The antibody and its fragments

The immunoglobulii (Ig) G molecule has a relatively large molecular weight of 150 kDa. Although IgG distributes throughout the body, its penetration into a tumor mass is slow, as demonstrated by autoradiography of tumor xenografts after intravenous injection of 125I-labeled antitumor IgG. The number of grains in areas on autoradiography sections that are more than 30 pm distant from vessel margins were shown to be increasing even after 72 h postinjection in one study. The whole IgG can also bind to tissue macrophages and circulating leukocytes through Fc receptors. Since immunoglobulins are highly immunogenic in different specics, injection of mouse antibodies raised against human tumors into humans results in the production of human anti-mouse Ig antibodies (HAMA). These antibodies accelerate the clearance of injected antitumor antibodies from the blood, and may cause a potentially fatal anaphylactic reaction and/or serum sickness. Replacement of mouse Ig constant domains with human counterparts by ligation of rearranged mouse Ig variable region with human Ig constant region genes results in the production of chimeric antibodies, which have much reduced immunogenicity.

Since Ig effector functions associated with the heavy chain constant region are not necessary for binding and accumulation of antitumor antibodies to neoplastic lesions, the use of smaller fragments of Ig that retain only the antigen-binding activity seems to be a reasonable way to reduce the immunogenicity and molecular mass of antitumor antibodies.

Enzymatic digestion of IgG with pepsin yields a F(ab')2 fragment, which is still divalent because the two antigen-binding portions are linked by disulfide bonds at the remaining hinge regions (Figure 1). Reduction of the disulfide bonds and the blocking of them with iodoacetamide generates the monovalent fragment called Fab', composed of a single light chain and an N-terminal half of a heavy chain. Another proteolytic enzyme, papain, cuts IgG between the antigen-binding portion and the hinge, resulting in the generation of a slightly different monovalent fragment called Fab. Divalent F(ab' usually retains the antigen-binding properties of the intact IgG, while binding affinities of monovalent Fab and Fab' are often much lower than those of the divalent counterparts. In a quantitative analysis, the affinity constant of Fab' was calculated to be one-seventh of those of F(ab')2 and whole IgG. Plasma

Whole IgG F(ab')2

Figure 1 The structure of IgG and its fragments.

Fab'

Fab scFv

Whole IgG F(ab')2

Figure 1 The structure of IgG and its fragments.

Fab'

Fab scFv clearance of Fab and Fab' is also much faster than that of F(ab')2 and whole IgG, leaving limited time for accumulation into the tumor. However, rapid clearance from the blood may conversely improve the contrast between the tumor and normal tissues. Because of the reduced binding affinity and rapid clearance, in vivo distribution of monomeric Fab is different from that of dimeric F(ab')2 and whole IgG. In general, a higher percentage of injected dose per gram of tissue is achieved with IgG but at a much later point after injection than with Fab'. On the other hand, because of the smaller molecular mass of 50 kDa, Fab' penetrates into tumor mass much more rapidly and evenly than F(ab')2 and whole IgG. Thus, tumor to normal tissue ratios of radioactivities are usually higher when a Fab or Fab' fragment is used as a radiolabeled antitumor targeting agent.

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