- Authors: Ereño-Orbea J. et al.
- Year: 2021
- Journal: J Biol Chem 100966
- Applications: in vitro / DNA / FectoPRO
- Cell types:
- Name: HEK-293F
- Name: HEK-293S
- Name: HEK-293S GnTI
1. CD22d6-d7 was tranfected in HEK 293 Gnt I−/− (HEK 293S) suspension cells for protein expression. Cells were incubated at 37 °C, 180 rpm, 8% CO2 in a Multitron Pro shaker for 6-7 days. After harvesting the cells by centrifugation supernatants were retained and filtered using a 0.22 μm Steritop filter. 2. The heavy chain and light chain of the Fab were co-transfected in HEK 293F cells at a 1:1 ratio of DNA:FectoPRO. Cells were transfected at a cell density of 0.8 × 106 cells ml-1 and incubated at 37 °C, 125 rpm, 8% CO2 in a Multitron Pro shaker for 5-7 days. Cells were harvested and supernatants retained and filtered with a 0.22 μm membrane. 3. CD22d6-d7 was transiently co-transfected with the heavy chain and light chain of the m971 Fab into HEK 293S cells at a 1:1.5:1 ratio of CD22:HC:LC DNA. Cells were co-transfected at a cell density of 0.8 × 106 cells ml-1 and incubated at 37 °C, 125 rpm, 8% CO2 in a Multitron Pro shaker for 5-7 days. After harvesting the cells by centrifugation at 6371×g for 20 min, supernatants were retained and filtered using a 0.22 μm Steritop filter.
CD22 belongs to the sialic acid-binding immunoglobulin-like lectins (Siglecs) family of receptors that is expressed on the surface of B cells. It has been classified as an inhibitory coreceptor for the B cell receptor due to its function in establishing a baseline level of B-cell inhibition. The restricted expression of CD22 on B cells and its inhibitory function makes it an attractive target for B-cell depletion in cases of B-cell malignancies. Genetically modified T cells with chimeric antigen receptors (CARs) derived from the m971 antibody have shown promise when used as an immunotherapeutic agent against B-cell acute lymphoblastic leukemia (B-ALL). A key aspect of the efficacy of this CAR-T was its ability to target a membrane-proximal epitope on the CD22 extracellular domain; however, the molecular details of m971 recognition of CD22 have thus far remained elusive. Here, we report the crystal structure of the m971 antigen-binding fragment (Fab) in complex with the two most membrane-proximal immunoglobulin-like domains of CD22 (CD22d6-d7). The m971 epitope on CD22 resides at the most proximal Ig domain (d7) to the membrane, and the antibody paratope contains electrostatic surfaces compatible with interactions with phospholipid head groups. Together, our data identify molecular details underlying the successful transformation of an antibody epitope on CD22 into an effective CAR immunotherapeutic target.