Handbook of Therapeutic Antibodies

Hybridoma production basically relies on the fusion of immunized lymphocytes from an experimental animal with immortal myeloma cells. The resulting cell hybrid contains the genetic material of both parents. From the tumor cell the hybrid acquires the capacity for indefinite growth while the B lymphocyte confers  the ability to synthesize a specific antibody. After stabilization by repeated cell cloning, hybridomas produce fairly large quantities of identical mAb for years (Fig. 2.1). The early development of the method is documented in a couple of scientific anthologies (Melchers et al. 1978; Kennett et al. 1980; Hämmerling et al. 1981). Importantly, the first patient suffering from non-Hodgkin’s lymphoma was treated in 1979 with a mouse mAb at the Dana Farber Cancer Center in Boston (Nadler et al. 1980). Taken together, mAbs have led to a revolution in basic sciences, medicine and industry during the past 30 years.

2.3 Antibody Structure and Function

2.3.1 Membrane-bound and Secreted Forms of Antibodies

The basic structure of an IgG antibody has been elucidated as a symmetric  monomer consisting of two identical heavy chains and two identical light chains, connected via disulfide bonds (Cohen and Porter 1964; Edelman et al. 1969). Five classes of immunoglobulins can be distinguished according to their distinct heavy chains: IgG, IgM, IgD, IgA, and IgE. Furthermore, each antibody contains one type of light chain, kappa or lambda. Both, heavy and light chains harbor a variable region of 110 amino acids at the N-terminus with three hypervariable segments called complementarity determining regions (CDRs). The hypervariable loops form the two antigen binding (or antigen-combining) sites
of an IgG molecule and determine its specificity. In contrast, the constant part of the immunoglobulin (named the Fc portion) is responsible for secondary effects like activation of the complement system or binding to cellular Fc receptors (Fig. 2.2).

 Importantly, immunoglobulins display a dual function since they are exposed on the surface membrane of B lymphocyte as antigen receptors and are also secreted by plasma cells. They circulate in the blood and other body fluids and are able to bind, neutralize and eliminate foreign antigens, such as viruses, bacteria or toxins. Antibodies represent the effector molecules of the humoral immune system. Transmembrane and secreted forms of an antibody are generated by differential splicing of a primary transcript RNA. In the course of an ongoing immune response specific B lymphocytes undergo isotype switching, that is the transition of the IgM+ and IgD+phenotype to IgG surface and secreted immunoglobulin. In addition, point mutations of the rearranged heavy and light
chain variable genes occur in response to T lymphocyte signaling, giving rise to somatic hypermutation necessary to increase antibody affinity. These processes take place in the germinal centers of secondary lymphoid tissues such as lymph node and spleen. The enzyme activation-induced cytidine deaminase (AID) has been identified as a key player for both class switching and affinity maturation (Barreto et al. 2005). Detailed information on how an effective antibody response
to antigen develops at a molecular level can be looked up in one of several excellent textbooks of immunobiology (Roitt et al. 2001; Abbas and Lichtman 2003; Janeway et al. 2005).

Immunoglobulin G (IgG, 150 kDa) is the most abundant immunoglobulin in serum, accounting for up to 80% of all secreted antibodies. There are four dif ferent IgG isotypes in humans (IgG1, IgG2, IgG3, and IgG4) and mouse (IgG1, IgG2a, IgG2b, and IgG3). Human IgG1 and IgG3 antibodies are potent activators of the complement system and also bind with high affinity to Fc receptors on phagocytic cells, resulting in ADCC. Immunoglobulin M (IgM, 900 kDa) accounts for approximately 10% of serum antibodies. It is expressed as monomer on B lymphocyte as antigen receptor, whereas the secreted form consists of a pentamer hold together by a J (joining) chain. Immunoglobulin D (IgD, 180 kDa) is only found in fairly low amounts in the serum but is, together with IgM, the major
membrane-bound form expressed on mature B cells. Immunoglobulin A (IgA, 160 kDa) constitutes about 10–15% of serum antibodies. It represents the major antibody class, being secreted into tears, saliva, and mucus of the bronchial, genitourinary, and digestive tracts. While the prevalent form in the serum is a monomer, the secretory IgA usually consists of dimers covalently connected via J (joining) chains together with an additional polypeptide called secretory component. Immunoglobulin E (IgE, 180 kDa) is found in the serum only in trace amounts. IgE antibodies are responsible for immediate hypersensitivity and cause the symptoms of hay fever, asthma and anaphylactic shock. Mast cells and basophils bind IgE via Fc receptors and subsequent contact with an allergen will cause
degranulation and release of histamine and other mediators.