| [Download PDF]
|Year : 2007 | Volume
| Issue : 1 | Page : 5--14
Monoclonal antibodies: Pharmacological relevance
Jasleen Kaur, DK Badyal, PP Khosla
Department of Pharmacology, Christian Medical College and Hospital, Ludhiana, India
Department of Pharmacology, Christian Medical College and Hospital, Ludhiana
Monoclonal antibodies (MAbs), a new class of biological agents, are used these days in therapeutics and diagnosis. MAbs also labeled as «SQ»magic bullets«SQ», are highly specific antibodies produced by a clone of single hybrid cells formed in the laboratory by fusion of B cell with the tumor cell. The hybridoma formed yields higher amount of MAbs. MAbs can be produced in vitro and in vivo . Animals are utilized to produce MAbs, but these antibodies are associated with immunogenic and ethical problems. Of late, recombinant DNA technology, genetic engineering,phage display and transgenic animals are used to produce humanized MAbs or pure human MAbs, which have fewer adverse effects. MAbs alone or conjugated with drugs, toxins, or radioactive atoms are used for treatment of cancer, autoimmune disorders, graft rejections, infectious diseases, asthma, and various cardiovascular disorders. New MAbs are being developed which are more specific and less toxic.
|How to cite this article:|
Kaur J, Badyal D K, Khosla P P. Monoclonal antibodies: Pharmacological relevance.Indian J Pharmacol 2007;39:5-14
|How to cite this URL:|
Kaur J, Badyal D K, Khosla P P. Monoclonal antibodies: Pharmacological relevance. Indian J Pharmacol [serial online] 2007 [cited 2021 Oct 20 ];39:5-14
Available from: https://www.ijp-online.com/text.asp?2007/39/1/5/30755
Humans and animals have the ability to make antibodies (Abs) to recognize any antigenic determinant (epitope) and to even discriminate between similar epitopes. These antibodies provide the basis for protection against various diseases. The use of vaccine for immunization is an excellent example of preventive use of Abs. Abs usage has now extended to treatment; hence, these are promoted as potential candidates for the management of various diseases. The Abs synthesized against a particular antigen (Ag) are known as monoclonal antibodies (MAbs). By definition, MAbs are a class of highly specific Abs produced by the clones of a single hybrid cell formed in the laboratory by the fusion of B-lymphocytes with a tumor cell.
MAbs are also known as 'magic bullet'. The idea of 'magic bullet' was first proposed by Paul Ehrlich who at the beginning of the 20th century figured 'if a compound could be made to selectively target a disease causing organism, then a toxin for that organism could be delivered along with the agent of selectivity'. The market for therapeutic MAbs is a most potential sector within the pharmaceutical industry. These antibodies are forecast to drive the market towards the $30 billion mark by the year 2011 due to a high level of innovation. Several MAbs are set to be launched in the next 5 years.
Experimental cancer studies have used various substances attached to MAbs such as radioactive material, drugs, immune killer cells, and so on, when injected into patients, home in on Ags that grow on the surface of killer cells. Therefore, MAbs are also labeled as biological response modifiers. Since they affect the immune system, they are also called immunotherapeutics as opposed to chemotherapeutics, which are drugs used to interfere cell growth (cancer). MAb therapy is a form of passive immune therapy because the antibodies are made in large quantities outside the body (in the laboratory) rather than by a person's immune system. Therefore, these MAbs do not require the person's immune system to take an 'active' role in fighting the cancer.
The development of the immortal hybridoma requires the use of animals. No method of generating a hybridoma that avoids the use of animals has been found. Recent in vitro techniques allow the intracellular production of antigen-binding antibody fragments, but such techniques are still experimental and have an uncertain yield, efficacy and antibody function. There are two methods for growing these cells: injecting them into the peritoneal cavity of a mouse or using in vitro cell-culture techniques.
Production of MAbs
The process of producing MAbs was invented by Kohler and Milstein in 1975. They shared the Nobel Prize in Physiology/Medicine in 1984 for the discovery of MAb. The key idea was to use a line of myeloma cells that had lost the ability to secrete antibodies. They came up with a technique to fuse these cells with healthy antibody producing B cells. This fusion resulted in a clone of cells that retained the myeloma cell lines and ability to live indefinitely in tissue culture. The procedure yielded a cell line capable of producing one type of antibody protein for a long period. The fused cell was called a hybridoma and produced large quantities of MAbs.
Production in animals [Figure 1]
When injected into a mouse, the hybridoma cells multiply and produce fluid (ascites) in its abdomen. This fluid contains a high concentration of antibody. The mouse ascites method is inexpensive and easy to use. However, if too much fluid accumulates or if the hybridoma is an aggressive cancer, the mouse will likely experience pain or distress. If a procedure produces pain or distress in animals, regulations call for a search for alternatives. The mouse ascites method usually produces very high MAbs concentration that often does not require further concentration procedures that can denature antibody and decrease effectiveness. It avoids the effects of contaminants as in in vitro batch-culture fluid method and no expert guidance is required to teach the method. However, it involves the continued use of mice requiring daily observation and the MAbs produced contains various mouse proteins and other contaminants that might require purification.
Production in cell-culture
One alternative is to grow hybridoma cells in a tissue culture medium, but this technique requires some expertise, special media and can be expensive and time-consuming. There has been considerable research on in vitro methods for growing hybridomas and these newer methods are less expensive, faster, and produce antibodies in higher concentration than has been the case in the past. Following are the in vitro production methods that are available.
Batch tissue-culture methods. The simplest approach for producing MAb in vitro is to grow the hybridoma cultures in batches and purify the MAbs from the culture medium. Fetal bovine serum is used in most tissue culture media. The MAb concentration achieved is low (around a few micrograms per milliliter) and some MAbs are denatured during concentration or purification process.
