To demonstrate the utility of phage display in generating highly specific antibodies, affinity selections were conducted on 20 related Src Homology 2 (SH2) domains (ABL1, ABL2, BTK, BCAR3, CRK, FYN, GRB2, GRAP2, LYN, LCK, NCK1, PTPN11 C, PIK3R1 C, PLCγ1 C, RASA1 C, SHC1, SH2D1A, SYK N, VAV1 and the tandem domains of ZAP70). The domains were expressed in Escherichia coli, purified and used in affinity selection experiments. In total, 1292/3800 of the resultant antibodies were shown to bind the target antigen. Of the 695 further evaluated in specificity ELISAs against all 20 SH2 domains, 379 antibodies were identified with unique specificity (i.e. monospecific). Sequence analysis revealed that there were at least 150 different clones with 1–19 different antibodies/antigen. This includes antibodies that distinguish between ABL1 and ABL2, despite their 89% sequence identity. Specificity was confirmed for many on protein arrays fabricated with 432 different proteins. Thus, even though the SH2 domains share a common three-dimensional structure and 20–89% identity at the primary structure level, we were able to isolate antibodies with exquisite specificity within this family of structurally related domains.

Approximately 10% of amyotrophic lateral sclerosis (ALS) cases are familial (known as FALS) with an autosomal dominant inheritance pattern, and ~25% of FALS cases are caused by mutations in Cu/Zn superoxide dismutase (SOD1). There is convincing evidence that mutant SOD1 (mtSOD1) kills motor neurons (MNs) because of a gain-of-function toxicity, most likely related to aggregation of mtSOD1. A number of recent reports have suggested that antibodies can be used to treat mtSOD1-induced FALS. To follow up on the use of antibodies as potential therapeutics, we generated single chain fragments of variable region antibodies (scFvs) against SOD1, and then expressed them as ‘intrabodies’ within a motor neuron cell line. In the present study, we describe isolation of human scFvs that interfere with mtSOD1 in vitro aggregation and toxicity. These scFvs may have therapeutic potential in sporadic ALS, as well as FALS, given that sporadic ALS may also involve abnormalities in the SOD1 protein or activity.

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