This article throws light upon the four types of applications of monoclonal antibodies. The four types of applications are: (1) Diagnostic Applications (2) Therapeutic Applications (3) Protein Purification and (4) Miscellaneous Applications.
Application # 1. Diagnostic Applications:
Monoclonal antibodies have revolutionized the laboratory diagnosis of various diseases. For this purpose, MAbs may be employed as diagnostic reagents for biochemical analysis or as tools for diagnostic imaging of diseases.
(A) MAbs in Biochemical Analysis:
Diagnostic tests based on the use of MAbs as reagents are routinely used in radioimmunoassay (RIA) and enzyme-linked immunosorbent assays (ELISA) in the laboratory. These assays measure the circulating concentrations of hormones (insulin, human chorionic gonadotropin, growth hormone, progesterone, thyroxine, triiodothyronine, thyroid stimulating hormone, gastrin, renin), and several other tissue and cell products (blood group antigens, blood clotting factors, interferon’s, interleukins, histocompatibility antigens, tumor markers). In recent years, a number of diagnostic kits using MAbs have become commercially available. For instance, it is now possible to do the early diagnosis of the following conditions/diseases.
Pregnancy by detecting the urinary levels of human chorionic gonadotropin.
Cancers estimation of plasma carcinoembryonic antigen in colorectal cancer, and prostate specific antigen for prostate cancer. Besides diagnosis, estimation of tumor markers is also useful for the prognosis of cancers. That is a gradual fall in a specific tumor marker is observed with a reduction in tumor size, following treatment.
Hormonal disorders analysis of thyroxine, triiodothyronine and thyroid stimulating hormone for thyroid disorders.
Infectious diseases by detecting the circulatory levels of antigens specific to the infectious agent e.g., antigens of Neisseria gonorrhoeae and herpes simplex virus for the diagnosis of sexually transmitted diseases.
(B) MAbs in Diagnostic Imaging:
Radiolabeled—MAbs are used in the diagnostic imaging of diseases, and this technique is referred to as immunoscintigraphy. The radioisotopes commonly used for labeling MAb are iodine—131 and technetium—99. The MAb tagged with radioisotope are injected intravenously into the patients.
These MAbs localize at specific sites (say a tumor) which can be detected by imaging the radioactivity. In recent years, single photon emission computed tomography (SPECT) cameras are used to give a more sensitive three dimensional appearance of the spots localized by radiolabeled— MAbs.
Immunoscintigraphy is a better diagnostic tool than the other imaging techniques such as CT scan, ultrasound scan and magnetic resonance. For instance, immunoscintigraphy can differentiate between cancerous and non-cancerous growth, since radiolabeled—MAbs are tumor specific. This is not possible with other imaging techniques. Monoclonal antibodies are successfully used in the diagnostic imaging of cardiovascular diseases, cancers and sites of bacterial infections.
The cardiac protein myosin gets exposed wherever myocardial necrosis (death of cardiac cells) occurs. Antimyosin MAb labeled with radioisotope indium chloride (111 In) is used for detecting myosin and thus the site of myocardial infarction. Imaging of radiolabeled MAb, is usually done after 24-48 hours of intravenous administration.
This is carried out either by planner gamma camera or single photon emission computed tomography (SPECT). It is possible to detect the location and the degree of damage to the heart by using radiolabeled antimyosin MAb. Thus, this technique is useful for the diagnosis of heart attacks.
Deep vein thrombosis (DVT):
DVT refers to the formation of blood clots (thrombus) within the blood veins, primarily in the lower extremities. For the detection of DVT, radioisotope labeled MAb directed against fibrin or platelets can be used. The imaging is usually done after 4 hours of injection. Fibrin specific MAbs are successfully used for the detection of clots in thigh, pelvis, calf and knee regions.
Thickening and loss of elasticity of arterial walls is referred to as atherosclerosis. Atherosclerotic plaques cause diseases of coronary and peripheral arteries. Atherosclerosis has been implicated in the development of heart diseases. MAb tagged with a radiolabel directed against activated platelets can be used to localize the atherosclerotic lesions by imaging technique.
Monoclonal antibodies against many types of human cancers are now available. A selected list of tumor markers (along with the associated cancers) that can be used for MAb imaging is given in Table 17.2. Tumors can be located in patients using radioisotope labeled MAbs specific to the protein(s), particularly of membrane origin.
It has been possible to detect certain cancers at early stages (lung cancer, breast cancer, ovariran cancer, malanoma, colorectal cancer) by employing MAbs. About 80 per cent specificity has been achieved for detecting cancers by this approach.