Semipermeable-membrane-based system. A barrier, either a hollow fiber or a membrane, with a low-molecular-weight cutoff (10,000-30,000 kD), called semipermeable-membrane-based system permits cells to grow at high densities in culture. The objective of this system is to isolate the cells and MAbs produced in a small chamber separated by a barrier from a larger compartment that contains the culture media. Culture can be supplemented with numerous factors that help optimize growth of the hybridoma. These methods produce MAbs in concentrations often as high as those found in ascitic fluid and are free of mouse ascitic fluid contaminants. These methods reduce the use of animals and are the methods of choice for large-scale production by the pharmaceutical industry because of the ease of culture.,
Although in vitro techniques can be used for more than 90% of MAb production, it must be recognized that there are situations in which in vitro methods will be ineffective, as some hybridomas do not grow well in culture or are lost in culture. Because hybridoma characteristics vary and MAb production needs are diverse, in vitro techniques are not suitable in all situations. These techniques might impede research, especially if large numbers of MAbs are to be screened for efficacy or specificity in the treatment of diseases. In vitro methods generally require the use of fetal calf serum, which limits some antibody uses and which is a concern from the animal-welfare perspective. The loss of proper glycosylation of the antibody (in contrast with in vivo production) might make the antibody product unsuitable for in vivo experiments because of increased immunogenicity, reduced binding affinity, changes in biologic functions or accelerated clearance in vivo . MAbs produced by membrane-based in vitro methods are contaminated with dead hybridoma cells and their products, thus require early and expensive purification. In vitro culture methods are generally more expensive than the ascites method for small or medium-scale production of MAbs.
Evolution of MAbs
The significance of MAbs lies in their specificity and immortality. Whereas hybridoma development of murine MAbs was the requisite for the development of MAbs as drugs, the inherent immunogenicity of rodent sequences in humans has presented obstacles to the clinical application of MAbs. Sensitization to MAb therapeutics poses significant risk to the patient and may blunt the efficacy of these therapies. The advent of chimeric antibodies lessened but did not eliminate the rodent content of MAbs. Thus, immunogenicity remained a concern. Further, elimination of rodent sequences enabled the production of humanized MAbs. This was followed by current technology using phage display and finally, transgenic mice technology, which allows for the generation of fully human therapeutic MAbs. The reduced immunogenicity of this new generation of MAbs is expected to enhance efficacy, safety, and ease of use. The MAbs are also classified into generations as per their evolution and immunogenicity as follows:
First generation MAbs. Majority of the earlier MAbs available were murine, rabbit, or rat proteins purified following immunization of the animal with an antigen preparation. These were labeled as first generation antibodies.
Patient often generated Abs to these foreign antigens. These host antibodies are referred to as human anti-mouse antibody (HAMA) or human anti-rat antibodies or human anti-rabbit antibodies (HARA). The host antibody blocked the effectiveness of therapy by prematurely clearing the treatment antibodies and limiting possibilities for future immunotherapy. HAMA or HARA responses could be associated with immune-complex related adverse events, such as serum sickness or anaphylaxis. In addition, these first generation MAbs are not able to recruit human effector functions such as antibody-dependent cellular cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC), which cause destruction of a malignant cell. Second generation MAbs. To overcome the obstacles of first generation antibodies, Winter et al. pioneered the techniques to humanize MAbs, by removing the reactions that many earlier MAbs caused in some patients. Nowadays, DNA technology or genetic engineering is used to construct hybrids composed of human antibody regions linked with a murine or primate backbone. These are labeled as second generation MAbs and are referred to as chimeric, humanized, primatized, or pure human MAbs.,
(i) Chimeric Abs . These are composite of antibodies from two different species. The Ab combines the Ag binding parts (variable region) of the mouse Ab with the effector parts (constant region) of a human Ab, e.g., infliximab, rituximab, abciximab.,
(ii) Humanized Abs . Human antibody containing the complementarity-determining region from a non-human source. The Ab combines only the amino acids responsible for making the Ag binding site (the hypervariable region) of a mouse Ab with the rest of human Ab molecule thus replacing its own hypervariable regions, e.g., daclizumab, herceptin, vitaxin.,
(iii) Primatized Abs . It is a composite of primate variable regions and human constant regions.,
(iv) Genetically engineered Abs . Fab portion from rodent Abs is attatched to Fc portion of human Abs. Human MAbs have been produced by immortalizing human B lymphocytes with the Epstein-Barr virus and using special immunodeficient mice immunologically reconstituted with immunocompetent cells of tissue. Using genetic engineering it is possible to make mouse-human hybrid antibodies.
(v) Human MAbs can be produced by the following techniques:
(a) Recombinant DNA technology. By inserting random human genes coding for variable Fab portion of human Abs into the genome of filamentous bacteriophages. As the bacteriophage replicate they display Fab portion Ab on their surface. The bacteriophages are subsequently mixed with an antigen to select those producing complementary Fabs. Those bacteriophage genomes are then converted into plasmids that can subsequently produce specific Fabs in bacteria.
(b) Transgenic mice . Transgenic technology has been exploited to make a transgenic mice that have human Ab gene loci inserted into their bodies (using embryo stem cell method) and their own genes for making antibodies 'knocked out'. Therefore, mouse can be immunized with the desired antigen and produces human Abs.
(c) Phage display . This is another technique for making human MAbs. It is used when MAbs do not directly recognize antigen or when antigen is undetectable normally and expressed only in disease. Phage display libraries are available.
Plant genetic engineering has also led to the production of plant-derived MAbs (MAbsP), which provides a safe and economically feasible alternative to the current methods of antibody production in animal systems. Transgenic plants have proven to be an efficient production system for the expression of functional therapeutic proteins. Plant-derived MAbP have the same advantages, namely, lack of animal pathogenic contaminants, low production cost and ease of agricultural scale-up compared with the conventional fermentation methods. Since the initial report of functional MAbs expressed in transgenic plants, therapeutic and diagnostic MAbsP have been successfully produced in transgenic tobacco, soybean, alfalfa, and other plants. Two MAbsP have recently been used for topical passive immunization against Streptococcus mutans and herpes simplex virus in animals. Till date, no study has reported the use of systemic administration of MAbP to provide immunoprotection.,
Large scale production of MAbs
The development of a commercial monoclonal antibody production process involves much more than just scaling-up the laboratory process and making it cost-effective. It involves establishing the hybridoma cell bank with cells that are free of adventitious agents such as viruses and mycoplasma, that have stability in continuous culture for antibody-production rate and cell viability and that do not have unusual or expensive media requirements. The style and mode of operation of the bioreactor used to produce the antibody must be explored. The antibody-based product must be processed to high levels of purity and specific contaminants such as DNA and endotoxin must be reduced to extremely low levels. Appropriate labeling or drug conjugation methods must also be developed. The product must be formulated, so that it has performance characteristics that are stable over a reasonable period of time. Adequate test procedures must be developed to assure product purity, activity, stability, and safety on a lot-to-lot-basis. Compliance with drug regulations, guidelines, and procedures must be guaranteed. In the coming decade, it is likely that the two arms of biotechnology, hybridoma technology and recombinant DNA technology, will be used together to generate unique protein molecules.
