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Figure 1 Methods of producing a monoclonal antibody.

Normal mouse

Normal murine fusion

Normal mouse

Manipulation of murine

Ab genes -

Human

Engineered mouse making human antibodies

Transformation of lymphocytes making human antibody

Manipulation of hybridoma Ab genes

Transfected cell making human antibody

Murine hybridoma making murine antibody

Engineered mouse making human antibodies

Normal murine fusion

Transformation of lymphocytes making human antibody

Transfected cell making human antibody

Murine hybridoma making human antibody

Human lymphocytes making human antibody

Figure 2 Strategies for generating a human monoclonal antibody. (A) From a conventional mouse hybridoma by manipulation of its constant region genes. (B) From a transgenic mouse carrying human immunoglobulin genes. (C) Transformation by Epstein-Barr virus. Note that in (A) and (B) the response reflects that of the mouse to a human antigen, whereas in (C) the human response to self-antigens (e.g. rhesus, autoantigens) is immortalized.

framework regions of the mouse antibody leaving only the CDRs (complementarity determining regions) of murine origin. Such antibodies elicit little or no immune response in humans.

(b) A genetically engineered mouse is hyperimmun-ized. Originally, the SCID (severe combined immunodeficiency) mouse was used as it could be, to some extent, populated with an artificial human immune system. More recently, two groups have produced mice with parts of the murine heavy and light chain immunoglobulin loci replaced by the human loci. Such mice, when hyperimmunized, produce murine B lymphocytes making human antibodies and these can then be used in a normal mouse fusion yielding a murine hybridoma making human antibodies.

(c) Use of Epstein-Barr virus (EBV) to immortalize human B lymphocytes which carry its CD21 receptor. This is the only method which gives hybridomas making a genuine human response to human antigens, as strategies (1) and (2) immortalize the murine response to the antigen. Clearly the mouse immune system may see many foreign determinants on a human cellular antigen and these may mask its response to the single antigen that the human immune system sees as foreign. The most obvious example is the rhesus blood group antigens which are masked in the mouse by its response to the human ABO system but which are strong antigens in humans. The EBV method can also be used to interrogate the antibody profile and V gene usage in autoimmune conditions by immortalizing the B lymphocytes present at the site of the lesion. EBV transformants grow well but tend to cease antibody secretion unless this is stabilized. They are therefore frequently 'backfused' after initial cloning to a more stably secreting myeloma line, frequently of mouse origin. The resultant antibody remains human.

Alternative recombinant methods

Newer recombinant methods involve the creation of gene expression libraries from (usually) immunized mice or humans. These may contain the variable regions, or variable plus partial constant region of the heavy or light immunoglobulin chains, and Fab libraries from a combination of the two. A very large number of clones can be screened by inexpensive filter binding methods. In addition, the antibodies can be expressed on the surface of filamentous DNA bacteriophage and selected by affinity chromatography on antigen.

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