Chimaeric and humanized antibodies

Recombinant DNA technology has provided an alternative (and successful) route of reducing the innate immunity of murine monoclonals. The genes for all human immunoglobulin sub-types have been cloned, and this has allowed generation of various hybrid antibody structures of reduced immunogenicity.

The first strategy entails production of 'chimaeric' antibodies, consisting of mouse variable regions and human constant regions (Figure 13.9). The chimaeric antibody would display the specificity of the original murine antibody, but would largely be human in sequence.

Figure 13.9 Production of chimaeric (a) and humanized (b) antibodies (via recombinant DNA technology). Chimaeric antibodies consist of murine monoclonal VH and VL domains grafted onto the Fc region of a human antibody. Humanized antibody consists of murine CDR regions grafted into a human antibody

Table 13.4 The serum half-life values of some IgG antibody preparations when administered to humans

Antibody type Serum half-life

Intact human monoclonal 14-21 days

Intact murine monoclonal 30-40 h

Chimaeric antibody 200-250 h

Murine F(ab)2 fragment 20 h

Murine Fab fragment 2 h

It was hoped that such chimaeric antibodies, when compared with murine antibodies, would be:

• significantly less immunogenic;

• display a prolonged serum half-life;

• allow activation of various Fc-mediated functions.

Reduced immunogenicity was expected, as only a minor part of the chimaeric antibodies is murine in origin. Furthermore, the HAMA response is normally directed largely at epitopes on the antibody's Fc domains. The variable region appears inherently less immunogenic. In practice, the expected reduced immunogenicity was observed. Early clinical trials with chimaerics have shown them to be generally safe and non-toxic. The rate of immune responses observed after single dose administration dropped from almost 80 per cent (murine) to in the region of 5 per cent (chimaeric). However, repeated administration of chimaerics did eventually raise an immune response in most recipients.

When compared with human monoclonals (half-life 14-21 days), murine monoclonals administered to humans display a relatively short half-life (30-40 h). Chimaerization increased serum half-life by fivefold, with typical values of 230 h being recorded (Table 13.4). A prolonged half-life is desirable if the antibody is to be used therapeutically, as it decreases the required frequency of product administration. Chimaeric antibodies also allow activation of Fc-mediated functions (e.g. activation of complement, etc.), as this domain displays human sequence.

Although chimaeric antibodies contain an entire murine-derived variable region, it is only the CDRs within this variable domain that actually dictate antigen specificity (Box 13.2). A method of reducing still further the antigenicity of murine antibodies is to 'humanize' them. This entails transferring the nucleotide sequences coding for the six CDR regions of the murine antibody of the desired specificity into a human antibody gene. The resulting hybrid antibody will, obviously, be entirely human in nature except for the CDRs (Figure 13.9).

Transfer of murine-derived CDR sequences into human antibody framework regions (Box 13.2), sometimes generates an antibody with greatly reduced antigen binding affinity. Selected murine framework sequences are often also included in the humanized antibody. This process (known as reshaping the human antibody) facilitates folding of the CDRs into their true native conformations. This, in turn, normally restores antibody-antigen-binding affinity. Over 95 per cent of antibodies are human in sequence. Clinical trials indicate that such proteins do indeed behave similarly to native human antibodies.

Humanization has overcome many of the major factors that limited the therapeutic effectiveness of first-generation (murine) monoclonals as therapeutic agents. Several such humanized products have now gained marketing authorization (Table 13.2), and one such product is featured in Box 13.3.

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