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Monoclonal antibody

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Title: Monoclonal antibody  
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Subject: Biological response modifiers, Breast cancer management, Antibody, Humanized antibody, Detumomab
Collection: Biotechnology, Immune System, Immunology, Monoclonal Antibodies, Therapeutic Antibodies
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Monoclonal antibody

A general representation of the method used to produce monoclonal antibodies.

Monoclonal antibodies (mAb or moAb) are monospecific antibodies that are made by identical immune cells that are all clones of a unique parent cell, in contrast to polyclonal antibodies which are made from several different immune cells. Monoclonal antibodies have monovalent affinity, in that they bind to the same epitope.

Given almost any substance, it is possible to produce monoclonal antibodies that specifically bind to that substance; they can then serve to detect or purify that substance. This has become an important tool in biochemistry, molecular biology and medicine. When used as medications, the non-proprietary drug name ends in -mab (see "Nomenclature of monoclonal antibodies"), and many immunotherapy specialists use the word mab anacronymically.


  • Discovery 1
  • Production 2
    • Hybridoma cell production 2.1
    • Purification of monoclonal antibodies 2.2
    • Antibody heterogeneity 2.3
    • Recombinant 2.4
    • Chimeric antibodies 2.5
    • "Fully" human monoclonal antibodies 2.6
  • Applications 3
    • Diagnostic tests 3.1
    • Therapeutic treatment 3.2
      • Cancer treatment 3.2.1
      • Autoimmune diseases 3.2.2
      • Examples of therapeutic monoclonal antibodies 3.2.3
  • See also 4
  • References 5
  • External links 6


The idea of a "Élie Metchnikoff received the 1908 Nobel Prize for Physiology or Medicine for this work, which led to an effective syphilis treatment by 1910.

In the 1970s, the B-cell cancer multiple myeloma was known, and it was understood that these cancerous B-cells all produce a single type of antibody (a paraprotein). This was used to study the structure of antibodies, but it was not yet possible to produce identical antibodies specific to a given antigen[1]:324

Building on the work of many others, in 1975, César Milstein succeeded in making fusions of myeloma cell lines with B cells to produce hybridomas that made antibodies to known antigens and that were immortalized.[2] They shared the Nobel Prize in Physiology or Medicine in 1984 for the discovery.[2]

In 1988, Greg Winter and his team pioneered the techniques to humanize monoclonal antibodies,[3] removing the reactions that many monoclonal antibodies caused in some patients.


Researchers looking at slides of cultures of cells that make monoclonal antibodies. These are grown in a lab and the researchers are analyzing the products to select the most promising of them.
Monoclonal antibodies can be grown in unlimited quantities in the bottles shown in this picture.
Technician hand-filling wells with a liquid for a research test. This test involves preparation of cultures in which hybrids are grown in large quantities to produce desired antibody. This is effected by fusing myeloma cell and mouse lymphocyte to form a hybrid cell (hybridoma).
Lab technician bathing prepared slides in a solution. This technician prepares slides of monoclonal antibodies for researchers. The cells shown are labeling human breast cancer.

Hybridoma cell production

Monoclonal antibodies are typically made by fusing myeloma cells with the spleen cells from a mouse that has been immunized with the desired antigen. However, recent advances have allowed the use of rabbit B-cells to form a rabbit hybridoma. Polyethylene glycol is used to fuse adjacent plasma membranes,[4] but the success rate is low so a selective medium in which only fused cells can grow is used. This is possible because myeloma cells have lost the ability to synthesize hypoxanthine-guanine-phosphoribosyl transferase (HGPRT), an enzyme necessary for the salvage synthesis of nucleic acids. The absence of HGPRT is not a problem for these cells unless the de novo purine synthesis pathway is also disrupted. By exposing cells to aminopterin (a folic acid analogue, which inhibits dihydrofolate reductase, DHFR), they are unable to use the de novo pathway and become fully auxotrophic for nucleic acids, thus requiring supplementation to survive.

The selective culture medium is called HAT medium because it contains hypoxanthine, aminopterin, and thymidine. This medium is selective for fused (hybridoma) cells. Unfused myeloma cells cannot grow because they lack HGPRT, and thus cannot replicate their DNA. Unfused spleen cells cannot grow indefinitely because of their limited life span. Only fused hybrid cells, referred to as hybridomas, are able to grow indefinitely in the media because the spleen cell partner supplies HGPRT and the myeloma partner has traits that make it immortal (similar to a cancer cell).