The United States adopted name (USAN) Council has outlined specific guidelines for the nomenclature of MAbs. These guidelines provide a foundation of knowledge about a specific MAb just by looking at the generic name.
All MAbs end in the suffix - mab . It is used to identify a class of medicines.The infix preceding - mab denotes the source of the product.
e.g., u = human i = primate
o = mouse e = hamster
a = rat xi = chimera
zu = humanizedThe infix preceeding the source of the Ab refers to medicine target.
e.g., vi(r) - viral ci(r) - cardiovascular
ba(c) - bacterial co(l) - colonic tumor
li(m) - immune le(s) - infectious lesions
me(l) - melanoma ma(r) - mammary tumor
go(t) - testicular tumor go(v) - ovarian tumor
pr(o) - prostate tumor tu(m) - miscellaneous tumor
When combining a target or disease infix stem with the source stem for chimeric or humanized MAb the last consonant of this target syllable is often dropped to make the name more pronounceable (e.g., -tum- changed to -tu- ).
Target source MAb stem
e.g., ci(r) -xi -mab Abciximab
tu(m) -zu -mab TrastuzumabThe starting prefix is a distinct syllable carrying no special meaning and unique for each drug. If the product is radiolabeled or conjugated to another chemical such as a toxin, a separate word is used to identify the conjugate.
Types of MAbs that are used in treatment
These are those without any drug or radioactive material attached to them. They attach themselves to specific Ag on cells, e.g. cancer cells. They mark the cancer cells for the immune system to destroy it. Others attach to certain Ag sites called receptors where other molecules that stimulate the cancer cell growth might otherwise attach. By blocking other molecules from attaching there, MAbs prevent cancer cells from growing rapidly. Some examples of naked MAbs available for use are:
Trastuzumab - For advanced breast cancerRituximab - For B cell non-Hodgkin's lymphoma Cetuximab - For advanced colorectal cancerBevacizumab - For metastatic colorectal cancerAlemtuzumab - For B cell chronic lymphocytic leukemia
MAbs, because of their inherent specificity, are ideal targeting agents. They can be used to deliver radionuclides, toxins, or cytotoxic drugs to a specific tissue or malignant cell population. These are attached to drugs toxins or radioactive atoms. They are also referred to 'tagged' 'labeled' or 'loaded' antibodies. MAbs with radioactive particles attached are referred to as radiolabeled and this type of therapy is known as radioimmunotherapy. MAbs attached to toxins are known as immunotoxins. The MAb acts as a homing device, circulating in the body until it finds a cancer cell with a matching antigen. It delivers the toxic substance to where it is needed most, minimizing damage to normal cell. Examples are:
Ibritumomab tiuxetan- Radiolabeled MAb to treat B cell non-Hodgkin's lymphoma. Tositumomab- Radiolabeled MAb for non-Hodgkin's lymphomaGemtuzumab ozogamicin (Mylotarg) - Immunotoxin MAb for AML. It is the only immunotoxin to receive FDA approval. It contains a toxin called calicheamicin. It is attached to an antibody directed against CD33 antigen present on leukemia cells,
Mechanism of action of Mabs
The mechanism by which MAbs achieve therapeutic effect is not very clear. Potential mechanisms include:
Blocking or steric hindrance of the function of target antigen i.e., T-lymphocytes, B lymphocytes, tumour necrosis factor-a (TNFa) and interleukin (IL) which are capable of transducing intracellular signals.Cytotoxicity to the cell expressing target AG by ADCC or CDC.Inhibition of growth factors: Epidermal growth factor receptor (EGFR) is a cell surface receptor involved in regulation of cell proliferation and survival. Also new vessels grow to feed the cancer cells through this factor. Theses factors can be inhibited to arrest growth of cancer cells e.g., cetuximab act as EGFR inhibitor.
MAbs are used by intravascular route and remain essentially intravascular. Intravenous injection may not always be appropriate for long-term treatment for a variety of reasons. Hour-long infusions require a hospital environment and are often associated with mild to very severe side effects. Continuous and sustained delivery of antibodies can lead to induction of neutralizing anti-idiotypic immune responses, which sometimes develop when massive doses of purified immunoglobulins are repeatedly injected into patients. Additionally, the bioavailability of therapeutic antibodies is often detrimental to the treatment efficacy. They have small volume of distribution and limited tissue penetration. They remain in circulation for 2 days to 2 weeks. Another limitation is the high cost of recombinant proteins certified for human use.
Antibodies can have exquisite specificity of target recognition and thus generate highly selective outcomes following their systemic administration. While antibodies can have high specificity, the doses required to treat patients, particularly for a chronic condition, are typically large. Fortunately, advances in production and purification capacities have allowed for the exceptionally large amounts of highly purified MAbs to be produced. Additionally, genetic engineering of antibodies has provided a stable of antibody-like proteins that can be easier to prepare.
Genetic manipulations of the immunoglobulin molecules are effective means of altering stability, functional affinity, pharmacokinetics, and biodistribution of the antibodies required for the generation of the 'magic bullet.'
Adverse effects with MAbs are related to one of three mechanisms:
Xenogenetic nature of MAb usedSuppression of physiological functionActivation of inflammatory cells or mediators after binding of MAb to its target
Adverse effects with naked MAbs are usually mild and often related to an 'allergic' reaction and occur while the drug is being first infused. This reaction is attributed to massive cytokine release resulting from transient activation of T lymphocytes. Reactions may include fever, chills, weakness, headache, nausea, vomiting, diarrhea, low blood pressure, and rashes. Some MAbs cause leucopenia, thrombocytopenia, and anemia. Conjugated MAbs cause more side effects and the actual effects depend to which substance it is attached. Sometimes it also causes suppression of physiological function depending on specificity of tissue targets. Thus anti-lymphocytes MAb cause immunosupression. There is also an increased risk of infection and cancer development. AntiTNF-a MAb treatment has been reported to increase the reactivation of tuberculosis because it interferes with the cellular response against mycobacteria and this adverse effect is more in areas with high incidence of tuberculosis. AntiTNF-a MAb treatment also leads to development of lymphomas. Rituximab may lead to depletion of plasma cells and cause hypogammaglobulinemia due to its action on B-cells.
