ROR1 targeting with the antibody-drug conjugate VLS-101 is effective in Richter syndrome patient-derived xenograft mouse models.

Richter syndrome (RS) represents the transformation of chronic lymphocytic leukemia (CLL), typically to an aggressive lymphoma. Treatment options for RS are limited and the disease is often fatal. Receptor tyrosine kinase-like orphan receptor 1 (ROR1) is expressed on CLL cells and other cancers but not on healthy adult tissues, making it an attractive, tumor-specific therapeutic target. VLS-101 is being developed as an antibody-drug conjugate (ADC) for therapy of ROR1-expressing (ROR1+) cancers. VLS-101 comprises UC-961 (a humanized immunoglobulin G1 monoclonal antibody that binds an extracellular epitope of human ROR1), a maleimidocaproyl-valine-citrulline-para-aminobenzoate linker, and the antimicrotubule cytotoxin monomethyl auristatin E (MMAE). VLS-101 binding to ROR1 results in rapid cellular internalization and delivery of MMAE to induce tumor cell death. We studied 4 RS patient-derived xenografts (RS-PDXs) with varying levels of ROR1 expression (11%, 32%, 85%, and 99% of cells). VLS-101 showed no efficacy in the lowest-expressing RS-PDX but induced complete remissions in those with higher levels of ROR1 expression. Responses were maintained during the posttherapy period, particularly after higher VLS-101 doses. In systemic ROR1+ RS-PDXs, VLS-101 dramatically decreased tumor burden in all RS-colonized tissues and significantly prolonged survival. Animals showed no adverse effects or weight loss. Our results confirm ROR1 as a target in RS and demonstrate the therapeutic potential of using an ADC directed toward ROR1 for the treatment of hematological cancers. A phase 1 clinical trial of VLS-101 (NCT03833180) is ongoing in patients with RS and other hematological malignancies.

Development of a modified lymphocyte transformation test for diagnosing drug-induced liver injury associated with an adaptive immune response.

Drug-induced liver injury (DILI) is a growing problem. Diagnostic methods to differentiate DILI caused by an adaptive immune response from liver injury of other causes or to identify the responsible drug in patients receiving multiple drugs, herbals and/or dietary supplements (polypharmacy) have not yet been established. The lymphocyte transformation test (LTT) has been proposed as a diagnostic method to determine if a subject with an apparent hypersensitivity reaction has become sensitized to a specific drug. In this test, peripheral blood mononuclear cells (PBMC) collected from a subject are incubated with drug(s) suspected of causing the reaction. Cell proliferation, measured by the incorporation of [3H]-thymidine into new DNA, is considered evidence of a drug-specific immune response. The objectives of the current studies were to: (1) develop and optimize a modified version of the LTT (mLTT) and (2) investigate the feasibility of using the mLTT for diagnosing DILI associated with an adaptive immune response and identifying the responsible drug. PBMC collected from donors with a history of drug hypersensitivity reactions to specific drugs (manifested as skin rash) were used as positive controls for assay optimization. Following optimization, samples collected from 24 subjects enrolled in the U.S. Drug-Induced Liver Injury Network (DILIN) were tested in the mLTT. Using cytokine and granzyme B production as the primary endpoints to demonstrate lymphocyte sensitization to a specific drug, most samples from the DILIN subjects failed to respond. However, robust positive mLTT responses were observed for two of four samples from three DILIN subjects with hepatitis due to isoniazid (INH). We conclude that the mLTT, as performed here on frozen and thawed PBMC, is not a reliable test for diagnosing DILI caused by all drugs, but that it may be useful for confirming the role of the adaptive immune response in DILI ascribed to INH.

Limitations of an ocular surface inflammatory biomarker in impression cytology specimens.

CONTEXT: A number of ocular conditions, such as dry eye, are associated with inflammation on the surface of the eye leading to irritation and ocular pain. Many drugs such as chemotherapeutics, beta blockers, angiotensin-converting enzymes and so forth also cause dry eye but currently there are no validated ocular surface biomarkers available. OBJECTIVE: We evaluated sample stability, assay sensitivity, reproducibility and overall performance of impression cytology (IC) utilizing the cellular surface biomarker human leukocyte antigen DR-1 (HLA-DR) as an ocular surface inflammatory biomarker by flow cytometry in a fit-for-purpose validation study. Additionally, subjects classified as normal or having various degrees of dry eye were evaluated to determine if HLA-DR could demonstrate a clear separation between normal and dry eye samples. RESULTS: The assay demonstrated high dynamic range detecting a broad range of fluorescent intensities in healthy donors. Additionally, inter, intra and stability assay results demonstrated strong concordance and low variability. Overall CV% for both assays were less than 25% for all measured parameters. However, high variability was observed for donor samples assayed beyond day 10 post IC sample collection (4.2-110.8 CV%). DISCUSSION: HLA-DR expression demonstrated a progressive increase in patients with mild to severe levels of dry eye disease providing sufficient evidence it is sensitive enough to monitor inflammatory effects of dry eye when coupled with additional biomarkers and/or methodologies such as cytokine analysis or ICAM-1. This biomarker can be used to monitor ocular surface disorders in patients and to evaluate potential treatment options during drug development. Although our results demonstrate this methodology is reproducible for routine evaluation, limitations around sample integrity exist. CONCLUSION: The ocular cell surface inflammatory biomarker, HLA-DR coupled with impression cytology is a simple non-invasive robust, specific and reproducible assay that can be utilized to measure inflammatory infiltrates on the surface of the eye in IC samples less than 10-days old.

