CD22L Conjugation to Insulin Attenuates Insulin-Specific B Cell Activation

Abstract

Pancreatic islet-reactive B lymphocytes promote Type 1 diabetes (T1D) by presenting an 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 the 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 (125Tg^SD) expressing anti-insulin BCRs provided cells for in vitro testing. Cells were cultured with constructs for 3 days, then assessed by flow cytometry. Duplicate wells with anti-CD40 simulated T cell help. A 2-insulin 4-arm PEG control caused robust proliferation and activation-induced CD86 upregulation. Anti-CD40 further boosted these effects. This may indicate that BCR-cross-linking occurs when antigens are tethered by the PEG backbone as soluble insulin alone has no effect. Addition of CD22L via the 2:2 insulin-CD22L conjugate restored B cell properties to that of controls without an 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 the WT B cells. Thus, multivalent antigen constructs tend to activate anti-insulin B cells, while monomeric antigen-CD22L conjugates reduce B cell activation in response to simulated T cell help and reduce pathogenic B cell numbers without harming normal cells. Therefore, monomeric antigen-CD22L conjugates warrant futher study and may be promising candidates for preclinical trials to prevent T1D without inducing immunodeficiency.

Figure 1.

Synthesis of insulin–mCD22L, a direct conjugate between insulin–alkyne and mCD22L–azide. A) Reaction scheme. B) HPLC analysis of reactants and reaction products. C) MALDI-TOF-MS analysis of insulin–alkyne and isolated insulin–mCD22L.

Figure 2.

Reaction scheme of 4-arm PEG–insulin(2)/mCD22L(2). First, insulin–azide is conjugated with 4-arm PEG–alkyne by copper-catalyzed azide–alkyne cycloaddition (CuAAC) to yield a mixture of 4-arm PEG–insulin (1–4) products, including 4-arm PEG–insulin(2). Second, isolated 4-arm PEG–insulin(2) is conjugated to mCD22L–azide by CuAAC to yield 4-arm PEG–insulin(2)/mCD22L(2).

Figure 3.

Analysis of 4-arm PEG–insulin products. A) Representation and molecular weights (MW) of the reaction products formed in the CuAAC reaction between 4-arm PEG–alkyne and insulin–azide. B) HPLC analysis of four product fractions following preparatory RP-HPLC purification of 4-arm PEG–insulin. The number in parentheses indicates the number of insulin molecules conjugated to the 4-arm PEG. C) MALDI-MS analysis of four product fractions following preparatory RP-HPLC purification of 4-arm PEG–insulin. The mode m/z ratio of the major peak is indicated.

Figure 4.

Analysis of 4-arm PEG–insulin/mCD22L products. A) HPLC analysis of the CuAAC reaction between 4-arm PEG–insulin(2) with mCD22L–azide before and 2 h after the addition of sodium ascorbate. Two equivalents of mCD22L–azide per alkyne were used. B) MALDI-MS analysis of the 4-arm PEG–insulin(2)/mCD22L(2) product following purification by RP-HPLC. The mode m/z ratio of the major peak is indicated. Representations and molecular weights (MWs) of the starting material and product are shown. C) Proton NMR analysis of 4-arm PEG–insulin(2)/mCD22L(2) and precursors. The aromatic region of the spectra is shown due to the distinctive peaks from both the aromatic side chains of insulin–azide and the bisphenyl group on mCD22L–azide.

Figure 5.

Anti-insulin B cell responses to the incubation of insulin and mCD22L conjugates in 125Tg B6 mouse splenocytes after 72 h. A) The percentage of total live cells that are B cells. B) Total live B cell number. C) CD22 expression levels on B cells as a ratio over untreated anti-insulin B cell CD22 expression. D) Number of proliferating B cells determined by dye-dilution assay. E) CD86 expression levels on B cells as a ratio over untreated anti-insulin B cell CD86 expression. n ≥ 6 mice per condition; standard deviation is indicated by error bars. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001 by one-way Anova with Tukey’s multiple comparison.

Figure 6.

Effects of insulin/mCD22L conjugates on anti-CD40 B cell stimulation in 125Tg B6 mouse splenocytes after 3 days. A) Representative histograms and quantification of B cell proliferation by dye-dilution assay following stimulation with anti-CD40 and treatment with mCD22L–azide or the insulin–mCD22L direct conjugate (*p < 0.05 by one-way Anova with Tukey’s multiple comparison). n ≥ 6 mice per condition. B) Representative histograms and quantification of B cell proliferation by dye-dilution assay following stimulation with anti-CD40 and treatment with 4-arm PEG–insulin(2) or 4-arm PEG–insulin(2)/mCD22L(2) (*p < 0.05, ****p < 0.0001 by one-way Anova with Tukey’s multiple comparison). n = 8 mice per condition.

Figure 7.

Surface marker expression on anti-insulin B cells after 3 days of anti-CD40 stimulation. A) Representative histograms of CD86 expression levels of anti-insulin B cells and quantification as a ratio over anti-CD40 stimulated WT B cells for treatment with mCD22L–azide, 4-arm PEG–insulin(2), 4-arm PEG–insulin(2)/mCD22L(2) or the insulin–mCD22L direct conjugate following 3 days of anti-CD40 stimulation. B) Representative histograms of IgM expression levels of anti-insulin B cells and quantification as a ratio over anti-CD40 stimulated WT B cells for treatment with mCD22L–azide or insulin–mCD22L following 3 days of anti-CD40 stimulation. C) Representative histograms of CD22 expression levels of anti-insulin B cells and quantification as a ratio over anti-CD40 stimulated WT B cells for treatment with unconjugated mCD22L–azide, 4-arm PEG–insulin(2), 4-arm PEG–insulin(2)/mCD22L(2) or the insulin–mCD22L direct conjugate following 3 days of anti-CD40 stimulation. n ≥ 6 mice per condition. Dotted line represents mean MFI of stimulated anti-insulin B cells with no construct. *p < 0.05, ** p < 0.01, ***p < 0.001, ****p < 0.0001 by one-way Anova with Tukey’s multiple comparison.