on the biological properties of 5T4, we designed the ADC by conjugating an internalizing humanized anti-5T4 antibody (Ab), A1, to the tubulin inhibitor monomethylauristatin F via a maleimidocaproyl (mc) linker (A1mcMMAF) with a drug/antibody ratio of ∼ 4. The A1 Ab has a Kd of 0.9 nM against the 5T4 antigen, binds to a broad range of 5T4 expressing cell lines, and rapidly internalizes (66% internalized within 4h). As an ADC, A1mcMMAF retains similar properties to the unconjugated Ab. In vitro, A1mcMMAF exhibited potent and target-specific cytotoxic activity against a panel of 5T4+ cell lines with an IC50 of 5 ng Ab/ml against high 5T4 expressing cells. In vivo, A1mcMMAF exhibited potent anti-tumor activity in all five tumor models tested (with a broad range of 5T4 expression) and caused long-term regressions monitored up to 100 days after the last dose. Strikingly, animals were cured of disease in every model with doses as low as 3 mg Ab/kg Q4d x 4. In a lung cancer patient-derived xenograft in which 5T4 is not uniformly expressed, but instead is preferentially expressed on the less differentiated sub-population of tumor cells, A1mcMMAF treatment resulted in sustained tumor regression. This result highlights the clinical potential of ADCs that target aggressive cell subpopulations such as TICs. In exploratory safety studies, A1mcMMAF exhibited no overt toxicities when administered at 10 mg Ab/kg/cycle x 2 to cynomolgus monkeys and had a half-life of ∼5 days. The encouraging efficacy and safety data of the conjugate resulting in high therapeutic index in preclinical models warrants further clinical testing of this ADC. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 2530. doi:1538-7445.AM2012-2530

The pharmacokinetics of a novel antibody (huA1)-drug (auristatin tubulin inhibitor MMAF) conjugate, targeting 5T4-expressing cells, were obtained in female nu/nu mice after a single IV dose of 1, 3 or 10 mg/kg and in male cynomolgus monkeys from a 2-cycle exploratory toxicity study at doses of 1, 3 or 10 mg/kg/cycle. Plasma/serum samples, collected over a 336 hr period after dosing, were analyzed using an ELISA-based method for antibody and conjugate (ADC) as well as for the Auristatin payload using an LC/MS/MS method. A comparison across doses (1 − 10 mg/kg) suggests lower CL values at the higher doses (3 and 10 mg/kg) in mice. Exposure in the mouse at anti-tumor doses was within the exposure observed at nontoxic doses with the monkeys. The huA1 and conjugate toxicokinetic data in monkeys dosed with A1mcMMAF indicates that exposure (AUC) increases with increasing dose although it appears to increase in a greater than proportional manner at the higher doses. In both mice and monkeys, the elimination half-life was longer as the dose increased from 1 to 10 mg/kg (from 4.2 to 8.7 days in monkeys, respectively). Systemic exposure (as assessed by Cmax and AUC) of the released payload increased with increasing dose although exposure was very low and its pharmacokinetics appeared to be formation rate limited. The analytical data from these studies are being used to inform PKPD models, to both quantitatively characterize the system and ultimately provide dose projections for this ADC construct Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2011 Nov 12-16; San Francisco, CA. Philadelphia (PA): AACR; Mol Cancer Ther 2011;10(11 Suppl):Abstract nr C117.