Revolutionizing Cytokine Research: InVivoKines™ from AdipoGen Life Sciences

Written by Emily Locke

The importance of antibodies, T and B lymphocytes, dendritic cells, macrophages and co. for the defense against pathogens is clear. However, in addition to these generally known defense components, there are also the silent heroes of our body's immune system, such as cytokines. These small signaling proteins are essential for cell communication, as they serve as messengers of the immune system and therefore play a crucial role in the regulation of immune responses, inflammation and hematopoiesis. They are also responsible for initiating the proliferation and differentiation of target cells [1]. Cytokines are produced by macrophages, B and T lymphocytes, natural killer cells and fibroblasts [2]. Due to their central role in the immune response, they represent an attractive target for preclinical and clinical research on a variety of immune diseases. However, despite their potential, the study of these dynamic molecules in living organisms is a challenging task. This is due to their short half-life in the blood, pleotropy and unfavorable biodistribution [3].

This is where our supplier AdipoGen Life Sciences comes into play with an innovative product line: InVivoKines™ are a new generation of fusion proteins with improved activity and stability for your in vivo research! They enable scientists to delve deeper into the complex world of cytokines in living organisms, gaining new insights and opening doors to effective therapies. Learn more about the technology behind InVivoKines™ and get an overview of the different product types and their applications.

These topics await you:

1) What are InVivoKines™?

2) What's Behind the InVivoKines™: Knobs-into-Holes Technology

3) "Silenced" InVivoKines™: LALA-PG Mutations

4) InVivoKines™: Proteins with Enhanced Activity and Stability for in vivo Research

5) Effective Monomeric Cytokines: Interleukin-2

6) Highly Active Heterodimeric Cytokines: Interleukin-27

7) Unique Homodimeric Cytokines: GDF-15

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What are InVivoKines™?

InVivoKines™ from AdipoGen Life Sciences are recombinant fusion proteins - but what exactly does that mean? A protein is called recombinant if it is produced artificially with the help of genetically modified microorganisms or in cell cultures [4]. A fusion protein is created by the co-expression of two different genes or gene segments that are located next to each other in the genome. The protein molecule therefore contains gene products from at least two different genes [5].

In many cases, the fusion of protein domains can help to increase the stability or half-life of the protein [3]. The Fc domain of antibodies is often used for this purpose. A brief refresher: antibodies consist of two fragments, the variable Fab domain and the constant Fc domain (Fig. 1). While the Fab domain is responsible for antigen binding, the Fc domain takes over the effector functions of the antibody by binding to Fcγ receptors on immune cells or activating the complement system [6]. In addition, the Fc domain ensures a long half-life of the antibody, which is why it is often used for fusion proteins in in vivo studies [3].

Figure 1: Comparison of the structure of an antibody and an Fc fusion protein. An antibody consists of a variable Fab domain and a constant Fc domain. A conventional Fc fusion protein consists of an Fc domain and a target protein. Due to the dimerization of the Fc domain, a homodimeric fusion protein is formed.

However, fusing a protein to an Fc fragment has more effects than just increasing the half-life: as dimerization is necessary for the activation of the Fc domain, fusing this fragment to a target protein creates a homodimeric protein (Fig. 1). In many cases this is advantageous, as some proteins are more active in the form of a homodimer. For other proteins, however, dimerization by Fc fragments can also drastically reduce their activity. This is a particular problem with cytokines or interleukins, as they are normally present as monomers or heterodimers [3].

What's Behind the InVivoKines™: Knobs-into-Holes Technology

How is it possible to develop a long-lived Fc fusion protein for in vivo research that is monomeric or heterodimeric, but not homodimeric? With the self-developed InVivoKines™, our supplier AdipoGen Life Sciences has found the answer to this question and successfully implemented it. Our immunology specialist utilizes a method called Fc-Knobs-into-Holes or Fc-KIH for short, which was introduced by Ridgway et al. in 1996 [7]. Traditionally used to develop bispecific antibodies, this groundbreaking technology enables the heterodimerization of two different Fc fragments, resulting in a structure with two different "arms". This enables the production of naturally occurring, active monomeric or heterodimeric fusion proteins - including cytokines (Fig. 2).

Figure 2: Comparison of the structure of a classic Fc fusion protein and an Fc-KIH fusion protein. While conventional Fc fusion proteins are available as homodimers, the KIH technology also enables the development of monomeric and heterodimeric fusion proteins.

But how exactly do you obtain the two different Fc arms? Important amino acid residues that are involved in the dimeric interaction of Fc are manipulated. Amino acid residues with a small side chain are replaced by those with larger ones, resulting in a knob in one chain. Conversely, in the partner chain, amino acid residues with a large side chain are replaced by smaller ones to create a hole. Typically, a T366Y mutation in one C_H3 domain is used to create a knob, while a Y407T mutation in the other C_H3 domain causes a hole. Such mutations lead to intermolecular interactions and promote the formation of heterodimers by pairing knob and hole [3].

