* I wish to thank all those who have heard or read various versions of this paper and whose comments have greatly encouraged and helped me.

Acute myeloid leukemia (AML) cells of subtypes M4 and M5 show enhanced expression of CD64 (Fc;RI), the highaffinity receptor for IgG, which is normally expressed at high levels only on activated cells of the myeloid lineage. CD64 is therefore a prime target for the specific delivery of cytotoxic agents. A promising toxin candidate is granzyme B, a human serine protease originating from cytotoxic granules of CD8 + T lymphocytes and natural killer cells. After evaluating the sensitivity of the AML-related cell line U937 toward cytosolic granzyme B, we genetically fused granzyme B to H22, a humanized single-chain antibody fragment (scFv) specific for CD64, to obtain Gb-H22(scFv), a fusion protein lacking the immunogenic properties of nonhuman immunofusions. Gb-H22(scFv) was successfully expressed in human 293T cells, secreted, and purified from cell culture supernatants. The purified protein bound specifically to CD64 + U937 cells. Despite linkage to the binding domain, the proteolytic activity of functional Gb-H22(scFv) was identical to that of free granzyme B. Target cell-specific cytotoxicity was observed with a half-maximal inhibitory concentration (IC 50 ) between 1.7 and 17 nmol/L. In addition, the induction of apoptosis in U937 cells was confirmed by Annexin A5 staining and the detection of activated caspase-3 in the cytosol. Finally, apoptosis was observed in primary CD64 + AML cells, whereas CD64 À AML cells were unaffected. This is the first report of a completely human granzyme B-based immunotoxin directed against CD64, with activity against an AML-related cell line and primary AML cells. [Mol Cancer Ther 2008;7(9):2924 -32]

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The most potent immunotoxins (ITs) developed to date contain bacterial or plant cytotoxic components. As these are potentially immunogenic in man, human proteins are preferred for the long-term treatment of cancer. We have developed the first humanized IT for the treatment of CD64 malignancies such as acute myeloid leukemia. The bacterially expressed IT is composed of a humanized anti-CD64 single chain fragment [h22(scFv)] genetically fused to the human RNase angiogenin. As angiogenin lacks a dedicated translocation domain responsible for the higher potency of bacterial and plant-derived toxins, we have incorporated a recombinant adapter that contains a synthetic translocation domain flanked by proteolytically cleavable endosomal and cytosolic consensus sites. Although insertion of the adapter increased the cytotoxicity by up to 20-fold, serum stability was markedly reduced. Therefore, we designed a modified adapter variant with the endosomal-cleavable peptide deleted. The IT containing the truncated adapter showed significantly higher cytotoxicity than the adapter-free IT and superior serum stability to facilitate the potential applications in patients.

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Transfection using the nonviral SB100X vector system avoids complications associated with viral gene delivery. SB100X-mediated transfer allows for stable PEDF gene integration into the cell's genome, ensuring continuous expression and secretion of PEDF. Stable expression of the therapeutic gene is critical for the development of cell-based gene addition therapies for retinal degenerative diseases.

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The central concern of this book is whether plural and incommensurable values necessarily result in unsolvable conflict and whether this conflict poses special problems for ethical theories. The view defended here is that plurality is no impediment to sound choice, and that a practicable ethics accounts for the fact that plurality and choice are ordinary and pervasive phenomena of moral life. Ethical theories, broadly divided into monistic and pluralistic, are pitted against each other in an attempt to show that the traditional understanding of the connection between value and conflict is flawed––a rationally grounded ethics does not require an evaluative monism purged of conflict, nor an algorithm to compare and choose between plural values. The book combines criticism of contemporary ethical theories with specific chapters on Aristotle's ethics. The first part focuses on the nature of moral conflict. Drawing on Aristotle's moral psychology, the author looks at dirty hands, moral immorality, and the nature of moral conflict in general. The second part focuses on whether plural values preclude sound moral judgement. The third part discusses whether conflict requires plural values, looking first at irrational conflict (akrasia or weakness of will) and next at rational conflict. The last part discusses maximization and plurality, and argues that maximization is mistaken, irrelevant, and parasitic. A central theme of the book is that ethics extends far beyond the guiding of action––‘our moral concern with acts goes beyond, primarily, what is to be done, and secondarily, what is better or best’.