title: Interrogation of individual intratumoral B lymphocytes from lung cancer patients for molecular target discovery.
abstract: Intratumoral B lymphocytes are an integral part of the lung tumor microenvironment. Interrogation of the antibodies they express may improve our understanding of the host response to cancer and could be useful in elucidating novel molecular targets. We used two strategies to explore the repertoire of intratumoral B cell antibodies. First, we cloned VH and VL genes from single intratumoral B lymphocytes isolated from one lung tumor, expressed the genes as recombinant mAbs, and used the mAbs to identify the cognate tumor antigens. The Igs derived from intratumoral B cells demonstrated class switching, with a mean VH mutation frequency of 4%. Although there was no evidence for clonal expansion, these data are consistent with antigen-driven somatic hypermutation. Individual recombinant antibodies were polyreactive, although one clone demonstrated preferential immunoreactivity with tropomyosin 4 (TPM4). We found that higher levels of TPM4 antibodies were more common in cancer patients, but measurement of TPM4 antibody levels was not a sensitive test for detecting cancer. Second, in an effort to focus our recombinant antibody expression efforts on those B cells that displayed evidence of clonal expansion driven by antigen stimulation, we performed deep sequencing of the Ig genes of B cells collected from seven different tumors. Deep sequencing demonstrated somatic hypermutation but no dominant clones. These strategies may be useful for the study of B cell antibody expression, although identification of a dominant clone and unique therapeutic targets may require extensive investigation.
authors.affiliation: Department of Radiology, Duke University Medical Center, Box 3808, Durham, NC, 27710, USA.
authors.forename: Michael J
authors.initials: MJ
authors.lastname: Campa
authors.affiliation: Department of Pediatrics, Duke University Medical Center, Durham, NC, 27710, USA.|Department of Duke Human Vaccine Institute, Duke University Medical Center, Durham, NC, 27710, USA.
authors.forename: M Anthony
authors.initials: MA
authors.lastname: Moody
authors.affiliation: Department of Duke Human Vaccine Institute, Duke University Medical Center, Durham, NC, 27710, USA.
authors.forename: Ruijun
authors.initials: R
authors.lastname: Zhang
authors.affiliation: Department of Duke Human Vaccine Institute, Duke University Medical Center, Durham, NC, 27710, USA.|Department of Medicine, Duke University Medical Center, Durham, NC, 27710, USA.
authors.forename: Hua-Xin
authors.initials: HX
authors.lastname: Liao
authors.affiliation: Department of Radiology, Duke University Medical Center, Box 3808, Durham, NC, 27710, USA.
authors.forename: Elizabeth B
authors.initials: EB
authors.lastname: Gottlin
authors.affiliation: Department of Radiology, Duke University Medical Center, Box 3808, Durham, NC, 27710, USA. patz0002@mc.duke.edu.|Department of Pharmacology and Cancer Biology, Duke University Medical Center, Durham, NC, 27710, USA. patz0002@mc.duke.edu.
authors.forename: Edward F
authors.initials: EF
authors.lastname: Patz
mesh_terms: D000368:Aged; D000595:Amino Acid Sequence; D000906:Antibodies / Q000737:chemistry / Q000235:genetics / Q000276:immunology / Q000302:isolation & purification; D000951:Antigens, Neoplasm / Q000276:immunology; D000970:Antineoplastic Agents / Q000494:pharmacology / Q000627:therapeutic use; D016175:B-Lymphocyte Subsets / Q000276:immunology* / Q000378:metabolism; D016022:Case-Control Studies; D060965:Clonal Evolution / Q000235:genetics / Q000276:immunology; D003001:Cloning, Molecular; D005260:Female; D015870:Gene Expression; D006801:Humans; D008175:Lung Neoplasms / Q000188:drug therapy / Q000235:genetics / Q000276:immunology* / Q000473:pathology*; D016246:Lymphocytes, Tumor-Infiltrating / Q000276:immunology* / Q000378:metabolism; D008297:Male; D008875:Middle Aged; D008969:Molecular Sequence Data; D058990:Molecular Targeted Therapy; D009367:Neoplasm Staging; D016415:Sequence Alignment; D017422:Sequence Analysis, DNA
chemical_list: D000906:Antibodies; D000951:Antigens, Neoplasm; D000970:Antineoplastic Agents
keywords: Intratumoral B lymphocytes; Non-small cell lung cancer; Target discovery; Tropomyosin 4; Tumor-infiltrating lymphocytes