This mixture of cells is then diluted and clones are grown from single parent cells on microtitre wells. The antibodies secreted by the different clones are then assayed for their ability to bind to the antigen (with a test such as ELISA or Antigen Microarray Assay) or immuno-dot blot. The most productive and stable clone is then selected for future use.

The hybridomas can be grown indefinitely in a suitable cell culture medium. They can also be injected into mice (in the peritoneal cavity, surrounding the gut). There, they produce tumors secreting an antibody-rich fluid called ascites fluid.

The medium must be enriched during in-vitro selection to further favour hybridoma growth. This can be achieved by the use of a layer of feeder fibrocyte cells or supplement medium such as briclone. Culture-medium conditioned by macrophages can also be used. Production in cell culture is usually preferred as the ascites technique is painful to the animal. Where alternate techniques exist, this method (ascites) is considered unethical.[5]

Purification of monoclonal antibodies

After obtaining either a media sample of cultured hybridomas or a sample of ascites fluid, the desired antibodies must be extracted. The contaminants in the cell culture sample would consist primarily of media components such as growth factors, hormones, and transferrins. In contrast, the in vivo sample is likely to have host antibodies, proteases, nucleases, nucleic acids, and viruses. In both cases, other secretions by the hybridomas such as cytokines may be present. There may also be bacterial contamination and, as a result, endotoxins that are secreted by the bacteria. Depending on the complexity of the media required in cell culture, and thus the contaminants in question, one method (in vivo or in vitro) may be preferable to the other.

The sample is first conditioned, or prepared for purification. Cells, cell debris, lipids, and clotted material are first removed, typically by centrifugation followed by filtration with a 0.45 µm filter. These large particles can cause a phenomenon called membrane fouling in later purification steps. In addition, the concentration of product in the sample may not be sufficient, especially in cases where the desired antibody is one produced by a low-secreting cell line. The sample is therefore condensed by ultrafiltration or dialysis.

Most of the charged impurities are usually anions such as nucleic acids and endotoxins. These are often separated by ion exchange chromatography.[6] Either cation exchange chromatography is used at a low enough pH that the desired antibody binds to the column while anions flow through, or anion exchange chromatography is used at a high enough pH that the desired antibody flows through the column while anions bind to it. Various proteins can also be separated out along with the anions based on their isoelectric point (pI). For example, albumin has a pI of 4.8, which is significantly lower than that of most monoclonal antibodies, which have a pI of 6.1. In other words, at a given pH, the average charge of albumin molecules is likely to be more negative. Transferrin, on the other hand, has a pI of 5.9, so it cannot easily be separated out by this method. A difference in pI of at least 1 is necessary for a good separation.

Transferrin can instead be removed by size exclusion chromatography. The advantage of this purification method is that it is one of the more reliable chromatography techniques. Since we are dealing with proteins, properties such as charge and affinity are not consistent and vary with pH as molecules are protonated and deprotonated, while size stays relatively constant. Nonetheless, it has drawbacks such as low resolution, low capacity and low elution times.

A much quicker, single-step method of separation is Protein A/G affinity chromatography. The antibody selectively binds to Protein A/G, so a high level of purity (generally >80%) is obtained. However, this method may be problematic for antibodies that are easily damaged, as harsh conditions are generally used. A low pH can break the bonds to remove the antibody from the column. In addition to possibly affecting the product, low pH can cause Protein A/G itself to leak off the column and appear in the eluted sample. Gentle elution buffer systems that employ high salt concentrations are also available to avoid exposing sensitive antibodies to low pH. Cost is also an important consideration with this method because immobilized Protein A/G is a more expensive resin.

To achieve maximum purity in a single step, affinity purification can be performed, using the antigen to provide exquisite specificity for the antibody. In this method, the antigen used to generate the antibody is covalently attached to an agarose support. If the antigen is a peptide, it is commonly synthesized with a terminal cysteine, which allows selective attachment to a carrier protein, such as KLH during development and to the support for purification. The antibody-containing media is then incubated with the immobilized antigen, either in batch or as the antibody is passed through a column, where it selectively binds and can be retained while impurities are washed away. An elution with a low pH buffer or a more gentle, high salt elution buffer is then used to recover purified antibody from the support.

Antibody heterogeneity

Product heterogeneity is common in monoclonal antibodies and other recombinant biological products and is typically introduced either upstream during expression or downstream during manufacturing.

