Entwicklung, Optimierung und Charakterisierung anti-CD64-basierter Immuntherapeutika

Immunotoxins (ITs) commonly are composed of modified antibodies that are linked to a toxin either chemically or through the generation of a fusion protein. The antibody directs the IT to specific target cells where it binds to surface molecules and is internalized, allowing the cytotoxic component to kill the cell. ITs are regarded as particularly suitable for the treatment of cancer, and several ITs containing plant or bacterial toxins have been evaluated for immunotherapy, the most potent toxins being Pseudomonas exotoxin A (ETA), diphtheria toxin and ricin. These toxins contain a catalytic domain responsible for the cytotoxic activity and a translocation domain that facilitates transfer of the catalytic domain into the cytosol of target cells, optimizing cytotoxicity. Recently, an IT based on ETA was reported, which was specific for acute myeloid leukemia (AML) cells. These cells are often characterized by overexpression of FcγR I (CD64), a receptor with high affinity for the Fc portion of immunoglobulins G1 and G3, which is normally expressed on monocytes and macrophages. These AML cells are targeted efficiently by the humanized anti-CD64 single chain antibody fragment (scFv) h22(scFv) and killed by ETA. However, the major drawback of such ITs is their potential immunogenicity. Repeated doses may cause hypersensitive reactions and lead to neutralization of the ITs by antibodies directed against the non-human domains. Further complications result from the non-specific binding of foreign proteins, leading to vascular leak syndrome and ultimately interstitial oedema and organ failure. The development of ITs containing human or humanized components may circumvent these problems. Neither should such ITs be immunogenic, nor should they show non-specific toxic effects. However, these human proteins do not possess their own translocation domain as mentioned above; thus, translocation of the toxic moiety into the cytosol is dependent on target structure internalization. The integration of a recombinant adapter that contains a synthetic translocation domain flanked by proteolytically cleavable endosomal and cytosolic consensus should compensate for these disadvantages and increase IT cytotoxicity. First, based on the well defined IT h22(scFv)-ETA, the functionality of this molecular adapter was compared to the properties of the natural translocation domain of ETA. Therefore a truncated version of the IT h22(scFv)-ETA was developed in which the natural translocation domain of ETA was replaced by the molecular adapter. In addition, an IT-version without adapter and natural tranlocation domain was designed to compare all resulting data. The ITs were expressed efficiently in E. coli and isolated from the periplasmic space for purification by IMAC. All variants bound specifically to CD64+ cells, as documented by flow cytometry, and exhibited specific toxicity towards CD64+ cell lines. With an IC50 of 0.5 nm, the toxicity of the IT containing the molecular adapter was comparable to the reference-IT h22(scFv)-ETA. The IT with no translocation domain and no adapter was 20 times less efficient (IC50=10nm). These results indicate that properties of the natural translocation domain and the adapter are similar. Based on these results a completely humanized IT and two IT-variants containing differently modified, cleavable adapter sequences were compared. As before, all ITs were based on the CD64 specific single chain antibody fragment h22(scFv) which is overexpressed on a subset of AML cells. The scFv was directly, or via recombinant adapters, coupled to the human RNase angiogenin, which exhibits cytotoxic activity following internalization. Two different adapter variants were used to optimize the translocation and toxicity of the ITs. All three ITs were expressed efficiently in E. coli and isolated from the periplasmic space for subsequent purification by IMAC. All variants also bound specifically to CD64+ cells, as documented by flow cytometry, and showed specific toxicity via apoptosis towards CD64+ cell lines. Most importantly, it was demonstrated that the insertion of a molecular adapter that contains cleavable peptides and a membrane transfer peptide increased the cytotoxicity up to 20-fold (from an IC50 of 0,2nm up to an IC50 of 0,01nm) but markedly decreased serum stability (from more than 24 hours stability down to 1 hour). The deletion of the endosomal-cleavable peptide within the molecular adapter dramatically enhanced serum stability while the cytotoxic potential was still more than 10-fold higher (IC50=0,03) in comparison to the IT with no adapter (IC50=0,2nm).

Grantor dc:publisher

Publikationsserver der RWTH Aachen University

Year dc:date

2009

Author dc:creator

• Hetzel, Christian

Contributors dc:contributor

• Fischer, Rainer

• info:eu-repo/classification/ddc/570
• Immunotoxin
• akute myeloische Leukämie
• Angiogenin
• Immuntherapie
• Biowissenschaften, Biologie
• CD64
• H22
• single chain fragment

Statement dc:rights

• info:eu-repo/semantics/openAccess

Language dc:language

ger

Identifier

https://publications.rwth-aachen.de/search?p=id:%22RWTH-CONV-113482%22

OAI record oai:identifier

https://publications.rwth-aachen.de/oai2d?verb=GetRecord&metadataPrefix=oai_dc&identifier=oai%3Apublications.rwth-aachen.de%3A51168