Polypeptides with enhanced anti-inflammatory and decreased cytotoxic properties and relating methods
An anti-inflammatory and active technology, applied in chemical instruments and methods, specific peptides, anti-inflammatory agents, etc., can solve problems such as undetermined meaning
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Embodiment 1
[0074] Example 1. IVIG with increased sialic acid content exhibits reduced cytotoxicity
[0075] To test whether IgG-specific glycoforms are involved in modulating antibody effector functions, the role of specific Asn297-linked glycoforms in mediating the cytotoxicity of specific IgG monoclonal antibodies was investigated. Specific carbohydrate composition and structure of antiplatelet antibody derived from 6A6 hybridoma detected by mass spectrometry ( figure 1 ), expressed by 293 cells as IgG12a or 2b switch variants, as previously described by Nitnmerjahn et al. in Immunity 23, 41 (2005). These antibodies contain the fewest sialic acid residues. Enrichment of sialic acid-containing species by Western elderberry agglutinin affinity chromatography to harvest antibodies enriched 60-80 times in sialic acid content figure 2 B and image 3 ). A comparison of the ability of sialylated and non-sialylated 6A6-IgG1 and 2b antibodies to mediate platelet clearance revealed that sia...
Embodiment 2
[0081] Example 2. Desialylation of IVIG reduces its anti-inflammatory effect in a mouse arthritis model
[0082] mouse
[0083] C57BL / 6 and NOD mice were purchased from Jackson Laboratories (Bar Harbor, ME). FcγRIIB - / - Mice were generated in the inventor's laboratory and backcrossed to the C57BL / 6 background for 12 generations. KRN TCR transgenic mice (K / B) of C57BL / 6 background were a kind gift of D. Mathis and C. Benoist (Harvard Medical School, Boston, MA) and bred with NOD mice to generate K / BxN mice. Female mice aged 8-10 weeks were used in all experiments and maintained at the Rockefeller University Animal Facility. All trials were conducted in accordance with federal law and institutional guidelines and were approved by The Rockefeller University (New York, NY).
[0084] Antibodies and soluble Fc receptors
[0085] A switch variant of the 6A6 antibody was prepared from transiently transfected 293T cells and subsequently purified with protein G according to Nimmerj...
Embodiment 3
[0087] Example 3. IVIG Fractions with Enriched Sialic Acid Content Reduce Inflammation in a Mouse Arthritis Model
[0088] Preparation of IVIG with increased sialic acid content
[0089] Since sialic acid is required to demonstrate the anti-inflammatory activity of IVIG, the basis for this high dose (1 g / kg) requirement for anti-inflammatory activity may be the limited concentration of sialylated IgG in the total IVIG preparation. IVIG was fractionated on a SNA-lectin affinity column to obtain IgG molecules enriched in sialic acid-modified glycan structures.
[0090] The protective effect of the sialic acid-rich fraction versus unfractionated IVIG was tested in a KxN serum-infused arthritis model. A 10-fold enhancement in the protective effect of the SNA-binding moiety was observed, so that 0.1 g / kg of SNA-enriched IVIG compared with 1 g / kg of unfractionated IVIG yielded equivalent protective results ( Figure 4 B, C). The distribution of serum half-life of IgG subtypes enr...
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