Bispecific antibodies and antibody fragments in various formats have been explored as a means to recruit cytolytic T cells to kill tumor cells. Encouraging clinical data have been reported with molecules such as the anti-CD19/CD3 bispecific T cell engager (BiTE) blinatumomab. However, the clinical use of many reported T cell-recruiting bispecific modalities is limited by liabilities including unfavorable pharmacokinetics, potential immunogenicity, and manufacturing challenges. We describe a B cell-targeting anti-CD20/CD3 T cell-dependent bispecific antibody (CD20-TDB), which is a full-length, humanized immunoglobulin G1 molecule with near-native antibody architecture constructed using "knobs-into-holes" technology. CD20-TDB is highly active in killing CD20-expressing B cells, including primary patient leukemia and lymphoma cells both in vitro and in vivo. In cynomolgus monkeys, CD20-TDB potently depletes B cells in peripheral blood and lymphoid tissues at a single dose of 1 mg/kg while demonstrating pharmacokinetic properties similar to those of conventional monoclonal antibodies. CD20-TDB also exhibits activity in vitro and in vivo in the presence of competing CD20-targeting antibodies. These data provide rationale for the clinical testing of CD20-TDB for the treatment of CD20-expressing B cell malignancies.

• Bispecific antibodies binding CD3 and CLL-1 deplete CLL-1 1 target cells in animal models.• An appropriately engineered CLL-1/CD3 bispecific antibody could be effective in treating AML.Acute myeloid leukemia (AML) is a major unmet medical need. Most patients have poor long-term survival, and treatment has not significantly changed in 40 years. Recently, bispecific antibodies that redirect the cytotoxic activity of effector T cells by binding to CD3, the signaling component of the T-cell receptor, and a tumor target have shown clinical activity. Notably, blinatumomab is approved to treat relapsed/refractory acute lymphoid leukemia. Here we describe the design, discovery, pharmacologic activity, pharmacokinetics, and safety of a CD3 T cell-dependent bispecific (TDB) full-length human IgG1 therapeutic antibody targeting CLL-1 that could potentially be used in humans to treat AML. CLL-1 is prevalent in AML and, unlike other targets such as CD33 and CD123, is not expressed on hematopoietic stem cells providing potential hematopoietic recovery. We selected a high-affinity monkey cross-reactive anti-CLL-1 arm and tested several anti-CD3 arms that varied in affinity, and determined that the high-affinity CD3 arms were up to 100-fold more potent in vitro. However, in mouse models, the efficacy differences were less pronounced, probably because of prolonged exposure to TDB found with lower-affinity CD3 TDBs. In monkeys, assessment of safety and target cell depletion by the highand low-affinity TDBs revealed that only the low-affinity CD3/CLL1 TDB was well tolerated and able to deplete target cells. Our data suggest that an appropriately engineered CLL-1 TDB could be effective in the treatment of AML. (Blood. 2017;129(5):609-618)

Trastuzumab emtansine (T-DM1) is an antibody-drug conjugate in clinical development for the treatment of human epidermal growth factor receptor 2 (HER2)-positive cancers. Herein, we describe a series of studies to assess T-DM1 absorption, distribution, metabolism, and excretion (ADME) in rats as well as to assess human exposure to T-DM1 catabolites. Following administration of unlabeled and radiolabeled T-DM1 in female Sprague Dawley rats as a single dose, plasma, urine, bile and feces were assessed for mass balance, profiling and identification of catabolites. In rats, the major circulating species in plasma was T-DM1, while DM1 concentrations were low (1.08 to 15.6 ng/mL). The major catabolites found circulating in rat plasma were DM1, [N-maleimidomethyl] cyclohexane-1- carboxylate-DM1 (MCC-DM1), and Lysine-MCC-DM1. These catabolites identified in rats were also detected in plasma samples from patients with HER2-positive metastatic breast cancer who received single-agent T-DM1 (3.6 mg/kg every 3 weeks) in a phase 2 clinical study. There was no evidence of tissue accumulation in rats or catabolite accumulation in human plasma following multiple dosing. In rats, T-DM1 was distributed nonspecifically to the organs without accumulation. The major pathway of DM1-containing catabolite elimination in rats was the fecal/biliary route, with up to 80% of radioactivity recovered in the feces and 50% in the bile. The rat T-DM1 ADME profile is likely similar to the human profile, although there may be differences since trastuzumab does not bind the rat HER2- like receptor. Further research is necessary to more fully understand the T-DM1 ADME profile in humans.

