Fully human antibodies against human 4-1BB (CD137)
A CDR2, monoclonal antibody technology, applied in the direction of anti-animal/human immunoglobulin, anti-receptor/cell surface antigen/cell surface determinant immunoglobulin, etc., to achieve a wide range of therapeutic effects
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[0048] The present invention relates to the preparation and characterization of antibodies and antigen-binding fragments thereof (including fusion proteins comprising antigen-binding fragments of the antibodies of the invention) for use in the treatment of diseases such as cancer, infectious diseases, inflammatory diseases or autoimmune diseases. The cancer can be, for example, prostate cancer, melanoma, or epithelial cancer.
[0049] The antibody can bind to H4-1BB, and can exhibit high affinity to H4-1BB and effectively enhance T cell response. In one aspect, the antibody induces IFN-γ production in costimulation assays, but does not affect the binding of H4-1BB to its corresponding ligand, H4-1BBL, and does not fix complement.
[0050] Antibodies of the invention can be prepared by methods known in the art. In one aspect, the antibodies can be produced by expression in transfected cells such as immortalized eukaryotic cells such as myeloma or hybridoma cells
[0051] Anti...
Embodiment 1
[0071] Example 1: Preparation of Antibodies
[0072] Materials and methods
[0073] A fully human monoclonal antibody to the human CD137 (4-1BB) receptor was raised in HuMAb-Mouse(R) (Medarex, Inc., Princeton, New Jersey). HuMAb mice were immunized five times intraperitoneally (i.p.) and subcutaneously (s.c.) with 25 μg of human CD137 extracellular domain in RIBI adjuvant (Ribi Immunochemical). Prior to infusion, mice were boosted intravenously (i.v.) with the same amount of antigen. Splenocytes from immunized mice with appropriate anti-huCD137 antibody titers were fused to mouse myeloma cells according to standard methods.
[0074] Anti-human CD3 mab (Kolon: HIT3a), ELISA kits for human and monkey IFN-γ, cytometric bead array (CBA) kits, and all conjugates for flow cytometry Antibodies for the technique were purchased from BD Pharmingen (San Diego, California). Human IgG 1 and human IgG 1 Kappa was purchased from Sigma-Aldrich (St. Louis, Missouri). CEM cells (ATCC-CRL...
Embodiment 2
[0091] Example 2: In vitro characterization of mab 20H4.9-IgG4
[0092] Based on its binding kinetics, inability to block CD137-CD137L action, and functional effect on human T cells, mab 20H4.9-IgG1 was selected to switch to IgG4 form. The IgG4 form of mab 20H4.9-IgG1 is 20H4.9-IgG4 (shown schematically in Figures 3 and 4).
[0093] The second phase of the study involved comparing the in vitro properties of mab 20H4.9-IgG4 and mab 20H4.9-IgG1. In this section, the binding kinetic properties and functional effects of the antibodies in human and monkey lymphocytes are described.
[0094] binding kinetics
[0095] The kinetic properties of the anti-human CD137 antibody were evaluated using surface plasmon resonance using a BIAcore3000 device. Antigen, human CD137-mouse IgG 2a Low-density covalent immobilization on the surface of CM5 sensor chips. Mab 20H4.9-IgG4 and mab 20H4.9-IgG1 were injected at a concentration of 25-200 nM. Figure 11 Injection of mab 20H4.9-IgG1h mab 2...
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