Humanized CD22 transgenic mouse model for in vivo analysis of anti‐CD22‐based immunotherapy

B cells are often involved in the development and pathogenesis of autoimmune diseases. In systemic lupus erythematosus (SLE) or rheumatoid arthritis, autoreactive B cells contribute to the pathogenesis by production of autoantibodies. To avoid autoreactive immune responses, Bcell activation needs to be strictly regulated. For this purpose, several regulatory receptors with inhibitory functions are expressed on B cells. One negative regulator for the control of B-cell activation is CD22. CD22 is a member of the Siglec family (sialic acid binding immunoglobulin-like lectins) and preferentially binds α2,6-linked sialic acids [1]. It is an inhibitory coreceptor of the BCR, which carries inhibitory ITIM-signalling motifs that recruit the tyrosine phosphatase SHP-1. SHP-1 binding results in the inhibition of the BCR signaling by subsequent dephosphorylation of signalling molecules. Dysregulated B-cell activation can also lead to the development of B-cell leukaemia or lymphoma. The majority of B-cell lymphomas and leukaemia’s express CD22 on the surface. Therefore, CD22 is a good target for immunotherapy in autoimmune diseases or leukaemia’s. We and others developed new high affine synthetic sialosides as ligands for human CD22 and coupled them to toxins in order to generate CD22-specific immunotoxins [2, 3]. CD22 can also be targeted with liposomal nanoparticles and several therapeutics based on anti-CD22 antibodies have been developed and tested in clinical trials [4]. Epratuzumab (Emab) is an antiCD22 antibody that modulates BCR signalling [5]. Emab has been evaluated in clinical trials for the treatment of several B-cell lymphomas, as well as in autoimmune diseases. Emab failed in Phase III clinical trials of SLE, however, Emab ameliorated disease in SLE patients with associated Sjögren ́s Syndrome [6]. CAR T cells are a promising new approach for the treatment of leukaemia’s and CD22 represents a good target for this kind of therapy. AntiCD22 CARs showed antileukeamic activity in mouse experiments and phase I clinical trials [7]. In order to analyse targeting of human CD22 in vivo, we previously developed a knockin mouse model expressing human CD22 (Huki CD22 mice) [8]. In this mouse model, we tested Emab effects in vivo [5]. Surprisingly, Huki CD22 mice showed a B-cell phenotype, with reduced numbers of mature recirculating B cells in the BM and reduced Transitional and Marginal zone (MZ) B cells in the spleen. Additionally, BCR-induced Ca2+ signalling was increased [8]. This resembled the phenotype of CD22-/mice [1]. We speculated that the CD22-/--like phenotype of the HuKi CD22 mouse was due to the very low expression of hCD22 on the B-cell surface. Therefore, we generated a new knockin mouse line expressing a chimeric CD22 molecule consisting of extracellular human (h) and intracellular murine (m) CD22. We generated this new h/mCD22 mouse line by insertion of a chimaeric h/m CD22 cDNA into the mouse Cd22 gene. By use of a targeting vector with a strong 6x poly-A sequence, we expected to obtain better mRNA stability and a higher h/mCD22 expression (Fig. 1). As expected, B cells from HuKi CD22 and h/mCD22 mice do not express murine CD22. Instead, they show expression of human CD22, with a much higher expression level in h/mCD22 mice compared to HuKi CD22 mice, indicating that the targeting strategy was successful and hCD22 expression could be increased (Fig. 2A-C). The hCD22 expression is still somewhat lower on B cells of h/mCD22 mice, when compared to human B cells (Fig. 2D). h/mCD22 mice show normal B-cell development in the BM (Fig. 2A). In contrast to HuKi CD22 mice, which had reduced numbers of mature recirculating B cells in the BM and transitional (T1 and T2) and MZ B cells in the spleen, h/mCD22 mice did not show any significant changes in these B-cell subsets (Fig. 2A-C). This indicates that the expression of the chimaeric CD22 does not interfere with the normal B-cell development in h/mCD22 mice. CD22-/mice show increased Ca2+ mobilization after stimulation of the BCR. HuKi CD22 mice have a low expression of hCD22 and also showed elevated calcium signalling. Huki CD22 mice carry a human cytoplasmatic CD22 tail that might not function properly. In the new h/mCD22 mouse, we expected comparable calcium signalling to WT mice, but we found even reduced calcium signalling (Fig. 2D). We speculate that by the increased expression of hCD22, the inhibitory effect on the BCR is higher, leading to a reduction of the calcium signalling. Alternatively, ligand bind-