Engineered Sialylation of Pathogenic Antibodies In Vivo Attenuates Autoimmune Disease

Abstract

Self-reactive IgGs contribute to the pathology of autoimmune diseases, including systemic lupus erythematosus and rheumatoid arthritis. Paradoxically, IgGs are used to treat inflammatory diseases in the form of high-dose intravenous immunoglobulin (IVIG). Distinct glycoforms on the IgG crystallizable fragment (Fc) dictate these divergent functions. IgG anti-inflammatory activity is attributed to sialylation of the Fc glycan. We therefore sought to convert endogenous IgG to anti-inflammatory mediators in vivo by engineering solubilized glycosyltransferases that attach galactose or sialic acid. When both enzymes were administered in a prophylactic or therapeutic fashion, autoimmune inflammation was markedly attenuated in vivo. The enzymes worked through a similar pathway to IVIG, requiring DC-SIGN, STAT6 signaling, and FcγRIIB. Importantly, sialylation was highly specific to pathogenic IgG at the site of inflammation, driven by local platelet release of nucleotide-sugar donors. These results underscore the therapeutic potential of glycoengineering in vivo.

Keywords: Antibody; autoimmune disease therapy; glycosylation; inflammation; platelets; sialic acid.

Figure 1. Solubilizing and engineering glycosyltransferase enzymes

(A) IgG Fab and Fc with a single, N-linked glycosylation site at N297. (B) Fc glycan core shown in the box consists of GlcNAc (blue squares), mannose (green circles); variable additions include fucose (red triangle), bisecting GlcNAc, galactose (yellow circles), or sialic acid (purple diamonds). (C) Trans-Golgi enzymes B4GALT1 and ST6GAL1 have cytoplasmic (cyto), transmembrane (TMD), and enzymatic luminal domains (Lumen). ST6GAL1 cleavage site EFQ41-43 is indicated by red line. (D, E) Lectin blots for terminal galactose (ECL) or sialic acid (SNA) on target glycoproteins fetuin(D), or mouse and human IgG Fcs (E). See also Figure S1.

Figure 2. Anti-inflammatory activity of in vivo sialylation

(A) Clinical scores of mice treated with K/BxN and PBS (black circles), ST6^Fc (pink triangles), B4^Fc (orange diamonds), or IVIG (blue squares). (B) Day 10 scores from (A) are plotted. (C) Clinical scores of K/BxN treated mice given PBS (black circles), IVIG (blue squares), or B4ST6^Fc (red triangles). (D) Day 9 scores from (C) are shown. (E) H&E of paw sections 7 days after K/BxN sera and PBS, IVIG, or B4ST6^Fc. Day 7 BUN levels (F) and survival (G) of mice induced with NTN, and treated with PBS (black circles), IVIG (blue squares), or B4ST6^Fc (red triangles). (H) H&E, Trichrome, and PAS staining of kidney sections from untreated or NTN-treated mice 7 days following PBS, IVIG, or B4ST6^Fc administration. Means and standard deviation are plotted. Results are representative of at least two independent repeats. *p<0.05, **p<0.01, ***p<0.005, ****p<0.001, ns (not significant), determined by two-way ANOVA followed by Tukey’s test. See also Figure S2.

Figure 3. Receptor requirements for in vivo sialylation

(A) Clinical scores of FcγRIIB^−/− mice given K/BxN sera and PBS (black circles), IVIG (blue squares), or B4ST6^Fc (red triangles). (B) Day 6 scores from (A) are plotted. (C) Clinical scores of STAT6^−/− mice given K/BxN sera and PBS (black circles), IVIG (blue squares), or B4ST6^Fc (red triangles). (D) Day 6 scores from (C) are shown. (E) Clinical scores of SIGN-R1^−/− and (F) hDC-SIGN⁺/SIGN-R1^−/− mice given K/BxN sera and PBS (black circles), IVIG (blue squares), or B4ST6^Fc (red triangles). (G) Day 6 scores from (E,F) are shown. Means and standard deviation are plotted. Results are representative of at least two independent repeats. **p<0.01, ***p<0.005, ****p<0.001, ns (not significant), determined by two-way ANOVA followed by Tukey’s test. See also Figures S3A–D.

