Antibody therapeutics
The use of monoclonal antibodies (mAbs) for cancer therapy has achieved considerable success in recent years due to their high specificity, activity, and favorable pharmacokinetics, as well as the availability of strategies to successfully engineer antibodies into humanized forms. Antibodies are capable of recruiting the immune system to attack cancer cells through complement-dependent cytotoxicity or antibody-dependent cellular cytotoxicity (Liu and Mardis 2017). Mechanisms of direct tumor cell killing by antibody-mediated apoptosis include neutralization of cell-surface-bound enzymes and induction of receptor agonist or antagonist activity.
In another approach, antibodies deliver drugs targeting cancer cells via antibody-drug conjugates (ADCs). ADCs direct cytotoxic compounds to tumor cells after selective binding to cancer cell surface antigens, internalization, and intracellular drug release (Wold et al. 2016). Moreover, ADCs are powerful new treatment options for lymphomas and solid tumors, and immunomodulatory antibodies have recently achieved remarkable clinical success. New advances in protein engineering technology have also generated multiple bispecific antibody (BsAb) formats capable of targeting numerous antigens as a single agent and directly targeting immune cells to tumors, thus reducing drug resistance and severe adverse side effects (Thakur et al. 2018).
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In-Fusion Cloning technology enhances antibody discovery and engineering workflows by speeding up the generation of antibody expression constructs. In-Fusion Cloning is fast (15 min), highly efficient (>95% cloning efficiency), sequence independent (any PCR insert can be cloned into any vector at any locus), seamless (no extra bp or amino acids added to target antibody), directional, and high-throughput (HTP) ready. Several publications have utilized the In-Fusion Cloning technology for HTP antibody fragment cloning due to its high cloning efficiency and accuracy (Spidel et al. 2016; Chen et al. 2014; Meng et al. 2015).
After expression construct generation and downstream expression of the target antibody, our next-gen Capturem Protein A and Capturem Protein G technologies can be utilized for rapid (5–15 min) and easy resin-free purification of high-quality and concentrated monoclonal/polyclonal antibodies. Our revolutionary Capturem Protein A and G technologies consist of spin columns or plates containing high-capacity membranes functionalized with Protein A or G (Figure 1), allowing antibody purification directly from complex matrices such as hybridoma supernatants or serum—within minutes. Capturem Protein A and Capturem Protein G technologies are available in miniprep, maxiprep, and 96-well/24-well-plate formats, which are ideal for rapid HTP screening of hybridoma clones.
Figure 1. Capturem Protein A and Protein G technology. Capturem Protein A and G mini/maxi spin columns or 96-well/24-well plates contain a high-capacity 3D nylon membrane (comprising immobilized Protein A or Protein G) with an increased surface area, providing much higher protein-binding capabilities per ml of the membrane than per ml of resin.
References for antibody therapeutics and selected publications citing the use of In-Fusion Cloning for HTP antibody cloning
Chen, C. G. et al. One-step zero-background IgG reformatting of phage-displayed antibody fragments enabling rapid and high-throughput lead identification. Nuc. Acids Res. 42, e26 (2014).
Meng, W. et al. Efficient generation of monoclonal antibodies from single rhesus macaque antibody-secreting cells. mAbs 7, 707–718 (2015).
Spidel, J. L. et al. Rapid high-throughput cloning and stable expression of antibodies in HEK293 cells. J. Immunol. Methods 439, 50–58 (2016).
Thakur, A. et al. Bispecific antibody-based therapeutics: strengths and challenges. Blood Revs. 2, 004 (2018).
Wold, E. D. et al. Antibody therapeutics in oncology. Immunother. Open 2, 108 (2016).
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