Therapeutic Potentials of MAbs
MAbs were being used in laboratory research and in medical tests since the mid 1970s, but their effectiveness in disease treatment was limited. MAbs created much excitement in the medical world and in the financial world in 1980s especially as a potential cure for cancer. Although this resulted in great optimism that a therapeutic 'magic bullet' could be engineered, success with MAbs was many years away. By early 21st century, several drugs based on MAbs were introduced for a wide variety of therapeutic uses. Herceptin, a humanized MAb for breast cancer treatment, became the first drug designed by biomolecular engineering approach to be approved by the FDA. A recent survey suggested that ¼ of all biotech drugs in development are MAb based. At least an additional 400 MAbs are under clinical trials to treat cancer, transplant rejection or to combat autoimmune or infectious diseases. It is now possible to obtain engineered antibodies, chimeric, or humanized or fully human MAbs via the use of phage display technology or of transgenic mice. Important therapeutic implications of MAbs are given in the preceding chapter.
Immunosupression - inhibition of alloimmune reactivity
In 1985, Muromonab CD-3 (OKT3) a murine MAb, was the first to be approved by the US FDA for clinical use in humans, for prevention of graft rejection in renal transplant patient. As first line or in steroid resistance rejection therapy, OTK3 has proved efficacious and improved graft survival. It specifically reacts with the T-cell receptor-CD3 complex on the surface of circulating human T cells. OKT3 binds to a glycoprotein (the 20-kd epsilon chain) on the CD3 complex to activate circulating T cells, resulting in transient activation of T cells, release of cytokines and blocking of T-cell proliferation and differentiation. Nearly all functional T cells are transiently eliminated from the peripheral circulation. Although T cells reappear in the circulation during the course of treatment, these cells are CD3-negative and are not capable of T-cell activation. However, the use of OKT-3 was hampered due to production of Ags and rapid clearance from circulation. This led to development of humanized OKT-3 which is under investigation.
Acute graft rejection is a T-cell mediated immune response and depends on presence of IL-2. IL-2 binds to IL-2 receptor. In the search for more specific immunosupression with MAb L- 2 Receptor (IL-2R), that is expressed on T-cells, which were chosen as target. Chimeric and humanized MAbs, basiliximab, and daclizumab were developed to bind to IL-2R. They competitively antagonized IL-2 or caused elimination of activated T-cells. They have been efficacious in preventing acute rejection episode after renal transplant.,, [Table 1] lists the MAbs used in immunosupression.
Autoimmune diseases - inhibition of autoimmune reactivity
For the treatment of autoimmune disease, MAbs need to target immune response cells, i.e., B or T-cells. MAb may function as an immunosuppressant by removing activated cells, blocking their function or normalizing elevated levels of proinflammatory cytokine. Therapeutic targets in this condition include T-cell surface Ags, T-cells activation Ags, molecules involved in T/B cell interaction, adhesion molecules, and cytokines.
The most promising result emerged from TNF-a blocking therapy in rheumatoid arthritis (RA) and Crohn's disease. TNF is a cytokine produced by activated monocytes and macrophages. The cytokine is actively produced at the synovial and mucosal sites of inflammation in RA and Crohn's disease. It is involved in vasodilation, increased vascular permeability, and activation of platelets and regulation of production of acute phase proteins involved in inflammation. TNF is also actively produced in various infectious diseases such as sepsis, malaria, adult respiratory disease syndrome, and AIDS.
TNF is considered to have an important role in autoimmune inflammatory disease., This led to the discovery of infliximab which is a chimeric MAb and found to be effective in various animal models and clinical trail for RA and Crohn's disease. It is clinically beneficial in Crohn's disease, reduces the response duration and also reduces fistula formation., The first trail of usefulness of infliximab [Table 1] in RA was shown in 1994. Infliximab halts radiographic progression of RA and also clinically cures the disease., Adalimumab, a humanized IgG1 MAb is also approved for the treatment of RA. It binds to soluble and cell membrane-bound TNF-a. It is proved to be efficacious and halts radiological progression of the disease., Anti TNF-a MAb treatment has shown promise in patients with seronegative spondyloarthropathies and psoriatic arthritis.,, Anti-IL-6 and anti-IL-6 receptor MAbs have also been found to be useful in RA as IL-6 is elevated in patients with RA and levels of RA correlates with disease activity and extent of joint erosion.,
Classical therapeutic modalities such as surgery, radiation, and chemotherapy not only fail to cure the great majority of malignant tumors, but also their employment often leads to severe and debilitating side effects. Immunotherapy as a fourth modality of cancer therapy has already been developed and proven to be quite effective. Strategies for the employment of antibodies for anti-cancer immunotherapy include: (1) Immune reaction directed destruction of cancer cell, (2) interference with the growth and differentiation of malignant cells, (3) antigen epitope directed transport of anti-cancer agents to malignant cells, (4) anti-idiotype vaccines, and (5) development of engineered (humanized) mouse monoclonals for anti-cancer therapy. In addition, a variety of different agents (e.g., toxins, radionuclides, chemotherapeutic drugs, etc.) have been conjugated to mouse and human MAbs for selective delivery to cancer cells.
Unconjugated antibodies show significant efficacy in the treatment of breast cancer, non-Hodgkin's lymphoma, and chronic lymphocytic leukemia. Promising new targets for unconjugated antibody therapy include cellular growth factor receptors, receptors, or mediators of tumor-driven angiogenesis and B cell surface antigens other than CD20. One immunoconjugate containing an antibody and a chemotherapy agent exhibits clinically meaningful anti-tumor activity in acute myeloid leukemia. Clinical trials of MAb therapy are in progress for almost every type of cancer. Rituximab was the first MAb used for treatment of cancer [Table 2]. It is chimeric IgG-1 MAb directed against CD20, which is a transmembrane protein on mature B-lymphocytes. Its efficacy has been demonstrated against low grade and follicular non-Hodgkin's lymphoma relapse. Rituximab has also been useful in Waldenstrom's macroglobulinemia, posttransplantation lymphoma, and multiple myeloma., CD52 MAb (Campath-1H) has also been studied to lyse malignant hemopoetic cells. CD52 MAb provides an effective therapy for chronic leukemia of T-cell or B-cell origin that is resistant to conventional chemotherapy. Anti-tumor therapy with MAbs targets growth factor receptor too.
