Pharmaceutical composition comprising the anti-4-1bb antibody for treating or preventing rheumatoid arthritis
A rheumatoid, composition technology, applied in the direction of drug combination, antibody, anti-animal/human immunoglobulin, etc., can solve the problems of hypotonia, hyperpigmentation, etc.
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Embodiment 1
[0057] [Example 1] Preparation of Antibody
[0058] Hybridoma cells producing 4-1BB (3H3) and 4-1BB (TKL-1) antibodies were kindly provided by Drs. Robert Mittler (Emory university, Atlanta, Georgia) and Hideo Yagita and Ko Okumura (Juntendo university, Tokyo, Japan), respectively. Antibodies were purified from Ehrlich ascites carcinoma cells by protein G-column (sigma, St. Louis, Missouri). LAL (Cambrex, Walkersville, Maryland) measured endotoxin levels below 0.05 units, and the binding activity of mAbs was tested in anti-CD3 mAb-stimulated T cells or 4-1BBL-transfected P815 cells. F(ab') of 3H3 was purified using a Sephacryl s-200HR column (sigma) after digesting the antibody with pepsin 2 Fragment, purified mouse IgG purchased from Sigma was used as a control antibody. Subsequent mAbs were purchased from BDPharMingen (San Diego, California) for flow cytometric analysis: FITC-, PE-.PerCP-, and biotin-anti-CD8 (53-6.7); PE-anti-CD4 (GK1. 5); PE- and biotin-anti-CD11c (HL3)...
Embodiment 2
[0059] [Example 2] CIA induction and anti-4-1BB treatment
[0060] To understand the role of 4-1BB in the CIA process, it was examined whether an agonistic anti-4-1BB antibody (3H3) or a blocking anti-4-1BB ligand could alter the CIA process.
[0061] CIA was induced by intradermal injection of 100D CFA-emulsified bovine collagen II (CII) (Chondrex, Redmond, Washington) into the base of the tail of 6-7 week old DBA / 1 male mice. Antigen was replenished by M. tuberculosis H37RA (2.0 mg / ml, Chondrex). Inflammation of the mouse joints was examined daily and scored as follows: 0, normal; 1, erythema and mild swelling confined to the ankle joint; 2, erythema and mild swelling extending outside the ankle joint; 3 , erythema and moderate swelling extending beyond the ankle joint; 4, erythema and severe swelling extending beyond the ankle joint; the maximum arthritis score per paw was 4, while the maximum disease score per mouse was 16.
[0062] Mice treated with control IgG develope...
Embodiment 3
[0063] [Example 3] Histology and immunostaining
[0064] Immunogenic hind paws were fixed in 10% formalin, decalcified and mounted in paraffin. Joint sections (5-7D) were prepared and stained with hematoxylin and eosin by conventional methods. Sections were examined by light microscopy. For double fluorescent staining, the purified CD11c + CD8 + T cells were incubated on L-lysine-coated slides at 37°C for 1 hour. Subsequent blocking was performed with 1D unlabeled 2.4G2 anti-FcyR Ab (BD PharMingen). Cells were stained with FITC-anti-CD3 plus PE-anti-CD8 or FITC-anti-CD3 plus PE-anti-CD11c or FITC-anti-CD8 plus PE-anti-CD11c. After final washing, slides were mounted on GVA use racks (Zymed, San Francisco, California) and viewed using a laser scanning confocal microscope (FV500, Olympus, Tokyo, Japan). For immunostaining of DLN, DLN sections (8D) were washed with PBS and stained with FITC-anti-CD3 plus PE-anti-CD11c or FITC-anti-CD8 plus PE-anti-CD11c. Slides were process...
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