Last week during a poster session in the Department of Statistics, I had an interesting discussing with Martin Buttenschoen (working on the other side of the group) regarding the difference between small molecules and antibodies as therapeutics. This discussion made me realise that even though I’m working on antibodies engineering and developability, I could use a little refresher on approved therapeutic antibodies and their mechanisms of action.
In case you also need this bigger picture, or want to get excited about therapeutic antibodies yourself, I will summarise the target, the development process, the molecular function, and the administration for three successful therapeutic antibodies.
Trastzumumab
Target
Trastuzumab targets HER2 (also named ErbB2), an epidermal growth factor receptor which is part of the receptor tyrosine kinases family. HER2 is overexpressed in 20-30% of human breast cancers and in some other cancers, such as gastric cancers and ovarian cancer, due to amplification (multiple copies) of the HER2-gene. Levels of HER2 correlate with poor disease prognoses. [1]
Development
In 1978 the first tyrosine kinase receptor was discovered, and in 1985 HER2 was identified as growth factor receptor. Antibodies against HER2 were identified in mice by researchers from Genentech. The variant that showed highest affinity to HER2 and minimal effect on cells with normal HER2 expression levels was humanised and further optimised. The resulting antibody, trastuzumab, has a binding affinity which is at least three times higher than the original murine antibody. Phase I clinical trials started in 1992 and in 1998 it became the first approved humanised antibody. [1, 2]
Molecular function
The binding of trastuzumab to the extracellular domain of HER2 results in regression of tumour cells overexpressing HER2. One of the effects of binding is blocking signalling via, among others, the mitogen-activated protein kinase (MAPK) cascade, which results in cell cycle arrest and apoptosis. Apoptosis is also promoted by antibody-dependent cellular cytotoxicity (ADCC) as natural killer cell activity is enhanced by trastuzumab. Trastuzumab also effects the interaction between HER2 and family member HER3 which effects the PI3K/AKT signalling regulating cell growth and proliferation. [3, 4]
Administration
Trastuzumab is administered via intravenous (via bloodstream) infusion or subcutaneous (via skin) injection. The original brand name is Herceptin, other trastuzumab-based therapies are Herzuma and Ontruzant.
Adalimumab
Target
Adimumab targets tumour necrosis factor-alpha (TNF-α), which is a pro-inflammatory cytokine. This protein is mainly produced by activated macrophages and stimulates inflammation. TNF-α is over expressed in many autoimmune diseases resulting in inflammation of joints (rheumatoid arthritis), the skin (psoriasis), and the gut (Crohn’s disease). [5]
Development
Before adalimumab, two therapeutics targeting TNF-α were developed. Infliximab is a humanised monoclonal antibody, and Etanercept is a fusion protein fusing the TNF receptor to the constant region of the IgG. Pharmaceutical BASF Bioresearch Corporation/Knoll developed adalimumab in the 1990s using phage display. The variable heavy (VH) and light (VL) chain of a murine antibody designed to target TNF during acute reactions, were combined with human VLs and VHs respectively. These constructed single chain variable fragments (scFvs) were selected based on TNF binding. A library of only the human chains of these hybrid selected scFvs resulted in a fully human antibody which was further optimised to increase affinity. In 2002 adalimumab was the first approved fully human anti-TNF therapeutic. [6]
Molecular function
Adalimumab binds to the soluble and membrane-bound form of TNF-α and inhibits the binding of TNF-α to its cell surface receptors p55/TNFR1 and p75/TNFR2. Binding of TNF-α to TNFR1, which is present on most cells, has a pro-inflammatory effect and mediates cell death. Binding to TNRF2, which is present on mainly immune cells, has the opposite effect. Preventing this binding with adalimumab decreases the production of proinflammatory cytokines and other inflammatory mediators and induces apoptosis of monocytes. Adalimumab thereby reduces symptoms and prevents disease progression. [7,8]
Administration
Trastuzumab is administered primarily via subcutaneous (via skin) injection. The original brand name is Humira, other adalimumab-based therapies are Amgevita, Hyrimoz, Idacio, Imraldi and Yuflyma.
