Monoclonal Antibodies



General information



Product Name
Monoclonal Antibodies
Inventors
Georges Köhler and Cesar Milstein
Patent holders
This product had not been patented
Area of Application
  • Diagnosis
  • Purification
  • Human therapy
  • Gene identification
  • Applied chemistry
Market Size
$41 billion (US$) per year, at 2010[1]

Monoclonal antibodies (mAB or moAB) have a specific target for an antigen and they are all the same due to their production by immune cells which are all clones from a unique parent cell.
The production of monoclonal antibodies is based on a line of cells named hybridomas , which are a product of the fusion between myeloma cells and spleen cells from a mouse that has been immunized with the desire antigen (although recent advances allowed the use of rabbit B-cells). However, nowadays (and mainly in the industry) this technique is used mostly for development and as a source of gene sequences for further cloning into a plasmid to be inserted into a cell (CHO or myeloma). So the main way of production of mABs is by cell culture and has the further steps[2] (the schematic representation is in the picture below) :
  1. Transfection - Delivery of the rDNA into the host cell nucleus for chromossomal random integration;
  2. Selection of the clones which incorporated the vector, recovery and expansion. The selection is made by growing the cells in a medium devoid of glycine, hypoxanthine and thymidine since the DHFR is involved with the synthesis of a co-factor which helps in the production of glycine and nucleic acid building blocks (purinesand thymidine). In this way all the cells that didn't incorporate the rDNA vector die;
  3. Selection of the clones whith high transcriptional activity and their amplification. In this case the selection is performed by adding methotrexate (MTX) which is a strong inhibitor of DHFR and in this way the selection pressure is enhanced. So in order to survive, cells undergo genomic rearragmentsand amplification of the rDNAintegration locus resulting in an enhanced copy numbers of both DHFR and protein of interest;
  4. Screening, expansion and cell banking.

production_mABs.JPG









History


The discovery was made by Georges Köhler (1946-1995) and Cesar Milstein (1927-2002), who shared the Nobel prize in Physiology or Medicine (1984), in the year of 1975, being the key idea of using a line of myeloma cells, which had lost their ability to secrete antibodies, and somehow fuse them with healthy antibody-producing B-cells and, at the end, select the successful ones.
The first monoclonal antibody to be made was antibody Sp1, a mouse IgM antibody specific for SRBC. The fusion experiment was carried out and the cell line cloned in late 1974, Kohler and Milstein submitted their paper to Nature in May 1975 and it was accepted for publication in June 1975, finally appearing in print in August 1975
The production of the first mAb to market was by Johnson & Johnson's Orthoclone OKT3 (muromonab) in 1986.
In 1988 Greg Winter and his team pioneered the techniques to humanize monoclonal antibodies, removing the reactions that many monoclonal antibodies caused in some patients[3] .

Market and Applications


The Monoclonal antibodies have various applications:
  • Identifications of cell surface markers
- Detection Assays/ Diagnosis
They are useful to diagnosis because they detect the presence of a specific substance which is the target for what the antibody was produced. They can also be attached to some visual probe so we can detect the signal. Some examples for this use are Western Blot, immune dot blot, immunohistochemistry, immunofluorescence and ELISA
- Purification techniques
This is another area where the monoclonals have had impact since as a given monoclonal antibody, which binds specifically to the product that we are interested in,can be attached to an insoluble surface and be used to purify the molecule of interest (affinity chromatography).In fact, this approach allows one to accomplish a several-fold purification in a single step.
It can be used as also for immunoprecipitation.
- Gene identification
The field of molecular genomics has benefited from the availability of monoclonal antibody technology. For example, a known monoclonal antibody that recognizes a molecule of interest can be used to identify its gene. Alternatively, if one has a newly identified gene with an unknown function, monoclonal antibodies can be generated against the predicted protein encoded by the gene. These reagents can then be used for expression and function studies. These avenues also open up new windows of investigative research. For example, one can use monoclonal antibodies to determine if the gene is abnormally expressed in certain disease states or has a different structure in different individuals[4] .
- Disease therapy
In medicinal treatments, the small variation (if any) in recognizing the antigen helps to reduce side effects. One of the actual uses of this product in therapy is for cancer treatment as the antibody can deliver the killing agents specifically to the cancer cells.


  • Applied Chemistry
It was appreciated that enzymes function in part by having a high affinity interaction with a short-lived transition state. It was reasoned that, if enzymes can interact with these transitional states, maybe an antibody can as well and potentially serve as an enzyme providing catalytic function. By generating antibody reagents against enzyme inhibitors that mimic the transitional state, one can isolate antibodies that can provide catalytic function. The catalytic reactions range from redox reactions to structural rearrangements. This initial success, together with the principles emerging in the field of combinatorial chemistry indicates that recognition molecules generated by the immune system have tremendous potential to be used as chemical tools[5] .

