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. 2016 Jan;15(1):114-24.
doi: 10.1158/1535-7163.MCT-15-0400. Epub 2015 Oct 29.

Dual Agonist Surrobody Simultaneously Activates Death Receptors DR4 and DR5 to Induce Cancer Cell Death

Snezana Milutinovic  1 , Arun K Kashyap  2 , Teruki Yanagi  3 , Carina Wimer  1 , Sihong Zhou  4 , Ryann O'Neil  5 , Aaron L Kurtzman  6 , Alexsandr Faynboym  2 , Li Xu  2 , Charles H Hannum  7 , Paul W Diaz  8 , Shu-ichi Matsuzawa  1 , Michael Horowitz  2 , Lawrence Horowitz  2 , Ramesh R Bhatt  9 , John C Reed  10
Affiliations

Dual Agonist Surrobody Simultaneously Activates Death Receptors DR4 and DR5 to Induce Cancer Cell Death

Snezana Milutinovic et al. Mol Cancer Ther. 2016 Jan.

Abstract

Death receptors of the TNF family are found on the surface of most cancer cells and their activation typically kills cancer cells through the stimulation of the extrinsic apoptotic pathway. The endogenous ligand for death receptors 4 and 5 (DR4 and DR5) is TNF-related apoptosis-inducing ligand, TRAIL (Apo2L). As most untransformed cells are not susceptible to TRAIL-induced apoptosis, death receptor activators have emerged as promising cancer therapeutic agents. One strategy to stimulate death receptors in cancer patients is to use soluble human recombinant TRAIL protein, but this agent has limitations of a short half-life and decoy receptor sequestration. Another strategy that attempted to evade decoy receptor sequestration and to provide improved pharmacokinetic properties was to generate DR4 or DR5 agonist antibodies. The resulting monoclonal agonist antibodies overcame the limitations of short half-life and avoided decoy receptor sequestration, but are limited by activating only one of the two death receptors. Here, we describe a DR4 and DR5 dual agonist produced using Surrobody technology that activates both DR4 and DR5 to induce apoptotic death of cancer cells in vitro and in vivo and also avoids decoy receptor sequestration. This fully human anti-DR4/DR5 Surrobody displays superior potency to DR4- and DR5-specific antibodies, even when combined with TRAIL-sensitizing proapoptotic agents. Moreover, cancer cells were less likely to acquire resistance to Surrobody than either anti-DR4 or anti-DR5 monospecific antibodies. Taken together, Surrobody shows promising preclinical proapoptotic activity against cancer cells, meriting further exploration of its potential as a novel cancer therapeutic agent.

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Conflict of interest statement

CONFLICT OF INTEREST: We declare no conflict of interest.