Angiogenesis also plays a central role in the growth and metastasis of cancers. Antibodies directed against EGFR directly inhibit the growth of tumors bearing such receptors. Trastuzumab, a humanized Ab targets HER2 receptor found in breast cancer. It is the first MAb approved by US FDA for the treatment of solid tumors. Trastuzumab blocks HER2 receptor action and this receptor is expressed on the surface membrane of tumor cells in 30% patients with breast cancer and signifies poor prognosis. Strategies aimed at interfering with tumor blood supply offer promise for new cancer therapies. Vitaxin (an anti-alpha-v/beta-3 antibody) interferes with blood vessel formation by inducing apoptosis in newly generated endothelial cells. In Phase II clinical trial it has shown promise in shrinking solid tumors. Bevacizumab which blocks the vascular endothelial growth factor (VEGF) receptor has been approved by US FDA for the treatment of colorectal cancer. Bevacizumab has shown promising results in clear-cell renal cancer in various clinical trials. The promising result of naked and conjugated type MAb in cancer may make MAb therapy significant in the management of these patients. It is expected that MAb-based immunotherapy may be accepted as a conventional form of therapy and employed not only in terminal cancer patients but also, in other instances like, during and following surgical resection.
[Table 2] shows various MAbs used in therapeutics. Acute coronary syndromes and percutneous coronary intervention share a common physiological mechanism of intimal disruption and platelet aggregation. Glycoprotein IIb/IIIa receptor antagonist which interrupt the final common pathway of platelet activation and aggregation are used for acute therapy. Abciximab was the first antagonist to be evaluated [Table 1]. It inhibits the clumping of platelets by binding to surface receptors that normally are linked by fibrinogen. It is helpful in preventing the reclogging of the coronary arteries.,,
Palivizumab, a humanized MAb directed against Respiratory Syncytial virus is used for the treatment of premature infants and infants with bronchopulmonary dysplasia [Table 1]. A MAb was also found to be useful to cure West Nile fever in mice.
Daclizumab has shown to be efficacious for noninfectious uveitis. Ranibizumab (Lucentis) is a recombinant humanized IgG1 kappa isotype monoclonal antibody fragment designed for intraocular use, which competitively binds and inhibits VEGF. Therefore, indicated for the treatment of neovascular (wet) age related macular degeneration.
Natalizumab, a humanized MAb was approved by FDA in November, 2004 for relapsing form of multiple sclerosis, but was withdrawn in February 2005 after three patients in the drug's developed progressive multifocal leukoencephalopathy (PML) during clinical trial., On 24th March, 2006 the FDA lifted the hold on clinical trials of natalizumab after confirming that there were no additional PML cases. In March, 2006, FDA consulted its advisory committee on drugs for peripheral and central nervous systems about the possibility of making natalizumab available to appropriate MS patients. The committee recommended a risk-minimization program with mandatory patient registration and periodic follow-up.
Omalizumab MAb has shown promise in allergic asthma [Table 1]. It acts by binding to IgE thus preventing IgE from binding to mast cells. Omalizumab has shown to reduce serum IgE levels, reduce inhaled steroid consumption and was also well tolerated by children and adults.
Data suggest that MAbs directed against T-cell mediated inflammation are clinically effective in the treatment of psoriasis. MAbs directed against key components of inflammatory process have been studied for safer, selective, and effective immunosuppressant agent as psoriasis is a T-cell mediated autoimmune disease in which proinflammatory Th-1 cytokine play an essential role. Efalizumab is the agent that is near the market launch [Table 1]. It is a humanized MAb that interrupts the interaction between the T-cell surface molecule lymphocyte function associated antigen LFA1 (composed of 2 subunits CD11a and CD18) and intercellular adhesion molecule1, which is found on the surface of antigen-presenting cells. It is used as a once weekly self-administered subcutaneous injection., It is administered every other week and carries a risk of tuberculosis reactivation, serious infections, and demyelinating disease.
Anti-CD3 MAb is in phase II trial for type I juvenile DM. This MAb targets an antigen expressed on T lymphocytes that is responsible for destruction of islet cells of pancreas and thus could slow the disease progression.
Refractory Wegener's granulomatosis
Humanized antilymphocyte MAbs may provide an effective treatment in patients with systemic vasculitis which is refractory or intolerant to steroids or cytotoxic agents.
Systemic lupus erythematosus (SLE)
IL-6 levels are elevated in human and murine SLE. Blocking the action of IL-6 ameliorates disease activity in murine model of SLE. A humanized MAb is in Phase I of clinical trial. T cells, B cells and monocytes from patients with SLE expressess CD40 L on their surface which have been found to produce autoantibodies in vitro . Therefore, humanized anti-CD40L IDEC-131 was tried for SLE but not found to be successful. Another humanized anti-CD40L Mab, ruplizumab was effective in SLE, but increased the incidence of myocardial infarction. Therefore the trials were discontinued.
Rituximab has also been shown to be effective in patients of SLE with glomerulonephritis, by causing B-cell depletion. B-cells in SLE display abnormal signaling, express aberrant cell surface markers and finally produce autoantibody and present auto antigen to T cells at increased rates.
Direct evidence of the importance of gangliosides as potential targets for active immunotherapy has been suggested by the observation that human MAbs against these glycolipids induce shrinkage of human cutaneous melanoma metastasis. Thus, the cellular over-expression and shedding of gangliosides into the interstitial space may play a central role in cell growth regulation, immune tolerance and tumor-angiogenesis, thereby representing a new target for anticancer therapy. Anti-iodiotype vaccine has been developed from proteins derived from the outer membrane proteins of Neisseria meningitidis B. 1E10 vaccine, is an anti-idiotype vaccine designed to mimic the N-Glycolyl-GM3 gangliosides. This monoclonal antibody is an Ab2-type-antibody which recognizes the Ab1 antibody called P3, the latter is a MAb that specifically recognize gangliosides as antigens. Results of the phase I clinical trials proved that the three vaccines were safe and able to elicit specific antibody responses. Phase II trials are being undertaken in several neoplastic diseases, with these vaccines. Vaccination of immunologically responding metastatic colorectal carcinoma patients with SCV 106 leads to slowing of disease progression, tumor dissemination and significantly prolongs survival time. Increased CMI responses to HIV-1 envelope glycoprotein measured by lymphocyte proliferation were associated with HIV-1 recombinant envelope glycoprotein vaccines. HIV-1-specific T helper cell responses can be successfully increased by therapeutic immunization in individuals with chronic infection on suppressive antiretroviral therapy. Further studies will be needed to determine whether the augmentation of these responses correlate with long-term clinical benefits.