These variants are typically aggregates, deamidation products, glycosylation variants, oxidized amino acid side chains, as well as amino and carboxyl terminal amino acid additions.[7] These seemingly minute changes in a monoclonal antibody’s structure can have a profound effect on preclinical stability and process optimization as well as therapeutic product potency, bioavailability, and immunogenicity. The generally accepted method of purification of process streams for monoclonal antibodies includes capture of the product target with Protein A, elution, acidification to inactivate potential Mammalian viruses, followed by Ion chromatography, first with anion beads and then with cation beads.

Displacement chromatography has been used to identify and characterize these often unseen variants in quantities that are suitable for subsequent preclinical evaluation regimens such as animal pharmacokinetic studies.[8][9] Knowledge gained during the preclinical development phase is critical for enhanced understanding of product quality and provides a basis for risk management and increased regulatory flexibility. The recent Food and Drug Administration’s Quality by Design initiative attempts to provide guidance on development and to facilitate design of products and processes that maximizes efficacy and safety profile while enhancing product manufacturability.[10]


The production of

  • Monoclonal Antibodies, from John W. Kimball's online biology textbook
  • Monoclonal antibodies at the US National Library of Medicine Medical Subject Headings (MeSH)
  • Antibodypedia, open-access virtual repository publishing data and commentary on any antibodies available to the scientific community.
  • Antibody Purification Handbook

External links

  1. ^ Tansey EM, Catterall PP (Jul 1994). "Monoclonal antibodies: a witness seminar in contemporary medical history". Med Hist. 38 (3): 322–7.  
  2. ^ a b Marks LV, A Healthcare Revolution in the Making: The Story of César Milstein and Monoclonal Antibodies: Making monoclonal antibodies
  3. ^ Riechmann L, Clark M, Waldmann H, Winter G (1988). "Reshaping human antibodies for therapy". Nature 332 (6162): 323–7.  
  4. ^ Yang J1, Shen MH. Polyethylene glycol-mediated cell fusion. Methods Mol Biol. 2006; 325:59-66.
  5. ^ National Research Council (US) Committee on Methods of Producing Monoclonal Antibodies. Recommendation 1: Executive Summary: Monoclonal Antibody Production. Washington (DC): National Academies Press (US); 1999. ISBN 978-0-309-07511-4
  6. ^ Vlasak J, Ionescu R (2008). "Hetergeneity of Monoclonal Antibodies Revealed by Charge-Sensitive Methods". Current Pharmaceutical Biotechnology 9 (6): 468–481.  
  7. ^ Beck A, Wurch T, Bailly C, Corvaia N (May 2010). "Strategies and challenges for the next generation of therapeutic antibodies". Nat. Rev. Immunol. 10 (5): 345–52.  
  8. ^ Khawli LA, Goswami S, Hutchinson R, Kwong ZW, Yang J, Wang X, Yao Z, Sreedhara A, Cano T, Tesar D, Nijem I, Allison DE, Wong PY, Kao YH, Quan C, Joshi A, Harris RJ, Motchnik P (2010). "Charge variants in IgG1: Isolation, characterization, in vitro binding properties and pharmacokinetics in rats". MAbs 2 (6): 613–24.  
  9. ^ Zhang T, Bourret J, Cano T (August 2011). "Isolation and characterization of therapeutic antibody charge variants using cation exchange displacement chromatography". J Chromatogr A 1218 (31): 5079–86.  
  10. ^ Rathore AS, Winkle H (January 2009). "Quality by design for biopharmaceuticals". Nat. Biotechnol. 27 (1): 26–34.  
  11. ^ Siegel DL (2002). "Recombinant monoclonal antibody technology". Transfusion clinique et biologique : journal de la Société française de transfusion sanguine 9 (1): 15–22.  
  12. ^ "Dr. George Pieczenik". LMB Alumni. MRC Laboratory of Molecular Biology (LMB). 17 September 2009. 
  13. ^ Schmitz U, Versmold A, Kaufmann P, Frank HG (2000). "Phage display: a molecular tool for the generation of antibodies—a review". Placenta 21 (Suppl A): S106–S112.  
  14. ^ Chadd HE, Chamow SM (April 2001). "Therapeutic antibody expression technology". Curr. Opin. Biotechnol. 12 (2): 188–94.  
  15. ^ Lonberg N, Huszar D (1995). "Human antibodies from transgenic mice". Int. Rev. Immunol. 13 (1): 65–93.  
  16. ^ Monoclonal antibody therapy
  17. ^ "Bristol-Myers Buys Medarex Drugmaker for $2.4 Billion (Update3)". 
  18. ^ "Amgen Completes Acquisition of Abgenix; Acquisition Provides Amgen with Full Ownership of Panitumumab and Eliminates a Denosumab Royalty". 
  19. ^
  20. ^ "Proprietary antibody platform". 
  21. ^ "Naturally optimized human antibodies". 
  22. ^  
  23. ^ Marks JD, Hoogenboom HR, Bonnert TP, McCafferty J, Griffiths AD, Winter G (1991). "By-passing immunization". Journal of Molecular Biology 222 (3): 581–97.  
  24. ^ Carmen S, Jermutus L (2002). "Concepts in antibody phage display". Briefings in Functional Genomics and Proteomics 1 (2): 189–203.  
  25. ^ Osbourn JK (2002). "Proximity-guided (ProxiMol) antibody selection". Methods Mol. Biol. 178: 201–5.  
  26. ^ Abeloff, MD; Armitage, JO; Niederhuber, JE; Kastan, MB; McKenna, G (2008). "Therapeutic Antibodies and Immunologic Conjugates". Abeloff's Clinical Oncology (4th ed.).  
  27. ^ "Cambridge Antibody Technology". 
  28. ^ Osbourn JK, Derbyshire EJ, Vaughan TJ, Field AW, Johnson KS (January 1998). "Pathfinder selection: in situ isolation of novel antibodies". Immunotechnology 3 (4): 293–302.  
  29. ^ "The Current State of Proteomic Technology". 
  30. ^ PhRMA Reports Identifies More than 400 Biotech Drugs in Development. Pharmaceutical Technology, August 24, 2006. Retrieved 2006-09-04.
  31. ^ FC Breedveld (2000). "Therapeutic monoclonal antibodies". The Lancet.  
  32. ^ Australian Prescriber (2006). "Monoclonal antibody therapy for non-malignant disease". 
  33. ^ Modified from Carter P (November 2001). "Improving the efficacy of antibody-based cancer therapies". Nat. Rev. Cancer 1 (2): 118–29.  
  34. ^ Takimoto CH, Calvo E. "Principles of Oncologic Pharmacotherapy" in Pazdur R, Wagman LD, Camphausen KA, Hoskins WJ (Eds) Cancer Management
  35. ^ a b c d e f g h i j Rang, H. P. (2003). Pharmacology. Edinburgh: Churchill Livingstone. pp. 241, for the examples infliximab, basiliximab, abciximab, daclizumab, palivusamab, gemtuzumab, alemtuzumab and rituximab, and mechanism and mode.  
  36. ^ "Growing body of published research (list)". Peregrine Pharmaceuticals, Inc. 
  37. ^ "Bavituximab Mechanism of Action" (PDF). Peregrine Pharmaceuticals, Inc. 


