Folate-Targeted Dinitrophenyl Hapten Immunotherapy: Effect of Linker Chemistry on Antitumor Activity and Allergic Potential

Abstract

Targeting of malignancies with folate-linked therapeutics has proven to be a promising endeavor due to the preferential expression of folate receptors (FR) on human tumors. We have shown that folic acid (pteroyl-glutamate) can be used to deliver an antigenic hapten, fluorescein, to the surface of tumor cells to promote their opsonization within a fluorescein-immunized host. Here, we investigate structure-activity relationships among members of another class of folate-hapten conjugates ( EC57, EC63, EC0293, and EC0294), namely, those containing the dinitrophenyl (DNP) group as the antigenic hapten. We report that despite exhibiting similar affinities for the FR, the antitumor activity and allergic potential of these DNP conjugates varied depending on their linker chemistries and abilities to bind anti-DNP IgG/IgE antibodies. Unlike EC57 and EC63, both EC0293 and EC0294 (i) share the identical DNP bridging chemistry to that found in keyhole limpet hemocyanin (KLH)-DNP (i.e., the immunogen), (ii) efficiently recognize DNP-specific IgG, and (iii) mediate more pronounced antitumor responses. However, EC0293 and EC0294 were also found to recognize DNP-specific IgE, and they displayed a greater risk of allergy when evaluated in a passive cutaneous anaphylaxis assay. Nonetheless, upon co-stimulation with the appropriate cytokines (IL-2/IFN-alpha), the folate-targeted "haptenization" process allowed for tumor rejection and protective antitumor immunity without causing any visible allergy in immunized mice. Our data further support the concept that folate-hapten-targeted immunotherapy may offer an effective therapeutic option for treatment of FR-positive cancers, but such treatment should proceed with caution given the risk of a potential allergic reaction.

Strategy to Prevent Drug-Related Hypersensitivity in Folate-Targeted Hapten Immunotherapy of Cancer

“…In a previous publication, DNPimmunized mice were found to produce anti-DNP IgE antibodies, and folate-DNP conjugates with identical hapten linker chemistry to that found in KLH-DNP (i.e. the immunogen) were found to cause allergy in a rat PCA assay (8). As most allergic and hypersensitivity reactions are associated with Th2-biased immune responses, the formation of allergen-specific IgE antibody is a hallmark of allergic sensitization (20,21).…”

“…While dosing folate-FITC early during immunization served the purpose of eliminating any allergic response in immunized animals regardless of immunogen dose, the impact that this maneuver had on antitumor activity was called into question. We have previously shown that the administration of folate-FITC supplemented with low to moderate doses of IL-2 ±IFN-α can generate cures or significantly extend the lifespan of FITC-immunized, M109 tumor-bearing mice (7,8). The M109 tumor is a weakly immunogenic FR-positive subclone of the Madison lung carcinoma cell line that requires regular passages in Balb/c mice (19).…”

“…Although the exact function is unclear, epithelial cancer cells tend to overexpress FR-α, which can bind folate-drug conjugates with nanomolar affinity and bring a fraction of the surface-bound molecules inside the cell via endocytosis (5). As therapeutic monoclonal antibodies have gained increasing popularity due to humanization, high specificity, and low toxicity (6), we developed an alternative strategy of targeting endogenous antibodies to FR-positive cancer via a low-molecular-weight folate-hapten conjugate, namely, folate-targeted hapten immunotherapy (7)(8)(9). Here, folate-hapten conjugates serve as a bispecific molecular "bridge" between tumor cells and the endogenous anti-hapten antibody induced by vaccination.…”

“…Although no allergic reaction to any folate-conjugated haptens [e.g., fluorescein (FITC) and dinitrophenyl (DNP)] was ever observed during therapy in preclinical modeling, we have reported that folate-DNP conjugates exhibited a greater risk of allergy if they shared an identical hapten linker chemistry to that found in its keyhole limpet hemocyanin (KLH)-based immunogen (8). Still, the allergic potential of folate-DNP conjugates was only detectable in a sensitive rat passive cutaneous anaphylaxis (PCA) assay.…”

Hapten-Induced Contact Hypersensitivity, Autoimmune Reactions, and Tumor Regression: Plausibility of Mediating Antitumor Immunity

“…Indeed, haptens have been tested as a treatment of cancer several times in the past. In this review, we examine the four main concepts of hapten-mediated antitumor treatment: (1) ex vivo haptenation [21–31], (2) in situ haptenation [32, 33], (3) epifocal hapten application [34–42], and (4) antigen-hapten administration [43–47]. Despite the wealth of experiments in this field, the mechanisms underlying these treatment approaches are largely unclear and require further study.…”