Pembrolizumab
Target
Pembrolizumab targets the programmed death receptor 1 (PD-1), a receptor on activated immune cells (T cells, B cells, monocytes, natural killer T cells, and antigen-presenting cells), down-regulating the immune response. The receptor regulates the threshold of the immune response based on the recognition of antigens which is, for example, important to prevent autoimmune diseases. This checkpoint protein also prevents recognition of cancer cells by the immune system. Targeting PD-1 with pembrolizumab has shown to be effective for solid tumours as well as haematology (blood) cancers. [9, 10]
Development
PD-1 was discovered in 1992 and after the success of therapeutic antibody ipilimumab, targeting another immune surveillance protein (CTLA-4), interest in other immune inhibitory checkpoints such as PD-1 grew. The original murine monoclonal antibody was discovered by accident by researchers from Organon who looked for drugs enhancing PD-1 to suppress the immune system for auto-immune diseases. Due to mergers and acquisitions of Organon to eventually Merck, optimising this antibody was given low priority. After the success of ipilumumab and nivolumab targeting immune inhibitory checkpoints, interest was regained. The murine antibody was humanised into a human IgG4 immunoglobulin and approved by the FDA in 2014. [11, 12]
Molecular function
Pembrolizumab binds to PD-1 and blocks the binding of ligands PD-L1 and PD-L2 to PD-1. Expression of PD-L1 on various cell types (mainly active immune cells) is regulated by cytokines during inflammation to protect these cells. PD-L2 is expressed on antigen-presenting cells. The binding of PD-1 to PD-L1 recruits a phosphatase (SHP-2) which dephosphorylates and inactivates important signalling proteins supressing T cell activity. PD-L1 is often over-expressed on tumour cells due to both existing in an inflammatory environment as well as genetic mutations. Blocking PD-1 with pembrolizumab prevent tumours from escaping the immune system. [9,13]
Administration
Pembrolizumab is administered via intravenous (via blood) infusion and is sold under the brand name Keytruda.
Final remarks
The three therapeutic antibodies described above are all successful therapeutics which act mainly by blocking a signalling pathway. Other mechanisms of action include recruiting the immune system (as also seen for trastuzumab), inducing cell death, and delivering a payload. The latter are called Antibody-Drug Conjugates (ADCs) and two examples are “trastuzumab rezetecan” and “trastuzumab botidotin”, which are currently in review for marketing applications. After binding of the trastuzumab part of these ADCs to HER2, the cytotoxic agent attacks this HER2 expressing cancer cell. Such variations on the standard monoclonal antibody are more common among new marketed therapeutics with non-canonical forms accounting for around 25% of antibodies approved in 2024. [14]
Bibliography
- Harries, M., & Smith, I. (2002). The development and clinical use of trastuzumab (Herceptin). Endocrine-related cancer, 9(2), 75-85.
- Swain, S. M., Shastry, M., & Hamilton, E. (2023). Targeting HER2-positive breast cancer: advances and future directions. Nature reviews Drug discovery, 22(2), 101-126.
- Nahta, R., & Esteva, F. J. (2006). HER2 therapy: molecular mechanisms of trastuzumab resistance. Breast Cancer Research, 8, 1-8.
- Maadi, H., Soheilifar, M. H., Choi, W. S., Moshtaghian, A., & Wang, Z. (2021). Trastuzumab mechanism of action; 20 years of research to unravel a dilemma. Cancers, 13(14), 3540.
- Lapadula, G., Marchesoni, A., Armuzzi, A., Blandizzi, C., Caporali, R., Chimenti, S., … & Salvarani, C. (2014). Adalimumab in the treatment of immune-mediated diseases. International Journal of Immunopathology and Pharmacology, 27(1_suppl), 33-48.
- Frenzel, A., Schirrmann, T., & Hust, M. (2016, October). Phage display-derived human antibodies in clinical development and therapy. In MAbs (Vol. 8, No. 7, pp. 1177-1194). Taylor & Francis.
- Wong, M., Ziring, D., Korin, Y., Desai, S., Kim, S., Lin, J., … & Singh, R. R. (2008). TNFα blockade in human diseases: mechanisms and future directions. Clinical immunology, 126(2), 121-136.
- Wajant, H., & Siegmund, D. (2019). TNFR1 and TNFR2 in the control of the life and death balance of macrophages. Frontiers in cell and developmental biology, 7, 91.
- Okazaki, T., Chikuma, S., Iwai, Y., Fagarasan, S., & Honjo, T. (2013). A rheostat for immune responses: the unique properties of PD-1 and their advantages for clinical application. Nature immunology, 14(12), 1212-1218.
- Kwok, G., Yau, T. C., Chiu, J. W., Tse, E., & Kwong, Y. L. (2016). Pembrolizumab (keytruda). Human vaccines & immunotherapeutics, 12(11), 2777-2789.
- McDermott, J., & Jimeno, A. (2015). Pembrolizumab: PD-1 inhibition as a therapeutic strategy in cancer. Drugs of today (Barcelona, Spain: 1998), 51(1), 7-20.
- https://www.science.org/content/blog-post/keytruda-story
- Wang, X., Teng, F., Kong, L., & Yu, J. (2016). PD-L1 expression in human cancers and its association with clinical outcomes. OncoTargets and therapy, 5023-5039.
- Crescioli, S., Kaplon, H., Wang, L., Visweswaraiah, J., Kapoor, V., & Reichert, J. M. (2025, December). Antibodies to watch in 2025. In mAbs (Vol. 17, No. 1, p. 2443538). Taylor & Francis.
