Maleimide conjugation markedly enhances the immunogenicity of both human and murine idiotype-KLH vaccines

Abstract

The collection of epitopes present within the variable regions of the tumor-specific clonal immunoglobulin expressed by B cell lymphomas (idiotype, Id) can serve as a target for active immunotherapy. Traditionally, tumor-derived Id protein is chemically-conjugated to the immunogenic foreign carrier protein keyhole limpet hemocyanin (KLH) using glutaraldehyde to serve as a therapeutic vaccine. While this approach offered promising results for some patients treated in early clinical trials, glutaraldehyde Id-KLH vaccines have failed to induce immune and clinical responses in many vaccinated subjects. We recently described an alternative conjugation method employing maleimide-sulfhydryl chemistry that significantly increased the therapeutic efficacy of Id-KLH vaccines in three different murine B cell lymphoma models, with protection mediated by either CD8 + T cells or antibodies. We now define in detail the methods and parameters critical for enhancing the in vivo immunogenicity of human as well as murine Id-KLH conjugate vaccines. Optimal conditions for Id sulfhydryl pre-reduction were determined, and maleimide Id-KLH conjugates maintained stability and potency even after prolonged storage. Field flow fractionation analysis of Id-KLH particle size revealed that maleimide conjugates were far more uniform in size than glutaraldehyde conjugates. Under increasingly stringent conditions, maleimide Id-KLH vaccines maintained superior efficacy over glutaraldehyde Id-KLH in treating established, disseminated murine lymphoma. More importantly, human maleimide Id-KLH conjugates were consistently superior to glutaraldehyde Id-KLH conjugates in inducing Id-specific antibody and T cell responses. The described methods should be easily adaptable to the production of clinical grade vaccines for human trials in B cell malignancies.

Microwave-promoted synthesis of cyclic imides

“…The activity of maleimides as antifungal, antimicrobial and insecticide compounds has been reported. 2,[20][21][22][23][24] These compounds have also proved to have analgesic and antispasmodic activities. 2 They can also form conjugates with peptides and antibodies and act as enzyme inhibitors.…”

“…2 They can also form conjugates with peptides and antibodies and act as enzyme inhibitors. [21][22][23][24][25] The maleimide moiety can be used as a versatile synthesis platform due to its Michael accepting [26][27][28][29] and dienophilic nature, as well as its dipolarophile characteristic in 1,3-dipolar cycloadditions. [30][31][32][33] A recent review shows the importance of maleimides as building blocks for the synthesis of polymers.…”

Tobacco derived cancer vaccines for non-Hodgkin’s lymphoma: Perspectives and progress

“…Other improvements, such as controlled chemistry conjugation may also help improve idiotype immunity. 45,46 Additionally, improved clinical trial design may also contribute to more successful vaccine trials. Bendandi's group used patient time to progression after chemotherapy as the benchmark for improved response to second line vaccine therapy plus chemotherapy.…”

Hemocyanins in the Immunotherapy of Superficial Bladder Cancer

“…Currently, hemocyanins are used as carrier-adjuvants for several tumor-associated antigens (TAAs), such as glycolipid and glycoprotein (mucin-like) antigens, in experimental therapeutic vaccines against certain cancers, including melanomas, sarcomas, breast, prostate, ovary and lung (Musselli et al, 2001;Schumacher, 2001;Zhu et al, 2009;Del Campo et al, 2011). Other therapeutic strategies that use hemocyanins include dendritic cell (DC) vaccines pulsed with tumor lysates to enhance interferon gammaIFN- production by tumor-reactive T cells (Timmerman & Levy, 2000;Shimizu et al, 2001;Millard et al, 2003;Lopez et al, 2009;Jacobs et al, 2010;Lesterhuis et al, 2011) and anti-idiotype vaccines for some types of B cell malignancies (Leitch & Connors, 2005;Kafi et al, 2009). KLH has been the gold standard for these applications for over 40 years simply because it was used in earlier studies instead of other hemocyanins (Harris & Markl, 1999).…”