An iodine (131l) labeled monoclonal antibody specific to breast cancer cells when administered to the patients detects (by imaging) the spread of cancer (metastasis) to other regions of the body. This is not possible by scanning techniques.
The imaging technique by using MAb can also be used to monitor therapeutic responses of a cancer. There are certain limitations in using MAb in cancer diagnosis and prognosis. These include the difficulty in the selection of a specific MAb and the access of MAb to the target site of the tumor which may be less vascularized.
MAbs in immunohistopathology of cancers:
The pathological changes of the cancerous tissue can be detected by immunohistochemical techniques. This can be done by using MAb against a specific antigen.
MAbs in hematopoietic malignancies:
Hematopoietic stem cells in bone marrow are the precursors for different blood cells, B- and T-lymphocytes which are produced in a stepwise transformation. During malignancy, transformation of lymphocytes stops at a particular stage of maturation. This can be detected by using stage- specific MAbs.
In recent years, attempts are made to detect the sites of infections by using MAbs. This is made possible by directing MAb against bacterial antigens. Further, monoclonal antibodies against inflammatory leucocytes which accumulate at infection site are also useful to specifically detect localized infections.
Application # 2. Therapeutic Applications:
Monoclonal antibodies have a wide range of therapeutic applications. MAbs are used in the treatment of cancer, transplantation of bone marrow and organs, autoimmune diseases, cardiovascular diseases and infectious diseases.
The therapeutic applications of MAbs are broadly grouped into 2 types:
(A) Direct use of MAbs as therapeutic agents
(B) MAbs as targeting agents.
(A) MAbs as Direct Therapeutic Agents:
Monoclonal antibodies can be directly used for enhancing the immune function of the host. Direct use of MAbs causes minimal toxicity to the target tissues or the host.
In destroying disease-causing organisms:
MAbs promote efficient opsonization of pathogenic organisms (by coating with antibody) and enhance phagocytosis. In fact, MAbs were found to protect chimpanzees against certain viral (hepatitis B-virus) and bacterial (E. coli Haemophilus influenza, Streptococcus sp and Pseudomonas sp) infections.
In the treatment of cancer:
MAbs, against the antigens on the surface of cancer cells, are useful for the treatment of cancer. The antibodies bind to the cancer cells and destroy them. This is brought out by antibody—dependent cell-mediated cytotoxicity, complement-mediated cytotoxicity and phagocytosis of cancer cells (coated with MAbs) by reticuloendothelial system.
The patients suffering from leukemia, colorectal cancer, lymphoma and melanoma have been treated with MAbs. However, there was a wide variation in the success rate. A monoclonal antibody specific to the cells of leukemia is used to destroy the residual leukemia cells without affecting other cells. MAbs are used in vitro to remove the residual tumor cells prior to autologous bone marrow transplantation (transplantation of the patient’s own bone marrow cells, due to non-availability of a suitable donor).
Limitations for direct use of MAbs in cancer:
1. The MAbs produced in mice and directly used for therapeutic purposes may lead to the development of anti-mouse antibodies and hypersensitivity reactions.
2. All the cancer cells may not carry the same antigen for which MAb has been produced. Thus, MAbs may not be attached to some cancer cells at all.
3. The free antigens (of target cells) present in the circulation may bind to MAbs and prevent them from their action on the target cells.
In the immunosuppression of organ transplantation:
In the normal medical practice, immunosuppressive drugs such as cyclosporin and prednisone are administered to overcome the rejection of organ transplantation. In recent years, MAbs specific to T-lymphocyte surface antigens are being used for this purpose. The monoclonal antibody namely OKT3, was the first MAb to be licensed by U.S.
Food and Drug Administration for use as immunosuppressive agent after organ transplantation in humans. OKT3 specifically directed against CD3 antigen of T-lymphocytes is successfully used in renal and bone marrow transplantations. In the normal course, CD3 antigen activates T-lymphocytes and plays a key role in organ transplant rejection (destroys the foreign cells in the host). This is prevented by use of MAb against CD3 antigen.
In the treatment of AIDS:
Immunosuppression is the hall mark of AIDS. This is caused by reduction in CD4 (cluster determinant antigen 4) cells of T-lymphocytes. The human immunodeficiency virus (HIV) binds to specific receptors on CD4 cells by using surface membrane glycoprotein (gp120).
Genetic engineers have been successful to attach Fc portion of mouse monoclonal antibody to human CD4 molecule. This complex has high affinity to bind to membrane glycoprotein gp120 of virus infected cells. The Fc fragment induces cell-mediated destruction of HIV infected cells (Fig. 17.7).
