Antibodies have become increasingly important agents in diagnostics and therapy, and there are growing demands for novel designer molecules that bind to a given site of a protein target. The existing methods allow production of antibodies against a given linear epitope. However, the majority of sites on the surface of a folded protein are conformational epitopes, and currently there are no technical means for obtaining antibodies to a pre-specified conformational epitope. This limitation is an important problem as this impedes development of novel antibody-based therapeutics. We propose a novel approach for engineering antibodies that bind to a pre-specified epitope of a folded protein. We wish to apply this technology for producing inhibitory antibodies to ectoenzymes that play a principal role in tumor progression and metastasis. In particular, we have shown that the cell surface ectonucleotidase CD39 hydrolyzes extracellular nucleotides to produce adenosine, which strongly suppresses anti-tumor immunity and promotes angiogenesis. Inhibition of CD39 with a small-molecule compound, polyoxometalate-1, significantly inhibits tumor growth, but poor selectivity and toxicity limit the therapeutic effects. The objective of thi application is to develop a universal technology for engineering antibodies that bind to a given epitope of a folded protein, and as an example, produce a potent and selective antibody inhibitor of mouse CD39. We hypothesize that inhibitory antibodies to CD39 and other enzymes can be engineered from a common precursor anti-fluorescein antibody by targeting active sites of enzymes. We will test this hypothesis as follows.
Aim 1 : Establish the inhibitory function of th anti-fluorescein antibody by targeting the active site of CD39. Based on the 3D structure of CD39, we will create artificial antibody-binding sites near the active site of CD39 by introducing cysteine residues via mutagenesis and labeling these with fluorescein. We will then identify a CD39 mutant where enzymatic activity is completely inhibited by the anti-fluorescein antibody, indicating the optimal position of the antibody for blocking the active site.
Aim 2 : Generate binding complementarity between the inhibitory anti-fluorescein antibody and CD39 independent of fluorescein label. We will use a traditional approach of """"""""affinity maturation"""""""" by randomization of complementarity determining regions of the antibody and selection for improved binding. We expect that selection for binding will preserve inhibitory functions and produce the inhibitory antibody to mouse CD39. We anticipate the following positive impacts: First, the inhibitory antibody to mouse CD39 will allow us to evaluate in subsequent animal studies the full therapeutic potential of targeting CD39 in cancer. Second, the derived technology will enable rational engineering of inhibitory antibodies to human CD39 and other key ectoenzymes implicated in cancer progression. In addition, the developed technology will meet needs in research, diagnostics and fundamentally advance the field of therapeutic antibody engineering.

Public Health Relevance

Cancer progression critically depends on activity of cell surface ectonucleotidases. We propose to develop a novel technology to produce antibody inhibitors of these enzymes for cancer treatment.

Agency
National Institute of Health (NIH)
Institute
National Cancer Institute (NCI)
Type
Exploratory/Developmental Grants (R21)
Project #
1R21CA164970-01A1
Application #
8309768
Study Section
Cancer Immunopathology and Immunotherapy Study Section (CII)
Program Officer
Yovandich, Jason L
Project Start
2012-04-01
Project End
2014-03-31
Budget Start
2012-04-01
Budget End
2013-03-31
Support Year
1
Fiscal Year
2012
Total Cost
$227,070
Indirect Cost
$96,570
Name
Beth Israel Deaconess Medical Center
Department
Type
DUNS #
071723621
City
Boston
State
MA
Country
United States
Zip Code
02215
Takenaka, Maisa C; Robson, Simon; Quintana, Francisco J (2016) Regulation of the T Cell Response by CD39. Trends Immunol 37:427-39
Miao, Ruoyu; Wu, Yan; Zhang, Haohai et al. (2016) Utility of the dual-specificity protein kinase TTK as a therapeutic target for intrahepatic spread of liver cancer. Sci Rep 6:33121
Gampe, Kristine; Haverkamp, Silke; Robson, Simon C et al. (2015) NTPDase2 and the P2Y1 receptor are not required for mammalian eye formation. Purinergic Signal 11:155-60
Bai, Aiping; Robson, Simon (2015) Beyond ecto-nucleotidase: CD39 defines human Th17 cells with CD161. Purinergic Signal 11:317-9
Mascanfroni, Ivan D; Takenaka, Maisa C; Yeste, Ada et al. (2015) Metabolic control of type 1 regulatory T cell differentiation by AHR and HIF1-α. Nat Med 21:638-46
Bai, Aiping; Moss, Alan; Rothweiler, Sonja et al. (2015) NADH oxidase-dependent CD39 expression by CD8(+) T cells modulates interferon gamma responses via generation of adenosine. Nat Commun 6:8819
Gampe, Kristine; Stefani, Jennifer; Hammer, Klaus et al. (2015) NTPDase2 and purinergic signaling control progenitor cell proliferation in neurogenic niches of the adult mouse brain. Stem Cells 33:253-64
Bai, Aiping; Moss, Alan; Kokkotou, Efi et al. (2014) CD39 and CD161 modulate Th17 responses in Crohn's disease. J Immunol 193:3366-77
Yu, Weiqun; Sun, Xiaofeng; Robson, Simon C et al. (2014) ADP-induced bladder contractility is mediated by P2Y12 receptor and temporally regulated by ectonucleotidases and adenosine signaling. FASEB J 28:5288-98
Vaughn, Byron P; Robson, Simon C; Longhi, Maria Serena (2014) Purinergic signaling in liver disease. Dig Dis 32:516-24

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