In this project we will explore V region nucleotide sequence variation or idiotype in human B cell tumors. Using murine anti-idiotype antibodies as probes, three patients have displayed heterogeneity of tumor surface immunoglobulin in populations with identical gene rearrangements. Somatic cell hybridization will be performed by fusing tumor cells from these patients with a mouse/human heteromyeloma (K6H6/B5). Through this highly efficient process, a heterogeneous population of tumor cells can be cloned into individual idiotype-producing hybrids. These will be screened for reactivity with anti-idiotype antibodies and sub-populations will be identified. A cDNA library will be created from each sub-population using isolated mRNA and synthetic olignucleotide primers created for this purpose. The cDNA will be screened with probes which are also generated from these primers and one specific for the constant region of the lambda light chain. Positive inserts will be cloned into M13 and sequenced using the dedeoxy chain termination method. Considerable heterogeneity may exist within populations defined as homogeneous by reactivity with anti-idiotype antibodies. We plan to use RNA probes created from cloned cDNA inserts to screen multiple hybrids using RNA mapping techniques. These methods provide a unique opportunity to examine the evolution of V region heterogeneity in vivo over time and in response to chemo/immunotherapy. Significant insight into the biology of these human tumors can be gained by examining the interaction between the host and the malignancy. In addition, information concerning the hypermutational mechanism so characteristic of normal B cell differentiation may be generated.