Comparison of 3 kidney injury multiplex panels in rats.

Kidney injury biomarkers have been utilized by pharmaceutical companies as a means to assess the potential of candidate drugs to induce nephrotoxicity. Multiple platforms and assay methods exist, but the comparison of these methods has not been described. Millipore's Kidney Toxicity panel, EMD/Novagen's Widescreen Kidney Toxicity panel, and Meso Scales Kidney Injury panel were selected based on published information. Kidney injury molecule 1, cystatin C, clusterin, and osteopontin were the 4 biomarkers common among all kits tested and the focus of this study. Rats were treated with a low and high dose of para-aminophenol, a known nephrotoxicant, and urine samples were collected and analyzed on the Bio-Plex 200 or MSD's Sector Imager 6000, according to manufacturers specifications. Comparatively, of the 3 kits, Millipore was the most consistent in detecting elevations of 3 out of the 4 biomarkers at both dose levels and indicated time points.

PD0325901, a mitogen-activated protein kinase kinase inhibitor, produces ocular toxicity in a rabbit animal model of retinal vein occlusion.

OBJECTIVE: PD0325901, a selective inhibitor of mitogen-activated protein kinase kinase (MEK), was associated with the occurrence of ocular retinal vein occlusion (RVO) during clinical trials in patients with solid tumors. As previous animal safety studies in rats and dogs did not identify the eye as a target organ of toxicity, this work was conducted to develop a rabbit model of ocular toxicity with PD0325901. METHODS: Dutch-Belted rabbits were administered a single intravitreal injection of PD0325901 (0.5 or 1 mg/eye) or saline control, and ophthalmic examinations and retinal angiography were conducted over a 2-week period post-dose. In addition, mechanism of ocular toxicity was further explored in rat with microarray analysis. RESULTS: PD0325901 treatment produced RVO with retinal vasculature leakage and hemorrhage within 48-h postinjection in Dutch-Belted rabbits. Subsequent retinal detachment and degeneration were also detected on day 8 postinjection. To evaluate the potential mechanism(s) of PD0325901-mediated RVO, male Brown Norway rats were orally administered PD0325901 (45 mg/kg/day) up to 5 days and retinal tissue was collected for gene array analysis. Although PD0325901 did not produce clinical evidence of RVO in rats, retinal gene expression suggested an increased oxidative stress and inflammatory response, endothelium and blood-retinal barrier damage, and prothrombotic effects. Moreover, soluble endothelial protein C receptor (sEPCR), a biomarker for RVO, was elevated in human umbilical vascular endothelial cells (HUVECs) cultured with PD0325901. CONCLUSIONS: This work has developed a rabbit model of PD0325901-induced RVO that may be used to characterize the cellular and molecular mechanisms of this effect in humans.

A lymphotoxin-IFN-beta axis essential for lymphocyte survival revealed during cytomegalovirus infection.

The importance of lymphotoxin (LT) betaR (LTbetaR) as a regulator of lymphoid organogenesis is well established, but its role in host defense has yet to be fully defined. In this study, we report that mice deficient in LTbetaR signaling were highly susceptible to infection with murine CMV (MCMV) and early during infection exhibited a catastrophic loss of T and B lymphocytes, although the majority of lymphocytes were themselves not directly infected. Moreover, bone marrow chimeras revealed that lymphocyte survival required LTalpha expression by hemopoietic cells, independent of developmental defects in lymphoid tissue, whereas LTbetaR expression by both stromal and hemopoietic cells was needed to prevent apoptosis. The induction of IFN-beta was also severely impaired in MCMV-infected LTalpha(-/-) mice, but immunotherapy with an agonist LTbetaR Ab restored IFN-beta levels, prevented lymphocyte death, and enhanced the survival of these mice. IFN-alphabetaR(-/-) mice were also found to exhibit profound lymphocyte death during MCMV infection, thus providing a potential mechanistic link between type 1 IFN induction and lymphocyte survival through a LTalphabeta-dependent pathway important for MCMV host defense.