InVivoKines™: Cytokines with Enhanced Activity & Stability

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The Role of InVivoKines™ Cytokines in Immune Communication

Cytokines are small proteins that facilitate communication among immune cells and orchestrate the response to infections and tumors as well as overall immune homeostasis, making them attractive for preclinical and clinical research for a variety of immune-related disorders. They have pivotal roles in immunity, and engineered cytokine-based therapies represent a new evolution of immunotherapeutics. However, the widespread use of cytokines has been limited by their short blood half-lives, pleiotropism, and unfavorable biodistribution. Increased knowledge of cytokine biology and innovative cytokine engineering and technologies allow to cope with such limitations, but often are not accessible for basic research purposes.

Innovative KIH Technology in InVivoKines™ Cytokines

InVivoKines™ are a new generation of recombinant Fc-fusion proteins using the Knobs-into-Holes (KIH) technology, for immuno-therapeutic, preclinical, and translational in vivo and in vitro research. They are developed and manufactured in-house by AdipoGen Life Sciences.

The Knobs-into-Holes (KIH) concept/format was a pioneering format proposed by Ridgway et al. in 1996, permitting heavy-chain heterodimerization, creating an ‘IgG with two different arms’, and traditionally it is used to engineer bispecific antibodies with an important role in drug development. Production of KIH bispecific biologicals to high purity is challenging due to the inherent complexity of the molecule. The efficient co-expression of KIH relies on approximately equal expression of both knob and hole. Therefore, the selection of a stable expression clone is a prerequisite for KIH production using mammalian cells and is a laborious and time-consuming process.

Enhancing Stability and Activity with Fc-KIH Technology

The Fc-KIH technology allows Fc heterodimerization to create a structure with two different arms, to design naturally occurring active monomeric, heterodimeric or homodimeric proteins (https://adipogen.com/invivokines#KIH). The Fc-KIH domain enhances the plasma half-life of the cytokine, which substantially improves the pharmacokinetics (PK) while maintaining activity. The Fc-KIH domain engages with the Fcγ receptors (FcγRs) and the complement C1q, which may result in ADCC, ADCP, and CDC, while Fc-KIH LALA-PG mutations inhibit binding to FcγRs and C1q while FcRn binding and Fc stability remain unaffected. The Fc-KIH proteins are developed for in vivo studies but are also suitable for in vitro experiments!

AdipoGen Life Sciences engineers the InVivoKines™ without or with silenced Fc-KIH domains (https://adipogen.com/invivokines#LALAPG) using the hIgG1-P329G LALA mutations comparable to the approach used for in vivo antibodies. The LALA-PG mutations show no detectable binding to Fcγ receptors or to C1q, are inactive in functional cell-based assays, and do not elicit inflammatory cytokine responses. They show FcRn interactions and keep the Fc long-acting effect.

Case Study: IL-12 (mouse):Fc-KIH (human) InVivoKine™

IL-12 acts as a bridge between innate and adaptive immunity and plays a crucial role in promoting the differentiation of T helper (Th)-1 cells and effector CD8+ T cells. IL-12 also enhances the effector function of activated T cells and natural killer (NK) cells, primarily by inducing interferon γ (IFN-γ) production.

Our new IL-12 (mouse):Fc-KIH (human) (rec.) InVivoKine™ (AG-40B-0240) shows strong activity when injected in mice, leading to a complete tumor growth stop (see Figures below). An optimized version with a silent Fc, IL-12 (mouse):Fc (LALA-PG)-KIH (human) (rec.) InVivoKine™ is also available.

Method: C57BL/6 (B6) mice are transferred with P14 TCR transgenic CD8+ T cells on day -1 (d-1) and implanted (subcutaneous) with B16-gp33 tumor cells on day 0 (d0) (A). When tumors become palpable at day 12 (d12), mice are treated with IL-12 (mouse):Fc-KIH (human) (rec.) or with Fc-KIH (human) IgG1 Control (rec.) (Prod. No. AG-35B-0015), 2µg each, 4 times, every 2 days, whereby each time half of the dose was injected intraperitoneal (i.p.) and the other half peritumorally. The tumor volume are measured over time (B) and the tumor weight are determined at sacrifice (d19) (C). The graphs show means ±SEM (tumor volume) or means ±SD (tumor weight). Statistics is based on 2-tailed students t-test, whereby ****p<0.0001.

Wide Range of InVivoKines™ Products Available

AdipoGen Life Sciences offers a broad range of InVivoKines™ for many important cytokines. A comprehensive list can be seen here (https://adipogen.com/invivokines#ALL-KIH).