Novel polypeptides
A bispecific, domain-binding technology for use in the field of peptides to address issues such as reducing tumor growth and/or spread
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example 1
[0582] Example 1: ELISA binding of CD40-EpCAM bsAb to hEpCAM
[0583] background and purpose
[0584]Binding was analyzed by ELISA. The bispecific antibodies 1132-005025.M, 1132-005038.M, 1132-3188.M, 1132-3174.M (in Morrison format) and 1132-3174.R (in RUBY TM format) binding to human EpCAM.
[0585] Materials and methods
[0586] Plates were coated with 0.5 μg / mL hEpCAM (R&D Systems, #9277-EP) in PBS overnight at 4°C. After washing in PBS / 0.05% Tween 20 (PBST), plates were blocked with PBS / 0.2% BSA for at least 30 minutes at room temperature and then washed again. Serially diluted samples from 50 nM in PBS / 0.02% BSA were then added and allowed to bind for at least 1 hour at room temperature. After washing, the plate was incubated with 0.5 μg / mL biotinylated hCD40 (504-030 from Ancell) or HRP-labeled goat anti-h-κ light chain (Abd Serotec, #STAR127P) at room temperature for at least 1 hour . Double-antigen-complexed bsAbs were detected with HRP-labeled streptavidin...
example 2
[0589] Example 2: Affinity measurements of EpCAM binding domains
[0590] background and purpose
[0591] Binding was measured by Octet. Bispecific antibodies 1132-005025.M, 1132-005038.M, 1132-3188.M, 1132-3174.M (in Morrison format) or 1132-3174.R (in RUBY TM format) binding to human EpCAM.
[0592] Materials and methods
[0593] Kinetic measurements were performed using the Octet RED96 platform (ForteBIo). Affinity assessment was performed with 3 different assays; Assay 1 was performed in solution with conjugated bsAb and dimeric antigen EpCAM-Fc (Sino hEpCAM_Fc (0.25 mg / ml in PBS) #10694-H02H); Assay 2 was performed in solution with conjugated bsAb and monomeric antigen EpCAM-his (R&DhEpCAM_His (500 ug / ml in PBS) #9277-EP); assay 3 was performed with conjugated antigen (SinohEpCAM_Fc (0.25 mg / ml) ml in PBS) #10694-H02H) and bsAb in solution.
[0594] Assays 1 and 2
[0595] BsAbs at 1.0 or 1.5 ug / ml were coupled to an anti-human Fab-CH1 Generation 2 (FAB2G) bi...
example 3
[0606] Example 3: Binding of CD40-EpCAM bispecific antibody to EpCAM expressing cell lines
[0607] background and purpose
[0608] 1132-3174.M, 1132-005025.M, 1132-005038.M and 1132-3188.M are CD40-EpCAM bispecific antibodies in Morrison format, where 1132 refers to the CD40 agonist domain and 3174, 005025, 005038 and 3188 refer to the EpCAM binding (tumor targeting) domain. These antibodies have been LALA mutated to silence Fcγ receptor binding.
[0609] The aim of this study was to evaluate the binding of CD40-EpCAM bispecific antibody to EpCAM expressed on cells.
[0610] Materials and methods
[0611] The human EpCAM gene was cloned into pcDNA3.1, and the vector was subsequently stably transfected into CHO cells. The tumor cell line JEG expressing high levels of EpCAM, BxPC3 and CHO-EpCAM cells expressing low levels of EpCAM were mixed with 1 μg / ml of 1132-3174.M, 1132-005025.M, 1132-005038.M or 1132-3188.M Incubated together. Antibody binding was detected using...
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