Structural details of monoclonal antibody m971 recognition of the membrane-proximal domain of CD22

June Ereño-Orbea,Xianglei Liu,Taylor Sicard,Iga Kucharska,Wei Li,Dorota Borovsky,Hong Cui,Yang Feng,Dimiter S Dimitrov,Jean-Philippe Julien

doi:10.1016/j.jbc.2021.100966

Abstract

Cluster of differentiation-22 (CD22) belongs to the sialic acid–binding immunoglobulin (Ig)-like lectin 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 because of its function in establishing a baseline level of B-cell inhibition. The restricted expression of CD22 on B cells and its inhibitory function make 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. 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 fragment antigen-binding in complex with the two most membrane-proximal Ig-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.

Highlights

To delineate the m971 epitope at high resolution, we obtained crystals of the membrane-proximal domains d6 and d7 of the extracellular portion (ECD) of human Cluster of differentiation-22 (CD22) (CD22d6– d7) in complex with the m971 fragment antigen-binding (Fab), which diffracted to 2.4 Å resolution (Table 1)
The antigen– antibody structure was solved by molecular replacement using the m971 Fab as an initial search model, which was derived from its 1.6 Å resolution crystal structure in complex with the crystallization chaperone anti-kappa VHH domain [15] (Table 1)
Previous studies on the development of anti-CD22 chimeric antigen receptors (CARs)-T cells showed the relevance of targeting a membraneproximal epitope on CD22 extracellular domain (ECD) [10]

Summary

Results

To delineate the m971 epitope at high resolution, we obtained crystals of the membrane-proximal domains d6 and d7 of the extracellular portion (ECD) of human CD22 (CD22d6– d7) in complex with the m971 fragment antigen-binding (Fab), which diffracted to 2.4 Å resolution (Table 1). To provide molecular insights into antibody recognition that leads into the development of effective anti-CD22 CAR-T cells, we structurally and biophysically delineated the binding site of m971 on CD22 [14]. The most membrane-proximal domain of the related Siglec-4 (MAG) showed a C1-type fold and two intradomain disulfide linkages (Fig. S1) [16]. Comparison of the variable domain of the CD22-liganded and -unliganded crystal structures of m971 Fab indicated that its paratope is largely preconfigured for binding its epitope (RMSD of 0.33 Å) (Fig. S2). Our data indicate that m971 recognizes the closest ECD to the membrane of CD22 and that N-linked glycosylation on CD22 does not impact the ability to access its epitope

A YH52AR mutation on m971 Fab increases affinity toward CD22 ECD

Discussion

Experimental procedures