Human vaccine studies regularly demonstrate vaccine-induced antibodies in the blood. Characterization of these antibodies shows that they exhibit an extensive range of bioactive mechanisms. Unfortunately, there is not yet a reliable method by which to produce these important antibodies. Human antibodies may not require any molecular modification for therapeutic application and have a much faster pipeline to clinical studies than animal-derived antibodies. Lymph nodes are the primary destination of tumor or vaccine antigens. In the node, B cells undergo clonal expansion and somatic hypermutation, leading to the affinity-matured populations of effector B cells secreting antibodies that bind to the tumor or vaccine. The cancer vaccine-draining node is the ideal source of B cells that produce anticancer antibodies. Despite multiple human cancer vaccine studies, very little research has been done to recover the B cells responsible for vaccine-induced anticancer antibodies. In the present study, we used mononuclear cells from surgically removed vaccine-draining lymph nodes of melanoma patients vaccinated with 6 melanoma peptides derived from cancer-testis antigens and from melanocytic differentiation proteins for generating anti-vaccine peptide antibodies. The lymph node draining the vaccination site was localized by lymphatic mapping with a radiotracer. The development and maturity of B cells were assessed by determining phenotypic characters of lymph node cells using multicolor flow cytometry. The results showed that these vaccine-draining nodes contain high numbers of class-switched (CD19+CD27+IgD-IgM-) B cells and plasmablasts (CD19+CD38+IgM-). B-cell ELISpot assay was used to quantify the proportion of B cells in vaccine-draining lymph nodes that secrete anti-melanoma peptide antibodies. Positive ELISpot responses were observed in patients who also showed serum antibody-reactivity towards the vaccine peptides. The identification of lymph node cell samples exhibiting strong B-cell responses allows efficient generation and screening of hybridomas that secrete antibodies against cancer vaccine antigens. This study establishes a step-wise protocol for generating anti-cancer antibodies from vaccine-draining lymph nodes. We anticipate that the ability to reliably generate in vitro the same antibodies observed in the blood of vaccinated patients will further stimulate research to understand mechanisms of human antibody activity.

Citation Format: Girja S. Shukla, Stephanie C. Pero, Yu-jing Sun, Chelsea L. Carman, Walter C. Olson, Craig L. Slingluff, David N. Krag. Vaccine-draining lymph nodes of cancer patients for generating anticancer antibodies. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3638. doi:10.1158/1538-7445.AM2014-3638