Chronic graft-versus-host-disease (cGVHD) is a major barrier to successful allogeneic hematopoietic stem cell transplantation (allo-HSCT), with highly variable clinical presentations. The pathophysiology of cGVHD remains relatively poorly understood. The utilization of murine models to study cGVHD encompasses experimental challenges distinct from those that have been successfully used to study acute GVHD (aGVHD). Nevertheless, despite these challenges, murine models of cGVHD have contributed to the understanding of cGVHD, and highlight its mechanistic complexity. In this article, insights into the pathophysiology of cGVHD obtained from murine studies are summarized in the context of their relevancy to clinical cGVHD. Despite experimental limitations, current and future models of murine cGVHD will continue to provide insights into the understanding of clinical cGVHD and provide information for new therapeutic interventions.

Interleukin 7 (IL-7) is critical in maintaining thymic-dependent and thymic-independent pathways of T-cell homeostasis. T-cell receptor (TCR) rearrangement excision circles (TRECs) have been used as markers for recent thymic emigrants (RTEs) in assessing human thymic function. To study the thymic and peripheral effects of IL-7 on RTEs, we measured TREC content and peripheral naive T-cell subsets and turnover in IL-7-treated mice. Short-term administration of IL-7 into thymus-intact mice resulted in increased total TREC numbers, consistent with RTE accumulation. Decreases in TREC frequency were attributable to dilution secondary to increased cell turnover. Significantly, IL-7 administration into thymectomized mice resulted in patterns of decreased TREC frequency and increased total TREC number similar to those in IL-7-treated thymus-intact mice. Distinct patterns of naive cell and RTE distribution among peripheral immune organs and altered expression of CD11a were observed following IL-7 treatment in thymus-intact and thymectomized mice. These results demonstrate (1) that total TREC number and not TREC frequency accurately reflects quantitative changes in RTEs; (2) that short-term IL-7 administration results in preferential accumulations of RTEs among peripheral immune organs, accounting for the increase in TRECs in the total peripheral lymphoid pool; and IntroductionIdentifying factors regulating thymic function is critical in developing strategies to enhance and/or preserve immune function in immunodeficient states associated with HIV infection and hematopoietic stem cell transplantation (HSCT). An important parameter of thymic function is the generation of T cells as recent thymic emigrants (RTEs). In the absence of phenotypic markers distinguishing RTEs, measurement of T-cell receptor (TCR) rearrangement excision circles (TRECs) has been developed for quantifying RTEs in humans. 1 TRECs are episomal DNA circles generated during TCR rearrangement. Most human TCR␣/␤ ϩ T cells contain TRECs formed by the rearrangement between the ␦-rec and J␣ loci during TCR␦ locus deletion and TCR ␣Ϫchain gene rearrangement. 2 Because episomal DNA does not replicate, TREC frequency (ie, TREC molecules per cell number) decreases with successive cell divisions, permitting its use not only as a marker of thymic function, but also as a quantitative marker for RTEs in the periphery.Interpretation of human TREC data expressed as a frequency is problematic because this value can be influenced by changes in peripheral cell turnover in the absence of altered RTE generation. [3][4][5][6] Importantly, sampling of human tissue for TREC analysis is almost exclusively limited to the peripheral blood. Even when quantitatively expressed, TREC numbers in blood cannot detect changes in RTE trafficking among multiple lymphoid compartments and extra-lymphoid tissue, nor does it provide information regarding the maintenance of RTEs in these sites. Thus, changes in TREC frequency in many cases may not reflect thymic function, and measuring TRECs in...