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Antibody Microarrays for Probing the Binding Cooperativity of Leukemia-Associated Cell Surface Antigens

Chaofang Yue, Chemical and Biomolecular Engineering, The Ohio State University, 125A Koffolt Laboratories, 140 west 19th Ave., Columbus, OH 43210, Yuan Yuan, Nanoscale Science and Engineering Center for Affordable Nanoengineering of Polymeric Biomedical Devices, The Ohio State University, 140 WEST 19TH AVE, Columbus, OH 43210, Bo Yu, Department of Chemical and Biomolecular Engineering, The Ohio State University, 140 West 19th Avenue, Columbus, OH 43210, J. James Lee, The Ohio State University, Room 125A, Koffolt Labs., 140W. 19th Ave., Columbus, OH 43210, and Michael E. Paulaitis, Department of Chemical engineering, The Ohio State University, 125 Koffolt Labs, 140, West 19th Ave, Columbus, OH 43210.

In the past several years, many novel targeted, immunosuppressive therapies have been developed for the treatment of leukemia. However, their therapeutic effects can deviate substantially from patient to patient due to variations in the levels of leukemia-associated cell surface antigens that are expressed across a diverse patient population. Efficient strategies for characterizing patient populations with heterogeneous clinical resistance to immunosuppressive therapies have not been established. One major challenge of targeted immunotherapies for leukemia treatment is designing targeting molecules or a repertoire of targeting molecules that can be applied across a heterogeneous population expressing a wide range of leukemia-associated cell surface antigens.

Recent studies have demonstrated that antibody microarrays can be effective tools for profiling leukemia-associated cell surface antigens [1,2]. In general, antibody microarrays quantify antigens expressed on a cell surface by measuring the density of cells captured on spots printed with the relevant antibodies. The level of cell capture depends primarily on the antigen concentration on surface of cells, and the antigen-antibody binding affinity/avidity. Most current strategies use only one targeting antibody for cell capture. The existence of positive cooperativity in the context of multivalent interactions is well recognized in the field of immunology. Thus, targeting strategies that can take advantage of multiple antibodies can potentially achieve higher targeting efficiency compared to single antibodies. The use of multiple targeting antibodies, however, requires the development of methods for rapidly and quantitatively characterizing cooperativity in binding cell surface antigens.

We have developing antibody microarrays to quantitatively characterize targeting efficiencies of multiple antibodies in a systematic, high-throughput fashion, which enables screening for optimal combinations of antibodies for targeted immunosuppressive therapies. Individual spots on the microarray are printed with different compositions of antibodies, and cell capture on each spot is measured to give the binding efficiency corresponding to that antibody composition. Comparing these results to cell capture on spots printed with the associated single antibodies gives the extent of binding cooperativity. Using a B-cell chronic lymphocytic leukemia cell line, we have shown that multiple antibodies can achieve much higher binding efficiencies compared to single antibodies printed at the same concentration. To investigate the kinetics of cell binding, a parallel-plate flow chamber has been designed to probe the kinetics of cell adhesion and the subsequent shear-induced detachment of bound cells. Time-tracked flow experiments can thus be carried out to estimate the dynamic range of cell binding to spots printed with multiple antibodies, compared to spots printed with single antibodies to provide important insights into the cooperative targeting of leukemia-associated cell surface antigens.

1. Belov, L., et al., Screening microarrays of novel monoclonal antibodies for binding to T-, B- and myeloid leukaemia cells. Journal of Immunological Methods, 2005. 305(1): p. 10-19.

2. Belov, L., et al., Analysis of human leukaemias and lymphomas using extensive immunophenotypes from an antibody microarray. British Journal of Haematology, 2006. 135(2): p. 184-197.