Development of a 10 color flow cytometric assay to assess binding of a monoclonal antibody (VB421) against IGF 1R in Peripheral Blood Mononuclear Cells (PBMCs) from patients with Thyroid Eye Disease

Introduction

ThyroidEye Disease ( also known as Graves’ ophthalmopathy, is adebilitating autoimmune disorder that occurs in patients with Graves’Disease in which inflammation in the muscle and fat tissue behind theeyes results in proptosis, diplopia, redness, pain, and swelling, leading tophotosensitivity, blurred vision, and in serious cases, blindness Themechanistic underpinnings of TED involve a complex interaction between autoantibody mediated stimulation of Thyroid Stimulating Hormone Receptor (and Insulin like growth factor 1 receptor (IGF 1 R)signaling in orbital fibroblasts that cause orbital tissue inflammation and expansion Current therapies include corticosteroids and teprotumumab, as well as surgical intervention to prevent vision loss VB 421L onigutamab) is a high affinity (KD 50 pM monoclonal antibody directed against IGF 1 R that induces rapid and efficient receptor internalizationVB 421 is being developed as a potential treatment for TED To support clinical development of VB 421 a multi color flow cytometric assay was developed to monitor the binding of VB 421 to IGF 1 R on the surface of human Peripheral Blood Mononuclear
Cells ( As VB 421 induces rapid IGF 1 R internalization upon binding, a traditional receptor occupancy assay format is less feasible Therefore, this assay is designed to detect both total IGF 1 R (free IGF 1 R and IGF 1 R/VB 421 complex) as well as free IGF 1 R This assay utilizes two anti IGF 1 R antibodies that bind different IGF 1 R epitopes and do not compete 1 H 7 competes with VB 421 while 33255 does not Together these two antibodies allow the assay to distinguish between unbound and total IGF 1 R This format was qualified using a cell line that constitutively expresses IGF 1 R at high levels (A 549 This assay will be used to monitor total and free amounts of IGF 1 R on live CD 3 Total T Cells, CD 4 T Cells, CD 8 T Cells, CD 19 B Cells, andMyeloid Cells expressing both CD 11 b and CD 16 after administration ofVB 421 This assay format has the potential to be applied to other situations where target receptor binding leads to rapid internalization and loss of
receptor binding epitopes.