The next table summarizes some of the products that are available and their application[6] :

Type
Application
Mechanism
Mode
infliximab
  • rheumatoid arthritis
  • Crohn's disease
  • Ulcerative Colitis
inhibits TNF-α
chimeric
brasiliximab
  • Acute rejection of kidney transplants
inhibits IL-2 on activated T cells
chimeric
bevacizumab
  • Anti-angiogenic cancer therapy
inhibits VEGF
humanized
abciximab
  • Prevent coagulation in coronary angioplasty
inhibits the receptor GpIIb/IIIa on platelets
chimeric
daclizumab
  • Acute rejection of kidney transplants
inhibits IL-2 on activated T cells
humanized
gemtuzumab
  • relapsed acute myeloid leukaemia
targets myeloid cell surface antigen CD33 on leukemia cells
humanized
alemtuzumab
  • B cell leukemia
targets an antigen CD52 on T- and B-lymphocytes
humanized
rituximab
  • non-Hodgkin's lymphoma
targets phosphoprotein CD20 on B lymphocytes
chimeric
palivizumab
  • RSV infections in children
inhibits an RSV fusion (F) protein
humanized
trastuzumab
  • anti-cancer therapy for a specific kind of breast cancer
targets the HER2/neu (erbB2) receptor
humanized
etanercept
  • rheumatoid arthritis
contains TNF receptor
fusion protein
adalimumab
  • rheumatoid arthritis
  • Crohn's disease
  • Ulcerative Colitis
inhibits TNF-α
human
nimotuzumab
  • Approved in squamous cell carcinomas, Glioma
  • Clinical trials for other indications underway
EGFR inhibitor
humanized
Note: Chimeric antibodies are the ones which contains the antigen-binding parts (variable regions) of the mouse and the effector parts (constant regions) of a human antibody, while the humanized ones only have the amino acids responsible for doing the biding sites (hypervariable regions) of the mouse antibody and the rest it's all of a human antibody.


Financial Information


Since the cost of production of the monoclonal antibodies is very high (due to the use of animals for development, pricey bioreactors and the rigorous downstream process when is for pharmaceutical uses) the selling cost also will be high, in a range of 100-600$ per 100µg.[7]

Perspectives for the future

The biotechnology field is committed to MAbs and continues to find new ways both to press onward with current processes and—to some extent in reaction to the economic pressures—to develop radically new concepts and technologies that could make monoclonals even more widespread.

Scientists are also trying to understand how MAbs aggregate during long-term storage. This is important if the industry is to achieve economies of scale and reduce costs to the end consumer. Like conventional drugs, antibodies are subject to oxidation and hydrolysis and demidation. Adding protective proteins to the MAbs might help preserve them.

Instead of wedding themselves to the pricey bioreactors, some biotech companies think the way of the future may be to use “transgenic” animals, such as goats and cows, and even corn or other plants, genetically engineered to carry genes for desired antibodies. With that technology, some companies are envisioning MAbs from the milk of transgenic cows or goats, while others are looking to transgenic plants. Both approaches remain in their infancy and will have to make their way through both whatever challenges science holds for them and whatever issues regulators will have.
It remains unclear just where MAb technology might head in the future.

Prepared by Márcia Mata

  1. bioinformatics
  2. biopharmaceutical
  3. company
  4. genetic tests
  5. monoclonal antibodies
  6. nanobiotechnology
  7. penicillin; antibiotics
  8. pharmaceutical
  9. poland
  10. portugal
  11. product
  12. technology
  13. tool
  14. vaccine

  1. ^ http://www.visiongain.com/Conference.aspx?cid=265
  2. ^ Dana C Andersen and Dorothea E Reilly, Production technologies for monoclonal antibodies and their fragments, Elsevier 2004; 15:456-462
    http://cmbi.bjmu.edu.cn/news/report/2004/biotech/60.pdf
  3. ^
    Riechmann L, Clark M, Waldmann H, Winter G. Reshaping human antibodies for therapy. Nature: http://www.nature.com/nature/journal/v332/n6162/pdf/332323a0.pdf
  4. ^
    W J Payne, Jr, D L Marshall, R K Shockley, and W J Martin, Clinical laboratory applications of monoclonal antibodies, Clin Microbiol Rev. 1988 July; 1(3):313-329: http://www.ncbi.nlm.nih.gov/pmc/articles/PMC358053/
  5. ^
    Schultz, P. G., and Lerner, R. A. From Molecular Diversity to Catalysis: Lessons from the Immune System. Science 269:1835-1842, 1995: http://www.sciencemag.org/cgi/content/abstract/269/5232/1835
  6. ^
    http://users.rcn.com/jkimball.ma.ultranet/BiologyPages/M/Monoclonals.html
  7. ^ www.genscript.com