Problems with monoclonal therapy
Antibody immunogenicity
remains one of the inherent therapeutic limitations associated with administration of murine monoclonals to human subjects. In most instances, single injections of the murine monoclonal will elicite an immune response in 50 to 80% of patients. Human anti-mouse antibodies (HAMA) will generally be detected within 14 days of antibody administration. Repeated administration of the monoclonal will increase the HAMA response significantly. In practice, therefore, therapeutic efficacy of murine monoclonals is limited to the first and at most, the second dose administration.
Monoclonal antibodies of murine origin
Murine monoclonal antibodies are obtained from murine hybridomas produced by fusion of B-lymphocytes from immunised mice or rats with murine myeloma cells.
Advantages
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Disadvantages
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- When compared to polyclonal, the rodent monoclonal antibodies (mAbs) of desired specificity and affinity can be mass produced reproducibly.
- The amount of antibody protein required for immunization is less with mAbs than with polyclonal antibodies.
- They can be more readily standardized and quality controlled.
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- All mAbs are primarily of rodent origin. When administered to humans, they elicit an immune response, human anti-mouse immunoglobulin (HAMA)
- Neutralization of murine mAb by HAMA can result in attenuation of its therapeutic effect and accelerated clearance from the blood.
- Production of human IgE antibodies may lead to hypersensitivity reactions.
- Short circulating half-life. Require repeated administrations.
- Rodent antibodies are not as effective as human antibodies with regard to eliciting human immune effector function.
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Why are there so few monoclonals being used in human therapy a quarter century after their discovery?
The main difficulty is that mouse antibodies are "seen" by the human immune system as foreign, and the human patient mounts an immune response against them, producing HAMA ("human anti-mouse antibodies"). These not only cause the therapeutic antibodies to be quickly eliminated from the host, but also form immune complexes that cause damage to the kidneys.
Solution to HAMA
An obvious strategy for overcoming the immunogenicity problem would be the generation and use of monoclonal antibodies of human origin.
Human monoclonal antibodies
The advantages of human monoclonal antibodies over murine monoclonal antibodies are that human recipients are less likely to develop antibodies against them and that human antibodies are likely to have the full range of biological functions.
Production of human monoclonal antibodies
a) Fusion of human lymphocytes (usually peripheral blood or lymph-node derived) with a murine myeloma or hybrid human-murine myeloma line. This procedure is essentially similar to the hybridoma technique used to produce murine monoclonal antibodies, but presents some technical problems in that lower fusion efficiency is usually found and human chromosomes are lost preferentially. (On long time storage) This procedure may be regarded as a compromise due to the absence of a suitable human myeloma fusion partner.
b) Transformation of human lymphocytes with Epstein-Barr virus (EBV). This procedure has been used for many years to produce continuous, rapidly growing human B cells. (It does not allow preferential immortalization of blastoids which generate Ab)
c) Fusion of human B-lymphocytes with a human lympho-blastoid B-cell line. (Produce cancer in human)
d) Fusion of an EBV-transformed human B-lymphocyte line with a mouse myeloma cell line.
- "Chimeric" antibodies. The antigen-binding part (variable regions) of the mouse antibody is fused to the effector part (constant region) of a human antibody using genetic engineering.
- "Humanized" antibodies. The amino acids responsible for making the antigen binding site (the hypervariable regions) are inserted into a human antibody molecule replacing its own hypervariable regions.
(1) Preparation of Chimeric Abs by Recombinant DNA Technology:
STEPS…..
i. Isolate DNA
ii. Locate the gene which is responsible for producing Ab.
iii. Gene isolate.
iv. Amino acids responsible for making antigen binding site are inserted in to human Ab molecule by replacing it’s over hypervariable region.
v. Modify the gene.
This chimeric Abs are humanized and it’s acceptable.
(2) Preparation of Humanized Antibodies:
STEPS……
i. Human antibody gene is inserted in to mice gene, the mice obtained in such a way called “Transgenic mice”.
ii. Transgenic mice loose the ability to produce mouse antibodies, but capable of producing humanized antibodies.
iii. They can be humanized with desired antigen to produce human Abs against antigen, it can be fused with myeloma cells to produce humanized Abs.
Chimeric antibody and Hyperchimeric antibody (humanized Abs)
The chimeric antibody has human heavy and light chain constant regions and mouse heavy and light chain variable regions. The first h