Antibody drug conjugates (ADCs) combine specific antibody targeting of tumour cells with the potent cytotoxic effect of a chemotherapeutic agent. Despite their conception in the early 1900’s, there are only two ADCs currently on the market. As we will explain, issues with their manufacture have posed a challenge to the industry but, with manufacturers starting to overcome these hurdles, ADCs may now realize their status as cancer’s magic bullet.
The Best of Both Worlds
Monoclonal antibodies target tumour cells with a high degree of accuracy and specificity. They can induce tumour cell death directly by binding tumour cell surface antigens to disrupt cell signalling, or indirectly, by marking the cells up for destruction by the body’s immune system, or by disrupting the tumour microvasculature.9
Combining the cell-targeting specificity of a monoclonal antibody with the tumour-cell-killing ability of a chemotherapy agent enables targeted destruction of tumours without the off-target effects of systemic chemotherapy.
How Do ADCs Work?
The concept for their mechanism of action is simple, the monoclonal antibody (mAb) part of the ADC will bind to its specific antigen on the surface of the tumour. Then, by endocytosis, the ADC will be internalised within the tumour cell where the cytotoxin will be released from its linker owing to the acidic environment of the internalized vesicle. The cytotoxin will then induce cell apoptosis. This will lead to the destruction of the cell and the tumour.
Precise targeting of the cytotoxin avoids the issues of systemic chemotherapy, meaning smaller volumes of more potent cytotoxins can be used whilst avoiding side effects.
Next generation of therapies animation from Lonza describes the structure and mechanism of action of ADCs.
The idea of combining a drug to a targeting molecule as a ‘Magic Bullet’ was first suggested by the German Physician Paul Erhlich in the early 1900’s.12,13 However, the technology to produce monoclonal antibodies was not developed until the 1970’s when Köhler and Milstein immortalised antibody producing cells by fusing them with a myeloma cell line.5 Humanized monoclonal antibodies such as trastuzumab and rituximab were then developed as therapies for the treatment of cancers in the 1990’s. The first ADC to go to market was Wyeth’s gemtuzumab ozogamicin (Mylotarg®) treatment for myeloid leukaemia, which was released in 2000. Although licensed by the FDA, this ADC failed a UK trial in which it was found not to improve survival rates and was [subsequently] removed from the market in 2010.7
In 2011, brentuximab vedotin (Adcetris®) an ADC for the treatment of Hodgkin’s lymphoma and anaplastic large-cell lymphomas was released, followed by trastuzumab emtansine (Kadcyla®) an ADC for the treatment of Her2-positive breast cancer in 2013.11
Even with just with these two approved ADCs available, the market for ADCs was estimated at $900m in 2015.6 This is set to grow, with 45 ADCs in various stages of development.1,3
Overcoming Manufacturing Challenges
There are three parts to an ADC that need to be manufactured, the parent mAb, the linker, and the cytotoxic payload. Each component comes with its own manufacturing challenges Then they must be combined.
Cell line production of IgG mAbs is followed by purification of the mAbs and quality control checks to ensure a pure, uncontaminated sample of protein.4 This must be done in sterile conditions, requiring specialist equipment and trained personnel.
The linker connects the targeting mAb to the payload cytotoxin. It must be designed to ensure it renders the payload unreactive until it is released in the target cells, whilst also not affecting the stability of the mAb. Given that most mAbs have a 72-hour half-life, the linker also needs to be stable in the blood plasma for several days. Given these requirements, four types of linker are currently used in ADC design and manufacture, and these fall into two categories, either cleavable or un-cleavable.
Hydrazone, peptide and disulphide linkers are cleavable linkers that are stable in the blood plasma, but are degraded in the vesicles of the cell following internalization. Thioether linkers are non-reducible and do not break down in the vesicles, but are destroyed by proteolytic cleavage.1,2
Hydrophilic poly(ethylene glycol) or PEG-linkers are also being developed. These linkers improve on the previously mentioned chemistry owing to their ability to link more payload molecules to the ADC, improving the drug:antibody ratio (DAR).1
Like the mAbs, these linkers need to be manufactured in a sterile environment to avoid contamination. This also requires specialist equipment and trained personnel.
Two types of cytotoxin are currently being incorporated into ADCs: microtubule disrupting agents which affect the tumour cell’s ability to divide and proliferate; and DNA modifying agents which induce cell death cascades. Auristatins and Maytansines are families of cytotoxins used in ADCs that prevent microtubule polymerisation. Duocarymycins, PBD dimers and α-Amanitin are types of cytotoxins that disrupt DNA transcription, inducing cell death (for detailed chemical information on these agents see ADC review1)
These extremely potent and dangerous toxins pose significant risk to the operators, and must be handled according to current good manufacturing practices (cGMP) in a sterile environment, ensuring an occupational exposure limit of less than 50ng/m3. The high level of containment for this requires specialist equipment and trained personnel.
