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Tissue expression datasets

File contains Supplementary Methods

Supplementary Figure 3 Evaluation of DLL3 expression in tumor types other than SCLC.

Tables S1 to S6

Affinity measurements and antibody panels

Supplementary Figure 6 A, Expression of human CD25, CD69, PD-1, and 4-1BB on human CD4+ T cells isolated from mouse lungs harboring SHP-77 tumors 168 hours after treatment with AMG 757 or a control HLE BiTE® molecule. B, Expression of human CD25, CD69, PD-1, and 4-1BB on human CD8+ T cells isolated from mouse lungs harboring SHP-77 tumors 168 hours after treatment with either AMG 757 or a control HLE BiTE® molecule. *P < 0.05; **P < 0.01; *** P< 0.001

Purpose:Small-cell lung cancer (SCLC) is an aggressive neuroendocrine tumor with a high relapse rate, limited therapeutic options, and poor prognosis. We investigated the antitumor activity of AMG 757, a half-life extended bispecific T-cell engager molecule targeting delta-like ligand 3 (DLL3)—a target that is selectively expressed in SCLC tumors, but with minimal normal tissue expression.Experimental Design:AMG 757 efficacy was evaluated in SCLC cell lines and in orthotopic and patient-derived xenograft (PDX) mouse SCLC models. Following AMG 757 administration, changes in tumor volume, pharmacodynamic changes in tumor-infiltrating T cells (TILs), and the spatial relationship between the appearance of TILs and tumor histology were examined. Tolerability was assessed in nonhuman primates (NHPs).Results:AMG 757 showed potent and specific killing of even those SCLC cell lines with very low DLL3 expression (Conclusions:AMG 757 has a compelling safety and efficacy profile in preclinical studies making it a viable option for targeting DLL3-expressing SCLC tumors in the clinical setting.

This file contains Supplementary Tables 1-5

Supplementary Figure 1 DLL3 is differentially expressed in SCLC tumors and cell lines compared with normal tissues.

Supplementary Figure 2 Representative photomicrographs of DLL3 immunostaining (brown color) in normal human tissues using immunohistochemistry.

Supplementary Figure 5 A, DLL3 RNA expression levels in a panel of SCLC patient-derived xenograft (PDX) models compared with positive (NCI-H82) and negative (NCI-H460) control SCLC cell lines. Data are reported as relative quantification normalized to NCI-H460 cell line expression. B, DLL3 protein expression levels in a panel of SCLC PDX models compared with positive (NCI-H82) and negative (NCI-H460) control SCLC cell lines. GAPDH, glyceraldehyde 3-phosphate dehydrogenase.

Purpose:Despite advances in the treatment of multiple myeloma, new therapies are needed to induce more profound clinical responses. T-cell–redirected lysis triggered by bispecific antibodies recruiting T cells to cancer cells is a clinically validated mechanism of action against hematologic malignancies and CD38 is a tumor-associated antigen with near-universal expression in multiple myeloma. Thus, an anti-CD38/CD3 bispecific T-cell–recruiting antibody has the potential to be an effective new therapeutic for multiple myeloma.Experimental Design:Anti-CD38/CD3 XmAb T-cell–recruiting antibodies with different affinities for CD38 and CD3 were assessed in vitro and in vivo for their redirected T-cell lysis activity against cancer cell lines, their lower levels of cytokine release, and their potency in the presence of high levels of soluble CD38. Select candidates were further tested in cynomolgus monkeys for B-cell depletion and cytokine release properties.Results:AMG 424 was selected on the basis of its ability to kill cancer cells expressing high and low levels of CD38 in vitro and trigger T-cell proliferation, but with attenuated cytokine release. In vivo, AMG 424 induces tumor growth inhibition in bone marrow–invasive mouse cancer models and the depletion of peripheral B cells in cynomolgus monkeys, without triggering excessive cytokine release. The activity of AMG 424 against normal immune cells expressing CD38 is also presented.Conclusions:These findings support the clinical development of AMG 424, an affinity-optimized T-cell–recruiting antibody with the potential to elicit significant clinical activity in patients with multiple myeloma.

Supplementary FIgure 4 AMG 757 demonstrated antitumor activity in the WM266-4 melanoma xenograft model.

Purpose: Small-cell lung cancer (SCLC) is an aggressive neuroendocrine tumor with a high relapse rate, limited therapeutic options, and poor prognosis. We investigated the antitumor activity of AMG 757, a half-life extended bispecific T-cell engager molecule targeting delta-like ligand 3 (DLL3)—a target that is selectively expressed in SCLC tumors, but with minimal normal tissue expression. Experimental Design: AMG 757 efficacy was evaluated in SCLC cell lines and in orthotopic and patient-derived xenograft (PDX) mouse SCLC models. Following AMG 757 administration, changes in tumor volume, pharmacodynamic changes in tumor-infiltrating T cells (TILs), and the spatial relationship between the appearance of TILs and tumor histology were examined. Tolerability was assessed in nonhuman primates (NHPs). Results: AMG 757 showed potent and specific killing of even those SCLC cell lines with very low DLL3 expression ( Conclusions: AMG 757 has a compelling safety and efficacy profile in preclinical studies making it a viable option for targeting DLL3-expressing SCLC tumors in the clinical setting.

