Monoclonal antibodies are collections of identical antibodies. All molecules of a monoclonal antibody are produced by the progeny of a single B-lymphocyte clone, and all the molecules therefore have the same V regions and bind to the same anti gen that originally triggered that B cell. A tumor of plasma cells (myeloma, or plasmacytoma), like most tumors of any cellular origin, is monoclonal and therefore produces antibodies of a single specificity. In most cases, the specificity of the tumor derived antibody is not known, so the myeloma antibody cannot be used to detect or bind to molecules of interest. However, the discovery of monoclonal antibodies produced by these tumors led to the idea that it may be possible to produce similar monoclonal antibodies of any desired specificity by immortalizing individual antibody-secreting cells from an animal immunized with a known antigen. A technique to accomplish this was described by Georges Kohler and Cesar Milstein in 1975 and this has proved to be one of the most valuable advances in biology and medicine. The method relies on fusing B cells from an immunized animal (typically a mouse) with an immortal myeloma cell line and growing the cells under conditions in which only the fused normal and tumor cells can survive (Fig. 1). The resultant fused cells that grow out are called hybridomas because they are hybrids of normal B cells and a myeloma tumor. Each hybridoma makes only one Ig, derived from one B cell from the immunized animal. The antibodies secreted by many hybridoma clones are screened for binding to the antigen of interest, and the clone with the desired specificity is selected and expanded. The products of these individual clones are monoclonal antibodies and each antibody is specific for a single epitope on the antigen used to immunize the animal.

Fig1. The generation of monoclonal antibodies. In this procedure, spleen cells from a mouse that has been immunized with a known antigen or mixture of antigens are fused with an enzyme-deficient partner myeloma cell line, with the use of chemicals such as polyethylene glycol that can facilitate the fusion of plasma membranes and the formation of hybrid cells that retain many chromosomes from both fusion partners. The myeloma partner used is one that does not secrete its own immunoglobulin. These hybrid cells are then placed in a selection medium that permits the survival of only immortalized hybrids; these hybrid cells are then grown as single cell clones and tested for the secretion of the antibody of interest. The selection medium includes hypoxanthine, aminopterin, and thymidine; it is therefore called HAT medium. There are two pathways of purine synthesis in most cells: a de novo pathway that needs tetrahydrofolate and a salvage pathway that uses the enzyme hypoxanthine-guanine phosphoribosyltransferase (HGPRT). Myeloma cells that lack HGPRT are used as fusion partners and they normally survive using de novo purine synthesis. In the presence of aminopterin, tetrahydrofolate is not made, resulting in a defect in de novo purine synthesis and also a specific defect in pyrimidine biosynthesis, namely, in generating thymidine monophosphate from deoxyuridine monophosphate. Hybrid cells receive HGPRT from the splenocytes and acquire the capacity for uncontrolled proliferation from the myeloma partner; if they are given hypoxanthine and thymidine, these cells can make DNA in the absence of tetrahydrofolate. As a result, only hybrid cells survive in HAT medium.

Monoclonal antibodies have many applications in research and in medical diagnosis and therapy. Some of their common applications include the following:

• Identification of phenotypic markers unique to particular cell types. The basis for the modern classification of lymphocytes and other leukocytes is the recognition of individual cell populations by specific monoclonal antibodies. These antibodies have been used to define clusters of differentiation (CD) markers for various cell types.

• Immunodiagnosis. The diagnosis of many infectious and systemic diseases relies on the detection of particular antigens or antibodies in the blood, urine, or tissues by use of mono clonal antibodies in immunoassays.

 • Tumor identification. Labeled monoclonal antibodies specific for various cell proteins are used to determine the tissue source of tumors by staining histological tumor sections.

 • Therapy. Advances in medical research have led to the identification of cells and molecules that are involved in the pathogenesis of many diseases. Monoclonal antibodies, because of their exquisite specificity, provide a means of targeting these cells and molecules. Many monoclonal antibodies are used therapeutically today (Table1). Some examples include anti bodies specific for the cytokine tumor necrosis factor (TNF) used to treat rheumatoid arthritis and other inflammatory diseases, antibodies against CD20 for the treatment of B cell derived tumors and for depleting B cells in certain autoimmune disorders, antibodies specific for the T-cell regulatory molecules PD-1 and CTLA-4 used in therapy for many types of cancers, antibodies that bind to epidermal growth factor receptors to target cancer cells, antibodies against vascular endothelial growth factor (a cytokine that promotes angiogenesis) in patients with macular degeneration, and so on.

 • Functional analysis of cell surface and secreted molecules. In biologic research, monoclonal antibodies that bind to cell surface molecules and either stimulate or inhibit particular cellular functions are invaluable tools for defining the functions of these molecules, including receptors for anti gens. Monoclonal antibodies are also widely used to purify selected cell populations from complex mixtures to facilitate the analysis of the properties and functions of these cells and to block or deplete secreted molecules and particular cells for studying their functions.

