Antibodies Expressed by Intratumoral B Cells as the Basis for a Diagnostic Test for Lung Cancer
Final rept. 1 Jul 2013-31 Mar 2015
DUKE UNIV DURHAM NC
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As described in our Annual Report, our initial work focused on the expression, purification, and characterization of monoclonal antibodies mAbs cloned from intratumoral B lymphocytes ITLs. While this work was successful in identifying a mAb with preferential binding to tropomyosin 4, all cloned mAbs exhibited polyreactivity. This suggested that the isolated ITLs that we used for mAb production were not members of clonal B cell populations arising in response to stimulation by intratumoral antigens. Hence, our most recent efforts have focused on methods to permit the cloning and expression of recombinant antibodies specifically from oligoclonal ITLs. We accomplished this by performing deep sequencing of VH and VL Ig genes from ITLs from seven additional lung cancer patients, We identified a total of 155,901 functional VH gene sequences with individual patient samples containing from 3,327 to 77,182 functional sequences. Although we obtained evidence for somatic hypermutation among these sequences, we did not identify any dominant clones. Sequences representing all VH gene families were identified and exhibited a distribution typically found in peripheral B cells. This suggests that it may be necessary to sequence Ig genes from microdissected intratumoral germinal centers in order to identify clonal ITL populations.
- Medicine and Medical Research