PDF file, 84K, Excess soluble DR4, but not DR5, blocks the cytotoxic effect of agonist DR4 Atrimer complexes. Atrimer complex 1C9 was pre-incubated with or without soluble DR4-Fc or DR5-Fc for 1 hour before addition to Colo205 cells. Cell viability was measured at 48 hours using the ViaLight Plus kit.

PDF file, 63K, Combining DR4 Atrimer complexes with TRAIL. Sub-G1 content in HCT116 cells treated with 1G2 (50 ng/mL) or TRAIL (25 ng/mL) for 24 hours (n=2).

PDF file, 131K,Atrimer complexes are not cytotoxic to TRAIL-resistant tumor cell lines. Cell viability with candidate DR4 Atrimer complex treatment for 48 hours in (A) HepG2 and (B) MDA-MB-231 cell lines (n=2).

PDF file, 157K, Representative example of screening affinity matured Atrimer complexes. Single point cytotoxicity assays (A) we performed with 293 PEAK supernatants at 1:2 and 1:10 dilution using the Vialight Plus kit and compared to previously identified agonists. DR4 ELISA (B) was performed concurrently with the same supernatants diluted 1:20 and also compared to previously identified binders.

PDF file, 80K, Schematic representation of different library designs for primary and affinity matured selections. The native sequence of the tetranectin loop regions is shown at the top. Alterations in the primary and affinity maturation libraries are shown below where an x indicates a position that was randomized in each library.

PDF file, 55K, Surface DR4 knockdown. Surface DR4 by flow cytometry in HCT116 cells transfected with a scramble control or DR4 siRNA.

PDF file, 30K, Combining DR4 Atrimer complexes with TRAIL. Sub-G1 content in HCT116 cells treated with 1G2 (50 ng/mL) or TRAIL (25 ng/mL) for 24 hours (n=2).

PDF file, 53K, TRAIL-receptor expression in SW620 and HCT15 cell lines. Western blot analysis of TRAIL receptors in SW620 and HCT15 cells.

TRAIL is a trimeric protein that potently induces apoptosis in cancer cells by binding to the trimeric death receptors (DR4 or DR5). Death receptors are attractive therapeutic targets through both the recombinant TRAIL ligand as well as receptor agonist monoclonal antibodies. Although efficacy of the ligand is hampered by its short half-life, agonistic antibodies have a much longer half-life and have shown some clinical efficacy as antitumor agents. However, the efficacy of these antibodies may be limited by their bivalent nature that does not optimally mimic the trimeric ligand. To overcome limitations of currently used death receptor-targeting agents, we engineered trimeric proteins called Atrimer complexes that selectively bind DR4 and potently induce apoptosis in a variety of cancer cells. Atrimer complexes are based on human tetranectin, a trimeric plasma protein of approximately 60 kDa. Loop regions within the tetranectin C-type lectin domains (CTLD) were randomized to create a large phage display library that was used to select DR4-binding complexes. A panel of unique and potent agonist DR4 Atrimer complexes with subnanomolar affinity to DR4 and no detectable binding to DR5 or the decoy receptors was identified. Mechanism of action studies with a selected Atrimer complex, 1G2, showed that Atrimer complexes induce caspase-dependent and DR4-specific apoptosis in cancer cells while sparing normal human fibroblasts and, importantly, hepatocytes. This proof-of-principle study supports the use of alternative proteins engineered to overcome limitations of therapeutically desirable molecules such as TRAIL. Mol Cancer Ther; 11(10); 2087–95. 2012 AACR.

Abstract TRAIL is a trimeric protein that potently induces apoptosis in cancer cells by binding to the trimeric death receptors (DR4 or DR5). Death receptors are attractive therapeutic targets through both the recombinant TRAIL ligand as well as receptor agonist monoclonal antibodies. Although efficacy of the ligand is hampered by its short half-life, agonistic antibodies have a much longer half-life and have shown some clinical efficacy as antitumor agents. However, the efficacy of these antibodies may be limited by their bivalent nature that does not optimally mimic the trimeric ligand. To overcome limitations of currently used death receptor-targeting agents, we engineered trimeric proteins called Atrimer complexes that selectively bind DR4 and potently induce apoptosis in a variety of cancer cells. Atrimer complexes are based on human tetranectin, a trimeric plasma protein of approximately 60 kDa. Loop regions within the tetranectin C-type lectin domains (CTLD) were randomized to create a large phage display library that was used to select DR4-binding complexes. A panel of unique and potent agonist DR4 Atrimer complexes with subnanomolar affinity to DR4 and no detectable binding to DR5 or the decoy receptors was identified. Mechanism of action studies with a selected Atrimer complex, 1G2, showed that Atrimer complexes induce caspase-dependent and DR4-specific apoptosis in cancer cells while sparing normal human fibroblasts and, importantly, hepatocytes. This proof-of-principle study supports the use of alternative proteins engineered to overcome limitations of therapeutically desirable molecules such as TRAIL. Mol Cancer Ther; 11(10); 2087–95. ©2012 AACR.

