Pancreatic islet-reactive B lymphocytes promote Type 1 diabetes (T1D) by presenting antigen to islet-destructive T cells. Teplizumab, an anti-CD3 monoclonal, delays T1D onset in patients at risk, but additional therapies are needed to prevent disease entirely. Therefore, bifunctional molecules were designed to selectively inhibit T1D-promoting anti-insulin B cells by conjugating a ligand for the B cell inhibitory receptor CD22 (i.e., CD22L) to insulin, which permit these molecules to concomitantly bind to anti-insulin B cell receptors (BCRs) and CD22. Two prototypes were synthesized: 2:2 insulin-CD22L conjugate on a 4-arm PEG backbone, and 1:1 insulin-CD22L direct conjugate. Transgenic mice (125TgSD) expressing anti-insulin BCRs provided cells for in vitro testing. Cells were cultured with constructs for three days then assessed by flow cytometry. Duplicate wells with anti-CD40 simulated T cell help. Surprisingly, a 2-insulin 4-arm PEG control caused robust proliferation and activation-induced CD86 upregulation. Anti-CD40 further boosted these effects. This was unexpected, as soluble insulin alone has no effect, and may indicate that BCR-crosslinking occurs when antigens are tethered by the PEG backbone. Addition of CD22L via the 2:2 insulin-CD22L conjugate restored B cell properties to that of controls, without additional beneficial effect. In contrast, the 1:1 insulin-CD22L direct conjugate significantly reduced anti-insulin B cell proliferation, CD86 upregulation, and cell number, even in the presence of anti-CD40. CD22L alone had no effect, and the constructs did not affect WT B cells. Thus, high valency constructs activate anti-insulin B cells, while low-valency antigen-CD22L conjugates co-ligate BCR and CD22, reducing pathogenic B cell numbers and function without harming normal cells. Thus, insulin-CD22L direct conjugate is a promising candidate for preclinical trials to prevent T1D without inducing immunodeficiency.

Type 1 diabetes (T1D) occurs when autoreactive T cells destroy insulin-producing beta cells. Anti-insulin B cells promote this disease, acting as essential antigen-presenting cells. Rituximab, which globally depletes B cells, provided temporary preservation of insulin production in early onset T1D, but increases susceptibility to infection. Antigen-specific immune therapy (ASIT) may be a viable option for treating T1D by targeting anti-insulin B cells for removal from the B cell repertoire. In this study we investigate the efficacy of soluble antigen arrays containing insulin (SAgAins) developed by the Berkland lab. These constructs consist of a polyethylene glycol (PEG) backbone with four linker arms for bioconjugation. We conducted in vitro experiments to investigate the effects of constructs containing insulin and a trisaccharide ligand (CD22L) for inhibitory B cell receptor CD22 on transgenic anti-insulin B cells. We found that incubating insulin-PEG with anti-insulin B cells stimulates proliferation and CD86 upregulation. However, when CD22L is added (CD22L-PEG-insulin) the expression of CD86 is dampened. This suggests that this novel construct harnesses the regulatory power of CD22 to dampen activation of B cells in an autoantigen-specific manner.