Domain II of Pseudomonas Exotoxin Is Critical for Efficacy of Bolus Doses in a Xenograft Model of Acute Lymphoblastic Leukemia.

Fabian Müller,Ira Pastan,Alan Wayne,Tyler Cunningham,Richard Beers,Tapan Bera

doi:10.3390/toxins10050210

Abstract

Moxetumomab pasudotox is a fusion protein of a CD22-targeting antibody and Pseudomonas exotoxin. Minutes of exposure to Moxetumomab achieves similar cell killing than hours of exposure to a novel deimmunized variant against some acute lymphoblastic leukemia (ALL). Because blood levels fall quickly, Moxetumomab is more than 1000-fold more active than the deimmunized variant in vivo. We aimed to identify which part of Moxetumomab increases in vivo efficacy and generated five immunotoxins, tested time-dependent activity, and determined the efficacy in a KOPN-8 xenograft model. Full domain II shortened the time cells had to be exposed to die to only a few minutes for some ALL; deimmunized domain III consistently extended the time. Against KOPN-8, full domain II accelerated time to arrest protein synthesis by three-fold and tripled PARP-cleavage. In vivo efficacy was increased by more than 10-fold by domain II and increasing size, and therefore half-life enhanced efficacy two- to four-fold. In summary, in vivo efficacy is determined by the time cells have to be exposed to immunotoxin to die and serum half-life. Thus, domain II is most critical for activity against some ALL treated with bolus doses; however, immunotoxins lacking all but the furin-cleavage site of domain II may be advantageous when treating continuously.

Highlights

Therapeutic antibodies have become the standard of care in the treatment of a variety of diseases, including cancer [1]
To test which protein domain was responsible for the substantial difference of activity of disulfide-stabilized Fv (dsFv)-PE38 and Fab-PE24(B) against the KOPN-8 cell line, we constructed a total of five molecules controlling for the three major differences, namely size of the antibody fragment, full domain II
We mathematically determined the fold-difference of cytotoxicity of the immunotoxin variants relative to dsFv-PE38

Summary

Introduction

Therapeutic antibodies have become the standard of care in the treatment of a variety of diseases, including cancer [1]. Toxins 2018, 10, 210 the constant fragment (Fc) of antibodies has been engineered to improve recruitment of complement and immune cells [2] and antibodies were armed with radionucleotides [3], chemotherapeutic drug conjugates [4,5], or plant and bacterial toxins [6]. The latter, called immunotoxins, show high clinical activity against hematologic malignancies [6,7]. Moxetumomab pasudotox (hereafter referred to as dsFv-PE38) produces response rates of 86% in patients with relapsed/refractory (r/r) hairy cell leukemia [8], and of 32% in r/r pediatric acute lymphoblastic leukemia (ALL) [9]

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Conclusion