See also

Main category Type Application Mechanism/Target Mode
infliximab[35] inhibits TNF-α chimeric
adalimumab inhibits TNF-α human
  • Acute rejection of kidney transplants
inhibits IL-2 on activated T cells chimeric
  • Acute rejection of kidney transplants
inhibits IL-2 on activated T cells humanized
  • moderate-to-severe allergic asthma
inhibits human immunoglobulin E (IgE) humanized
Anti-cancer gemtuzumab[35] targets myeloid cell surface antigen CD33 on leukemia cells humanized
alemtuzumab[35] targets an antigen CD52 on T- and B-lymphocytes humanized
rituximab[35] targets phosphoprotein CD20 on B lymphocytes chimeric
  • breast cancer with HER2/neu overexpression
targets the HER2/neu (erbB2) receptor humanized
nimotuzumab EGFR inhibitor humanized
cetuximab EGFR inhibitor chimeric
bevacizumab inhibits VEGF humanized
Anti-cancer and anti-viral bavituximab[36]
  • cancer, viral infections
immunotherapy, targets phosphatidylserine[37] chimeric
Other palivizumab[35]
  • RSV infections in children
inhibits an RSV fusion (F) protein humanized
abciximab[35] inhibits the receptor GpIIb/IIIa on platelets chimeric

Monoclonal antibodies for research applications can be found directly from antibody suppliers, or through use of a specialist search engine like CiteAb. Below are examples of clinically important monoclonal antibodies.