Purpose: Despite advances in the treatment of multiple myeloma, new therapies are needed to induce more profound clinical responses. T-cell–redirected lysis triggered by bispecific antibodies recruiting T cells to cancer cells is a clinically validated mechanism of action against hematologic malignancies and CD38 is a tumor-associated antigen with near-universal expression in multiple myeloma. Thus, an anti-CD38/CD3 bispecific T-cell–recruiting antibody has the potential to be an effective new therapeutic for multiple myeloma. Experimental Design: Anti-CD38/CD3 XmAb T-cell–recruiting antibodies with different affinities for CD38 and CD3 were assessed in vitro and in vivo for their redirected T-cell lysis activity against cancer cell lines, their lower levels of cytokine release, and their potency in the presence of high levels of soluble CD38. Select candidates were further tested in cynomolgus monkeys for B-cell depletion and cytokine release properties. Results: AMG 424 was selected on the basis of its ability to kill cancer cells expressing high and low levels of CD38 in vitro and trigger T-cell proliferation, but with attenuated cytokine release. In vivo, AMG 424 induces tumor growth inhibition in bone marrow–invasive mouse cancer models and the depletion of peripheral B cells in cynomolgus monkeys, without triggering excessive cytokine release. The activity of AMG 424 against normal immune cells expressing CD38 is also presented. Conclusions: These findings support the clinical development of AMG 424, an affinity-optimized T-cell–recruiting antibody with the potential to elicit significant clinical activity in patients with multiple myeloma.

Objective Systemic lupus erythematosus (SLE) is a complex autoimmune disease that is driven in part by chronic B and T lymphocyte hyperresponsiveness to self antigens. A deficiency of interleukin-21 (IL-21) or IL-21 receptor (IL-21R) in mice dramatically reduces inflammation and B and T cell activation in models of autoimmunity, including SLE. However, whether IL-21 is essential for the maintenance and amplification of preestablished inflammation has not been widely examined in various animal models. The purpose of this study was to examine the impact of novel mouse IL-21R neutralizing antibodies on recall responses to antigen challenge and on disease progression in the (NZB × NZW)F1 (NZB/NZW) mouse model of SLE. Methods Humoral and cellular immune responses to immunization with sheep red blood cells (SRBCs) were measured in mice dosed with IL-21R blocking antibodies. Progression of nephritis and markers of immune activation was monitored in NZB/NZW mice following different anti–IL-21R treatment regimens. Results IL-21R blockade specifically inhibited secondary IgG responses to SRBC immunization. In NZB/NZW mice, IL-21R blockade completely inhibited the onset of nephritis, which was associated with dramatic reductions in splenomegaly and in B cell and T cell activation. When administered to mice with preexisting disease, anti–IL-21R antibody halted the disease progression and mortality and reversed the nephritis in a subset of mice. Furthermore, treatment cessation was not followed by rapid reemergence of disease. Conclusion Our results highlight the importance of IL-21 in promoting humoral recall responses and in sustaining autoimmune inflammation.

The metabolic stability of a drug is an important property that should be optimized during drug design and development. Nitrogen incorporation is hypothesized to increase the stability by coordination of nitrogen to the heme iron of cytochrome P450, a binding mode that is referred to as type II binding. However, we noticed that the type II binding compound 1 has less metabolic stability at sub-saturating conditions than a closely related type I binding compound 3. Three kinetic models will be presented for type II binder metabolism; (1) Dead-end type II binding, (2) a rapid equilibrium between type I and II binding modes before reduction, and (3) a direct reduction of the type II coordinated heme. Data will be presented on reduction rates of iron, the off rates of substrate (using surface plasmon resonance) and the catalytic rate constants. These data argue against the dead-end, and rapid equilibrium models, leaving the direct reduction kinetic mechanism for metabolism of the type II binding compound 1.

Systemic lupus erythematosus (SLE) is a complex autoimmune disease that is driven in part by chronic B and T lymphocyte hyperresponsiveness to self antigens. A deficiency of interleukin-21 (IL-21) or IL-21 receptor (IL-21R) in mice dramatically reduces inflammation and B and T cell activation in models of autoimmunity, including SLE. However, whether IL-21 is essential for the maintenance and amplification of preestablished inflammation has not been widely examined in various animal models. The purpose of this study was to examine the impact of novel mouse IL-21R neutralizing antibodies on recall responses to antigen challenge and on disease progression in the (NZB × NZW)F1 (NZB/NZW) mouse model of SLE.Humoral and cellular immune responses to immunization with sheep red blood cells (SRBCs) were measured in mice dosed with IL-21R blocking antibodies. Progression of nephritis and markers of immune activation was monitored in NZB/NZW mice following different anti-IL-21R treatment regimens.IL-21R blockade specifically inhibited secondary IgG responses to SRBC immunization. In NZB/NZW mice, IL-21R blockade completely inhibited the onset of nephritis, which was associated with dramatic reductions in splenomegaly and in B cell and T cell activation. When administered to mice with preexisting disease, anti-IL-21R antibody halted the disease progression and mortality and reversed the nephritis in a subset of mice. Furthermore, treatment cessation was not followed by rapid reemergence of disease.Our results highlight the importance of IL-21 in promoting humoral recall responses and in sustaining autoimmune inflammation.