"Silenced" InVivoKines™: LALA-PG Mutations

As mentioned above, Fc fragments bind to Fcγ receptors (FcγRs) on immune cells or activate the complement system. As a result, they can trigger various immunological mechanisms, including antibody-dependent cell mediated cytotoxicity (ADCC), antibody-dependent cell mediated phagocytosis (ADCP) and complement-dependent cytotoxicity (CDC) [3]. In addition to FcγRs, Fc fragments also bind to the neonatal Fc receptor, FcRn for short. Binding to this receptor prevents the degradation of the antibodies or fusion proteins and thus prolongs their half-life in the blood [8].

Although the effector functions of the Fc domain are an important part of our immune defense and play a decisive role in fighting tumor cells, for example, it can be interesting for certain research experiments to use Fc domains that lack these functions. For this reason, AdipoGen also offers the InVivoKines with silenced Fc-KIH domains. For this purpose, hlgG1 P329G LALA mutations, LALA-PG for short, are used, which suppress the interactions with FcγRs and the complement system - however, the interaction with the FcRn remains unaffected (Fig. 3) [3]. Therefore, the advantage of the InVivoKines™ LALA-PG is that on the one hand the immunological side effects due to the Fc-KIH fusion domain are avoided, but on the other hand the increased half-life in vivo is retained.

Figure 3: Inhibition of Fc effector functions by LALA-PG mutations. LALA-PG mutations prevent the triggering of various immunological mechanisms (including ADCC and ADCP) as well as the activation of the complement system (CDC). However, binding to the neonatal Fc receptor and the associated increased half-life in vivo is retained.

InVivoKines™: Proteins with Enhanced Activity and Stability for in vivo Research

Now you know the background to InVivoKines™. Have we sparked your interest? Then discover the different products that this new product line brings with it and that can support your immunotherapeutic, preclinical and translational in vivo research. All InVivoKines™ from AdipoGen have the following 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

Please also take a look at AdipoGen's brochure (see right), where you can find more detailed information about the InVivoKines^TM.

Effective Monomeric Cytokines: Interleukin-2

Interleukin-2 (IL-2) is a cytokine from the interleukin family whose main effect is the autocrine stimulation of T helper cells [9]. In addition, it promotes the proliferation and differentiation of effector T cells (CD4 and CD8) and natural killer cells (NKs) and is therefore frequently used in cell therapy with tumor-infiltrating lymphocytes (TILs) to promote T cell expansion. AdipoGen Life Sciences' IL-2-Superkine (monomeric):Fc-KIH induces an effective antitumor response and can be used both in vitro and in vivo. The IL-2-InVivoKine™ is particularly unique for in vivo studies due to its high activity and long half-life in blood [3].

Highly Active Heterodimeric Cytokines: Interleukin-27

Interleukin-27 (IL-27) is a cytokine from the IL-12 family and can activate various signaling pathways, including the JAK-STAT and p38 MAPK signaling pathways. Both pro- and anti-inflammatory responses can be elicited [10]. IL-27 consists of two subunits, IL-27p28 and EBI3. AdipoGen Life Sciences' IL-27 (mouse):Fc-KIH (human) (rec.) is a heterodimeric Fc-KIH construct that binds to the mouse and human IL-27 receptor to activate Stat3 phosphorylation. Due to its heterodimeric structure, the IL-27-InVivoKine™ shows higher activity than conventional IL-27-Fc constructs [3].

Unique Homodimeric Cytokines: GDF-15

Growth differentiation factor 15 (GDF-15) has multiple effects, including on appetite regulation, metabolism, pregnancy and immune responses. GDF-15 is currently studied for the treatment of many metabolic diseases, including obesity and type 2 diabetes mellitus, as well as cardiovascular diseases. AdipoGen Life Sciences' Fc (LALA-PG)-KIH (human):GDF15 (mouse) (rec.)  is a unique protein with long-lasting high activity for in vivo studies. While other classical GDF-15-Fc proteins form large, inactive aggregates, GDF-15-InVivoKine™ forms a biologically active homodimer [3].

Are you looking for cytokines with higher activity and half-life for your in vivo or in vitro studies? Then the new InVivoKines™ product line from our supplier AdipoGen Life Sciences is just right for you! Discover the quality and benefits of these innovative solutions for your cytokine research.

Sources

[1] https://de.wikipedia.org/wiki/Zytokin, 03.06.2024

[2] https://flexikon.doccheck.com/de/Zytokin, 03.06.2024

[3] https://adipogen.com/invivokines/, 03.06.2024

[4] https://flexikon.doccheck.com/de/Rekombinant, 03.06.2024

[5] https://flexikon.doccheck.com/de/Fusionsprotein, 03.06.2024

[6] https://flexikon.doccheck.com/de/Fc-Fragment, 03.06.2024

[7] Ridgway JB, Presta LG, Carter P. 'Knobs-into-holes' engineering of antibody CH3 domains for heavy chain heterodimerization. Protein Eng. 1996 Jul;9(7):617-21.

[8] https://flexikon.doccheck.com/de/Neonataler_Fc-Rezeptor, 03.06.2024

[9] https://flexikon.doccheck.com/de/Interleukin-2, 03.06.2024

[10] https://en.wikipedia.org/wiki/Interleukin_27, 03.06.2024