A platform for phenotypic discovery of therapeutic antibodies and targets applied on Chronic Lymphocytic Leukemia

A Ljungars,N Persson,J Mattsson,B Jansson,M Ohlin,M Hansson,B Frendéus,M Mattsson,S Ek,U-C Tornberg,G Juliusson,I Teige,L Mårtensson,M Kovacek,A Sundberg,M Jerkeman,V Kuci Emruli

doi:10.1038/s41698-018-0061-2

Abstract

Development of antibody drugs against novel targets and pathways offers great opportunities to improve current cancer treatment. We here describe a phenotypic discovery platform enabling efficient identification of therapeutic antibody-target combinations. The platform utilizes primary patient cells throughout the discovery process and includes methods for differential phage display cell panning, high-throughput cell-based specificity screening, phenotypic in vitro screening, target deconvolution, and confirmatory in vivo screening. In this study the platform was applied on cancer cells from patients with Chronic Lymphocytic Leukemia resulting in discovery of antibodies with improved cytotoxicity in vitro compared to the standard of care, the CD20-specific monoclonal antibody rituximab. Isolated antibodies were found to target six different receptors on Chronic Lymphocytic Leukemia cells; CD21, CD23, CD32, CD72, CD200, and HLA-DR of which CD32, CD200, and HLA-DR appeared as the most potent targets for antibody-based cytotoxicity treatment. Enhanced antibody efficacy was confirmed in vivo using a patient-derived xenograft model.

Highlights

A pool of Chronic Lymphocytic Leukemia (CLL)-specific antibodies was generated by differential cell panning, applying positive selection pressure to primary CLL cells and concomitant negative selection pressure to peripheral blood mononuclear cells (PBMC) from healthy donors, using the phagedisplay human antibody library n-CoDeR® 5 (Fig. 2a)
PBMC from healthy donors were chosen as non-target cells as they are easy to obtain in large cell-numbers, contain critical immune effector cells, e.g. CD8+ T cells that one may not want to target, and express many general hematopoietic antigens with limited therapeutic potential
Unique scFvs were analyzed by flow cytometry for binding to primary patient CLL cells, various cell lines, and PBMC from healthy donors

Summary

INTRODUCTION

Drug discovery is either phenotypic or target based. Molecules with a desired effect on the phenotype of a cell or an organism are isolated followed by identification of their targets. For antibodies, which are the fastest growing class of drugs, only a few have been isolated using phenotypic discovery (for a comprehensive review see Minter et al 20173). It enables, depending on the selected phenotypic assay, both identification of antibodies that mediate their mechanism of action through the biology of the target receptor and through effector mechanisms such as Fcreceptor binding and engagement of immune effector cells. By the use of primary patient cells, phenotypic discovery enables development of personalized drugs. In essence, (1) isolation of 100s–1000s of antibodies that, ideally, comprise specificity for all therapeutically relevant cell surface receptors, (2) high-throughput phenotypic screening of antibodies in clinically predictive in vitro and in vivo assays, and (3) target deconvolution of antibodies

RESULTS

Methods used

METHODS