Immunization of mice with fucosyl-GM1 conjugated with keyhole limpet hemocyanin results in antibodies against human small-cell lung cancer cells

Abstract

Fucosyl-GM1 (Fuc-GM1) [Fucalpha1 --> 2Galbeta1 --> 3GalNAcbeta1 --> 4(NeuAcalpha2-3)Galbeta1 --> 4Glcbeta1 --> O-Cer] is a small-cell-lung-cancer (SCLC)-associated ganglioside initially defined by the murine monoclonal antibody F12. On the basis of its known distribution, Fuc-GM1 is a potential target for active immunotherapy in SCLC patients. Fuc-GM1 has been extracted and purified from bovine thyroid. The immunogenicity of Fuc-GM1 was tested in mice either alone, mixed with carrier protein keyhole limpet hemocyanin (KLH) or covalently linked with KLH, plus immunological adjuvant QS-21. The Fuc-GM1-KLH conjugate plus QS-21 adjuvant was found to be optimal. It induced consistent IgM and IgG enzyme-linked immunosorbent assay (ELISA) titers against Fuc-GM1. These antibodies were strongly reactive with the strongly Fuc-GM1-positive rat hepatoma cell line H4-II-E, and they were moderately reactive with the moderately positive human SCLC cell line H146 by flow cytometry and complement-mediated lysis. Both ELISA and fluorescence-activated cell sorting (FACS) reactions were inhibited with Fuc-GM1or H4-II-E but not with the structurally related ganglioside GM1 or Fuc-GM1-negative colon cancer cell line LS-C. On the basis of these results, a vaccine comprising Fuc-GM1-KLH plus QS-21 is being prepared for testing in patients with SCLC.

Constructing an adenocarcinoma vaccine: Immunization of mice with synthetic KH‐1 nonasaccharide stimulates anti‐KH‐1 and anti‐Ley antibodies

“…18 The final issue addressed in our study is how the nature of antigen conjugation to KLH and vaccine formulation affects antibody production. Consistent with our prior experience involving other glycolipid antigens 13,17,18 was that little, if any, antibody production was induced by immunization with a mixture consisting of KH-1 ceramide and QS-21 adjuvant. We have demonstrated previously that this is also the case when KLH is included in the vaccine formulation.…”

“…While Le y and Le x have a normal tissue distribution largely restricted to the secretory borders of a variety of epithelial tissues (a location apparently poorly accessible to the immune system), Le x is also expressed on peripheral blood polymorphonucleocytes. 16,17 From the outset of our investigation, we were wary of the possibility that antibody production in direct response to the Le x epitope of KH-1 would be detrimental to any immuno-therapeutic approach that involved KH-1. Fortunately, as demonstrated here through inhibition studies involving Le y allyl glycoside, Le x and anti-sera raised in response to our synthetic KH-1-based…”

Carbohydrate vaccines as immunotherapy for cancer

“…As seen in the other ganglioside vaccine in mice, the Fuc‐GM1‐KLH conjugate plus QS‐21 was optimal for IgM and IgG antibody induction. These antibodies were strongly reactive with fucosyl‐positive cells and mediated complement‐dependent cell lysis 46 .…”

Increasing the Antigenicity of Synthetic Tumor‐Associated Carbohydrate Antigens by Targeting Toll‐Like Receptors

“…3, 58, 59 It has been established that the use of KLH as a carrier protein in combination with the powerful adjuvant QS‐21 gives the best results. However, a drawback of this approach is that KLH is a very large and cumbersome protein that can elicit high titers of anti‐KLH‐antibodies,60 leading to immune suppression of the tumor‐associated carbohydrate epitope. Furthermore, the conjugation chemistry is often difficult to control because it results in conjugates with ambiguities in composition and structure, which may affect the reproducibility of immune responses.…”