Isolation of monoclonal antibodies from anti-synthetase syndrome patients and affinity maturation by recombination of independent somatic variants

Abstract

The autoimmune disease known as Jo-1 positive anti-synthetase syndrome (ASS) is characterized by circulating antibody titers to histidyl-tRNA synthetase (HARS), which may play a role in modulating the non-canonical functions of HARS. Monoclonal antibodies to HARS were isolated by single-cell screening and sequencing from three Jo-1 positive ASS patients and shown to be of high affinity, covering diverse epitope space. The immune response was further characterized by repertoire sequencing from the most productive of the donor samples. In line with previous studies of autoimmune repertoires, these antibodies tended to have long complementarity-determining region H3 sequences with more positive-charged residues than average. Clones of interest were clustered into groups with related sequences, allowing us to observe different somatic mutations in related clones. We postulated that these had found alternate structural solutions for high affinity binding, but that mutations might be transferable between clones to further enhance binding affinity. Transfer of somatic mutations between antibodies within the same clonal group was able to enhance binding affinity in a number of cases, including beneficial transfer of a mutation from a lower affinity clone into one of higher affinity. Affinity enhancement was seen with mutation transfer both between related single-cell clones, and directly from related repertoire sequences. To our knowledge, this is the first demonstration of somatic hypermutation transfer from repertoire sequences to further mature in vivo derived antibodies, and represents an additional tool to aid in affinity maturation for the development of antibodies.

Anti-Cancer Auto-Antibodies: Roles, Applications and Open Issues

“…A third point to consider is that what is often defined as a “single” antibody detected by ELISA or similar tests against an antigen, is in fact likely to be a signal generated by multiple antibodies derived from a polyclonal population of B cells. This is the scenario that is emerging in the field of auto-immune diseases [ 149 ], and dissecting this complexity will not be easy. The current availability of suitable techniques allowing isolation of single antibody specificities towards specific antigens from human B cells will be central in this task.…”

Longitudinal assessment of reactivity and affinity profile of anti-Jo1 autoantibodies to distinct HisRS domains and a splice variant in a cohort of patients with myositis and anti-synthetase syndrome

“…Strikingly, the majority of anti-Jo1 + patients presented anti-HisRS-FL antibodies with a high-affinity profile already at the time close to diagnosis. Recently, another study showed that individual anti-Jo1 monoclonal antibodies, selected based on somatic hypermutation using single-cell isolation and sequencing, also displayed affinities from low nM K D and below [ 35 ]. Considering the generally high reactivity against all HisRS domains at diagnosis observed in this study, in combination with the fact that high-affinity autoantibodies were retrieved from patients with a recent diagnosis of IIM/ASSD, one can speculate that affinity maturation of anti-HisRS antibodies through somatic hypermutation together with epitope spreading has already happened before the onset of specific symptoms, ultimately leading to the diagnosis of IIM/ASSD.…”

Longitudinal assessment of reactivity and affinity profile of anti-Jo1 autoantibodies to distinct HisRS domains and a splice variant in a cohort of patients with myositis and anti-synthetase syndrome

“…Strikingly, the majority of anti-Jo1 + patients presented anti-HisRS-FL antibodies with a high-a nity pro le already at the time close to diagnosis. Recently, another study showed that individual anti-Jo1 monoclonal antibodies, selected based on somatic hypermutation using single cell isolation and sequencing, also displayed a nities from low nM K D and below (35). Also, in patients with RA, high a nity circulating autoantibodies (targeting the citrullinated peptide Cit573) have previously been observed (36).…”