Figure 4. Enzymatic requirements for in vivo sialylation

(A) B4GALT1 and enzymatically inactive ST6GAL1 (B4ST6^Fc_CACA). (B) Clinical scores of mice given K/BxN sera and PBS (black circles), IVIG (blue squares), or B4ST6^Fc (red triangles), or B4ST6^Fc_CACA (black crosses, red dotted line). (C) Day 6 scores from (B) are shown. (D) B4^Fc and ST6^Fc Fc glycan removal by EndoS (B4ST6Fc-Endo). (E) Clinical scores of mice given K/BxN sera and PBS (black circles), IVIG (blue squares), or B4ST6^Fc (red triangles), B4ST6^Fc-Buffer (red triangle with black edge, red dotted line) or B4ST6^Fc-Endo (red triangle with black edge, red solid line). (F) Day 6 scores from (E) are shown. Means and standard deviation are plotted. Results are representative of at least two independent repeats. *p<0.05, **p<0.005, ****p<0.001, ns (not significant), determined by two-way ANOVA followed by Tukey’s test. See also Figure S3E.

Figure 5. Characterizing in vivo sialylation during autoimmune inflammation

(A) HPLC glycan traces and ratios of monosialylated and agalactosylated glycans (S1/G0) (B) of serum IgG after K/BxN and PBS, IVIG, or B4ST6^Fc treatment. (C) HPLC glycan traces and ratios of monosialylated and agalactosylated glycans (S1/G0) (D) of joint-deposited IgG after K/BxN and PBS, IVIG, or B4ST6^Fc treatment. (E) HPLC glycan traces and ratios of monosialylated and agalactosylated glycans (S1/G0) (F) from serum IgG of NTN-treated mice following administration of PBS, IVIG, or B4ST6^Fc. (G) HPLC glycan traces and ratios of mono-sialylated and agalactosylated glycans (S1/G0) (H) of kidney-deposited IgG of NTN-treated mice are shown. (A,C,E,F) Shading corresponds to retention time of terminal sugar (blue, G0; yellow, G1; orange, G2; pink, S1; purple, S2). Means and standard deviation are plotted. Results are representative of at least two independent repeats. **p<0.01, ns (not significant), as determined by two-way ANOVA followed by Tukey’s test. See also Figure S5.

Figure 6. Platelet activation and in vivo sialylation

(A) CD41-specific IHC in kidneys of untreated and day 7 NTN-induced mice following treatment with PBS, IVIG and B4ST6^Fc. Black asterisks indicate CD41⁺ platelet accumulation. (B) Kidneys of untreated and day 7 NTN-treated mice following treatment with PBS, IVIG and B4ST6^Fc were examined for glomeruli (mNephrin, green), mouse IgG (blue), platelets (CD41, red), activated platelets (CD62, yellow). Representative individual and overlaid images are shown. (C, D) Clinical scores of control- and clopidogrel-treated mice given K/BxN sera and PBS (black circles), IVIG (blue squares), or B4ST6^Fc (red triangles). (E) Day 6 scores of (C, D) are shown. NTN was induced in control- (F, G) and clopidogrel-treated (F, H) animals, and day 7 BUN levels (mg/dL) and survival was monitored. Means and standard deviation are plotted. Results are representative of at least two independent repeats. *p<0.05, ***p<0.005, ****p<0.001, ns (not significant), determined by two-way ANOVA followed by Tukey’s test. See also Figures S6, 7.

Figure 7. Therapeutic in vivo sialylation

(A) HPLC glycan traces of IgG recovered from joint after K/BxN and PBS or B4ST6^Fc treatment with or without clopidogrel. (B) Ratios of monosialylated and agalactosylated glycans (S1/G0) from IgG described in (A). (C,D) Human platelets were untreated, activated (Thrombin+), or activated after clopidogrel treatment (Thrombin+, Clopidogrel+), and assayed for UPD-Gal (C) and CMP-SA (D). (E) Clinical scores of mice treated with K/BxN sera on day 0, and PBS (black circles), IVIG (blue squares), or B4ST6^Fc (red triangles) on day 3. (F) Day 7 scores from (E) are shown. Means and standard deviation are plotted. Results are representative of at least two independent repeats. ns (not significant), **p<0.01, ***p<0.005, ****p<0.001, determined by two ANOVA followed by Tukey’s test. See also Figures S7D,E.