Journal Club: Human Germline Antibody Gene Segments Encode Polyspecific Antibodies

This week’s paper by Willis et al. sought to investigate how our limited antibody-encoding gene repertoire has the ability to recognise the unlimited array of antigens. There is a finite number of V, D, and J genes that encode our antibodies, but it still has the capacity to recognise an infinite number of antigens. Simply, the authors’ notion is that an antibody from the germline (via V(D)J recombination; see entry by James) is able to adopt multiple conformations, thus allowing the antibody to bind multiple antigens.

Three antibodies derived from the germline gene 5*51-01, all binding to very different antigens.

Three antibodies derived from the germline gene 5*51-01 bind to very different antigens.

To test this hypothesis, the authors performed a multiple sequence alignment for the amino acid sequence between the mature antibodies and the germline antibody sequence from which the antibodies are derived from. if a single position from ONE mature antibody showed a difference to the germline sequence, it was identified as a ‘variable’ position, and allowed to be changed by Rosetta’s multi-state design (MSD) and single-state design (SSD) protocols.

Pipeline: align mature antibodies (2XWT, 2B1A, 3HMX) to the germline sequence (5-51) , identify 'variable' positions from the alignment, then allow Rosetta to change those residues during design.

Figure 1) from Willis et al., showing the pipeline: align mature antibodies (2XWT, 2B1A, 3HMX) to the germline sequence (5-51) , identify ‘variable’ positions from the alignment, then allow Rosetta to change those residues.

Surprisingly, without any prior information of the germline sequence, the MSD yielded a sequence that was closer to the germline sequence, and the SSD for each mature antibody had retained the mature sequence. In short, this indicated that the germline sequence is a harmonising sequence that can accommodate the conformations of each of the mature antibodies (as proven by MSD), whereas the mature sequence was the lowest energy amino acid sequence for the particular antibody’s conformation (as proven by SSD).

To further demonstrate that the germline sequence is indeed the more ‘flexible’ sequence, the authors then aligned the mature antibodies and determined the deviation in ψ-ϕ angles at each of the variable positions that were used in the Rosetta study. They found that the ψ-ϕ angle deviation in the positions that recovered to the germline residue was much larger than the other variable positions along the antibody. In other words, for the positions that tend to return to the germline amino acid in MSD, the ψ-ϕ angles have a much larger degree of variation compared to the other variable positions, suggesting that the positions that returned to the germline amino acid are prone to lots of movement.

In addition to the many results that corroborate the findings mentioned in this entry, it’s neat that the authors took a ‘backwards’ spin to conventional antibody design. Most antibody design regimes aim to find amino acid(s) that give the antibody more ‘rigidity’, and hence, mature its affinity, but this paper went against the norm to find the most FLEXIBLE antibody (the most likely germline predecessor*). Effectively, they argue that this type of protocol can be exported to extract new antibodies that can bind to multiple antigens, thus increasing the versatility of antibodies as potential therapeutic agents.

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