In the treatment of autoimmune diseases:
Autoimmune diseases like rheumatoid arthritis and multiple sclerosis are of great concern. Some success has been reported in the clinical trials of rheumatoid arthritis patients by using MAbs directed against T-lymphocytes and B-lymphocytes.
(B) MAbs as Targeting Agents in Therapy:
Toxins, drugs, radioisotopes etc., can be attached or conjugated to the tissue-specific monoclonal antibodies and carried to target tissues for efficient action. This allows higher concentration of drugs to reach the desired site with minimal toxicity. In this way, MAbs are used for the appropriate delivery of drugs or isotopes.
MAbs in use as immunotoxins:
The toxins can be coupled with MAbs to form immunotoxins and used in therapy e.g., diphtheria toxin, Pseudomonas exotoxin, toxins used for cancer treatment. Anti-Tac MAb raised against IL2-R (T-cell growth factor receptor) can be conjugated with exotoxin of Pseudomonas sp. This immunotoxin can be used to destroy the malignant T-cells in the patients suffering from T-cell leukemia (Note: IL2-R is expressed in abnormal T-cells with lymphoid malignancies).
Ricin is a cytotoxic protein derived from castor oil plant. It is composed of two polypeptide chains (A and B) held together by a disulfide linkage. The B-chain of ricin binds to the cell surface. This binding facilitates the A-chain of ricin to enter the cell and inhibit the function of ribosomes (i.e. biosynthesis of all proteins is blocked).
This results in the death of cells (Fig. 17.8A). Ricin can be subjected to oxidation to separate to A and B chains. The toxic A-chain can be conjugated to MAb that is specific to cancer cells. The tumor- specific MAb bound to A-chain of ricin binds to cancer cells and not to normal cells. Once the A-chain enters the cells, it blocks ribosomal function, leading to the death of cancer cells (Fig. 17.8B).
MAbs in drug delivery:
In general, the drugs are less effective in vivo (in the living body) when compared to in vitro (in laboratory when tested with cultured cells). This is mainly due to the fact that sufficient quantity of the drug does not reach the target tissue. This problem can be -solved by using tissue-specific MAbs. The drugs can be coupled with MAb (directed against a cell surface antigen of the cells, say a tumor) and specifically targeted to reach the site of action (Fig. 17.9A).
In the treatment of certain diseases, a pro-drug (an inactive form of the drug) can be used. This can be enzymatically converted to active drug in the target tissues. For this purpose, the enzyme (that converts pro-drug to drug) is coupled with MAb that is directed against a specific cell surface antigen (Fig. 17.9A). This approach, referred to as antibody-directed enzyme pro-drug therapy (ADEPT), allows an effective delivery of the drug to the cells where it is required.
The following are some examples of enzymes that have been used in ADEPT:
i. Alkaline phosphatase for the conversion of phosphate pro-drugs.
ii. Carboxy peptidase for converting inactive carboxyl pro-drugs to active drugs.
iii. Lactamase for hydrolyzing β-lactam ring containing antibiotics.
MAbs in the dissolution of blood clots:
A great majority of natural deaths are due to a blockage in cornary or cerebral artery by a blood clot (thrombus). Fibrin is the major constituent of blood clot which gets dissolved by plasmin. Plasmin in turn is formed by the activation of plasminogen by plasminogen activator. The blockage of arteries occurs due to inadequate dissolution of blood clots. Tissue plasminogen activator (tPA) can be used as a therapeutic agent to remove the blood clots.
A monoclonal antibody directed against fibrin can be coupled to tPA and used for degradation of blood clots. MAb-tPA complex due to a high affinity gets attached to fibrin (Fig. 17.10). Due to the concentration of tPA at the target spots, there is more efficient conversion of plasminogen to plasmin which in turn dissolves blood clot (fibrin). Good success of clot lysis has been reported by using MAb-tPA complex in experimental animals.
Drug delivery through liposomes coupled to tissue-specific MAbs:
Liposomes are sacs or vesicles formed spontaneously when certain lipid molecules are exposed to aqueous environment. Drug entrapped in liposomes that are coated with MAbs directed against tissue-specific antigens are being tried for drug delivery. Unfortunately, the progress in this approach has been limited, since such liposomes do not reach the target cells. They are retained mostly in the liver and spleen (reticuloendothelial cells), and degraded.
MAbs in radio immunotherapy (RAIT):
The radioisotopes can be coupled to MAbs that are directed against tumor cells. This allows the concentration of radioactivity at the desired sites and a very efficient killing of target cells (tumor cells). The advantage with radio immunotherapy is that conjugated complex need not penetrate the cells, as is required in immunotoxin therapy.
