Vaccine-draining lymph nodes of cancer patients for generating anti-cancer antibodies

Abstract

Background: Our research is focused on using the vaccine draining lymph node to better understand the immune response to cancer vaccines and as a possible source of anti-cancer reagents. We evaluated vaccine draining lymph nodes archived from a clinical study in melanoma patients and determined the reaction of B cells to the vaccine peptides.

Methods: Mononuclear cells (MNCs) were recovered from cryopreserved lymph nodes that were directly receiving drainage from multi-peptide melanoma vaccine. The patients were enrolled on a vaccine study (NCT00089219, FDA, BB-IND No. 10825). B cell responses in the vaccine-draining lymph nodes were studied under both stimulated and un-stimulated conditions. Cryopreserved cells were stimulated with CD40L, stained with multiple human cell-surface markers (CD19, CD27, IgM) to identify different categories of B cell sub populations with flow cytometry. Hybridomas were generated from the lymph node cells after CD40L-stimulation. Cells were fused to murine plasmacytoma P3X63.Ag8.653 using Helix electrofusion chamber. ELISA was used to evaluate hybridoma derived antibody binding to vaccine peptides.

Results: Viable MNCs were satisfactorily recovered from lymph nodes cryopreserved from six vaccine study patients 8-14 years previously. B cell ELISPOT demonstrated responses for each patient to multiple vaccine peptides. CD40L stimulation of lymph node cells increased the proportion of CD19+ CD27+ cells from 12 to 65% of the sample and increased the proportion of class-switched cells. Screening of IgG secreting clones demonstrated binding to melanoma vaccine peptides.

Conclusions: B cells were successfully recovered and expanded from human cryopreserved vaccine-draining lymph nodes. Individual B cells were identified that secreted antibodies that bound to cancer vaccine peptides. The ability to reliably generate in vitro the same antibodies observed in the blood of vaccinated patients will facilitate research to understand mechanisms of human antibody activity and possibly lead to therapeutic antibodies.

Keywords: Antibodies; B cells; Cancer vaccine; Human; Melanoma.

Figures

Fig. 1

B cell ELISPOT analyses showing the number of vaccine-draining lymph node B cells per patient that secreted IgG Abs against individual and pooled (6MEL) vaccine peptides. The number of spots were plotted after subtracting the values of irrelevant peptide negative control

Fig. 2

Representative density plots and histograms from flow cytometric analyses of vaccine-draining lymph node cells, showing enrichment of a CD19+CD27+ cells and b class-switched IgM− CD19+ CD27+ cells following subsequent rounds of CD40L-stimulated amplification

Fig. 3

Screening of IgG secreting clones and their binding to vaccine antigens. a ELISA screening of a limited set of hybridoma culture supernatant samples identified high numbers of IgG secreting clones. b Further analysis of these supernatant samples identified two clones, A11 and C3, for their binding to the mixture of six vaccine peptides

Fig. 4

Specificity of the clones that bind to vaccine antigens. Hybridoma culture supernatant samples from clones A11 and C3 specifically bind to FLL peptide and not to RNG peptide