Polyclonal antibodies from patients immunized with a globo H-keyhole limpet hemocyanin vaccine: isolation, quantification, and characterization of immune responses by using totally synthetic immobilized tumor antigens

Abstract

We have previously reported on a carbohydrate-based vaccine program for immunotherapy in cancer patients. One such vaccine, based on the globo H antigen conjugated to the protein keyhole limpet hemocyanin (KLH), has been in clinical evaluation. Although this and other carbohydrate vaccines have been shown to induce antibody responses, there are currently no quantitative data on the antibody levels achieved in immunized patients by these or other anti-cancer vaccines. We report herein an efficient route to complex synthetic oligosaccharides attached to an affinity matrix for identifying and isolating antibodies elicited against such a carbohydrate-based vaccine in humans. Pre- and postvaccination profiles from serum samples of patients immunized with globo H-KLH were compared. All anti-globo H antibody activity was efficiently separated from other serum constituents. The isolated antibodies were readily quantified, and their specificities were analyzed. Since no comparable data were available on antibodies resulting from the vaccination of other cancer patients, we compared the observed levels with those quoted in studies with bacterial polysaccharide vaccines that had been quantified. Remarkably, cancer patients immunized with globo H-KLH produce anti-globo H antibody levels often exceeding those formed by immunization with bacterial polysaccharides. In addition, substantial quantities of both IgG and IgM antibodies were elicited, clearly indicating a class switch to IgG. Taken together, these analyses serve to clarify several aspects of the immune response to the vaccine and give several new insights to the carbohydrate-based vaccination strategy. Furthermore, antibodies so isolated could well have applications in clinical therapy.

Scheme 1

Figure 1

Affinity chromatography of sera from a patient (no. 4) on globo H-agarose column. (A) Postvaccination serum. (B) Prevaccination serum. Fractions were assayed for protein levels (■, OD₂₈₀) and reactivity with globo H-ceramide by ELISA (▴). Initial wash buffer: PBS; second wash (arrow 1): 1 M NaCl/PBS; final elution buffer (arrow 2): 0.05 M glycine⋅HCl (pH 2.5).

Figure 2

SDS/PAGE analysis of fractions isolated from sera from six patients, immunized with globo H-KLH, by affinity chromatography on a globo H-agarose column. Lanes 1–6, low pH-eluted fractions from patients 1–6. Lane 7, eluted fraction from patient 3 further purified to remove HSA. Lane S, protein standards (220, 130, 90, 70, 60, 40, 30, and 20 kDa). The migration rates of IgM, IgG, and HSA are indicated. The samples were separated on a 7% polyacrylamide gel under nonreducing conditions and then stained with Coomassie blue.

Figure 3

Analysis of specificity of antibody fractions isolated from five immunized patients: patient 2 (A), patient 4 (B); patient 8 (C), patient 13 (D); and patient 14 (E) (as listed in ref. 10). Reactivity was measured with a direct ELISA using rabbit anti-human IgG (H and L chains) conjugated to alkaline phosphatase as the second antibody. (F) Anti-globo H monoclonal antibody VK-9. Test compounds: 1, globo H-Cer; 2, gal-globoside (SSEA-3 antigen); 3, globoside; 4, Le^y (Fucα1–2Galβ1–4[Fucα1–3]GlcNAcβ1–3Gal)-BSA; 5, Le^b (Fucα1–2Galβ1–3[Fucα1–4]GlcNAcβ1–3Gal)-BSA; 6, Le^x (Galβ1–4[Fucaα1–3]GlcNAc)-PAA; 7, Le^x-Cer; 8, Le^a (Galβ1–3[Fucaα1–4]GlcNAc)-PAA; 9, H type 2 (Fucα1–2Galβ1–4GlcNAc)-PE; 10, H type 2-PAA; 11, H type 1 (Fucα1–2Galβ1–3GlcNAcβ1–3Gal)-BSA; 12, H type 1-PAA; 13, lactose-CETE; 14, Galα1–4Galβ1–4Glc-CETE; 15, Galα1–4GlcNAc-CETE-BSA. Abbreviations: Cer, ceramide; BSA, bovine serum albumin; PAA, polyacrylamide; PE, phosphatidylethanolamine; CETE, 2-(carbomethoxyethylthio)ethyl.