Table1. Examples of Monoclonal Antibodies in Clinical Use

One of the limitations of monoclonal antibodies for therapy is that these antibodies are most easily produced by immunizing mice and fusing B cells from the spleen of the mouse with a mouse myeloma cell line. But patients treated with mouse antibodies will make antibodies against the mouse Ig, called human-anti-mouse antibodies (HAMA). These anti-Ig antibodies block the function and enhance the clearance of the injected monoclonal antibody and may also cause serum sickness. Genetic engineering techniques have been used to replace the mouse sequences in the monoclonal antibody with human sequences to avoid an anti-Ig response. The complementary DNAs (cDNAs) that encode the polypeptide chains of a monoclonal antibody can be isolated from a hybridoma and these genes can be manipulated in vitro. As discussed earlier, only small portions of the antibody molecule are responsible for binding to antigen; the remainder of the antibody molecule can be thought of as a framework. This structural organization allows the DNA segments encoding the antigen-binding sites from a mouse monoclonal antibody to be inserted into a cDNA encoding a human Ig, creating a hybrid gene. When it is expressed, the resultant protein, which retains the antigen specificity of the original mouse monoclonal but has the core structure of a human Ig, is referred to as a humanized antibody.

Fully human monoclonal antibodies are also in clinical use. These can be derived using phage display methods or by immunization of engineered mice in which human Ig genes replace their murine counterparts. The phage display technology for generating humanized monoclonal antibodies against specific targets involves screening bacteriophage libraries in which each bacteriophage expresses a different human VH and VL gene combination so that each phage particle expresses a different synthetically generated antigen-binding site on its sur face. By screening millions of such synthetic binding sites for binding to the antigen of interest, the ones that are specific for the target antigen can be identified and the DNA for each of these VH and VL domains can then be inserted into appropriate Ig gene cassettes that include all framework regions and constant domains to create a humanized monoclonal antibody.

Humanized antibodies are far less likely than mouse monoclonals to appear foreign in humans and to induce anti-anti body responses. However, a proportion of subjects receiving fully humanized monoclonal antibodies for therapy develop blocking anti-antibodies, known as human-anti-human anti bodies (HAHAs). Why this happens in some individuals is not understood.

Beyond the humanization of antibodies, many other innovations in antibody engineering have contributed to improvements in therapeutic efficacy. Monoclonal antibodies can be modified in their Fab portions to enhance affinity and in their Fc segments to reduce their inflammatory potential or to increase half-life. Antibodies can be engineered to be bifunctional—to have two nonidentical Fabs; typically, one engineered Fab may bind a cell surface antigen on a target cell— for instance on a tumor cell, and the other Fab might have been engineered to recognize T cells. The antigen binding variable domains only of the heavy and light chain of an antibody can be joined by a flexible linker peptide sequence, to form what is called an scFv (single chain fragment variable) which is about half the size of a Fab fragment. Two scFvs can also be joined together to form a bifunctional antibody. These kinds of bifunctional antibodies, sometimes called BiTES (for bifunctional T-cell engagers), are already in use as antitumor immunotherapeutics. They bring T cells in apposition to tumor targets, activate these T cells, and thus con tribute to more efficient tumor killing.

اخر الاخبار

اخبار العتبة العباسية المقدسة

شعبة فاطمة بنت أسد (عليها السلام): ملتقى الكوثر منصةً لتحقيق وعي أسري مستند للقرآن الكريم

شعبة فاطمة بنت أسد (عليها السلام) تطلق النسخة الثالثة من ملتقى الكوثر

استقبال المشاركات في ملتقى الكوثر الثقافي الثالث إيذانًا بانطلاق برامجه التوعوية

في ذكرى زواج النورين.. شعبة التوجيه النسوي تنظم جلسات ثقافية للزائرات

المزيد

الآخبار الصحية

طريقة مبتكرة للكشف المبكر عن أمراض القلب والشرايين

منظمة الصحة تحذر من خطر إدمان الشباب لأكياس النيكوتين

خبراء يحذرون من عادة شائعة تهدد صحة الملايين

خبير يكشف 5 حقائق طبية صادمة وغير معروفة

المزيد

الآخبار التكنلوجية

الأولى في العالم.. الصين تجري تجربة رائدة في الفضاء باستخدام "جنين اصطناعي"

دراسة حديثة تفند ربط التستوستيرون بالمخاطرة لدى الرجال

ميتا تضيف وضع "المحادثة المخفية" إلى تطبيق "واتساب"

فوبيا المرتفعات ليست من الدماغ.. دراسة تكشف السبب

المزيد

مواضيع ذات صلة

Structural Features of Antibody Constant Regions

Structural Features of Antibody Variable Regions

General Features of Antibody Structure

Stages of T-Cell Maturation

Mechanisms that Limit Innate Immune Responses

Stimulation of Adaptive Immunity

Systemic and Pathologic Consequences of Inflammation

Immuno microscopy

V(D)J Recombination

Role of Macrophages in Tissue Repair

Ingestion and Killing of Microbes by Activated Phagocytes

Sequence of Events in Inflammation: Vascular Changes and Leukocyte Migration Into Tissues