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

Abstract

(2015) Phage display-based generation of novel internalizing antibody fragments for immunotoxin-based treatment of acute myeloid leukemia, mAbs, 7:2, 390-402, DOI: 10.1080DOI: 10. /19420862.2015 To link to this article: https://doi.org/10. 1080/19420862.2015 The current standard treatment for acute myeloid leukemia (AML) is chemotherapy based on cytarabine and daunorubicine (7 C 3), but it discriminates poorly between malignant and benign cells. Dose-limiting off-target 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 M2-derived Kasumi-1 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 AML-selective 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.

Antibody Engineering for Pursuing a Healthier Future

“…In addition, they are used in the diagnosis of immunological interpolation with passive antibodies, anti-idiotype suppression, or magic bullet treatments with cytotoxic agents coupled to anti-mouse specific antibodies (Keller and Stiehm, 2000). Similarly, recombinant DNA technology (rDNA) has revolutionized the reconstruction of mAbs by genetic engineering using chimeric antibodies, humanized antibodies, CDR grafted antibodies for therapeutic use (Dimitrov and Marks, 2009;Shen et al, 2014;Fitting et al, 2015).…”

Antibody Fragments and Their Purification by Protein L Affinity Chromatography

“…While mAbs are effective therapeutic agents when the antigenic target is well-defined and accessible, such as in some cancers and autoimmune diseases, many diseases offer less accessible (e.g., intracellular), cryptic, or hypoallergenic targets [40,62]. In such circumstances, antibody fragments may provide advantages due to their smaller size and structure, and the ease with which they can be produced and genetically modified [11,21,27,47,[63][64][65][66]. Ab-fragment-related biomedical applications include tumor treatment and imaging [67], viral replication suppression [68][69][70], toxin and venom neutralization [41,71], and receptor blockage [36,72].…”

“…As such scFv's are currently viewed as promising therapeutic fragments, with several having been in clinical trials [3,7,9,46]. Recently scFv fragments which can be internalized by cells were suggested as potentially novel agents for tumor modulation [47].…”

Phage display-based on-slide selection of tumor-specific antibodies on formalin-fixed paraffin-embedded human tissue biopsies

“…4A). The sequences were transferred to the pETderived pMT2 vector and introduced into E. coli BL21(DE3) cells for expression and purification [31]. The purified antibodies were quantified using the BCA assay, revealing concentrations in the range 150-250 g/ml, and their integrity was checked by SDS-PAGE and western blot (Fig.…”