Generally, MAbs are being used as invaluable reagents in diagnostics. In fact, they have played a major role in deciphering the functions of various bio-molecules in cryptic biosynthetic pathways. These have also become the reagents of choice for identification and characterization of tumor specific antigens and have become a valuable tool in the classification of cancer. The ability of MAb to accumulate at tumor sites, led to its approval for localization of cancer, for example, igorvomab for ovarian cancer, teenamab K-1 for melanoma, votumab, and acrolunumab for colorectal cancer and sulemab for detection of infection. MAbs will be useful agents for diagnostic imaging of prostate cancer. MAbs are used commercially in pregnancy tests where they are directed against proteins in urine, determine glucose level in diabetes, detect antibiotic residues in milk, and to detect salmonella too.
MAbs are new biological agents that have good clinical effects and an extended choice in the treatment spectrum to the patients who were not responding to the existent treatments. The use of MAbs for the treatment of autoimmune diseases, infections, and malignancies is an evolving field. New therapeutic approaches are rapidly emerging and further studies may help in designing more specific MAbs that would spare the normal tissue, have less adverse effects and improve the patient's quality of life. Soon, new therapeutic drugs and high-value biomolecules will be designed and produced by biomolecular engineering for the treatment and prevention of not-so-easily cured diseases such as cancers, genetic diseases, age-related diseases, and other metabolic diseases.
|1||Baker DD. Monoclonal antibodies. Indian J Med Sci 2001;55:651-4.|
|2||The future of monoclonal antibody therapeutics: Innovation in antibody engineering, key growth strategies and forecasts to 2011; 2006: Available from http://www.research and markets.com/report.|
|3||Frenken LG, Hessing JG, Van den Hondel CA, Verrips CT. Recent advances in the large-scale production of antibody fragments using lower eukaryotic microorganisms. Res Immunol 1998;149:589-99.|
|4||de Geus B, Hendriksen CF. In vivo and in vitro production of MAb - Introduction. Res Immunol 1998;149:533-4.|
|5||Kohler G, Milstein C. Continuous cultures of fused cells secreting antibody of predefined specificity. Nature 1975;256:495-7.|
|6||Hendriksen CF, de Leeuw W. Production of MAbs by the ascites method in laboratory methods. Res Immunol 1998;149:535-42.|
|7||Evans TL, Miller RA. Large scale production of murine MAb using hollow fibre bioreactors. Biotechniques 1988;6:762-7.|
|8||Federspiel G, McCullough KC, Kihm U. Hybridoma Ab production in vitro in type II serum free medium using Nutridoma-SP supplements. Comparison with in vivo methods. J Immunol 1991;145:213-21.|
|9||Weiner LM. Fully human therapeutic monoclonal antibodies. J Immunother 2006;29:1-9.|
|10||White CA, Weaver RL, Grillo-Lopez AJ. Antibody-targeted immunotherapy for treatment of malignancy. Ann Rev Med 2001;52:125-45.|
|11||Riechmann L, Clark M, Waldmann H, Winter G. Reshaping human antibodies for therapy. Nature 1988;332:323-7.|
|12||Newman R, Alberts J, Anderson D, Carner K, Heard C, Norton F, et al . "Primatization" of recombinant antibodies for immunotherapy of human diseases: A macaque/human chimeric antibody against human CD4. Biotechnology 1992;10:1455-60.|
|13||Reff ME, Carner K, Chambers KS, Chinn PC, Leonard JE, Raab R, et al . Depletion of B cells in vivo by a chimeric mouse human monoclonal antibody to CD20. Blood 1994;83:435-45.|
|14||Hiatt A, Cafferkey R, Bowdish K. Production of antibodies in transgenic plants. Nature 1989;342:76-8.|
|15||Ma JK, Hikmat BT, Wycoff K, Vine ND, Chargelegue D, Yu L, et al . Characterization of a recombinant plant monoclonal secretory antibody and preventive immunotherapy in humans. Nat Med 1998;4:601-6. |
|16||Bogard WC Jr, Dean RT, Deo Y, Fuchs R, Mattis JA, McLean AA, et al . Practical considerations in the production, purification and formulation of monoclonal antibodies for immunoscintigraphy and immunotherapy. Semin Nucl Med 1989; 19:202-20.|
|17||Van Laan S. Nomenclature for biological products: Monoclonal antibodies. American medical association. [cited 2006 Feb]. Available from: www.ama-assn.org.|
|18||Weiner LM. An overview of MAb therapy of cancer. Semin Oncol 1999;26: 41-50.|
|19||Breedveld FC. Therapeutic monoclonal antibodies. Lancet 2000;355:735-40.|
|20||Pelegrin M, Gros L, Dreja H, Piechaczyk M. Monoclonal antibody-based genetic immunotherapy. Curr Gene Ther 2004;4:347-56.|
|21||Keane J, Gershon S, Wise RP, Mirabile-Levens E, Kasznica J, Schwieterman WD, et al . Tuberculosis associated with infliximab, a tumor necrosis factor alpha-neutralizing agent. N Engl J Med 2001;345:1098-104.|
|22||Wolfe F, Michaud K. Lymphoma in rheumatoid arthritis: The effect of methotrexate and anti tumor necrosis factor therapy in 18572 patients. Arthritis Rheum 2004;50:1740-51.|
|23||Tsokos GC. B cells-be gone. B-cell depletion in the treatment of rheumatoid arthritis. N Engl J Med 2004;350:2546-8.|
|24||Ryu DD, Nam DH. Recent progress in biomolecular engineering. Biotechnol Prog 2000;16:2-16.|
|25||Desgranges C. Monoclonal antibodies and theapeutics. Pathol Biol 2004;52:351-64.|
|26||A randomized clinical trial of OKT3 monoclonal antibody for acute rejection of cadaveric renal transplants. Ortho multicenter transplant study group. N Engl J Med 1985;313:337-42.|
