Custom Antibody Services Case Study: Antibody-Cytokine Fusion Proteins
A pharmaceutical company’s internal development group requested the development of an immunocytokine from an existing mouse hybridoma clone to serve as a proof of concept to induce an anti-tumor response and prolonged immune cell survival in their model.
Antibody-cytokine fusion proteins, also known as immunocytokines, are modular agents that are being investigated as potential cancer immunotherapeutics. Cytokines have been used in cancer therapies as they activate and regulate the immune system. However, complications arise with cytokines as they don’t specifically target cancer cells and only initiate broad immune reactions. Additionally, adverse side effects and unfavorable pharmacokinetics limit the escalation of cytokine doses to therapeutically active levels. However, using antibodies to deliver cytokines to tumor cells can increase the therapeutic index of cytokines. Cytokines can be fused to antibodies that are directed against antigens expressed on the surface of tumor cells. This allows for the targeted delivery of these therapeutic agents to tumors, thereby causing a localized immune reaction directed at tumor cells while reducing systemic immune responses that can toxicity.
The company contacted us for this custom project as the development of immunocytokines can be complicated. The antibody format used to develop an antibody-cytokine fusion protein is important because the pharmacokinetic properties of immunocytokines directly influence their performance. Typically, the structure of the cytokine dictates the antibody format. For this project, our scientists used the conventional antibody structure to direct a payload of two monomeric cytokines to the target (Figure 1).
Figure 1. Structure of the newly developed antibody-cytokine fusion protein.
An existing mouse hybridoma clone was first converted to a recombinant antibody and humanized for use in our customer’s model system. We then utilized our modular cloning platform to develop a panel of antibody-cytokine fusion proteins with different linkers connecting a specific cytokine to the newly humanized antibody. The immunocytokine constructs were tested with flow cytometry for antibody binding using HEK293 EGFP cells that expressed the cell-surface target antigen (Figure 2). The immunocytokine panel was also tested for cytokine activity. The validated antibody-cytokine fusion proteins were passed to the customer for testing in their model system and they chose the best configuration for scale up production.
Figure 2. Antibody binding of an antibody-cytokine fusion protein to transfected HEK293 EGFP cells.