Bispecific Hexavalant Antibodies With Enhanced Trogocytosis For Treatment Of Lupus
Authors: Rossi Edmund A , Michel Rosana , Chang Chien-Hsing , Goldenberg David M 
Published: October 2019

Abstract

Background
The humanized anti-CD22 antibody, epratuzumab, has demonstrated therapeutic activity in clinical trials of lymphoma and autoimmune diseases (AIDs), treating currently over 1500 cases of non-Hodgkin lymphoma (NHL), acute lymphoblastic leukemias, Waldenström's macroglobulinemia, Sjögren's syndrome, and systemic lupus erythematosus (SLE). Because epratuzumab, which is currently in worldwide Phase III registration trials for SLE, reduces on average only 35% of circulating B cells in patients, and has minimal antibody-dependent cellular cytotoxicity (ADCC) and negligible complement-dependent cytotoxicity (CDC) when evaluated in vitro, its therapeutic activity may not result completely from B-cell depletion. Instead, ligation of epratuzumab to CD22 could modulate other surface molecules involved in regulating B-cell antigen receptor (BCR) signaling, activation, homing, and re-circulation, leading to altered B-cell functions that ultimately mitigate symptoms of the underlying diseases. We reported recently that epratuzumab mediates Fc/FcR-dependent membrane transfer from B cells to effector cells via trogocytosis, resulting in a substantial reduction of multiple BCR modulators, including CD22, CD19, CD21, and CD79b, as well as key cell adhesion molecules, including CD44, CD62L, and b7 integrin, on the surface of B cells in peripheral blood mononuclear cells (PBMCs) obtained from normal donors or SLE patients, and of NHL cells spiked into normal PBMCs (Rossi et al., Blood 2013 PMID: 23821660). Rituxmab has clinical efficacy in SLE, but failed to achieve primary endpoints in a Phase III trial. Here we show for the first time that a bispecific hexavalent antibody (bsHexAb), comprising epratuzumab and veltuzumab (humanized anti-CD20), exhibits enhanced trogocytosis compared to epratuzumab, with considerably less B-cell depletion than observed with anti CD20 mAbs.

Methods and Results
A pair of bsHexAbs were generated using DOCK-AND-LOCKTM (DNLTM) to comprise epratuzumab fused with four additional Fab fragments of either veltuzumab [designated 22*-(20)-(20)] or of a humanized anti-CD19 mAb [22*-(19)-(19)]. PBMCs were incubated with the bsHexAbs or the parental mAbs (10 µg/mL) overnight, and the relative surface levels of the key antigens were analyzed by flow cytometry. The 22*-(20)-(20) exhibited the broadest and most extensive trogocytosis, reducing each of CD22, CD20, CD19, CD21, CD79b, CD44, CD62L, and Beta-7 integrin more than epratuzumab, and to a similar extent as veltuzumab, except for CD22, which was much lower with the 22*-(20)-(20) (Table 1). In general, 22*-(19)-(19) showed intermediate trogocytosis, with less antigen reduction than 22*-(20)-(20), but more than epratuzumab. Veltuzumab and rituximab caused considerable (40-50%) B-cell depletion in the ex-vivo assay. Alternatively, epratuzumab, hA19, and both bsAbs did not significantly deplete B cells. ADCC, which is presumably, the primary mechanism of B-cell depletion in the ex-vivo assay, is less potent for 22*-(20)-(20), compared to veltuzumab. CDC, which along with ADCC is an important mechanism for B-cell depletion in vivo, is ∼25-fold less potent for 22*-(20)-(20) compared to veltuzumab. Epratuzumab has minimal CDC and ADCC.

Conclusion
The bsHexAb 22*-(20)-(20) is an excellent candidate for treatment of SLE and other AIDs due to its ability to mediate potent trogocytosis without wholesale depletion of B cells, which leads to increased risk of serious infections associated with anti-CD20 therapy.

Disclosures:
Rossi: Immunomedics, Inc.: Employment. Michel:Immunomedics, Inc.: Employment. Chang:Immunomedics, Inc: Employment, Stock option Other; IBC Pharmaceuticals, Inc.: Employment, Stock option, Stock option Other. Goldenberg:Immunomedics: Employment, stock options, stock options Patents & Royalties.