Examples of therapeutic monoclonal antibodies

Monoclonal antibodies used for rejection of kidney transplants.[35] Omalizumab inhibits human immunoglobulin E (IgE) and is useful in moderate-to-severe allergic asthma.

Autoimmune diseases

MAbs approved by the FDA include[34]

The illustration below shows all these possibilities:

Monoclonal antibodies for cancer. ADEPT, antibody directed enzyme prodrug therapy; ADCC, antibody dependent cell-mediated cytotoxicity; CDC, complement dependent cytotoxicity; MAb, monoclonal antibody; scFv, single-chain Fv fragment.[33]

One possible treatment for cancer involves monoclonal antibodies that bind only to cancer cell-specific antigens and induce an immunological response against the target cancer cell. Such mAb could also be modified for delivery of a toxin, radioisotope, cytokine or other active conjugate; it is also possible to design bispecific antibodies that can bind with their Fab regions both to target antigen and to a conjugate or effector cell. In fact, every intact antibody can bind to cell receptors or other proteins with its Fc region.

Cancer treatment

Therapeutic monoclonal antibodies act through a number of mechanisms, such as blocking of targeted molecule functions, inducing apoptosis of cells which express the target, or by modulating signalling pathways.[31][32]

Therapeutic treatment

Once monoclonal antibodies for a given substance have been produced, they can be used to detect the presence of this substance. The Western blot test and immuno dot blot tests detect the protein on a membrane. They are also very useful in immunohistochemistry, which detect antigen in fixed tissue sections and immunofluorescence test, which detect the substance in a frozen tissue section or in live cells.

Diagnostic tests


In August 2006 the Pharmaceutical Research and Manufacturers of America reported that U.S. companies had 160 different monoclonal antibodies in clinical trials or awaiting approval by the Food and Drug Administration.[30]

Monoclonal antibodies have been generated and approved to treat cancer, cardiovascular disease, inflammatory diseases, macular degeneration, transplant rejection, multiple sclerosis, and viral infection (see monoclonal antibody therapy).

Phage display can be used to express variable antibody domains on filamentous phage coat proteins.[22][23][24] These phage display antibodies can be used for various research applications.[25][26] and ProAbTM. ProAb was announced in December 1997[27] and involved high throughput screening of antibody libraries against diseased and non-diseased tissue, whilst Proximol used a free radical enzymatic reaction to label molecules in proximity to a given protein.[28][29]

  • Medarex — which marketed the UltiMab platform. Medarex was acquired in July 2009 by Bristol Myers Squibb[17]
  • Abgenix — which marketed the Xenomouse technology. Abgenix was acquired in April 2006 by Amgen.[18]
  • Regeneron's VelocImmune technology.[19]
  • Kymab - who market their Kymouse technology.[20]
  • Open Monoclonal Technology's OmniRat™ and OmniMouse™ platform.[21]

A number of commercial organizations have used transgenic mice:

Transgenic mice technology is by far the most successful approach to making "fully" human monoclonal antibody therapeutics: 7 of the 9 "fully" human monoclonal antibody therapeutics on the market were derived in this manner.[16]

Ever since the discovery that monoclonal antibodies could be generated, scientists have targeted the creation of "fully" human antibodies to avoid some of the side effects of humanised or chimeric antibodies. Two successful approaches have been identified: transgenic mice[15] and phage display.

"Fully" human monoclonal antibodies

In an effort to overcome this obstacle, approaches using recombinant DNA have been explored since the late 1980s. In one approach, mouse DNA encoding the binding portion of a monoclonal antibody was merged with human antibody-producing DNA in living cells. The expression of this chimeric DNA through cell culture yielded partially mouse, partially human monoclonal antibodies. For this product, the descriptive terms "chimeric" and "humanised" monoclonal antibody have been used to reflect the combination of mouse and human DNA sources used in the recombinant process.[14]

Early on, a major problem for the therapeutic use of monoclonal antibodies in medicine was that initial methods used to produce them yielded mouse, not human antibodies. While structurally similar, differences between the two were sufficient to invoke an immune response when murine monoclonal antibodies were injected into humans, resulting in their rapid removal from the blood, as well as systemic inflammatory effects, and the production of human anti-mouse antibodies (HAMA).

Chimeric antibodies

Fermentation chambers have been used to produce these antibodies on a large scale. [13] These techniques can be used to enhance the specificity with which antibodies recognize antigens, their stability in various environmental conditions, their therapeutic efficacy, and their detectability in diagnostic applications.[12]

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