|27||Norman DJ, Vincenti F, de Mattos AM, Barry JM, Levitt DJ, Wedel NI, et al . Phase I trial of HuM 291, a humanized anti-CD3 antibody, in patients receiving renal allografts from living donors. Transplantation 2000;70:1707-12.|
|28||Nashan B, Moore R, Amlot P, Scmidt AG, Abeywickrama K, Soulillu JP. Randomized trial of basiliximab versus placebo for control of acute ceelular rejectionin renal allograft recepints. Lancet 1997;350:1193-8.|
|29||Vincenti F, Kirkman R, Light S, Bumgardner G, Pescovitz M, Halloran P, et al . Interleukin-2 receptor blockade with daclizumab to prevent acute rejection in renal transplantation. N Engl J Med 1998;338:161-5.|
|30||Nashan B, Light S, Hardie IR, Lin A, Johnson JR. Reduction of acute renal allograft rejection with daclizumab. Transplantation 1999;67:110-5.|
|31||Feldmann M, Elliott MJ, Woody JN, Maini RN. Anti-tumor necrosis factor-alpha therapy of rheumatoid arthritis. Adv Immunol 1997;64:283-350.|
|32||Van Devennter SJ. Tumor necrosis factor and CD. Gut 1997;40:443-8.|
|33||Targan SR, Hanauer SB, van Deventer SJ, Mayer L, Present DH, Braakman T, et al. A short term study of chimeric monoclonal antibody cA2 to tumor necrosis factor alpha for Crohn's disease. N Engl J Med 1997;337:1029-35.|
|34||Present DH, Rutgeerts P, Targan S, Hanauer SB, Mayer L, van Hogezand RA, et al . Infliximab for the treatment of fistulas in patients with Crohn's disease. N Engl J Med 1999;340:1398-405.|
|35||Elliott MJ, Maini RN, Feldmann M, Kalden JR, Antoni C, Smolen JS, et al . Randomised double-blind comparison of chimeric monoclonal antibody to tumour necrosis factor alpha (cA2) versus placebo in rheumatoid arthritis. Lancet 1994;344:1105-10.|
|36||Lipsky PE, van der Heijde DM, St Clair EW, Furst De, Breedveld FC, Kalden Jr, et al . Infliximab and methotrexate in the treatment of rheumatoid arthritis. N Engl J Med 2000;343:1594-602.|
|37||Maini R, St Clair EW, Breedveld F, Furst D, Kalden J, Weisman M, et al . Infliximab (chimeric anti-tumour necrosis factor alpha monoclonal antibody) versus placebo in rheumatoid arthritis patients receiving concomitant methotrexate: A randomized phase III trial. ATTRACT Study Group. Lancet 1999;354:1932-9.|
|38||Weinblatt ME, Keystone EC, Furst DE, Moreland LW, Weisman MH, Birbara CA, et al . Adalimumab, a fully human anti-tumor necrosis factor alpha monoclonal antibody, for the treatment of rheumatoid arthritis in patients taking concomitant methotrexate: The ARMADA trial. Arthritis Rheum 2003;48: 35-45.|
|39||Keystone EC, Kavanaugh AF, Sharp JT, Tannenbaum H, Hua Y, Teoh LS, et al. Radiograhic, clinic and functional outcomes of treatment with adalimumab (a fully human anti-tumor necrosis factor monoclonal antibody) in patients with active rheumatoid arthritis receiving concomitant methotrexate therapy. Arthritis Rheum 2004;50:1400-11.|
|40||Braun J, BrandtJ, Listing J, Zink A, Alten R, Goldwer W, et al . Treatment of active ankylosing spondylitis with infliximab-a double blind placebo controlled multicenter trial. Lancet 2002;359:1187-93. |
|41||Antoni C, Kavanaugh A, Kirkham B. The infliximab multinational psoriatic arthritis controlled study. Ann Rheum 2002;46:S381.|
|42||Antoni C, Kavanaugh A, Kirkham B. The infliximab multinational psoriatic arthritis controlled study. Substantial efficacy on synovitis and psoriatic lesion with or without concomitant DMARD therapy. Ann Rheum Dis 2003;62:90.|
|43||Guerne PA, Zuraw BL, Vaughan JH, Carson DA, Lotz M. Synovium as a source of interleukin 6 in vitro . Contribution to local and systemic manifestations of arthritis. J Clin Invest 1989;83:582-92.|
|44||Nishimoto N, Yoshizaki K, Miyasaka N, Yamamoto K, Kawai S, Takeuchi T, et al . Treatment of rheumatoid arthritis with humanized anti-interleukin-6 receptor antibody: A multicenter, double-blind, placebo-controlled trial. Arthritis Rheum 2004;50:1761-9.|
|45||Bodey B, Bodey B, Siegel SE, Kaiser HE. Genetically engineered monoclonal antibodies for direct anti-neoplastic treatment and cancer cell specific delivery of chemotherapeutic agents. Curr Pharm Des 2000;6: 261-76.|
|46||von Mehren M, Adams GP, Weiner LM. Monoclonal antibody therapy for cancer. Ann Rev Med 2003;54:343-69.|
|47||McLaughlin P, Grillo-Lopez AJ, Link BK, Levy R, Czuczman MS, Williams ME, et al. Rituximab chimeric anti-CD 20 monoclonal antibody therapy for relased indolent lymphoma: Half of patients respond to a four dose treatment programme. J Clin Oncol 1998;16:2824-33.|
|48||Dimopoulos MA, Zomas A, Viniou NA, Grigoraki V, Galani E, Matsouka C, et al . Treatment of Waldenstrom's macroglobulinemia with thalidomide. J Clin Oncol 2001;19:3596-601.|
|49||Treon SP, Anderson KC. The use of Rituximab in the treatment of malignant and non-malignant plasma cell disorders. Semin Oncol 2000;27:79-85.|
|50||Dyer MJ. The role of CAMPATH-1antibodies in the treatment of lymphoid malignancies. Semin Oncol 1999;26:52-7.|
|51||Mass R. The role of HER-2 expression in predicting response to therapy in breast cancer. Semin Oncol 2000;27:46-52.|
|52||Gutheil JC, Campbell TN, Pierce PR, Watkins JD, Huse WD, Bodkin DJ, et al . Targeted antiangiogenic therapy for cancer using vitaxin: A humanized monoclonal antibody to the Integrin αvβ3. Clin Cancer Res 2000;6:3056-61.|
|53||Hurwitz H, Fehrenbacher L, Novotny W, Cartwright T, Hainsworth J, Heim W, et al . Bevacizumab plus irinotecan, fluorouracil and leucovorin for metastatic colorectal cancer. N Engl J Med 2004;350:2335-42.|
