Immunotherapy with Long-Lived Anti-CD38 × Anti-CD3 Bispecific Antibodies Stimulates Potent T Cell-Mediated Killing of Human Myeloma Cell Lines and CD38+ Cells in Monkeys: A Potential Therapy for Multiple Myeloma
Authors: Chu Seung Y. , Miranda Yvonne , Phung Sheryl , Chen Hsing , Rashid Rumana , Endo Nancy A. , Chan Emily W. , Pong Erik , Bonzon Christine , Muchhal Umesh S. , Leung Irene W.L. , Bernett Matthew J. , Moore Gregory L. , Szymkowski David E. , Desjarlais John R. 
Published: October 2019

Abstract
CD38, being highly expressed on malignant plasma cells, is an attractive target of new therapies for multiple myeloma (MM). Several anti-CD38 antibodies including daratumumab are in clinical development; however, a limitation of such monospecific antibodies is their inability to stimulate cytotoxic T cell killing of myeloma cells. To exploit the potent mechanism of T cell immunotherapy yet preserve the favorable drug and dosing properties of therapeutic antibodies, we designed bispecific antibodies that recruit T cells to CD38+ MM cells. Such bispecifics act via a "redirected T cell-cytotoxicity" (RTCC) mechanism because they stimulate targeted T cell-mediated killing regardless of T cell receptor antigen specificity. Unlike other bispecific formats, these antibodies possess a full Fc domain and spontaneously form stable heterodimers that are readily manufactured. Their Fc domain was also engineered to abolish binding to Fcγ receptors (to reduce the potential for nonselective T cell activation), yet preserve binding to human FcRn (to maintain long serum half-life).
We first generated a library of humanized and affinity-optimized anti-CD38 × anti-CD3 antibodies and measured their potency using RTCC assays in which antibodies stimulated killing of the human MM cell line RPMI8226 by human T cells. From this screen, we selected two candidates for further assessment. XmAb13243 and XmAb13551 have 21 and 0.2 nM affinities, respectively, for human CD38, and have identical T cell-engaging domains with 8 nM affinity for human CD3. XmAb13243 stimulated RTCC with an EC50 of 2.5 ng/ml (20 pM) after 24 hr, while XmAb13551 had an EC50 of ~100 pg/ml (~1 pM).
In contrast to bispecific formats lacking an Fc domain, XmAb13243 and XmAb13551 had long half-lives in mice of ~7.6 and 8.3 days, respectively. Because these bispecifics were optimized for human CD38 and CD3 binding and do not crossreact with mouse antigens, we next evaluated efficacy in immunodeficient SCID mice engrafted with human PBMCs. In this model, engrafted human B cells differentiate into CD38+ plasma cells, which produce high levels of human Ig. Bispecific antibodies dosed at 0.2, 1, and 5 mg/kg, 7 and 15 days after engraftment, suppressed human IgG2, IgM, and IgE to below detectable levels by Day 14 (> 50-fold for IgG2, > 1,000-fold for IgM, and > 80-fold for IgE). Daratumumab at 5 mg/kg was markedly less potent than bispecifics, reducing IgG2 by 2-fold, IgM by 6-fold, and IgE by 3-fold. The control bispecific anti-RSV × anti-CD3 (which binds to T cells but not to CD38+ cells) had no effect on IgG2, IgM, or IgE levels. To investigate activity against an immune response requiring production of new human plasma cells, mice were vaccinated with tetanus toxoid 8 days after engraftment. Anti-CD38 × anti-CD3 bispecifics suppressed human anti-tetanus antibody titers to baseline (> 100-fold), while daratumumab suppressed titers by only 2-fold.
We next assessed efficacy in cynomolgus monkeys. Unlike daratumumab, which does not crossreact with monkey CD38, XmAb13243 and XmAb13551 bind to both CD38 and CD3 in monkeys (23 and 0.3 nM, respectively, to CD38, and 6 nM to CD3 for both). We treated monkeys with a single dose of XmAb13243 or XmAb13551 at 2, 5, and 20 μg/kg. T cells were activated within 1 hr, as measured by dramatic increases in CD25 and CD69 activation markers. Within 8 hr, T cells depleted circulating CD38+ cells by > 95% at the 20 μg/kg dose.
Our results demonstrate that XmAb13243 and XmAb13551 effectively recruit T cells to kill CD38+ cells in vivo. Our preclinical data in monkeys and humanized mice provide a rationale for clinical testing of anti-CD38 × anti-CD3 bispecific antibodies in patients with multiple myeloma and other CD38+ malignancies.

Disclosures
Chu: Xencor: Employment, Equity Ownership. Miranda:Xencor, Inc.: Employment, Equity Ownership. Phung:Xencor, Inc.: Employment, Equity Ownership. Chen:Xencor, Inc.: Employment, Equity Ownership. Rashid:Xencor, Inc.: Employment, Equity Ownership. Endo:Xencor, Inc.: Employment, Equity Ownership. Chan:Xencor, Inc.: Employment, Equity Ownership. Pong:Xencor, Inc.: Employment, Equity Ownership. Bonzon:Xencor, Inc.: Employment, Equity Ownership. Muchhal:Xencor, Inc.: Employment, Equity Ownership. Leung:Xencor, Inc.: Employment, Equity Ownership. Bernett:Xencor, Inc.: Employment, Equity Ownership. Moore:Xencor, Inc.: Employment, Equity Ownership. Szymkowski:Xencor, Inc.: Employment, Equity Ownership. Desjarlais:Xencor, Inc.: Employment, Equity Ownership.