4-1bb binding molecule
A competitive binding, H-CDR1 technology, applied in the field of antibodies binding to human 4-1BB, can solve unmet problems
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Embodiment 1
[0228] Example 1: Generation of Fab fragments that bind 4-1BB
[0229] Some of the antibodies provided herein were originally generated from Fabs that bind human 4-1BB. The Fab was selected from a phage display library based on reciprocal panning on 4-1BBFC and cells expressing human 4-1BB, MorphoSysHuCAL Phagemid library. These Fabs include those Fabs referred to as "Fab-6032", "Fab-7361", "Fab-7480" and "Fab-7483". The 4-1BB antibodies MOR-6032, MOR-7361, MOR-7480 and MOR-7483 disclosed in this application were produced from "Fab-6032", "Fab-7361", "Fab-7480" and "Fab-7483", respectively . The amino acid sequences of the light and heavy chain variable regions of a given Fab are identical to the amino acid sequences of the light and heavy chain variable regions of an exemplary antibody whose name shares the name of the Fab Same number number. For example, Fab-7480 has the same amino acid sequence as the light and heavy chain variable regions of antibody MOR-7480.
[02...
Embodiment 2
[0232] Example 2: Identification of Fab
[0233] Identification of the four Fabs described in Example 1 was determined in the assay described below using a monovalent Fab-format comprising a Fab with a Flag / His-tag.
[0234] 2A. Determination of affinity using the solution equilibrium titration (SET) method
[0235] The affinity of the four Fabs (in K D denoted) was determined using the SET method, performed using instrumentation from MesoScale Discovery ("MSD"). Monomer fractions of antibody proteins (at least 90% monomer content by analytical SEC analysis; Superdex75 (AmershamPharmacia) for Fab, or TosohG3000SWXL (TosohBioscience) for IgG) were used.
[0236] Affinity determinations in solution were performed essentially as described in the literature (Frigue et al. 1985). In order to improve the sensitivity and precision of the SET method, it was changed from the traditional ELISA to an ECL-based technique (Haene et al. 2005).
[0237] 1 mg / ml goat anti-human (Fab) 2...
Embodiment 3
[0259] Example 3: Identification of IgG
[0260] Some of the Fabs obtained from panning as described in the present invention, including Fab-6032, Fab-7361, Fab-7480 and Fab-7483, were selected to be converted to full-length antibodies in IgG1 and IgG4 formats for further use as described in this example. Identification. The four exemplary antibodies identified in this example, MOR-6032, MOR-7361, MOR-7480, and MOR-7483, were transformed from Fab-6032, Fab-7361, Fab-7480, and Fab-7483, respectively. Antibodies in IgG form were expressed and purified, then characterized in ELISA, FACS, and luciferase reporter gene assays.
[0261] 3A. Conversion to IgG
[0262] To express full-length IgG, the variable domain fragments of the heavy (VH) and light (VL) chains were subcloned from Fab expression vectors into the appropriate in the carrier.
[0263] 3B. Transient expression and purification of human IgG
[0264] Transient expression of full-length human IgG was performed i...
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