The complicated nature of the ADC manufacture requires high levels of containment to protect both the product and the personnel, and staff need to be specialists in the use of the equipment and manufacture of the ADC to generate the finished product.8
Read more: CMO’s dedicated to ADC development
Testing the ADC
ADCs are both drug and biological molecules, therefore the analytical tests need to be appropriate for the entire molecule. As yet, there is no single confirmatory test available to determine both the DAR and activity of the ADC.10
Once conjugated, the ADCs must be tested to assess drug loading, i.e. how many cytotoxic molecules have bound to the antibody, to investigate the DAR. This is achieved by mass spectrometry, hydrophobic interaction chromatography, and reversed-phase HPLC.
The mAb activity also needs to be assessed to check that conjugation does not affect its biological activity, such as its antigen binding specificity. Comparisons with the un-conjugated form of the mAb provide evidence for affected functionality.
Quality control for the cytotoxic drug should identify the total drug content and purity. The free drug concentration in a sample needs to be determined as these could prove toxic in a patient.
The first toxicology test for the ADC determines if it kills target-antigen expressing cells, whilst leaving other cells unharmed.
Looking to the future
With 45 molecules in development the future of ADCs is bright and exciting. As more ADCs are developed and manufactured, it is anticipated that manufacturing practices will also advance. Specialist CMO’s for the development of ADCs and strategic alliances between biotech companies are driving this growth.
Read more: strategic alliances in the development of ADCs
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2. Carter, P. and Senter, P. (2008). Antibody-Drug Conjugates for Cancer Therapy. The Cancer Journal, 14(3), pp.154-169.
3. de Goeij, B. and Lambert, J. (2016). New developments for antibody-drug conjugate-based therapeutic approaches. Current Opinion in Immunology, 40, pp.14-23.
4. Kelley, B. (2017). Industrialization of mAb production technology: The bioprocessing industry at a crossroads.
5. Köhler, G. and Milstein, C. (1975). Continuous cultures of fused cells secreting antibody of predefined specificity. Nature, 256(5517), pp.495-497.
6. ltd, R. (2017). Antibody Drug Conjugates Market (3rd Edition) - Research and Markets. [online] Researchandmarkets.com. Available at: http://www.researchandmarkets.com/research/c2ctf5/antibody_drug [Accessed 24 Mar. 2017].
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8. Rohrer, T. (2013). Industry Perspective: Consideration for the Safe and Effective Manufacturing of Antibody-drug Conjugates (Update). ADC Review / Journal of Antibody-drug Conjugates, 1(1).
9. Scott, A., Wolchok, J. and Old, L. (2012). Antibody therapy of cancer. Nature Reviews Cancer, 12(4), pp.278-287.
10. Shapiro, and Chen, (2017). Regulatory Considerations When Developing Assays for the Characterization and Quality Control of Antibody-Drug Conjugates. [online] Americanlaboratory.com. Available at: http://www.americanlaboratory.com/913-Technical-Articles/119843-Regulatory-Considerations-When-Developing-Assays-for-the-Characterization-and-Quality-Control-of-Antibody-Drug-Conjugates/ [Accessed 28 Mar. 2017].
11. Slamon, D., Leyland-Jones, B., Shak, S., Fuchs, H., Paton, V., Bajamonde, A., Fleming, T., Eiermann, W., Wolter, J., Pegram, M., Baselga, J. and Norton, L. (2001). Use of Chemotherapy plus a Monoclonal Antibody against HER2 for Metastatic Breast Cancer That Overexpresses HER2. New England Journal of Medicine, 344(11), pp.783-792.
12. Stern, F. (2004). Paul Ehrlich: The Founder of Chemotherapy. ChemInform, 35(45).
13. Strebhardt, K. and Ullrich, A. (2008). Paul Ehrlich's magic bullet concept: 100 years of progress. Nature Reviews Cancer, 8(6), pp.473-480.
14. Younes, A., Bartlett, N., Leonard, J., Kennedy, D., Lynch, C., Sievers, E. and Forero-Torres, A. (2010). Brentuximab Vedotin (SGN-35) for Relapsed CD30-Positive Lymphomas. New England Journal of Medicine, 363(19), pp.1812-1821.