Abstract TRAIL death receptor DR4 is a promising therapeutic target in oncology with expression in a wide variety of tumors. DR4 agonists, including TRAIL and monoclonal antibodies, can induce p53-independent apoptosis and are currently being evaluated in clinical trials in combination with chemotherapy. We aimed to surpass currently available therapeutics by developing trimeric death receptor agonists with properties expected to significantly exceed that of recombinant human TRAIL. The agonists can mimic the natural trimer-trimer interaction of the native ligand/receptor, but do not cross-react with the decoy receptors. Potent DR4 agonist AtrimersTM were engineered using human tetranectin, a trimeric human serum protein of 60 kDa, as a scaffold. A panel of unique DR4 binders was selected from novel phage libraries displaying the C-type lectin domain (CTLD) of tetranectin containing randomized loop sequences. DR4 Atrimers have sub-nanomolar affinity to recombinant DR4-Fc and showed no detectable binding to recombinant forms of DR5 or the decoy receptors. In vitro, the DR4 Atrimers efficiently killed DR4-positive cancer cell lines with sub-nanomolar EC50, but did not kill DR4-negative cell lines. DR4 Atrimers induced cell death of DR4-expressing tumor cells through the caspase pathway, but did not kill primary human B cells and hepatocytes. Interestingly, DR4 Atrimers have differential activities on various cell lines, and also vary in their degree of internalization. While some Atrimers show potent killing of Colo-205 and are rapidly internalized, other agonist Atrimers did not show measurable internalization. These unique properties open the possibility of developing potent naked Atrimers with prolonged half-lives due to lack of internalization, as well as leveraging rapidly internalizing DR4 Atrimers for design of Atrimer-drug conjugates. Further characterization of DR4 Atrimers is ongoing in Colo-205 xenograft models. DR4 agonist AtrimersTM with their superior potency and expected improvement in tumor penetration (vs. antibodies) represent a novel class of targeted cancer therapeutics for efficient induction of apoptosis and provide a promising approach for the treatment of a broad range of cancer types. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 1774. doi:10.1158/1538-7445.AM2011-1774

Traditional strategies for the identification of cell-surface cancer targets often fall short of their objective. For example, whole-cell panning of antibody libraries to isolate a diverse panel of antibodies directed against targets on cancer cells often identifies all immunogenic and/or abundant cell-surface antigens, not simply tumor-specific or tumor-associated antigens. Here we describe the use of stringent negative selection in combination with positive panning to increase tumor specificity and clinical relevance of selected antibodies. Sera from cancer cell-immunized mice showed strong binding to immunizing cancer cell lines but also cross-reacted strongly with human blood cells. Antisera blood cell binding was considerably decreased after stringent subtraction with human red blood cells (RBCs) and white blood cells (WBCs), yet cancer cell specificity was retained. In order to select for a higher percentage of clinically relevant antibodies for potential therapeutic use, stringent negative selection by RBC subtraction was employed in whole-cell panning of a disease-specific phage displayed antibody library on the prostate cancer cell line, PC-3. Isolated antibodies were found to bind to target antigens implicated in tumorigenicity and cancer cell migration and/or invasion, and included CD26, CDCP1, and the integrin complexes alpha2/beta1, alpha3/beta1, alpha5/beta1, and alpha6/beta4. Compared with traditional cell panning, this method considerably increased the selectivity of antibodies to tumor-associated antigens.

Through a whole-cell panning approach, we previously identified a panel of antibodies that bound to prostate cancer cell surface antigens. One such antigen, CUB domain-containing protein 1 (CDCP1), was recognized by monoclonal antibody 25A11 and is a single transmembrane molecule highly expressed in several metastatic cancers as well as on CD34(+) CD133(+) myeloid leukemic blast cells. We show CDCP1 expression on prostate cancer cell lines by real-time quantitative PCR (RT-qPCR), flow cytometry, and immunohistochemistry and on prostate cancer patient samples by RT-qPCR and immunohistochemical staining. In cell-based assays, antibody 25A11 inhibited prostate cancer cell migration and invasion in vitro. Further characterization showed that CDCP1 is internalized on antibody binding. When 25A11 was coupled to the cytotoxin saporin either directly or via a secondary antibody, both resulted in prostate cancer cell killing in vitro. In vivo targeting studies with an anti-CDCP1 immunotoxin showed significant inhibition of primary tumor growth as well as metastasis in a mouse xenograft model. These data provide support for continued evaluation of anti-CDCP1 therapy for potential use in cancer in primary and metastatic disease.

By using rational design, antibody fragments (Fabs) that mimic thrombopoietin (TPO) were created. A peptide with cMpl receptor-binding capability was grafted into different complementarity-determining regions of a fully human Fab scaffold. Functional presentation of the peptide was optimized by using phage display and cell-based panning. Select antibodies and fragments containing two grafted peptides were assayed for their ability to stimulate the cMpl receptor in vitro . Several candidates demonstrated agonist activity in an in vitro cMpl receptor signaling reporter assay, including Fab59, which was estimated to be equipotent to TPO. Fab59 additionally was able to effectively stimulate platelet production in normal mice. These rationally designed mimetic Fabs may provide a therapeutic intervention for thrombocytopenia while avoiding the potential generation of neutralizing antibodies to endogenous TPO. Furthermore, this study demonstrates a method by which short-lived linear peptides with binding activity may be converted to more stable and potent agonists capable of activating cell surface receptors.