Generation of IL‐2‐Fc‐antibody conjugates by click chemistry

Abstract

BackgroundImmunocytokines (ICKs) are antibody directed cytokines produced by genetic fusion of an antibody to a cytokine.MethodsWe now show that antibodies conjugated by click chemistry to interleukin‐2 (IL‐2)‐Fc form fully active conjugates, and in one example, equivalent activity to a genetically produced ICK.ResultsAn IL‐2‐Fc fusion protein was optimized for click chemistry at hinge cysteines using protein stabilizing IL‐2 mutations at Lys35 and Cys125 and Fc hinge mutations at Cys142 and Cys148. The IL‐2‐Fc fusion protein with K35E and C125S mutations with 3 intact hinge cysteines, designated as IL‐2‐Fc Par, was selected based on its minimal tendency to aggregate. IL‐2‐Fc‐antibody clicked conjugates retained high IL‐2 activity and bound target antigens comparable to parent antibodies. An IL‐2‐Fc‐anti‐CEA click conjugate showed comparable anti‐tumor activity to an anti‐CEA‐IL‐2 ICK in immunocompetent CEA transgenic mice bearing CEA positive orthotopic breast tumors. Significant increases in IFNγ+/CD8+ and decreases in FoxP3+/CD4+ T‐cells were found for the clicked conjugate and ICK therapies, suggesting a common mechanism of tumor reduction.ConclusionThe production of antibody targeted IL‐2 therapy via a click chemistry approach is feasible with comparable activity to genetically produced ICKs with the added advantage of multiplexing with other monoclonal antibodies.

AGuIX nanoparticle-nanobody bioconjugates to target immune checkpoint receptors

“…This reaction has been widely utilized in various applications due to its simplicity in terms of preparation and purification steps, enabling rapid generation of new products with high reaction rates, such as bioconjugates. 60,[63][64][65] The formation of the triazole linkage is irreversible and quantitative, providing excellent reaction stability. Azide and dibenzocyclooctyne (DBCO) are selected as strain-promoted azidealkyne cycloaddition reactions (SPAAC) tools due to their relatively small functional groups with a favorable rate constant (k 2 = 0.2-0.5 M −1 s −1 ) 39 which have been shown to be effective.…”

Antibody mimetics: The next generation antibody engineering, a retrospective and prospective analysis

“…Two compelling instances involve the fusion of antibody mimetics with the bacterial toxin colicin Ia, [39] resulting in the creation of "pheromonicins" that specifically inhibit tumor growth and the synthesis of antibody mimetics, achieved through site-specific binding of small molecules or proteins to Fc fragments with high affinity and specificity for disease-associated antigens. [40,41] These advancements above, fortified by an expanding interdisciplinary approach, not only accelerate the discovery and production of antibody mimetics but also augment their applicability in a wide array of scientific domains (Table 2), illustrating the potential for antibody mimetics to replace outdated antibody-based drugs or revitalize obsolete ones and positioning antibody mimetics on a trajectory toward dominance in the coming years. Nevertheless, concerns about offtarget effects and toxicity necessitate careful evaluation, [42] with each case requiring extensive reassessment of the safety profile before reintroduction into the market.…”

“…Two compelling instances involve the fusion of antibody mimetics with the bacterial toxin colicin Ia, [ 39 ] resulting in the creation of “pheromonicins” that specifically inhibit tumor growth and the synthesis of antibody mimetics, achieved through site‐specific binding of small molecules or proteins to Fc fragments with high affinity and specificity for disease‐associated antigens. [ 40,41 ]…”

Comparison of IL‐2‐antibody to IL‐2‐Fc with or without stereotactic radiation therapy in CEA immunocompetent mice with CEA positive tumors

“…Most notably, the fusion of IL-2 to Fc derived from antibody subtypes with high effector function (such as murine IgG2 or human IgG1) yields therapeutic agents with low toxicity and high antitumor activity mediated by Fc-mediated Treg depletion and moderate systemic immune cell activation. 9,10 In addition, IL-2 fusion proteins must also be screened for aggregation due to the natural propensity of IL-2 to aggregate. To minimize aggregation commonly seen with IL-2-Fc, the K35E mutation in the IL-2 moiety was incorporated in a fully human IL-2 IgG1 Fc fusion protein construct.…”

“…13 Based on previous data, we know that an IL-2-Fc (containing K35E in the human IL-2 moiety; composed of human IL-2 and human IgG1 Fc) and M5A-IL-2 ICK have minimal aggregation and maintain CD25 binding and IL-2 activation. 10,13 We now compare IL-2-Fc to tumor-targeted M5A-IL-2 ICK in CEA + solid breast and colon cancer models in CEA transgenic mice. Early and late time points were assessed for changes in immune landscape and potential toxicities.…”