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Chapter 35: Monoclonal Antibodies and Recombinant Cytokines Monoclonal antibodies Two key regions of antibodies have made

e them attractive molecules for biotechnologists to develop as drugs: 1. The Fab region: confers specificity for antigen and could act as a "magic bullet" directing its effects against pathogens or tumor cells. This specificity results in predictable therapeutic effects and reduced side effects. 2. The Fc region: effector region e.g. activation of the complement, macrophages,NK, and mast cells. The Fc region can also be altered to deliver drugs and toxins to specific cells. Historical Approaches to Antibody Therapy - Animal polyclonal immunoglobulin was previously used to treat infections and as antivenom. - Horse serum was frequently used, but frequently induced antihorse antibodies immune complexes trigger a type III hypersensitivity reaction serum sickness. Polyclonal human immunoglobulin is sometimes given as a form of passive immunity Anti-D immunoglobulin is given to rhesus-negative women pregnant with rhesus-positive fetuses, to prevent hemolytic disease of the newborn. Obtaining large quantities of human Igs from donors is expensive and always carries a risk for transmitting blood-borne infection.

Monoclonal Antibody Technologies Mouse monoclonal antibodies (murine mAbs) were invented in 1975. They have the advantage that they can be raised against antigens that could not be used to immunize humans (e.g. dangerous pathogens or tumor cells). The conventional approach has been to fuse spleen cells from immunized mice with mouse myeloma cells production of high levels of murine mAb. Disadvantages of murine mAbs: 1. They have mouse Fc regions 2. They do not always interact well in vivo with human complement molecules or Fc receptors on human macrophages and NK cells. 3. They stimulate the production of antimouse antibodies.


Antimouse antibodies can cause immune complex disease (IgG antimouse antibodies) or anaphylaxis. Antimouse IgG antibodies also neutralize the effects of the murine mAb and are produced in about 40% of patients. Approaches that have been used to increase the proportion of human genetic sequences used in manufacturing mAbs: 1. Chimeric mAbs: use mouse immunoglobulin variable region gene sequences and human constant region gene sequences. Chimeric mAbs contain only about 30% mouse amino acid sequence but still tend to elicit antibodies when used therapeutically. 2. Humanized mAbs: use a technique similar to chimeric antibodies, but only the mouse hypervariable region gene sequence is used. Only about 10% of the amino acids are derived from mouse sequences. 3. Transgenic mice: can be manufactured to make human-like mAbs. Two strains of mice have been created by knocking out mouse constant region genes and inserting synthetic minichromosomes containing human heavy-chain and and lightchain loci. After immunization, these mice process the human genes leading to the synthesis of human-like antibodies. -

4. Phage libraries. These are libraries of random and slightly differing VH and VL genes expressed in bacteriophages. Many different phages can be produced, each of which express different variable region molecules. These can be screened for binding to the antigen of interest and then cloned into a mammalian cell (along with constant-region genes) for expression.

New genetic sequences produced by chimeric, humanized, transgenic, or phage library techniques can be expressed in different ways: Hybridoma technology. But this was a low-yield technology, and expensive. Manufacture transgenic animals that express the mAb in breast milk. The mAb-containing milk can be collected in a dairy and then purified at reduced cost. Plants have also been transfected with genes to produce high levels of mAbs and may provide low-cost mAbs in the future. These technologies have been used to develop mAbs with humanlike amino acid sequences and reduce the risk of evoking antimouse responses.


A further complication is provided by the differences in glycosylation (addition of oligosaccharide side chains to proteins) of immunoglobulin produced in human and nonhuman cells. The structure of oligosaccharide side chains has species specificity due to species-specific enzymes involved in their synthesis. When mAbs are expressed in nonhuman cells, there is a tendency for them to be recognized by anticarbohydrate antibodies. Increasing the Effects of Monoclonal Antibodies Various approaches have been used to increase the effects of mAbs: 1. Bispecific Antibodies These combine the specificities of two different antibodies. In the case of cancer immunotherapy, these have been manufactured against the T-cell receptor on CTLs and target antigens on tumor cells. The bi-specific antibody brings the CTL and target cell together and activates the CTL to initiate cell killing. 2. Conjugates mAbs can be combined with substances that would otherwise be too toxic when administered at higher concentration alone. E.g. mAbs can be conjugated to toxins or radioactive isotopes to deliver them to the target cells. Conjugation thus increases the specificity of the toxin or isotope for the target cells.

Some Successes and Failures in mAb Therapy Provide useful therapy in cancer, where there is little evidence that antibodies are part of the normal human immune response to cancer. mAbs have been successful in the treatment of drug overdose and substance abuse (e.g. digoxin are very dangerous in overdose; treatment with antidigoxin mAbs leads to rapid clearance of the offending drug. . Although antibody has a very clear role in the physiologic defense against infection, mAbs have so far proved to be rather unsuccessful. It is possible to produce mAbs against LPS, but the diversity of antigens in wild-type organisms means that these mAbs are not always effective in a strain causing infection. So, the very high specificity of mAbs limits their use in treating infections. mAbs have been used in drug-dependent patients. The drug and mAb form an immune complex, preventing the drug from reaching specific receptors in the brain and instead directing the drug to the spleen. Patients do not receive the normal reward from drug consumption, and hence dependency can be broken. 3/5

Recombinant cytokines The two major immunologic cytokines in relatively frequent use are interferon- (IFN-) and IL-2. Interferon- IFN- is used to treat some forms of viral hepatitis (treatment needed up to a year). IFN- induces a mild acute-phase response, and this makes most patients feel slightly unwell during treatment. Some patients produce anti-IFN- which neutralize the effects of the treatment. It has very short, 4-hour half-life of IFN-. The major problem has been the cost of treatment. Some of these problems can be overcome by PEGylation. PEGylation: Addition of polyethylene glycol (PEG) molecules to the IFN- structure. During PEGylation, PEG molecules are covalently joined onto the IFN- molecule. This increases the half-life of the molecule by increasing its molecular weight so that it is not filtered at the kidney. PEGylation also effectively surrounds the IFN- molecule so that it is not digested by proteases. These effects increase the half-life to 40 hours, meaning treatment can be given weekly in stead of daily. PEGylation also protects the IFN- molecule from the patient's immune system and reduces the risk for inducing anti- IFN- antibodies.

Interleukin-2 The biological activity of IL-2 is lost when it is PEGylated it has to be given as a daily intravenous infusion. Recombinant IL-2 has been used in HIV infection in combination with HAART. Compared with HAART alone, the use of IL-2 results in faster and greater restoration of Tcell numbers.


Effects of IL-2: 1. IL-2 induces production of Bcl-2, so has antiapoptotic effects prevent destruction of CD4+ T cells through apoptosis in HIV infection. 2. IL-2 boosts T-cell proliferation and thus helps T-cell numbers recover in HIV infection. IL-2 is pleiotropic; they affect many different cell types. At moderately high doses IL-2 affects multiple cell types and triggers a low-grade acute phase reaction. Hence, patients experience low-grade fevers, fatigue, and muscle ache, mimicking the symptoms of chronic infection.

IL-7 is another growth factor for T cells. It appears to be less pleiotropic and it may help increase T-cell numbers in HIV with fewer side effects. IL-2 also has effects in some cancers. It is thought to act by overcoming anergy or apoptosis in tumor-specific T cells and by activating NK cells. IL-2 is licensed for use in renal cancer and in melanoma. In cancer treatment, IL-2 works most effectively when given at very high doses at high doses; IL-2 has effects on the endothelium leading to the capillary leak syndrome pulmonary edema and hypotension.