The limitation is that the neighbouring normal cells may also get damaged or killed. This can be minimized by using radioisotopes with short half-lives. Yttrium-90 with a half-life of 64 hours is a suitable isotope to be employed in RAIT. Due to shortage in the supply of yttrium-90, indium-111 is more commonly used.
Application # 3. Protein Purification:
Monoclonal antibodies can be produced for any protein. And the so produced MAb can be conveniently used for the purification of the protein against which it was raised. MAbs columns can be prepared by coupling them to cyanogen bromide activated Sepharose (chromatographic matrix). The immobilized MAbs in this manner are very useful for the purification of proteins by immunoaffinity method.
There are certain advantages of using MAbs for protein purification. These include the specificity of the MAb to bind to the desired protein, very efficient elution from the chromatographic column and high degree of purification. Immunoaffinity chromatography is routinely used for the purification of recombinant interferon’s. The efficiency of this technique will be obvious from the fact that by a single step, it is possible to achieve more than 5,000 fold purification of interferon-α2.
It is not possible to achieve 100% purity of the target protein by immunoaffinity. This is due to the fact that a small quantity of MAb leaks into the elution. Further, MAbs cannot distinguish between the intact target protein and a fragment of it with the antigenic site.
Application # 4. Miscellaneous Applications:
(A) Catalytic MAbs (ABZYMES):
Catalysis is the domain of enzymes. The most important common character between enzymes and antibodies is that both are proteins. Further, the binding of an antibody to its antigen is comparable to the binding of an enzyme to its substrate. In both instances, the binding is specific with high affinity and involves weak and non-covalent interactions (electrostatic, hydrogen and van der Waals forces). The striking difference is that the enzyme alters the substrate (to a product) while the antigen bound to antibody remains unaltered.
Certain similarities between enzyme-substrate interaction and antibody-antigen interaction have tempted researchers to explore the possibility of using antibodies in catalysis. The antibody enzymes, appropriately regarded as abzymes, are the catalytic antibodies. There is a difference in the antibody recognition of an antigen and enzyme recognition of a substrate. While the antibodies recognize in ground state, the enzymes recognize in a transition state (associated with a conformational change of protein).
In fact, it is in the transition condition the catalysis occurs. If a molecule resembling the transition state and conformation (between substrate and product) could be used as a hapten the antibodies so produced should bring about catalysis. This is what precisely is done to create abzymes.
Researchers have produced a hapten-carrier complex which resembles the transition state of an ester undergoing hydrolysis. This hapten conjugate is used to generate anti-hapten monoclonal antibodies. These MAbs could bring about hydrolysis of esters with great degree of specificity (to the transition state to which MAbs were raised).
Besides ester hydrolysis, there are several other types of reactions wherein antibodies can be used. These include hydrolysis of amides and carbonates, cyclization reactions, elimination reactions and bio-molecular chemical reactions. Certain enzymes require cofactors for their catalytic function. MAbs incorporating metal ions have been developed to carry out catalysis.
Lerner and his associates carried out pioneering work in the development of abzymes. They could create a large number of immunoglobulin-gene libraries for the production of antibodies that could be screened for their catalytic function. Abzymes represent a major biotechnological advancement that will have a wide range of applications (cutting of peptides and DNAs, dissolution of blood clots, killing of viruses etc.)
Advantages of abzymes:
The number of naturally occurring enzymes and their catalytic functions are limited. Antibodies, on the other hand, are unlimited, and may be developed to possess recognized site structures, appropriate for the catalytic functions. This makes abzymes versatile with wide ranging catalytic applications. Thus, the area of abzyme technology is very promising, although the studies are at preliminary stages.
Limitations of abzymes:
Despite the progress made in the production and utility of abzymes, it is doubtful whether they will ever match the natural enzymes in their catalytic function. However, the abzymes will be certainly useful for a variety of reactions where the natural enzymes do not have the desired specificities.
(B) Autoantibody Fingerprinting:
The occurrence of autoantibodies and their involvement in certain diseases is well known (e.g. rheumatic arthritis). A new category of individual specific (IS) autoantibodies have been discovered in recent years. These IS-autoantibodies are produced after birth and reach maximum in number by 2 years, and then remain constant for the later part of life.
Monoclonal antibodies produced against IS-autoantibodies can be used for their detection, and identification of individuals. This technique referred to as autoantibody fingerprinting, is particularly useful for the detection of criminals, rapists etc. The autoantibodies collected from samples such as blood, saliva, semen and tears can be used.