In vivo efficacy of the recombinant anti‐CD64 immunotoxin H22(scFv)‐ETA′ in a human acute myeloid leukemia xenograft tumor model

Abstract

Target-specific acute myeloid leukemia (AML) immunotherapy requires selective cell-surface antigens on AML blast cells. CD64 is a promising candidate antigen because it is abundantly expressed on monocytoid differentiated AML subtypes. In previous studies, a chemically linked full-length anti-CD64 immunotoxin based on ricin A showed promising results in several animal models, but further development has been hindered by its substantial, dose-limiting off-target effects. We recently constructed the recombinant immunotoxin H22 ( Acute myeloid leukemia (AML) is a heterogeneous collection of neoplasms characterized by the aberrant growth of myeloid precursor cells in bone marrow and peripheral blood.

Monomeric IgG1 Fc molecules displaying unique Fc receptor interactions that are exploitable to treat inflammation-mediated diseases

“…Indeed, a number of anti-FcgRI immunotoxins, composed of anti-FcgRI antibodies attached to various toxins, have recently been developed, and their efficacy has been demonstrated in vitro and in mouse models of different diseases, such as chronic cutaneous inflammation, rheumatoid arthritis, acute myeloid leukemia. 11,12,25,26,36,37 In this regard, the use of mFc to deliver therapeutic molecules could be particularly valuable because it provides extended circulating half-life and improved biodistribution by interacting with FcRn, 38,39 and it offers additional targeting to FcgRI-overexpressed cells, which in certain cases could lead to greater therapeutic benefit. For instance, one immediate application is to fuse mFc with anti-FcgRI immunotoxins.…”

“…9,10,[13][14][15]24 Indeed, elimination of macrophages using anti-FcgRI scFv immunotoxins has been shown to effectively resolve inflammation in vitro and in animal models. 11,12,25,26 Given the finding that mFc can selectively and potently bind to FcgRI, we therefore sought to determine whether mFc-based fusion proteins could be capable of FcgRI-mediated targeting of macrophages, offering additional therapeutic benefits in treatment of chronic inflammation-related diseases.…”

Antimalarial Activity of Granzyme B and Its Targeted Delivery by a Granzyme B–Single-Chain Fv Fusion Protein

“…The concept of recombinant immunotoxins has been extensively investigated in the field of targeted tumor therapy with various bacterial and human effector domains (30)(31)(32), including Gb (11,13,27,33). After binding to a disease-specific cell surface antigen, these immunotoxins are internalized, e.g., by receptor-mediated endocytosis, released from endosomal compartments into the cytoplasm, and efficiently kill the malignant cell by their catalytic activity.…”

Phage display-based generation of novel internalizing antibody fragments for immunotoxin-based treatment of acute myeloid leukemia

“…The scFv-ETA' immunotoxin was expressed under stress in the presence of compatible solutes as previously described. 33 The periplasmic fraction was recovered by pelleting the bacteria and re-suspending in buffer (75 mM Tris-HCl, 300 mM NaCl, 10% glycerol, one tablet protease inhibitor per 50 ml) then sonicating for 9 £ 1 min at 70% intensity. The periplasmic fraction was recovered after centrifugation at 30,000 x g for 30 min and the recombinant immunotoxin was purified in a batch procedure using Ni-NTA Sepharose (QIAGEN, Hilden, Germany).…”

“…The resin was washed once with buffer 1 (50 mM NaH 2 PO 4 , 300 mM NaCl, 20 mM imidazole), twice with buffer 2 (50 mM NaH 2 PO 4 , 300 mM NaCl, 40 mM imidazole) and bound protein was stripped with elution buffer (50 mM NaH 2 PO 4 , 300 mM NaCl, 250 mM imidazole). The eluted protein was finally purified using size exclusion chromatography as described 33 and analyzed quantitatively by SDS-PAGE. Functional binding activity was confirmed by flow cytometry using the anti-ETA' monoclonal antibody TC-1 (kindly provided by Dr Galloway, Ohio, USA).…”