Synthesis of sialyl Lewis(a) (sLe (a), CA19-9) and construction of an immunogenic sLe(a) vaccine

Abstract

Sialyl Lewis(a) (sLe(a)), also termed CA19-9 antigen, is recognized by murine mAb19-9 and is expressed on the cancer cell surface as a glycolipid and as an O-linked glycoprotein. It is highly expressed in a variety of gastrointestinal epithelial malignancies including colon cancer and pancreatic cancer, and in breast cancer and small cell lung cancer, but has a limited expression on normal tissues. sLe(a) is known to be the ligand for endothelial cell selectins suggesting a role for sLe(a) in cancer metastases and adhesion. For these reasons, sLe(a) may be a good target for antibody mediated immunotherapy including monoclonal antibodies and tumor vaccines. However, sLe(a) is structurally similar to sLe(x) and other blood group related carbohydrates which are widely expressed on polymorphonucleocytes and other circulating cells, raising concern that immunization against sLe(a) will induce antibodies reactive with these more widely expressed autoantigens. We have shown previously both in mice and in patients that conjugation of a variety of carbohydrate cancer antigen to keyhole limpet hemocyanin (KLH) and administration of this conjugate mixed with saponin adjuvants QS-21 or GPI-0100 are the most effective methods for induction of antibodies against these cancer antigens. We describe here for the first time the total synthesis of pentenyl glycoside of sLe(a) hexasaccharide and its conjugation to KLH to construct a sLe(a)-KLH conjugate. Groups of five mice were vaccinated subcutaneously four times over 6 weeks. Sera were tested against sLe(a)-HSA by ELISA and against sLe(a) positive human cell lines adenocarcinoma SW626 and small cell lung cancer (SCLC) DMS79 by FACS. As expected, mice immunized with unconjugated sLe(a) plus GPI-0100 or unconjugated sLe(a) mixed with KLH plus GPI-0100 failed to produce antibodies against sLe(a). However, mice immunized with sLe(a)-KLH conjugate without GPI-0100 produced low levels of antibodies and mice immunized with sLe(a)-KLH plus GPI-0100 produced significantly higher titer IgG and IgM antibodies against sLe(a) by ELISA. These antibodies were highly reactive by FACS and mediated potent complement mediated cytotoxicity against sLe(a) positive SW626 and DMS79 cells. They showed no detectable cross reactivity against a series of other blood group-related antigens, including Le(y), Le(x), and sLe(x) by dot blot immune staining. This vaccine is ready for testing as an active immunotherapy for treating sLe(a) positive cancer in clinical settings.

Fig. 1

Scheme for the synthesis of pentenyl glycoside of sLe^a hexasaccharide

Fig. 2

Preparation of sLe^a-KLH conjugate involves ozone cleavage of sLe^a pentenyl glycoside and use of the heterobifunctional cross linker 4-(4-N-maleimidomethyl) cyclohexane-1-carboxyl hydrazide (MMCCH)

Fig. 3

IgM and IgG FACS profiles for the five mice immunized with 5 μg sLe^a-KLH conjugate plus 100 μg GPI-0100 (group 4) tested on SW626 ovarian cancer cells. Median pre and post immunization results are shown. The percent positive cells (% of Max) and (MFI) and shown, “au” is arbitrary units of FITC positive cells

Fig. 4

Complement dependant cytotoxicity results against SW626 using sera from mice immunized with 5 μg sLe^a-KLH conjugate plus 100 μg GPI-0100 (group 4) plus human complement. Sera was drawn pretreatment (pre) 1 week after the third vaccination (post), or 1 week after the forth vaccination (boost)

Fig. 5

Dot blot immune stains for four of the five mice immunized with 5 μg sLe^a-KLH conjugate plus 100 μg GPI-0100 (group 4)