The use of therapeutic monoclonal antibodies is constrained because single antigen targets often do not provide sufficient selectivity to distinguish diseased from healthy tissues. We present HexElect®, an approach to enhance the functional selectivity of therapeutic antibodies by making their activity dependent on clustering after binding to two different antigens expressed on the same target cell. lmmunoglobulin G (lgG)-mediated clustering of membrane receptors naturally occurs on cell surfaces to trigger complement- or cell-mediated effector functions or to initiate intracellular signaling. We engineer the Fc domains of two different lgG antibodies to suppress their individual homo-oligomerization while promoting their pairwise hetero-oligomerization after binding co-expressed antigens. We show that recruitment of complement component C1q to these hetero-oligomers leads to clustering-dependent activation of effector functions such as complement mediated killing of target cells or activation of cell surface receptors. HexElect allows selective antibody activity on target cells expressing unique, potentially unexplored combinations of surface antigens.
© 2022. The Author(s).

Red blood cells (RBCs) are attractive tools for surface modification to adhere specifically to molecules, cellular fragments (e.g., microvesicles), or whole cells for potential use in bioanalytical assays or as a delivery vehicle in targeted therapy. Within this study, we have loaded RBCs with fluorochrome-conjugated antibodies (Ab) against CD45 and CD22 leukocyte markers and evaluated the conjugation process by microscopy. We have assessed the potential application of RBCs fragments generated from conjugated RBCs for targeting Cyto-Trol control cells by flow cytometric (FCM) approaches. Based on their scattering and fluorescence characteristics (FITC and PE expression), modified RBCs and their fragments, Cyto-Trol cells, and clusters of both were distinguished by two color FCM analysis. Fragments with anti-human Kallestad Ab as a nonspecific FITC conjugate had less than 20% binding to Cyto-Trol controls compared to CD45-FITC Ab conjugate with nearly 100% binding capacity. Cyto-Trol-microvesicle-clusters were more than 45% positive for either FITC or PE. Anti-CD22-PE modified RBCs fragments were also useful in staining and showing about 19.5% positively stained events in the Cyto-Trol region. The proof-of-concept shows, that specific antibody can be attached to RBCs, and generated fragments can be useful to stain target cells for FCM analysis. © 2018 International Society for Advancement of Cytometry.
© 2018 International Society for Advancement of Cytometry.

The use of targeted therapy is growing in the setting of hematopoietic neoplasms. Flow cytometry is a cornerstone of residual disease monitoring post therapy in this group of malignancies. Often, there is overlap between antigens targeted by immunotherapies and gating reagents utilized for population identification by flow cytometry. Such overlap can render a previously excellent gating reagent inadequate for disease detection. Recently, several anti-CD19 T cell-engaging immunotherapeutic agents and an anti-CD22 immunotoxin have been FDA approved for use in B lymphoblastic leukemia (B-LL), with an anti-CD22 T cell-engaging agent in development. In the setting of such targeted therapies, CD19 and CD22 expression may be altered, compromising the use of these reagents for identification of abnormal blasts. We describe herein a strategy for flow cytometric monitoring for residual disease in patients with B-LL post T cell-engaging anti-CD19 and anti-CD22 therapies. © 2018 by John Wiley & Sons, Inc.
© 2018 John Wiley & Sons, Inc.

CD22 is a member of the Sialic acid-binding Ig-like lectin (Siglec) family of lectins described to be exclusively present in B lymphocytes and B cell-derived neoplasms. Here, we describe a novel splice form of CD22 (designated CD22∆N), which lacks the N-terminal domain as demonstrated by exon-specific RT-PCR and differential recognition by anti-CD22 antibodies. Importantly, CD22∆N mRNA is expressed in skin lesions from 39 out of 60 patients with cutaneous T cell lymphoma (CTCL), whereas few patients (6 out of 60) expresses full-length, wild type CD22 (CD22wt). In addition, IHC staining of tumor biopsies confirmed the expression of CD22 in CD4+ T cells. Moreover, four out of four malignant T cell lines express CD22: Two cell lines express CD22∆N (MyLa2059 and PB2B) and two express CD22wt (MAC-1 and MAC-2A). siRNA-mediated silencing of CD22 impairs proliferation and survival of malignant T cells, demonstrating a functional role for both CD22∆N and CD22wt in these cells.In conclusion, we provide the first evidence for an ectopic expression of CD22 and a novel splice variant regulating malignant proliferation and survival in CTCL. Analysis of expression and function of CD22 in cutaneous lymphomas may form the basis for development of novel targeted therapies for our patients.