CD22L Conjugation to Insulin Attenuates Insulin-Specific B cell Activation

Abstract

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 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 B cell activation in response to simulated T cell help and reducing pathogenic B cell numbers without harming normal cells. Thus, the insulin-CD22L direct conjugate is a promising candidate for preclinical trials to prevent T1D without inducing immunodeficiency