(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.

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.

Antibody-based immunotherapy of leukemia requires the targeting of specific antigens on the surface of blasts. The Fc gamma receptor (CD64) has been investigated in detail, and CD64-targeting immunotherapy has shown promising efficacy in the targeted ablation of acute myeloid leukemia (AML), acute myelomonocytic leukemia (AMML) and chronic myeloid leukemia cells (CML). Here we investigate for the first time the potential of FcaRI (CD89) as a new target antigen expressed by different myeloid leukemic cell populations. For specific targeting and killing, we generated a recombinant fusion protein comprising an anti-human CD89 single-chain Fragment variable and the well-characterized truncated version of the potent Pseudomonas aeruginosa exotoxin A (ETA'). Our novel therapeutic approach achieved in vitro EC 50 values in range 0.2-3 nM depending on the applied stimuli, that is, interferon gamma or tumor necrosis factor alpha. We also observed a dose-dependent apoptosis-mediated cytotoxicity, which resulted in the elimination of up to 90% of the target cells within 72 hr. These findings were also confirmed ex vivo using leukemic primary cells from peripheral blood samples of three previously untreated patients. We conclude that CD89-specific targeting of leukemia cell lines can be achieved in vitro and that the efficient elimination of leukemic primary cells supports the potential of CD89-ETA' as a potent, novel immunotherapeutic agent.Under normal physiological conditions, the Fc-alpha receptor (FcaR, CD89) is present on monocytes, macrophages, neutrophils and eosinophils. Its biological function is to interact with IgA-opsonized targets, triggering several immunological defense processes, e.g. phagocytosis, antibody-dependent cellmediated cytotoxicity and the stimulation of inflammatory mediators. 1 In many human tissues, most of the CD89 1 cells are neutrophils and monocytes/macrophages. Furthermore, although monocytes in the blood express relatively high levels of CD89, most tissue macrophages (particularly those located in the gut lamina propria) tend not to express CD89 on the surface, with a parallel downregulation of CD14. 2 This suggests that the abundance of CD89 depends on the differentiation stage of myeloid cells. The biological relevance of CD89 downregulation during the maturation of myeloid precursor cells is unknown.On the other hand, the presence of anomalous numbers of myeloid cells in the blood is indicative of several myelogenous hematological malignancies, with AML and CML being the most common. 3 The different forms of myeloid leukemia have been categorized using the French-American-British (FAB) system divided into eight subtypes (M0-M7). The World Health Organisation (WHO) uses expanded criteria. 4 A standard panel of markers is used for detailed diagnosis and classification, including Fc gamma receptor (FcgRI) (CD64) which is present on AML subtypes M0-M5 in varying degrees. 5 Thus far, there is no evidence that other Fc receptors can be used as diagnostic antigens or therapeutic targets in A...

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