Figures

Figure 1
Figure 1. Death receptor dual agonist Surrobody is a more potent inducer of tumor cell death than DR4 or DR5 monospecific antibodies
Various TRAIL-sensitive cancer cell lines were plated at a density of 750 cells/well in 384 wells. The next day cells were cultured with increasing concentrations of monospecific DR4 antibody (pink), monospecific anti-DR5 antibody (orange), the combination of anti-DR4 and anti-DR5 antibodies (black), death receptor dual agonist Surrobody (green), or TRAIL (blue) for 48 hr. Before addition to cells, all the antibodies and Surrobody were incubated for 5 minutes with 0.5× molar ratio of protein G to facilitate clustering. Relative cell viability was estimated from cellular ATP measurements using the Cell Titer Glo reagent (Promega), expressing data as % inhibition of cell survival (mean ± SEM; n= 4). Data are shown for Jurkat (A), Ramos (B), MDA-MB-231 (C), Colo-205 (D), Ovcar-3 (E) and Ovcar-5 (F) and HCT116 (G) cell lines.
Figure 2
Figure 2. Death receptor dual agonist Surrobody shows superior activity to monospecific anti-DR4 and anti-DR5 antibodies in 3D tumor spheroid culture model
MDA-MB-231 cells were cultured in ultralow attachment 96 well plates and grown for 8 days to reach a tumor spheroid diameter of ~750 um. The spheroids were treated with indicated DR agonists at 1000 ng/ml (~6nM) or with TRAIL at 20 ng/ml (~1nM) for 2 days and survival assessed by measuring cellular ATP (using Cell Titer Glow reagents). Before addition to the spheroids, all the monospecific antibodies and Surrobody were incubated for 5 minutes with 0.5× molar ratio of protein G to facilitate clustering. Data represent mean ± SD of three spheroids. Representative phase-contrast images of spheroids are shown. Treatment with DR agonists left a core of residual surviving cells surrounded by dead and dying cells.
Figure 3
Figure 3. Death receptor dual agonist inhibits tumor xenograft growth in mice
A. Three million Colo-205 cells were implanted into flanks of nude mice bilaterally and tumors were allowed to reach approximately 100 mm3 (5 days) before starting intravenous treatment with either PBS vehicle or 3 mg/kg of DR4 antibody, DR5 antibody or dual agonist Surrobody. The mice were treated on day 5, 8, 11 and 15 (indicated by arrows) for a total of four treatments and the tumor size was measured twice weekly. Mean tumor volumes and SEMs are shown for PBS treated (squares), DR4 treated (diamonds), DR5 treated (circles) and Surrobody treated (triangles) mice (10 mice per treatment group). The tumors below the palpitation limit were assigned a volume of 0 mm3. The PBS treated mice were sacrificed at day 18 when the tumors reached the endpoint volume of 1000 mm3, while the mice treated with DR4 antibody, DR5 antibody and Surrobody were sacrificed at 25 days post-implantation. B. The comparison of DR5 antibody- and Surrobody-treated tumor growth curves is shown at a higher volume resolution for easier comparison.
Figure 4
Figure 4. Death receptor dual agonist Surrobody is a potent inducer of caspase activation
MDA-MB-231 cells were cultured in 384 wells (2000 cells per well) and treated with increasing concentrations of DR agonists for 6 hr. The activity of CASPASE 8 (A) and CASPASE 3/7 (B) was assessed using CASPASE-8 Glo (contains IETD peptide) and ApoLive-Glo (contains DEVD peptide) reagents, respectively, from Promega, expressing data as relative luminescence units (RLU) generated per hour per 2000 cells (mean ± SEM; n= 4) (C) MDA-MB-231 cells were plated at a density of 70,000 cells/well and the next day they were treated for 3 h with DR agonists at 1 nM. Before addition to cells, all the monospecific antibodies and Surrobody were incubated for 5 minutes with 0.5× molar ratio of protein G to facilitate clustering. Cells were lysed into SDS-sample buffer and lysates were analyzed by SDS-PAGE/immunoblotting using antibodies specific for cleaved CASPASE-8, cleaved CASPASE-3, PARP, or beta-actin. Molecular weight markers are indicated in kilo-Daltons (kDa). Processed CASPASE-8 large (44/42 kDa) and small (18 kDa) subunits are shown. Processed CASPASE-3 large subunits (17/19 kDa) are shown.
Figure 5
Figure 5. Smac mimics and bortezomib sensitize TRAIL-resistant tumor cells
A. TRAIL resistant cells MDA-MB-468 were cultured at a density of 750 cells/well in 384 well plates. The next day cells were cultured with increasing concentrations of monospecific DR4 antibody (pink), monospecific anti-DR5 antibody (orange), the combination of anti-DR4 and anti-DR5 antibodies (black), death receptor dual agonist Surrobody (green), or TRAIL (blue) for 48 hr. Before addition to cells, all the monospecific antibodies and Surrobody were incubated for 5 minutes with 0.5× molar ratio of protein G to facilitate clustering. Relative cell viability was estimated from cellular ATP measurements using the Cell Titer Glo reagent (Promega), expressing data as % inhibition of cell survival (mean ± SEM; n= 4). B and C. The cells were cultured as in A, but they were pre-treated with 10 nM smac mimic AEG40730 or 1 nM bortezomib for 2h before addition of DR agonists. D. TRAIL resistant cells PC3 were cultured, treated and assayed for cell viability as in A. In E and F, PC3 cells were cultured, treated and assayed as in A, but they were pre-treated with 10 nM smac mimic AEG40730 or 1 nM bortezomib for 2h before addition of DR agonists.
Figure 6
Figure 6. DR4 and DR5 resistant tumor cell lines remain sensitive to death receptor dual agonists Surrobody
MDA-MB-231 cell lines resistant to DR4 (A), DR5 (B), DR4+DR5 (C), or TRAIL (D) were created by culturing them for 3 weeks in 500 ng/ml of anti-DR4 antibody, 1000ng/ml of anti-DR5 antibody, 250 ng/ml each of anti-DR4 and anti-DR5, or 50 ng/ml TRAIL. MDA-MB-231 cells that are deficient for either DR4 (E) or DR5 (F) receptors were generated using Crispr gene knockout technology. To assess the sensitivity of these resistant cells to various DR agonists, the cells were plated at a density of 750 cells/well into 384 wells and cultured with increasing concentrations of monospecific anti-DR4 antibody (pink), monospecific anti-DR5 antibody (orange), death receptor dual agonist Surrobody (bright green), or TRAIL (blue) for 48 hr. Before addition to cells, all the monospecific antibodies and Surrobody were incubated for 5 minutes with 0.5× molar ratio of protein G to facilitate clustering. Relative cell viability was estimated from cellular ATP measurements using the Cell Titer Glo reagent (Promega), expressing data as % inhibition of cell survival (mean ± SEM; n= 4).
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