Antibody engineering to develop new antirheumatic therapies

Abstract

There has been a therapeutic revolution in rheumatology over the past 15 years, characterised by a move away from oral immuno-suppressive drugs toward parenteral targeted biological therapies. The potency and relative safety of the newer agents has facilitated a more aggressive approach to treatment, with many more patients achieving disease remission. There is even a prevailing sense that disease 'cure' may be a realistic goal in the future. These developments were underpinned by an earlier revolution in molecular biology and protein engineering as well as key advances in our understanding of rheumatoid arthritis pathogenesis. This review will focus on antibody engineering as the key driver behind our current and developing range of antirheumatic treatments.

Figure 1

Basic antibody structure and the different types of therapeutic antibody. (a) Basic antibody structure. (b) Basic structure of a murine, chimeric, humanised, and human monoclonal antibody. Red indicates murine sequence and black indicates human sequence. CDR, complementarity-determining region.

Figure 2

The domain structures of an antibody molecule and its derivatives. (a) An antibody molecule. (b) A fragment antigen-binding (Fab) fragment. (c) A non-covalently linked VH and VL domains (Fv). (d) A single-chain Fv. (e) A receptor-immunoglobulin fusion protein. CH, heavy chain constant domain; CL, light chain constant domain; Fc, fragment crystallisable; VH, heavy chain variable domain; VL, light chain variable domain.

Figure 3

Antibody heavy-chain gene rearrangement, transcription, and translation. In step 1, any V segment (in this case, V2) rearranges to any D segment (in this case, D1). In step 2, the VD segment rearranges to one of the six J segments (in this case, J5). Primary RNA transcripts extend from the rearranged VDJ segments through to the Cδ gene (step 3). Finally, RNA processing results in the incorporation of either Cμ or Cδ by the transcripts, encoding for an IgM or IgD antibody, respectively.

Figure 4

Developing a fully human monoclonal antibody (mAb) using (a) phage display technology and (b) transgenic mouse technology. (a) Step 1: A suitable source of starting material (for example, human blood) is subjected to polymerase chain reaction using appropriate primers, providing 'libraries' of heavy chain V domain (VH) and light chain V domain (VL) sequences. Step 2: Randomly combined VH and VL sequences, connected via a short linker, are incorporated into the genome of a bacteriophage such that they will be expressed at the phage surface. The combination marked with an asterisk encodes the desired specificity. Step 3: The phage library is used to infect a bacterial culture, and the resulting supernatant, containing single-chain Fv-expressing phage particles, is incubated with an appropriate source of target antigen (panning). This can be on a column, Petri dish, and so on. Phage with appropriate specificity adheres to the antigen source. Step 4: Adherent phage is eluted and enriched for the appropriate specificity by further rounds of panning. Step 5: After several rounds of panning, adherent phage is sequenced. A successful procedure should lead to the presence of just one or a few Fv specificities, which can be individually cloned and their specificity checked. At this stage, in vitro affinity maturation procedures can be performed if required (see 'Human antibodies' section for details). Ultimately, the desired specificity is recloned into an appropriate vector containing full-length mAb sequence for expression in a mammalian cell line. (b) Step 1: A transgenic mouse that produces human antibodies is created by targeted disruption of the endogenous murine immunoglobulin heavy- and light-chain genetic loci and their replacement by the equivalent human sequences. Step 2: The mouse, now containing human immunoglobulin genes, is immunised in a conventional manner using the target antigen. Step 3: Splenocytes from the immunised mouse are used to generate hybridomas via conventional fusion technology. Step 4: Resulting hybridomas are screened, leading to isolation and cloning of a hybridoma-secreting high-affinity mAb against the target antigen. Note: In theory, phage display rather than fusion technology can be applied from stage 3 onwards.