RESUMO
The current standard treatment for acute myeloid leukemia (AML) is chemotherapy based on cytarabine and daunorubicine (7 + 3), but it discriminates poorly between malignant and benign cells. Dose-limiting offtarget effects and intrinsic drug resistance result in the inefficient eradication of leukemic blast cells and their survival beyond remission. This minimal residual disease is the major cause of relapse and is responsible for a 5-year survival rate of only 24%. More specific and efficient approaches are therefore required to eradicate malignant cells while leaving healthy cells unaffected. In this study, we generated scFv antibodies that bind specifically to the surface of AML blast cells and AML bone marrow biopsy specimens. We isolated the antibodies by phage display, using subtractive whole-cell panning with AML M2derived Kasumi1 cells. By selecting for internalizing scFv antibody fragments, we focused on potentially novel agents for intracellular drug delivery and tumor modulation. Two independent methods showed that 4 binders were internalized by Kasumi-1 cells. Furthermore, we observed the AMLselective inhibition of cell proliferation and the induction of apoptosis by a recombinant immunotoxin comprising one scFv fused to a truncated form of Pseudomonas exotoxin A (ETA'). This method may therefore be useful for the selection of novel disease-specific internalizing antibody fragments, providing a novel immunotherapeutic strategy for the treatment of AML patients.
Assuntos
ADP Ribose Transferases , Anticorpos Antineoplásicos , Toxinas Bacterianas , Crise Blástica/tratamento farmacológico , Exotoxinas , Imunotoxinas , Leucemia Mieloide Aguda/tratamento farmacológico , Anticorpos de Cadeia Única , Fatores de Virulência , ADP Ribose Transferases/genética , ADP Ribose Transferases/imunologia , ADP Ribose Transferases/farmacologia , Anticorpos Antineoplásicos/genética , Anticorpos Antineoplásicos/imunologia , Anticorpos Antineoplásicos/farmacologia , Especificidade de Anticorpos/genética , Toxinas Bacterianas/genética , Toxinas Bacterianas/imunologia , Toxinas Bacterianas/farmacologia , Crise Blástica/imunologia , Crise Blástica/patologia , Linhagem Celular Tumoral , Exotoxinas/genética , Exotoxinas/imunologia , Exotoxinas/farmacologia , Humanos , Imunotoxinas/genética , Imunotoxinas/imunologia , Imunotoxinas/farmacologia , Leucemia Mieloide Aguda/imunologia , Leucemia Mieloide Aguda/patologia , Anticorpos de Cadeia Única/genética , Anticorpos de Cadeia Única/imunologia , Anticorpos de Cadeia Única/farmacologia , Fatores de Virulência/genética , Fatores de Virulência/imunologia , Fatores de Virulência/farmacologia , Exotoxina A de Pseudomonas aeruginosaRESUMO
Over the past few years, the SNAP-tag technology has become a methodology with great potential in a variety of applications, e.g. the (specific) visualization of individual proteins and studies of protein interaction in living cells. Furthermore, the tag can be used for immunopurification and detection of recombinant proteins or site-specific coupling of recombinant proteins to surfaces. Next to the in vitro applications, it also enables detection of tagged proteins in vivo. This review gives an overview of the SNAP-tag technology in different fields of research and its potential for future developments.
Assuntos
Imagem Molecular/métodos , Proteínas/química , Proteínas/metabolismo , Animais , Regulação da Expressão Gênica/fisiologia , Ligação Proteica , Proteínas/genética , Proteínas RecombinantesRESUMO
Although current cancer treatment strategies are highly aggressive, they are often not effective enough to destroy the collectivity of malignant cells. The residual tumor cells that survived the first-line treatment may continue to proliferate or even metastasize. Therefore, the development of novel more effective strategies to specifically eliminate also single cancer cells is urgently needed. In this respect, the development of antibody-based therapeutics, in particular example immunotoxins, has attracted broad interest. Since the internalization of immunotoxins is essential for their cytotoxic effectivity, it is of crucial importance to study their internalization behavior to assess the potential for their therapeutic use. In this study, we determined the internalization behavior of four different single-chain fragments variable (scFv) when binding to the corresponding target antigen as expressed on solid or non-solid tumor cell lines. The scFvs were recombinantly fused to the SNAP-tag, an engineered variant of the human repair enzyme O(6)-alkylguanine-DNA alkyltransferase that covalently reacts with benzylguanine derivatives. Since a large number of highly sensitive organic fluorescent dyes are already available or can easily be derivatized to react with the self-labeling SNAP-tag, this system provides versatile applications for imaging of intraand extracellular compartments of living cells. The fusion proteins were coupled to SNAP-surface(®) Alexa Fluor(®) 488 or SNAP-surface(®) Alexa Fluor(®) 647 and binding as well as internalization was monitored by flow cytometry and confocal microscopy, respectively. Depending on the respective target antigen, we could distinguish between slow and rapid internalization behavior. Moreover, we detected increased internalization rate for bivalent scFv constructs. Our approach allows for rapid and early stage evaluation of the internalization characteristics of new antibodies designated for further therapeutic development.