Fc fusions are a growing class of drugs comprising an antibody Fc domain covalently linked to a protein or peptide and can pose manufacturing challenges. In this study we evaluated three synthetic approaches to generate Fc fusions, using Fc-insulin as a model drug candidate. Engineered human IgG1 was digested with HRV3C to produce an Fc fragment with a C-terminal sortase tag (Fc-LPETGGH 6 ). The synthesis of Fc-insulin 2 from Fc-LPETGGH 6 was evaluated with direct sortase-mediated ligation (SML) and two chemoenzymatic strategies. Direct SML was performed with triglycine-insulin, and chemoenzymatic strategies used SML to fuse either triglycine-azide or triglycine-DBCO prior to linking insulin with copper-catalyzed or strain-promoted azidealkyne cycloaddition. Reaction conditions were optimized by evaluating reagent concentrations, relative equivalents, temperature, and time. Direct SML provided the most effective reaction yields, converting 60−70% of Fc-LPETGGH 6 to Fc-insulin 2 , whereas our optimized chemoenzymatic synthesis converted 30−40% of Fc-LPETGGH 6 to Fc-insulin 2 . Here we show that SML is a practical and efficient method to synthesize Fc fusions and provide an optimized pathway for fusion drug synthesis.

No abstract

Autoimmune diseases are characterized by aberrant immune responses toward self-antigens. Current treatments lack specificity, promoting adverse effects by broadly suppressing the immune system. Therapies that specifically target the immune cells responsible for disease are a compelling strategy to mitigate adverse effects. Multivalent formats that display numerous binding epitopes off a single scaffold may enable selective immunomodulation by eliciting signals through pathways unique to the targeted immune cells. However, the architecture of multivalent immunotherapies can vary widely, and there is limited clinical data with which to evaluate their efficacy. Here, we set forth to review the architectural properties and functional mechanisms afforded by multivalent ligands and evaluate four multivalent scaffolds that address autoimmunity by altering B cell signaling pathways. First, we address both synthetic and natural polymer backbones functionalized with a variety of small molecule, peptide, and protein ligands for probing the effects of valency and costimulation. Then, we review nanoparticles composed entirely from immune signals which have been shown to be efficacious. Lastly, we outline multivalent liposomal nanoparticles capable of displaying high numbers of protein antigens. Taken together, these examples highlight the versatility and desirability of multivalent ligands for immunomodulation and illuminate strengths and weaknesses of multivalent scaffolds for treating autoimmunity.

Kifunensine is a known inhibitor of type I α-mannosidase enzymes and has been shown to have therapeutic potential for a variety of diseases and application in the expression of high-mannose N-glycan bearing glycoproteins; however, the compound’s hydrophilic nature limits its efficacy. We previously synthesized two hydrophobic acylated derivatives of kifunensine, namely, JDW-II-004 and JDW-II-010, and found that these compounds were over 75-fold more potent than kifunensine. Here we explored the effects of these compounds on different mice and human B cells, and we demonstrate that they affected the cells in a similar fashion to kifunensine, further demonstrating their functional equivalence to kifunensine in assays utilizing primary cells. Specifically, a dose-dependent increase in the formation of high-mannose N-glycans decorated glycoproteins were observed upon treatment with kifunensine, JDW-II-004, and JDW-II-010, but greater potency was observed with the acylated derivatives. Treatment with kifunensine or the acylated derivatives also resulted in impaired B-cell receptor (BCR) signaling of the primary mouse B cells; however, primary human B cells treated with kifunensine or JDW-II-004 did not affect BCR signaling, while a modest increase in BCR signaling was observed upon treatment with JDW-010. Nevertheless, these findings demonstrate that the hydrophobic acylated derivatives of kifunensine can help overcome the mass-transfer limitations of the parent compound, and they may have applications for the treatment of ERAD-related diseases or prove to be more cost-effective alternatives for the generation and production of high-mannose N-glycan bearing glycoproteins.

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.