Date of Award

January 2020

Document Type

Thesis

Degree Name

Medical Doctor (MD)

Department

Medicine

First Advisor

Gary V. Desir

Abstract

The pharmacokinetics of a humanized anti-RNLS monoclonal antibody used for the treatment of melanoma in a congenic mouse species

Authors: Oriyomi Alimi1, Gary Desir1

1Department of Internal Medicine, Yale School of Medicine, New Haven, CT

Cancer cells can overcome signaling that restrains their growth and promotes senescence and cell death. Renalase (RNLS) is a secreted flavoprotein that functions as a survival factor after ischemic and toxic injury, signaling through the plasma calcium channel PMCA4b to activate the PI3K/AKT and MAPK pathways. In addition, recent studies, indicate that dysregulated RNLS signaling promotes survival of melanoma cells due to its capacity to augment expression of growth-related genes and to promote macrophage polarization toward a tumor promoting phenotype. Preliminary data show single agent efficacy of the inhibitory rabbit monoclonal m28- RNLS in PD1 resistant tumors. We humanized m28-RNLS and selected a variant (K5), formatted to human IgG1 containing an Fc portion that promotes complement binding. The purpose of these experiments is to determine the pharmacokinetic (PK) profile in C57BL6 mouse of K5, a therapeutic candidate for cancer therapy. K5 was produced by transfecting human embryonic 293 kidney cells using standard methods. To determine the pharmacokinetics of K5, mice (n=28) were dosed with 6.4 mg/kg K5 either as a single intravenous injection (i.v) via tail vein or subcutaneous (s.c) injection. Blood was collected at 14 time points and stored at -20°C until assayed for K5 concentrations. K5 concentration was determined using a direct-sandwich ELISA and uses His-tag purified recombinant human renalase as capture antigen and goat anti- human Fc IgG as detection antibodies. Standards were made in duplicate using known concentrations of antibody diluted into mouse serum. Standard curves were interpolated using GraphPad Prism. Concentrations were determined from the standard curves. Concentration

versus time data were analyzed using a non-compartmental analysis in R Studio. PK data indicate that K5 may behave in vivo like other commercially available IgG1 molecules. It exhibits high bioavailability, with a rapid distribution phase. Rapid elimination of the humanized antibody may reflect formation of anti-tumor antibodies. Pharmacokinetic data in mice can be helpful in predicting the pharmacokinetics of K5 in humans using allometric scaling but may not be useful in C57/BL6 mice. Pharmacokinetic testing of K5 in transgenic mice with human neonatal receptors (e.g Tg32 homozygous mice) or other species such as non-human primates is needed before first in human doses.

Comments

This thesis is restricted to Yale network users only. It will be made publicly available on 09/10/2022

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