Improvement of Idiotype Vaccine Efficacy Using a Sulfhydryl-Based Carrier Protein Conjugation Method: Results in Multiple Murine Lymphoma Models.

Abstract

Therapeutic vaccination of lymphoma patients with tumor-specific immunoglobulin (idiotype, Id) chemically-coupled to the highly immunogenic foreign carrier protein keyhole limpet hemocyanin (Id-KLH) has shown promising results in phase I/II clinical trials, and phase III trials are underway. However, many patients in these trials fail to mount detectable anti-Id immune responses. Id proteins are traditionally coupled to KLH using glutaraldehyde (glut), which primarily crosslinks proteins via lysine and cysteine residues, with secondary reactions at tyrosine and histidine. This extensive crosslinking could potentially inhibit proteosomal processing or destroy critical immunogenic epitopes. In contrast, maleimide (mal) crosslinks proteins only via cysteine sulfhydryl groups, limiting the potential for epitope destruction. This method generates a thioether bond which is reversible at acidic pH, and may allow more efficient lysosomal processing in antigen presenting cells, whereas glut conjugation irreversibly links Id to KLH. The murine B cell lymphoma A20 has been reported to be unresponsive to vaccination with glut/Id-KLH. We hypothesized that the epitopes of some Id proteins, including A20, might be damaged by glut, but preserved using the gentler sulfhydryl-based mal conjugation method. Id proteins were pre-treated with a reducing agent to generate free sulfhydryl groups, followed by reaction with maleimide-activated KLH. Mice with 4-day established A20 lymphoma were vaccinated with 3 weekly glut/Id-KLH or mal/Id-KLH plus granulocyte-macrophage colony-stimulating factor (GM-CSF) injections. As previously reported, tumor eradication was uncommon in glut/Id-KLH-treated mice (16-25% in 4 experiments), with no statistical advantage over control-treated mice (p>0.05). In contrast, tumor was eradicated in 33–66% of mice vaccinated with mal/Id-KLH (4 experiments), with tumor-free survival being highly significant compared to control mice (p=0.0001) and glut-Id-KLH-vaccinated mice (p=0.0084). Combined vaccination with mal/Id-KLH plus glut/Id-KLH did not improve survival over mal/Id-KLH alone (both 58%, p=0.99). The superior survival after mal/Id-KLH was not simply due to the pre-reduction of Id prior to KLH conjugation, as reduction prior to glut conjugation did not improve outcome. Mice vaccinated with A20 mal/Id-KLH generated anti-Id antibody titers 7-fold higher than those given glut/Id-KLH, and had superior survival after lethal tumor challenge (71% vs. 25%, respectively). This approach was validated in the prototypical lymphoma model 38C13, used in many early Id vaccine studies. Mice vaccinated with 38C13 mal/Id-KLH developed higher anti-Id antibody titers than glut/Id-KLH-treated animals (161 +/−54 μg/ml vs. 52 +/−23 μg/ml, respectively, p>0.0001) and displayed superior protection from tumor challenge (88% vs. 43%, respectively). These results demonstrate that the method of Id conjugation to carrier protein can substantially affect the level of tumor protection elicited by Id-KLH vaccination for B cell lymphoma, and may explain why some patients fail to respond to traditional glut/Id-KLH vaccines. Future studies will determine the roles of antibody versus T cell anti-Id effectors in these and other murine lymphoma models. These findings have important implications for the ongoing development of therapeutic vaccines targeting tumor Id.