The Human Combinatorial Antibody Library (HuCAL) was screened for antibodies specific to human leukocyte antigen-DR (HLA-DR) that induce programmed death of lymphoma/leukemia cells expressing the target antigen. The active Fab fragments were affinity-matured, and engineered to IgG(4) antibodies of sub-nanomolar affinity. The antibodies exhibited potent in vitro tumoricidal activity on several lymphoma and leukemia cell lines and on chronic lymphocytic leukemia patient samples. They were also active in vivo in xenograft models of non-Hodgkin lymphoma. Cell death occurred rapidly, without the need for exogenous immunological effector mechanisms, and was selective to activated/tumor-transformed cells. Although the expression of HLA-DR on normal hematopoietic cells is a potential safety concern, the antibodies caused no long-lasting hematological toxicity in primates, in vivo. Such monoclonal antibodies offer the potential for a novel therapeutic approach to lymphoid malignancies.

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The anaphylatoxic peptide C3a is part of a basic immunological defense mechanism, the complement system. Research on the human C3a receptor and signal transduction is hampered by the lack of a suitable human cell or cell line. We screened tumor cell lines and human blood cells for a C3a-dependent increase in cytosolic Ca2+ ([Ca2+]i) and analyzed this reaction in a fura-2/AM fluorescence assay for cells in suspension. U937 cells, when differentiated with dibutyryl-cAMP (Bt2cAMP), and purified human neutrophils reacted in a dose-dependent fashion to C3a and a C3a analogue synthetic peptide. We found complete homologous desensitization of this response and no heterologous desensitization to human C5a. Pertussis toxin totally blocked the increase in [Ca2+]i, indicating the possible involvement of a G-protein. Single-cell analysis by digital imaging fluorescence microscopy indicated that neutrophilic granulocytes responded to C3a. In binding studies with Bt2cAMP-differentiated U937 cells and human granulocytes, the 125I-C3a binding was displaced by C3a, yielding one class of C3a binding sites with dissociation constants (Kd) in the low nanomolar range. We identified myo-inositol 1,4,5-trisphosphate (IP3) as the second messenger possibly causing the [Ca2+]i increase and the release of N-acetyl-beta-D-glucosaminidase as one secretory cell response. By functional and binding studies we demonstrated the expression of the C3a receptor on Bt2-cAMP-differentiated U937 cells and human neutrophils and characterized parts of the C3a signal pathway. Our data support a physiological concept in which C3a might be more important than presently thought.

The human combinatorial antibody library Fab 1 (Hu-CAL®-Fab 1) was generated by transferring the heavy and light chain variable regions from the previously constructed single-chain Fv library (Knappik, A., Ge, L., Honegger, A., Pack, P., Fischer, M., Wellnhofer, G., Hoess, A., Wö lle, J., Plü ckthun, A., and Virnekä s, B. (2000) J. Mol. Biol. 296, 57-86), diversified in both complementarity-determining regions 3 into a novel Fab display vector, yielding 2.1 ؋ 10 10 different antibody fragments. The modularity has been retained in the Fab display and screening plasmids, ensuring rapid conversion into various antibody formats as well as antibody optimization using prebuilt maturation cassettes. Hu-CAL®-Fab 1 was challenged against the human fibroblast growth factor receptor 3, a potential therapeutic antibody target, against which, to the best of our knowledge, no functional antibodies could be generated so far. A unique screening mode was designed utilizing recombinant functional proteins and cell lines differentially expressing fibroblast growth factor receptor isoforms diversified in expression and receptor dependence. Specific Fab fragments with subnanomolar affinities were isolated by selection without any maturation steps as determined by fluorescence flow cytometry. Some of the selected Fab fragments completely inhibit targetmediated cell proliferation, rendering them the first monoclonal antibodies against fibroblast growth factor receptors having significant function blocking activity. This study validates HuCAL®-Fab 1 as a valuable source for the generation of target-specific antibodies for therapeutic applications.

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