Bcma binding molecules and methods of use thereof
An antigen binding molecule, B cell technology, applied in the field of treating cancer or other diseases or conditions in patients, which can solve problems such as adverse side effects
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Embodiment 1
[0551] BCMA expression was measured in various cell lines. BCMA was found to be expressed (fragments / kilobase-long exons / million mapped reads (FPKM) greater than 35) in 99% of multiple myeloma tumor cell lines tested (Figure 2A). BCMA expression was higher than that of CD70, CS-1, CLL-1, DLL-1 and FLT3 (Fig. 2A). To further characterize the expression of BCMA, EoL-1 (Sigma) was stained with an anti-BCMA antibody conjugated to PE (Biolegend, San Diego, CA) in staining buffer (BD Pharmingen, San Jose, CA) at 4°C. , NCI-H929 (Molecular Imaging) and MM1S (Molecular Imaging) staining for 30 minutes. Cells were then washed and resuspended in staining buffer with propidium iodide (BD Pharmingen) prior to data acquisition. Then, samples were obtained by flow cytometry and data were analyzed ( Figures 2B to 2C ). In the myeloma cell line MM1S( Figure 2C ) and NCI-H929 ( Figure 2D ) but not in the human eosinophil cell line EoL-1 ( Figure 2B ). Furthermore, little to no BCMA...
Embodiment 2
[0553] A third-generation lentiviral transfer vector containing the BCMA CAR construct was combined with the ViraPower TM Lentiviral packaging mix (Life Technologies, FIX TM ) to generate the lentiviral supernatant. Briefly, a transfection mixture was generated by mixing 15 μg of DNA and 22.5 μl of polyethileneimine polyethileneimine (Polysciences, 1 mg / ml) in 600 μl of OptiMEM medium. The transfection mixture was incubated for 5 minutes at room temperature. At the same time, 293T cells (ATCC) were trypsinized and counted. Then, add up to 10x10 6 293T cells were plated together with the transfection mixture in a T75 flask. After three days of cultivation, the supernatant was collected and filtered through a 0.45 μm filter before being stored at −80°C.
[0554] Peripheral blood mononuclear cells (PBMCs) were isolated from leukocyte concentrates (leukopak) (Hemacare) from two different healthy donors using ficoll-paque gradient density centrifugation according to the manufa...
Embodiment 3
[0560] Antigens were biotinylated using the EZ-Link Sulfo-NHS-Biotinylation Kit from Pierce / ThermoFisher (Waltham, MA). Goat anti-human F(ab')2κ-FITC (LC-FITC), Extravidin-PE ( EA-PE) and streptavidin 633 (SA-633). Streptavidin microbeads and MACS LC separation columns were purchased from Miltenyi Biotec (Gladbachn, Germany).
[0561] initial discovery
[0562]Eight initial human synthetic yeast pools each with a diversity of approximately 109 were propagated as described herein (see WO2009036379, WO2010105256 and WO2012009568 by Xu et al.). For the first two selection rounds, the Miltenyi MACs system was used for magnetic bead sorting as described ((Siegel et al., 2004). Briefly, yeast cells (approximately 1010 cells / pool) were mixed with 3 ml of 100 nM Biotinylated monomeric antigen or 10 nM biotinylated Fc fusion antigen was incubated for 15 minutes at room temperature in FACS wash buffer (phosphate-buffered saline (PBS) / 0.1% bovine serum albumin (BSA)). Use 50 ml Aft...
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