|54||Yang J, Haworth L, Sherry RM, Hwu P, Schwartzentruber DJ, Topalian SL, et al . A randomized trial of bevacizumab, an anti-vascular-endothelial growth-factor antibody for metastatic renal cancer. N Engl J Med 2003;349:427-34. |
|55||Platelet glycoprotein IIb/IIIa receptor blockade and low-dose heparin during percutaneous coronary revascularisation. EPILOG Investigators. N Engl J Med 1997;336:1689-96.|
|56||Randomized placebo-controlled trial of abciximab before and during coronary intervention in refractory unstable angina: The CAPTURE study. CAPTURE Investigators. Lancet 1997;349:1429-35.|
|57||Hamm CW, Heeschen C, Goldmann B, Vahanian A, Adgey J, Miguel CM, et al. Benefit of abciximab in patients with refractory unstable angina in relation to serum troponin T levels. c7E3 Fab Antiplatelet Therapy in Unstable Refractory Angina (CAPTURE) Study Investigators. N Engl J Med 1999;340:1623-9.|
|58||Palivizumab, a humanized respiratory syncytial virus monoclonal antibody, reduces hospitalization from respiratory syncytial virus infection in high-risk infants. lmpact-RSV Study Group. Pediatrics 1998;102:531-7.|
|59||Oliphant T, Engle M, Nybakken GE, Doane C, Johnson S, Huang L, et al. Development of a humanized monoclonal antibody with therapeutic potential against West Nile virus. Nat Med 2005;11:522-30.|
|60||Nussenblatt RB, Peterson JS, Foster CS, Rao NA, See RF, Letko E, et al . Initial evaluation of subcutaneous daclizumab treatments for noninfectious uveitis: A multicenter noncomparative interventional case series. Ophthalmology 2005;112:764-70.|
|61||Chan WM, Ohji M, Lai TY, Liu DT, Tano Y, Lam DS. Choroidal neovascularisation in pathological myopia: An update in management. Br J Ophthalmol 2005;89:1522-8.|
|62||FDA News. First Monoclonal antibody treatment for multiple sclerosisapproved. [cited 2004 Nov 23]. Available from: http://www.fda.gov/cder/drug/infopage/natalizumab. |
|63||Drug information. Natalizumab Information: [cited 2006 Jun 5]. available from: http://www.fda.gov/cder/drug/infopage/natalizumab/default.htm|
|64||Walker S, Monteil M, Phelan K, Lasserson TJ, Walters EH. Anti-IgE for chronic asthma in adults and children. Cochrane Database Syst Rev 2004;CD003559.|
|65||Leonardi CL, Papp KA, Gordon KB, Menter A, Feldman SR, Caro I, et al . Extended efalizumab therapy improves chronic plaque psoriasis: Results from a randomized phase III trial. J Am Acad Dermatol 2005;52:425-33.|
|66||Gordon KB, Papp KA, Hamilton TK, Walicke PA, Dummer W, Li N, et al . Efalizumab for patients with moderate to severe plaque psoriasis: A randomized controlled trail. J Am Med Ass 2003;290:3073-80.|
|67||Keymeulen B, Vandemeulebroucke E, Ziegler AG, Mathieu C, Kaufman L, Hale G, et al . Insulin needs after CD3-antibody therapy in new-onset type 1 diabetes. N Engl J Med 2005;352:2598-608.|
|68||Lockwood CM, Thiru S, Stewart S, Hale G, Isaacs J, Wraight P, et al . Treatment of refractory Wegner's granulomatosis with humanized monoclonal antibodies. QJM 1996;89:903-12.|
|69||Desai-Mehta A, Lu L, Ramsey-Goldman R, Datta SK. Hyperexpression of CD40 ligand by B and T cells in human lupus and its role in pathogenic autoantibody production. J Clin Invest 1996;97:2063-73.|
|70||Kalunian KC, Davis JC Jr, Merril JT, Totoritis MC, Wofsy D. Treatment of systemic lupus erythematosus by inhibition of T cell costimulation with anti-CD154: A randomized, double-blind, placebo-controlled trial. Arthritis Rheum 2002;46:3251-8.|
|71||Boumpas DT, Furie R, Mnzi S, Illei GG, Wallace DJ, Balow JE, et al . A short course of BG9588 (anti-CD40 ligand antibody) improves serological activity and decreases hematuria in patients with proliferative lupus glomerulonephritis. Arthritis Rheum 2003;48:719-27. |
|72||Sfikakis PP, Boletis JN, Lionaki S, Vigklis V, Fragiadaki KG, Iniotaki A, et al. Remission of proliferative lupus nephritis following B cell depletion therapy is preceded by down-regulation of the T cell costimulatory molecule CD40 ligand. Arthritis Rheum 2005;52:501-13.|
|73||Tsokos GC, Liossis SN. Immune cell signalling in lupus: Activation, anergy and death. Immunol Today 1999:20:119-24.|
|74||Bitton RJ, Guthmann MD, Gabri MR, Carnero AJ, Alonso DF, Fainboim L, et al . Cancer vaccines: An update with special focus on ganglioside antigens. Oncol Rep 2002;9:267-76.|
|75||Samonigg H, Wilders-Truschnig M, Kuss I, Plot R, Stoger H, Schmid M, et al . A double-blind randomized-phase II trial comparing immunization with antiidiotype goat antibody vaccine SCV 106 versus unspecific goat antibodies in patients with metastatic colorectal cancer. J Immunother 1999;22:481-8.|
|76||Gorse GJ, Simionescu RE, Patel GB. Cellular immune responses in asymptomatic human immunodeficiency virus type 1 (HIV-1) infection and effects of vaccination with recombinant envelope glycoprotein of HIV-1. Clin Vaccine Immunol 2006;13:26-32.|
|77||Robbins GK, Addo MM, Troung H, Rathod A, Habeeb K, Davis B, et al . Augmentation of HIV-1-specific T helper cell responses in chronic HIV-1 infection by therapeutic immunization. AIDS 2003;17:1249-51.|
|78||Nelson PN, Reynolds GM, Waldron EE, Ward E, Giannopoulos K, Murray PG. Monoclonal antibodies. Mol Pathol 2000;53:111-7.|
|79||Elsasser-Beile U, Wolf P, Gierschner D, Buhler P, Schultze-Seemann W, Wetterauer U. A new generation of monoclonal and recombinant antibodies against cell-adherent prostate specific membrane antigen for diagnostic and therapeutic targeting of prostate cancer. Prostate 2006;66:359-70.|