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Humanized Antibodies - Production and Applications in Cancer Therapy

BY: Kanya Sasi | Category: Genetics | Submitted: 2011-04-15 02:43:51
 

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Humanized Antibodies
Humanized antibodies are the name given to antibodies derived from non human species whose proteins have been modified to enhance their resemblance to natural human antibodies. "Humanization" is generally carried out in non-human monoclonal antibodies developed for therapeutic applications in humans.

When therapeutic antibodies are produced in non human immune system (for example mice) their protein sequence are to some extent different from that of natural human antibodies. These antibodies on administration to humans can be possibly immunogenic and dangerous eliciting severe and sometimes fatal reactions. Therefore it is important to humanize these antibodies before use. However not all monoclonal antibodies are required to be humanized, as most antibodies therapies are for short terms.

Production of humanized antibodies
Antibodies for human use are produced in the following ways:

1. Human lymphocytes are fused with mouse myeloma cells. But sometimes these Human mouse hybrid cells exhibit loss of human chromosomes due to which they become unstable.


2. Imortalization of human lymphocytes with Epstein Barr virus. Most of the times EBV provides no scope to carry out preferential immortalization of blasts cells in antibody production phase.

Since both the above mentioned methods have some constraints, humanization of monoclonal antibodies is carried out via genetic engineering. Some strategies have already been developed and applied in antibodies used in cancer therapy.

Use of Genetic engineering to humanise antibodies:
The production protocol of monoclonal antibodies makes use of recombinant DNA technology. Gene fragments which possess information to produce antibodies are isolated and cloned into cells (mouse cells) which become antibody producing factories and the produce is mass harvested. The step involving recombinant DNA offers a point to also carry out the alterations required to humanize the antibodies. This basically involves insertion or deletion at the genetic level.

Humanization using chimeras as intermediates:
To produce monoclonal antibodies, human antibody producing gene segments (Fc) is fused with the Fab portion of mouse antibodies. This is called a mouse-human chimera. In the usual process, the Fc portion of mouse antibody is replaced with that of a human antibody which humanizes the antibody to an extent. However further humanization includes production of a mouse-human chimera which selectively modifies the amino acids sequence in the Fab portion of the chimera. Only those portions of the mouse Fab region should be modified which differ from that of humans. This selective modification must be carried out in such a way that the specificity of the antibody must be retained. Especially the CDR region of the mouse FAB is never altered as it enables the antibody to bind to the target.
A good example of humanized chimeras, designed for use as therapeutic agents is Otelixizumab for arthritis and diabetes mellitus.

Humanization by sequencing and inserting CDRs:
Humanised antibodies can also be generated directly without intervening in the chimeric intermediate. This can be done by using recombinant DNA techniques. Once the desired antibody is produced in a mouse or other non human systems, the antibody encoding DNA may sequenced after isolating and cloning in a vector. This way the genetic sequence of the antibody's CDR region can be determined. Once this is known, a strategy could be developed wherein these sequences can be inserted into a construct containing the DNA for a human antibody variant. This method may require synthesis of linear DNA fragments based on the CDR sequences.

A good example of an antibody product humanized in this way is Alemtuzumab, which is an approved product for therapy of B-cell chronic lymphocytic leukemia.

Monoclonal antibodies preparations sans mouse systems:
Many techniques have been developed which allow derivation of monoclonal antibodies without using any mouse or any other non human systems. A good example of this method is "phage display". These methods make use of elevated B-cells levels exhibited during immune response in humans.

These techniques make use of the principle of specific antibody production and employ phages (phage display) or cell free extracts (ribosome display). Gene libraries of various antibodies are derived human RNA which is isolated from peripheral blood cells. Using this information Fab or scFv fragments. However, though these antibody fragments are derived completely from human sequences, they are not full antibodies. They require further processes similar to humanization to incorporate and express the full antibodies. A good example of antibodies developed through phage display and approved for human use, is Adalimumab.

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