InVivoKines™ – Cytokines with Enhanced Activity & Stability

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.

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

InVivoKines™ Quality Features

• Native Conformation − Production in HEK 293 or CHO Cells
• Production under Animal-Free Conditions
• High Bioactivity tested by ELISA/Cell-based Assays
• Verified Purity & Homogeneity by SEC
• Low Endotoxin Levels
• Batch-to-Batch Consistency

Naturally Occuring Cytokines Fused to Fc (KIH-Technology)

InVivoKines™ are Fc-based fusion proteins using the Knobs-into-Holes (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.
• 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*.
• The Fc-KIH LALA-PG mutations inhibit binding to FcγRs and C1q while FcRn binding and Fc stability remain unaffected.
• The Fc-KIH-constructs are produced in mammalian cells with low endotoxin content.
• The Fc-KIH proteins are developed for in vivo studies but are also suitable for in vitro experiments!

AdipoGen Life Sciences leveraged the KIH-technology to develop monomeric, heterodimeric or homodimeric Fc fusion proteins.

*Antibody-dependent cellular cytotoxicity (ADCC), Antibody-dependent cellular phagocytosis (ADCP), Complement-dependent cytotoxicity (CDC)

Background on the Knobs-into-Holes Technology

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. It allows the generation of complementary interacting interfaces by manipulating key amino acid residues that participate in the Fc dimeric interaction. Amino acids with a small side chain are replaced by ones with larger side chains, thereby creating a knob or protrusion in one chain and vice versa to create a hole or socket in the partner chain. Traditionally, a T366Y mutation in one CH3 domain has been used to create a knob while an Y407T mutation in the other CH3 domain gives rise to a hole. Such mutations establish intermolecular interactions and promote the heterodimer formation due to knob/hole pairing. 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.