Superior T Cell and Humoral Immunity Generated Against B Cell Lymphomas Using a Sulfhydryl-Based Idiotype Carrier Protein Conjugation Method.
Authors: Betting David J. , Kafi Kamran , Abdollahi-Fard Alireza , Hurvitz Sara A. , Timmerman John M. 
Published: October 2019

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 fail to mount anti-Id immune responses. Traditionally Id is coupled to KLH using glutaraldehyde (glut), which crosslinks via lysine, cysteine, tyrosine and histidine residues. Maleimide (mal) crosslinks only via cysteine sulfhydryl groups, thus limiting the potential for epitope destruction, and potentially allowing more efficient lysosomal processing in antigen presenting cells. The murine B cell lymphoma A20 was reported to be unresponsive to vaccination with glut/Id-KLH. We hypothesized that Id epitopes might be damaged by glut, but preserved using the more specificmal conjugation method. Id proteins were partially reduced to generate free sulfhydryl groups, and reacted with mal-activated KLH. Mice with 4-day established A20 lymphoma were vaccinated with 3 weekly glut/Id-KLH or mal/Id-KLH plus GM-CSF. As previously reported, tumor eradication was uncommon in glut/Id-KLH-treated mice (8–25%), with no statistical advantage over control-treated mice. In contrast, tumor was eradicated in 33–66% of mice vaccinated with mal/Id-KLH, 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 glut/Id-KLH+mal/Id-KLH did not improve survival over mal/Id-KLH alone (p=0.99). KLH conjugated to an irrelevant antibody did not result in any long-term survivors when challenged with A20. The length of time Id and KLH were allowed to conjugate with glut showed a statistically significant time dependence that affected survival (highest 58%) and anti-Id titers, while mal conjugation showed no such time dependence, and gave superior protection (83%). Mice vaccinated with A20 mal/Id-KLH generated 14-fold higher anti-Id antibody titers compared to glut/Id-KLH, and had superior survival after lethal tumor challenge (58% vs. 25%, respectively). A20 immune sera stained A20 cells via FACS analysis and the dominant isotype was shown to be IgG1 by ELISA. Adoptive transfer of high titer A20 anti-Id immune sera resulted in no protective advantage compared to naïve sera indicating antibodies are not the dominant protective mechanism against A20. Depletion of CD8+ T cells demonstrated their critical role in A20 tumor eradication (p<0.0001). In the prototypical 38C13 model, mice vaccinated with mal/Id-KLH developed 3-fold higher anti-Id antibody titers compared to glut/Id-KLH treated animals, and had superior survival after tumor challenge (85% vs. 47%, respectively). Depletion of T cell subsets did not affect animal survival when challenged with 38C13. Here protection was dependent on antibodies rather than T cells. In a third model (BCL-1), 50% of mal/Id-KLH mice survived compared to 25% for glut/Id-KLH, with 3-fold higher titers. Human IgG could be efficiently bound to mal-activated KLH, demonstrating the feasibility of this method for human applications. These results show 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 vaccinations. These findings have important implications for the ongoing development of vaccines targeting tumor Id.