Evaluation of human antibody responses to keyhole limpet hemocyanin on a carbohydrate microarray

Abstract

Purpose: Keyhole limpet hemocyanin (KLH) is used as a vaccine adjuvant, as a carrier protein for small haptens, and as a treatment for bladder cancer. Immunization with KLH produces antibodies to tumor-associated carbohydrate antigens (TACAs) in animals, and these antibodies have been postulated as the basis of efficacy for bladder cancer treatment. The purpose of this study was to evaluate antibody responses to KLH in humans.

Experimental design: A carbohydrate microarray was used to profile antibody responses in 14 individuals immunized with KLH plus alum adjuvant.

Results: Eight out of fourteen individuals produced antibodies to at least one TACA. Increases to Lewis X, Lewis Y, GA1di, GM3, and sialyl Lewis A were observed in certain individuals, but, in general, antibody profiles were highly variable. Pre-immunization antibody levels to a subset of array antigens had a statistically significant correlation with the magnitude of the antibody response to KLH.

Conclusions and clinical relevance: Antibodies to TACAs can be produced in humans, but antibody profiles differ considerably from person to person, which may contribute to variable clinical responses with KLH. Pre-treatment antibody levels to certain antigens may be useful for predicting which patients will respond favorably to KLH.

Figure 1. Proportion of antibody response targeting carbohydrates

(a) Comparison of serum antibody binding to KLH or periodate oxidized KLH, (b) comparison of serum antibody binding to KLH in the presence or absence an inhibitory cocktail of sugars (100 mM each of methyl-α-D-galactopyranoside, methyl-β-D-galactopyranoside, methyl-α-D-mannopyranoside, methyl-α-D-glucopyranoside, β-lactose, D-cellobiose, D-maltose monohydrate, L-fucose, N-acetyl-D-galactosamine, and N-acetyl-D-glucosamine). The level of induced antibody was calculated as the difference in signal between pre-immune and post-immune serum samples.

Figure 2. Antibody responses to KLH plus alum

Changes in antibody profiles between preand post-immune sera measured on the carbohydrate microarray. Carbohydrates are indicated in the rows and subjects are indicated in the columns. Each rectangle represents the difference between the pre- and post-immune normalized signal [log-transformed (base 2)]. The color of the rectangle corresponds to values on the color scale to the left of the heat map. Gray rectangles represent no change or changes that were not statistically significant.

Figure 3. Changes in total antibody levels

Differences between pre- and post-immune sera for total IgG and IgM measured by ELISA. Each bar represents the ratio of the pre- and post-immune antibody concentrations, presented in log scale (base2).

Figure 4. Correlations between pre-immune antibody levels and the KLH response

Pre-immune antibody signals to LSTc, GlcNAca1-3Gal, asialo-fetuin, KLH, ovalbumin, and asialo-OSM were averaged and the average value (log base 2) for each subject was plotted versus the magnitude of the antibody response to KLH (x-axis; log base 2) for that subject. The correlation was highly statistically significant (p < 0.000073).